CN106011184A - Noncellular synthetic biology based preparation method of 2-phenylethanol and application - Google Patents

Abstract

The invention discloses a non-cell synthetic biology preparation method and application of 2-phenylethanol. The present invention utilizes the three recombinases ARO8, ARO10 and ADH1 to form a co-reaction system, and simplifies the Ehrlich pathway in microorganisms that requires multi-step continuous reactions and the participation of multiple enzymes to synthesize such a complex metabolic process as 2-phenylethyl alcohol. It is carried out in the same reaction system in vitro, thereby reducing environmental pollution and shortening the reaction process. Further, the present invention uses glutaraldehyde as a cross-linking agent to carry out carrier-free co-immobilization of three recombinant single enzymes to prepare co-cross-linked enzyme aggregates (Combi-CLEAs), and synthesize target products in vitro, thereby achieving the above-mentioned beneficial effects On the basis of further realizing the recycling of enzymes and reducing production costs, the present invention fills in the technical gap in the preparation of 2-phenylethanol in vitro by multiple enzymes, and provides important references for the biological preparation of 2-phenylethanol.

Description

Preparation method and application of 2-phenylethanol by non-cell synthetic biology

technical field

The invention relates to the technical field of biosynthesis, more specifically, to a method for preparing 2-phenylethanol through non-cell synthetic biology and its application.

Background technique

2-Phenylethanol (2-Phenylethanol, 2-PE) is also called β-Phenylethanol (β-Phenylethanol), its chemical name is 2-Phenylethanol (2-Phenylethyl alcohol, 2-PEA), its molecular weight is 122.16, and its structural formula is:

2-Phenylethyl alcohol is an aromatic alcohol with a light and delicate smell of roses. It naturally exists in the essential oils of many plants. 2-Phenylethyl alcohol has a soft and sweet aroma and is very popular. It is widely used in the fields of food, cosmetics, tobacco and daily chemical products. It is not only the basic component of all rose fragrances, but also has become a variety of fragrances because of its synergistic and synergistic effects. The components required by the formula are spice substances with high value; in medicine, 2-phenylethanol is a traditional antibacterial agent.

At present, the global annual output of 2-phenylethanol is nearly 10,000 tons, which are basically chemically synthesized from cheap chemical raw materials benzene or styrene, and only a small part is extracted from rose oil. Most of the raw materials used in chemical synthesis have great harm to human health and the environment, and the synthesized product 2-phenylethanol often contains impurities that have bad odor and are difficult to remove, such as biphenyl, β-chloroethylbenzene, chloroethanol, etc. , seriously affecting product quality. With the change of consumption concept, people are more and more advocating "green", "natural" food and additives. In European and American countries, flavorings and fragrances that can be labeled as "natural" must be extracted from natural materials by physical methods and produced by enzymatic or microbial fermentation. If natural 2-phenylethanol is extracted from roses, only 1 kg of rose essential oil can be extracted per 5 t of rose flowers, and the production cost is extremely expensive and cannot be produced on a large scale. Internationally, natural 2-phenylethanol is basically produced by microbial transformation of L-phenylalanine, mainly concentrated in France, Germany, Japan and other developed countries, but the production volume is far from meeting the consumption demand.

An important production route of 2-phenylethanol is the use of microbial fermentation. The raw materials and synthesis process of this route are green and environmentally friendly, with mild reaction conditions, safe products, short production cycle, and large-scale production. There are three pathways for the biosynthesis of 2-phenylethanol in microorganisms: the first pathway is the shikimic acid pathway of de novo synthesis, which widely exists in microorganisms, but the metabolic pathway is long, has many branches, and has multiple inhibitions. The yield is extremely low; the second is the decarboxylation of L-phenylalanine to generate phenethylamine, and the reduction of deamination to generate 2-phenylethanol, which rarely occurs; the third is the Ehrlich pathway, through which L-phenylalanine Ammonia reacts to generate phenylpyruvate, then decarboxylates to form phenylacetaldehyde, and finally reduces to 2-phenylethanol. The Ehrlich pathway is the preferred pathway for microorganisms to synthesize 2-phenylethanol, which is carried out under the action of three enzymes, namely, aromatic aminotransaminase, phenylpyruvate decarboxylase and alcohol dehydrogenase. In-depth research was carried out, focusing on the structural genes encoding the three enzymes and their transcription and regulation.

The metabolism of all substances in the biological world is completed by the joint action of multiple enzymes. These different enzymes can self-assemble in a highly ordered manner to form multienzyme complexes (multienzyme complexes, MECs) to participate in the reaction, by improving the transport of intermediate products between different enzymes Concentration, to achieve efficient catalysis (Lopez-Gallego et al., 2010; Schoffelen et al., 2012; Zhang, 2011). However, after multi-enzyme co-reaction, there are problems such as difficulty in enzyme separation and recovery, low reuse rate, and poor stability. There are also many shortcomings such as low yield of 2-phenylethanol, increased cost, and affected aroma quality.

So far, no relevant research and literature reports on the preparation of 2-phenylethanol in vitro by multi-enzymes have been found.

Contents of the invention

The technical problem to be solved in the present invention is aimed at the deficiency of the existing non-cellular synthetic biology preparation technology of 2-phenylethanol. It intends to use the advantage of the high-efficiency catalysis of the multi-enzyme complex to artificially prepare a multi-enzyme reaction system in vitro, which will be highly efficient. The three enzymes and three recombinant enzymes expressing the Ehrlich pathway were placed in a suitable reaction system, and the optimal reaction system for the in vitro co-reaction of the three recombinant enzymes to prepare 2-phenylethanol was constructed. The target product 2-phenylethanol was prepared, and a method for the preparation of 2-phenylethanol by non-cell synthetic biology was established.

The purpose of the present invention is achieved through the following technical solutions:

Provided is a non-cell synthetic biological preparation method of 2-phenylethanol, which is catalyzed by a co-catalyzed system composed of recombinant transaminase IARO8, phenylpyruvate decarboxylase ARO10 and alcohol dehydrogenase ADH1 in the same reaction system. Alanine is converted into 2-phenylethanol via phenylpyruvate and phenylacetaldehyde; wherein, in the reaction system, L-phenylalanine and oxaloacetate are used as substrates, and the recombinases ARO8, ARO10 and ADH1 press The enzyme activity is determined by the ratio of 1:0.25:1, 1:1:1, 1:0.5:1 or 1:0.25:2 (U/U/U). In a reaction system with a Mg ²⁺ concentration of 1.0-5mM and a water bath temperature of 30-40°C, after 4-7 minutes of reaction conversion, the reaction is terminated with an equal volume of acetonitrile to obtain 2-phenylethanol.

The present invention creatively summarizes and obtains a free multi-enzyme system composed of recombinant aromatic transaminase I, phenylpyruvate decarboxylase and alcohol dehydrogenase, which can use L-phenylalanine and oxaloacetic acid as substrates to prepare 2- Phenylethyl alcohol has the advantages of high catalytic efficiency, simple operation, and no need to purify intermediate products.

Under the conditions of the free multi-enzyme system, the molar ratio of L-phenylalanine and oxaloacetate is preferably 0.75:1.

Preferably ARO8, ARO10 and ADH1 are mixed in a ratio of 1:0.25:1 (U/U/U). Preferably the Mg ²⁺ concentration is 1.0-2.5 mM; more preferably 1.5 mM. Preferably, the temperature of the water bath is 33-40°C; more preferably 37°C, preferably reacting in the water bath at 37°C for 5 minutes. Preferably, the pH value is 5.5-6.5; more preferably, the pH value is 6. Most preferably, under the conditions of the free multi-enzyme system, the molar ratio of L-phenylalanine and oxaloacetate is 0.75:1, and the recombinase ARO8, ARO10 and ADH1 are 1:0.25:1 according to the enzyme activity Proportion, the addition amount is 100 μL, the pH value is 6.0, the Mg ²⁺ concentration is 1.5 mM, and the reaction is terminated with an equal volume of acetonitrile in a 37°C water bath for 5 minutes.

In practical application, the multi-enzyme system has not completely solved the problems of insufficient yield caused by unstable properties of intermediates, certain difficulties in separation and recovery of enzymes after the reaction, low reutilization rate, and poor stability. In order to further improve In the technical solution of the present invention, the present invention further immobilizes the three kinds of recombinant enzymes in the same carrier at the same time, improves the stability and reutilization rate of the enzymes, and increases the local concentration of the reaction to increase the yield of the product. In the actual research and experiment process, only the optimal temperature, optimal pH, enzyme co-immobilization ratio, immobilization sequence and other factors' effects and mechanisms as well as the rules and relationships of mutual influence and restriction can be determined scientifically. Only then can the co-immobilization technical scheme of the recombinase in the above 3 be summarized.

Therefore the present invention provides a kind of preferred technical scheme is first described recombinant transaminase I ARO8, phenylpyruvate decarboxylase ARO10 and alcohol dehydrogenase ADH1 to make cocatalytic system Combi-CLEAs by co-immobilization method, then utilize Combi-CLEAs to L-phenylalanine is catalytically converted to 2-phenylethanol via phenylpyruvate and phenylacetaldehyde.

Under the conditions of the co-immobilization technical scheme, wherein, in the reaction system, L-phenylalanine and oxaloacetic acid are used as substrates, the preferred pH value is 5.0, the ^Mg2+ concentration is 1.0-5mM, and the water bath temperature is 40°C, the reaction conversion time is 5min.

The preparation method of the co-catalyst system Combi-CLEAs is as follows: ARO8, ARO10 and ADH1 are mixed according to the ratio of 1:1:1, 1:0.5:1 or 1:0.25:1, and then precipitated by precipitant ammonium sulfate to form poly For enzyme aggregates, add glutaraldehyde to cross-link the multi-enzyme aggregates to prepare a multi-enzyme co-immobilization system; wherein, the saturation of ammonium sulfate is 60-90%, the pH value is 4-6, and the addition of glutaraldehyde The amount is determined according to its final concentration of 0.05-0.25% (V/V).

Preferably, the saturation of the precipitating agent ammonium sulfate is 70%. Preferably, the pH value of the precipitant ammonium sulfate is 5. Preferably, the enzyme ratio of ARO8, ARO10 and ADH1 is 1:0.5:1. Preferably, the added amount of glutaraldehyde is determined according to its final concentration of 0.1% (V/V). Preferably, the crosslinking temperature is 20-35°C, more preferably 30°C. Preferably, the cross-linking time is 30-150 min, more preferably 120 min.

The Combi-CLEAs of the present invention have excellent repeatability, and can still maintain 66% of the initial enzyme activity after repeated use for 4 times.

The present invention has the following beneficial effects:

The present invention provides a new non-cellular synthetic biological preparation method of 2-phenylethanol. Three kinds of recombinant enzymes are used to form a co-reaction system, and the Ehrlich pathway in microorganisms requires multi-step continuous reactions and the participation of multiple enzymes to synthesize 2-phenylethanol. The complex metabolic process of phenethyl alcohol is simplified to be carried out in the same reaction system in vitro, thereby reducing environmental pollution and shortening the reaction process. Further, the present invention uses glutaraldehyde as a cross-linking agent to carry out carrier-free co-immobilization of three recombinant single enzymes to prepare co-cross-linked enzyme aggregates (Combi-CLEAs), and synthesize target products in vitro, thereby achieving the above-mentioned beneficial effects On the basis of further realizing the recycling of enzymes, the production cost is reduced. The invention fills up the technical blank of preparing 2-phenylethanol in vitro by multiple enzymes, provides important reference for the biological preparation of 2-phenylethanol, and provides innovative ideas for preparing natural 2-phenylethanol.

Description of drawings

Fig. 1 is a schematic diagram of the route of synthesizing 2-phenylethanol from L-phenylalanine catalyzed by multi-enzyme system. Figure 2 is the result of high performance liquid chromatography detection of 2-phenylethanol after adding three enzymes in sequence. Fig. 3 The result of high performance liquid chromatography detection of 2-phenylethanol in three enzyme co-reactions. Fig. 4 Analysis results of the reaction product 2-phenylethanol in the three-enzyme co-reaction by gas chromatography-mass spectrometry. Fig. 5 The result of ion mass spectrometry analysis of the reaction product 2-phenylethanol in the three-enzyme co-reaction. Figure 6 The influence of the reaction time of the three-enzyme co-reaction on the multi-enzyme activity. Figure 7 The effect of the multi-enzyme ratio on the reaction of the three-enzyme co-reaction. Fig. 8 Effect of Mg ²⁺ concentration in the three-enzyme co-reaction on multi-enzyme activity. Figure 9 The influence of the temperature of the three-enzyme co-reaction on the multi-enzyme activity. Figure 10 The influence of the pH value of the three-enzyme co-reaction on the multi-enzyme activity. Figure 11 The effect of the substrate concentration of the three-enzyme co-reaction on the multi-enzyme activity. Fig. 12 The results of high-performance liquid chromatography determination of 2-phenylethanol under the conditions of optimized three-enzyme co-reaction. Figure 13 The effect of precipitant on the activity of multi-enzyme aggregates in the co-immobilization of three enzymes. Figure 14 The effect of ammonium sulfate saturation on the activity of multi-enzyme aggregates in the co-immobilization of three enzymes. Figure 15 The effect of pH on the activity of multi-enzyme aggregates in the co-immobilization of three enzymes. Figure 16 Effect of multi-enzyme ratio on the activity of Combi-CLEAs in three-enzyme co-immobilization. Figure 17 The effect of glutaraldehyde concentration on the activity of Combi-CLEAs in the co-immobilization of three enzymes. Figure 18 Effect of cross-linking temperature on the activity of Combi-CLEAs in the co-immobilization of three enzymes. Figure 19 Effect of cross-linking time on the activity of Combi-CLEAs in three-enzyme co-immobilization. Figure 20 Effect of temperature on co-immobilized multienzyme and free multienzyme activity in three enzyme co-immobilization. Figure 21 Temperature stability of co-immobilized multienzyme and free multienzyme. Figure 22 Effect of pH on co-immobilized multienzyme and free multienzyme activity. Figure 23 Effect of substrate ratio on co-immobilized multienzyme and free multienzyme activity. Figure 24 Storage stability of co-immobilized multienzyme and free multienzyme. Figure 25 Operational stability of co-immobilized multienzymes. Fig. 26 Cloning and identification results of Aro8 gene. Fig. 27 Cloning and identification results of Aro10 gene. Fig. 28 Cloning identification results of ADH1 gene. Fig. 29 Electrophoresis analysis diagram of expression vector p32-YT0801-Aro8. Fig. 30 Electrophoresis analysis diagram of expression vector p32-YT0801-Aro10. Fig. 31 Electrophoresis analysis diagram of expression vector p32-DV10-Adh1.

detailed description

The present invention will be further described below in conjunction with specific examples. The following examples are for illustrative purposes only, and should not be construed as limiting the present invention. Unless otherwise specified, the raw materials used in the following examples are commercially available or commercially obtained raw materials, and unless otherwise specified, the methods and equipment used in the following examples are methods and equipment routinely used in the art.

Example 1

Main reagents: recombinant aromatic aminotransferase I, phenylpyruvate decarboxylase and alcohol dehydrogenase, conventional commercially available or constructed with reference to prior art documents or constructed with reference to the method of Example 5 of the present invention. Chemical standards: L-phenylalanine, oxaloacetic acid, and 2-phenylethanol were purchased from Sigma-Aldrich, USA. All other chemical reagents were domestic analytically pure or chromatographically pure. Main instruments: digital display constant temperature water bath HH-2 (Jiangsu Changzhou Guohua Electric Co., Ltd.), constant temperature metal bath CHB-100 (Jiangsu Hangzhou Bioer Technology Co., Ltd.), precision pH meter PHS-3C (Shanghai Precision Scientific Instrument Co., Ltd. ), Mettler-Toledo electronic balance AB204-N (Shanghai Mettler-Toledo Instrument Co., Ltd.), ultrasonic breaker (Ningbo Science and Technology Co., Ltd.), liquid chromatography (Shanghai Tianmei Company), gas chromatography mass spectrometer GC-MSQP2010Plus (Japan Island Tsu Corporation).

Figure 1 shows the conversion of L-phenylalanine to 2-phenylethanol. In the same reaction system, L-phenylalanine is converted into 2-phenylethanol through phenylpyruvate and phenylacetaldehyde through the conversion of recombinant transaminase IARO8, phenylpyruvate decarboxylase ARO10 and alcohol dehydrogenase ADH1 in sequence. All reactions were 0.5 mL in buffer containing 0.01 mM pyridoxal phosphate, 0.2 mM thiamine pyrophosphate and 0.1 mM NADPH.

1. Activity determination of multi-enzyme co-reaction: using L-phenylalanine and oxaloacetate as substrates, HPLC detection of catalytic activity shared by multiple enzymes. The total volume of the reaction system is 500μL, containing: 10mM L-phenylalanine, 10mM oxaloacetate, 0.5mM Mg ²⁺ , 0.01mM pyridoxal phosphate, 0.1mM thiamine pyrophosphate, 0.1mM NADPH, ARO8, ARO10 and 10 μL each of the three ADH1 recombinases. Add enzyme solution and mix evenly, start timing, react at 33°C for 3 minutes, add 500 μL of acetonitrile to terminate the reaction, filter the supernatant, and measure the content of 2-phenylethanol by HPLC.

The reaction was divided into two groups: the first group added three enzymes sequentially according to the action time of the single enzyme in Chapter 3, that is, ARO8 was added for 10 minutes, ARO10 was added to react for 30 minutes, and finally ADH1 was added for 3 minutes; the second group was added with three recombinases at the same time , and react for 3 minutes after mixing evenly. Determination of the content of 2-phenylethanol in the two groups.

Enzyme activity definition of multi-enzyme co-reaction: under optimal conditions, the amount of 2-phenylethanol catalyzed by per milliliter of multi-enzyme mixture per minute. The unit of enzyme activity is μmol/(min·mL).

2. Determination conditions of 2-phenylethanol:

Instrument: Tianmei liquid chromatography; Chromatographic column: Phenomenex luna C18 250mm×4.6mm; Reference method, mobile phase is acetonitrile-water (50/50, V/V), filtered through a 0.22μm microporous membrane and ultrasonicated before use Degassing; the detection wavelength is 216nm; the flow rate is 1.0mL/min; the column temperature is room temperature. 20μl quantitative loop quantification.

3. GC-MS detection conditions of 2-phenylethanol

The instrument adopts GC-MSQP2010PlusD, column (0.25mm ID, 30m), split injection. Heating program: injection temperature 250°C, initial temperature 40°C, hold for 5 minutes, raise to °C at 4.0°C/min, then heat to 250°C at 30.0°C/min and hold for 5 minutes.

4. Determination of reaction conditions and test methods for multi-enzyme co-reaction

(1) Multi-enzyme co-reaction time: The enzyme activities of the multi-enzymes were measured at different reaction times, and the measured enzyme activities of the recombinant enzymes ARO8, ARO10, and ADH1 were: 0.87, 0.22, and 1.75 μmol/(min mL) (Measurement method refers to existing routine). Taking the maximum enzyme activity as 100%, the result is expressed by relative enzyme activity.

(2) Enzyme ratio of multi-enzyme co-reaction: When designing multi-enzyme ratio, fix the amount of one of the recombinant enzymes, such as the amount of fixed ARO8, and match the enzymes in different ratios according to the enzyme activity: 1:1:1, 1:0.5:1, 1:0.25:2, 1:0.25:1, 1:0.25:0.5, 1:0.15:0.5 and 1:0.15:0.25, respectively measure the enzyme activity under different ratios, the maximum enzyme activity 100%, the result is expressed by relative enzyme activity.

(3) Concentration of Mg ^{2+ in multi-enzyme co-reaction: set different final concentrations of Mg 2+ in} the reaction system, and measure the enzyme activity under different conditions. For example, the final concentration of Mg ²⁺ is set to 0, 0.3, 0.5, 0.6, 1.0, 1.5 and 2.0 mM respectively, and the results of enzyme activity determination under different conditions are expressed by relative enzyme activity, and the maximum enzyme activity determined is 100%.

(4) Optimum temperature for multi-enzyme co-reaction: measure the enzyme activity of multi-enzyme at 30°C, 33°C, 35°C, 37°C and 40°C respectively, with the maximum enzyme activity as 100%. Live representation.

(5) Optimum pH for multi-enzyme co-reaction: Add the three recombinant enzymes ARO8, ARO10, and ADH1 in different ratios according to enzyme activity into 0.1M sodium phosphate buffer with different pH, mix well, and place at 4°C for 30 minutes. React under optimal conditions and measure enzyme activity. The pH system is 5.0, 5.5, 6.0, 6.5 and 7.0 respectively, and the results are expressed by relative enzyme activity, taking the maximum enzyme activity determined as 100%.

(6) The influence of substrate concentration on enzyme activity: measure the multienzyme enzyme activity under the different molar ratio conditions of substrate L-phenylalanine and oxaloacetate, the result is represented by relative enzyme activity, take maximum enzyme activity as 100%. In order to avoid repeating, this embodiment is carried out with the molar ratio of the substrate L-phenylalanine and oxaloacetate being 0.2:1, 0.5:1, 0.75:1, 1:1, 1.5:1 and 2:1 respectively illustrate. All independent experiments were repeated at least three times.

5. Test results:

(1) Activity identification of multi-enzyme co-reaction: In this example, the substrates L-phenylalanine and oxaloacetate in the enzymatic reaction of ARO8 are used as the initial substrates, and the catalyzed product 2-phenylethanol of ADH1 is used as the final product , react together in the same system, and detect the catalytic activity of the three enzymes to verify the feasibility of synthesizing 2-phenylethanol in vitro. Two groups were set up for the reaction at the same time: the first group added three enzymes sequentially according to the action time of single enzyme (the reaction time of ARO8 at 30°C was 10 min, and the action time of ARO10 and ADH1 were 30 min and 3 min respectively); the second group simultaneously Add three recombinases and react for 3 min. With reference to 2-phenylethanol standard substance, detect reaction product with HPLC, the first group only has substrate peak and other intermediate product miscellaneous peaks, does not detect the existence of reaction product, and analysis is because the intermediate product that forms is unstable; A small peak appeared at the same position as the peak time of the standard 2-phenylethanol, that is, at 4.650 min, and the substrate peak area decreased, indicating that the system produced the target product 2-phenylethanol with a concentration of 11.51 μM, as shown in the figure 2 and Figure 3. It shows that ARO8, ARO10 and ADH1 have catalytic activity when they co-exist in the reaction system.

The reaction product of the three-enzyme co-reaction system was further detected by GC-MS, and the presence of the target product 2-phenylethanol was found at 32.563min, as shown in Figure 4. The ion fragment analysis was carried out on this peak, and by comparison, It was further confirmed that the product was 2-phenylethanol, as shown in Figure 5. The results showed that the co-reaction of ARO8, ARO10 and ADH1 could successfully catalyze the conversion of the substrate L-phenylalanine into 2-phenylethanol.

(2) Accurate determination of the time of multi-enzyme co-reaction

The reaction time of the single-enzyme system is different, the reaction time of ARO8 is 10min at 30℃, while the action time of ARO10 and ADH1 is 30min and 3min respectively. In the present invention, the properties of the intermediate products obtained by analyzing the co-reaction of multiple enzymes are unstable. If the enzyme action time is too long, it is easy to polymerize, thicken or even be oxidized. Therefore, it is necessary to investigate the optimal co-reaction time of the multi-enzyme system.

Using equimolar L-phenylalanine and oxaloacetic acid as substrates, the concentration of Mg ²⁺ in the reaction system is 0.5mM, adding three recombinant enzymes with equal enzyme activities, reacting at 33°C for different times, using the HPLC method The content of 2-phenylethanol in the reaction liquid was measured, and the relative enzyme activity was calculated, and the reaction time of the three-enzyme system was 5 min, as shown in FIG. 6 .

(3) Precise determination of the enzyme ratio of the multi-enzyme co-reaction

In the multi-enzyme reaction system, L-phenylalanine is sequentially catalyzed by transaminase I, phenylpyruvate decarboxylase and alcohol dehydrogenase to generate 2-phenylethanol, because the enzyme activities and enzymatic reaction speeds of the three enzymes are different , in order to maximize the conversion rate of L-phenylalanine, this embodiment further determines the optimal ratio of the three enzymes. Since transaminase Ⅰ ARO8 is the first enzyme in the reaction, when discussing the ratio of multiple enzymes, the enzyme amount of ARO8 was fixed, and the three enzymes were matched in different proportions according to the enzyme activity, and a total of 7 reactions with different proportions were determined. All reaction systems were 0.5 mL, containing 0.01 mM pyridoxal phosphate, 0.2 mM thiamine pyrophosphate, 0.1 mM NADPH and 0.5 mM Mg ²⁺ . After bathing in water at 33°C for 5 minutes, the content of 2-phenylethanol was measured by HPLC, and the relative enzyme activity was calculated. Fig. 7 is a graph showing the main experimental results of the present invention. If ARO8 is insufficient, the phenylpyruvate concentration that produces is low, and the productive rate of 2-phenylethanol is also low; If ARO8 is excessive and ARO10 and ADH1 are insufficient, then the concentration of phenylpyruvate increases, easily causes product inhibition, to L-phenylpropanol The conversion rate of amino acid is affected; therefore, the appropriate multi-enzyme ratio is conducive to the formation of products. It can be seen from Figure 7 that the activity of the three enzymes in the ratio of 1:0.25:1 is the highest according to the enzyme activity; while the enzyme activities in the ratio of 1:1:1, 1:0.5:1 and 1:0.25:2 respectively are higher than 1 :0.25:1 is next, but it can also guarantee sufficient activity, it just means that the ARO10 and ADH1 enzymes in these three groups are saturated or excessive; while 1:0.25:0.5, 1:0.15:0.5 and 1:0.15:0.25 Enzyme activity is lower when proportioning, indicating that the amount of ADH1 and ARO10 enzymes in these three groups is insufficient. Therefore, when multiple enzymes co-react, ARO8, ARO10 and ADH1 are 1:1:1 (U/U/U), 1:0.5:1 (U/U/U) and 1:0.25:2 (U/U/U ) or 1:0.25:1 (U/U/U) is preferred, and further, 1:0.25:1 (U/U/U) is the optimal ratio.

(4) Mg ²⁺ concentration determination test for multi-enzyme co-reaction

The present inventor studies and analyzes because Mg ²⁺ is a magnesium ion with two positive charges, which can participate in the catalytic activity of the enzyme, so that Mg ²⁺ has a stronger catalytic effect than acid, which is beneficial to the formation of the enzyme active center and the formation of the spatial structure. Stablize. In addition, Mg ²⁺ can promote the reaction by electrostatically shielding negative charges. But at the same time, a higher concentration of Mg ²⁺ has an inhibitory effect on the enzyme. The inventors' research analysis believes that Mg ²⁺ forms a coordination bond with the electron-rich center, which blocks the combination of the substrate and the active center of the enzyme. At the same time, a large number of previous experiments by the inventors concluded that the concentration of Mg ²⁺ in the ARO8 single-enzyme catalytic system is 0.6mM, the Mg ²⁺ in the ARO10 system is 0.5mM, and 5mM Mg ²⁺ is beneficial to the activity of ADH1, which further confirms the Inventor's research analysis conclusion. Therefore, the optimal concentration range of Mg ²⁺ must be considered in the multi-enzyme reaction system composed of a certain proportion. According to the suitable range of Mg ²⁺ concentration for the action of the three recombinant single enzymes, several groups of reactions with different Mg ²⁺ concentrations were determined. Figure 8 shows the reaction results of 6 groups of different Mg ²⁺ concentrations. The ratio of ARO8/ARO10/ADH1 according to the enzyme activity was 1:0.25:1, bathed in water at 33°C for 5 minutes, and the relative enzyme activity of each group was measured. It can be seen from Figure 8 that when Mg ²⁺ is not added to the reaction system, the recombinant enzymes ARO8 and ARO10 cannot exert their catalytic activity; when the concentration of Mg ²⁺ is in the range of 1.0-2.5mM, the multi-enzyme activity has always maintained a high and stable level , no obvious change; when the Mg ²⁺ concentration continued to increase to 5.0mM, the enzyme activity decreased slightly, indicating that the enzyme activity was inhibited by Mg ²⁺ ; when the Mg ²⁺ concentration was 1.5mM, the catalytic activity of the multi-enzyme system maximum.

(5) Optimum temperature determination test for multi-enzyme co-reaction

Test summary, the optimum temperature of the recombinase ARO8, ARO10 and ADH1 is 30°C, 37°C and 35°C respectively, and Figure 9 shows the change of enzyme activity at different temperatures. In the multi-enzyme reaction system, the ratio of ARO8/ARO10/ADH1 was 1:0.25:1 according to the enzyme activity, the concentration of ^Mg2+ was 1.5mM, the reaction was terminated by adding an equal volume of acetonitrile after 5min in water bath.

The results showed that within the temperature range of 30-40°C, the effect of temperature on the activity of the three-enzyme system was not obvious. 30°C is the optimum temperature for ARO8, but it slightly affects the activities of ARO10 and ADH1, and the relative enzyme activity of the multi-enzyme system is 75%. 65%. The optimum reaction temperature shared by multiple enzymes is consistent with that of ARO10, which is both 37°C. The analysis concludes that ARO10 is the key enzyme of the three-enzyme system and plays a stronger role in the temperature range of 30-40°C.

(6) Optimum pH determination test for multi-enzyme co-reaction

The optimum pH of test summary three recombinant single enzymes has difference, is respectively 7.0, 6.0, 6.0, so the inventor analyzes, the optimum pH of the multi-enzyme system that three forms, can be affected by the pH difference of three kinds of single enzyme systems. Influence, for this reason, carried out different experiments, adjusted the pH in the range of pH5.5～7.5, studied the optimal pH of the three-enzyme system. The test results in Figure 10 are illustrated under the conditions of Mg ²⁺ concentration of 1.5mM, ARO8/ARO10/ADH1 ratio of 1:0.25:1 according to enzyme activity, and 37°C water bath for 5 minutes. Summary of Experiments It can be seen from Figure 10 that the optimum pH range of the multi-enzyme system is 5.5-6.5, and the relative enzyme activity is the largest at pH 6.0. In the pH range of 5.5-7.5, the catalytic activity of the multi-enzyme does not decrease significantly, and the lowest relative activity can maintain about 66%.

(7) Effect test of substrate concentration on enzyme activity

The study concluded that the concentration ratio of the substrates L-phenylalanine and oxaloacetate will affect the conversion rate of ARO8 to the substrate, and then affect the conversion rate of ARO10 and ADH1. Therefore, under the condition that the substrate oxaloacetate concentration was kept constant at 10 mM, different molar ratios were used for the substrates L-phenylalanine and oxaloacetate, and after 5 min under the optimal conditions of the three-enzyme co-reaction , measure the content of 2-phenylethanol, calculate relative enzyme activity, the result is shown in Figure 11. As can be seen from Figure 11, when the mol ratio of L-phenylalanine and oxaloacetate is lower than 0.75: 1, the relative enzymatic activity of multi-enzyme co-reaction increases with the increase of L-phenylalanine proportioning; When the concentration of the substrate L-phenylalanine was further increased, the multi-enzyme activity did not change significantly and remained at the highest level, indicating that the concentration of L-phenylalanine had reached saturation; the molar ratio increased from 1.5:1 At 2:1, the relative enzyme activity decreased slightly to 96%, which was due to the inhibitory effect of L-phenylalanine. This shows that, when the mol ratio of L-phenylalanine and oxaloacetate is 0.75: 1, relative enzyme activity is maximum.

Multi-enzyme co-reaction synthesis of 2-phenylethanol under the optimal conditions of embodiment 2

Refer to Example 1 for the instruments, reagents and detection methods of this embodiment. Using L-phenylalanine and oxaloacetate as substrates, the optimal synthesis of 2-phenylethanol by recombinant aromatic aminotransferase ⅠARO8, phenylpyruvate decarboxylase ARO10 and alcohol dehydrogenase ADH1 was studied. Conditions, all reaction systems were 0.5 mL, containing 0.01 mM pyridoxal phosphate, 0.2 mM thiamine pyrophosphate and 0.1 mM NADPH.

Using L-phenylalanine and oxaloacetate in a molar ratio of 0.75:l as substrates, 2-phenylethanol was synthesized according to the aforementioned optimal conditions. Recombinant enzymes ARO8, ARO10 and ^ADH1 were mixed according to enzyme activity at a ratio of 1:0.25:1, and the amount added was 100 μL. Acetonitrile was used to terminate the reaction, and the content of 2-phenylethanol was measured by HPLC, which was 40.92 μM, as shown in FIG. 12 . Under the optimal multi-enzyme co-reaction condition of the present invention, the concentration of 2-phenylethanol reaches a higher value. The enzyme activity of the recombinant multienzyme was 0.082 μmol/(min·mL).

Example 3 Co-reaction test in which the three-enzyme co-immobilization system participates

Main reagents: recombinant aromatic aminotransferase I, phenylpyruvate decarboxylase and alcohol dehydrogenase are commercially available or constructed by referring to conventional techniques in the field, or constructed by referring to the method of Example 5 of the present invention. Chemical standards: L-phenylalanine, oxaloacetic acid, and 2-phenylethanol were purchased from Sigma-Aldrich, USA. Other reagents were routinely purchased from the market, and the above chemical reagents were all domestic analytical or chromatographically pure. Main instruments: digital display constant temperature water bath HH-2 (Jiangsu Changzhou Guohua Electric Appliance Co., Ltd.), electric blast drying oven 101A-3S (Guangdong Guangzhou Fumin Measurement and Control Technology Co., Ltd.), constant temperature metal bath CHB-100 (Jiangsu Hangzhou Bioer Technology Co., Ltd.), Electrolux Refrigerator Freezer BCD-263C (Elite (China) Co., Ltd.), Eppendorf Centrifuge 5810R (Germany Eppendorf Company), Precision pH Meter PHS-3C (Shanghai Precision Scientific Instrument Co., Ltd.), Mettler -Toledo electronic balance AB204-N (Shanghai Mettler-Toledo Instrument Co., Ltd.), ultrasonic breaker (Ningbo Science and Technology Co., Ltd.), liquid chromatograph (Shanghai Tianmei Company). 1. Test method:

Enzyme activity assay method: L-phenylalanine and oxaloacetate were used as substrates, and the catalytic activity of co-immobilized multienzymes (Combi-CLEAs) was detected by HPLC. The total volume of the reaction system is 500 μL, containing: 10 mM L-phenylalanine, 10 mM oxaloacetate, 1.5 mM Mg ²⁺ , 0.01 mM pyridoxal phosphate, 0.1 mM thiamine pyrophosphate, 0.1 mM NADPH, co-immobilized poly Enzyme 30 μL. After adding the enzyme solution and mixing evenly, start timing, react at the optimum temperature for 5 minutes, quickly add 500 μL of acetonitrile to terminate the reaction, filter the supernatant with a 22 μm filter membrane, measure the absorbance at 216 nm wavelength by HPLC, and calculate the content of 2-phenylethanol.

The definition of the enzyme activity of the three-enzyme co-immobilization system Combi-CLEAs: under the optimal conditions, the amount of μmol of 2-phenylethanol catalyzed per milliliter of Combi-CLEAs mixture per minute. The unit of enzyme activity is μmol/(min·mL).

2-phenylethanol assay conditions are the same as in Example 1.

Enzyme activity recovery rate calculation: Enzyme activity recovery rate refers to the percentage of the activity expressed by Combi-CLEAs to the total activity of the initial free multi-enzyme solution.

1. Preparation of Co-immobilized Multiple Enzymes (Combi-CLEAs)

Preparation of multi-enzyme aggregates: Mix recombinant transaminase Ⅰ, phenylpyruvate decarboxylase and alcohol dehydrogenase at a ratio of 1:0.25:1 according to enzyme activity, take 100 μL of mixed enzyme solution in a test tube, and slowly drop 900 μL of precipitate into it At the same time, add it dropwise and gently stir, take a sample every 30 minutes, centrifuge at 8000 rpm for 5 minutes at low temperature, take the supernatant to measure the enzyme activity, until all the proteins are precipitated.

Cross-linking of multi-enzyme aggregates: Slowly add a certain concentration of cross-linking agent glutaraldehyde to the multi-enzyme aggregate suspension added with precipitant, continue to stir slightly, cross-link at a certain temperature for a certain time, centrifuge at 4000rpm for 10min, discard The supernatant was washed with 1 mL of pH 7.0 disodium hydrogen phosphate/sodium dihydrogen phosphate buffer and the precipitate was centrifuged. The precipitate was resuspended in 1 mL of buffer and stored at 4°C for use.

2. Study on the co-immobilization conditions of multiple enzymes

Determination of the type of precipitant: use 90% ammonium sulfate, 90% isopropanol, 90% butanol, 60% PEG6000, 90% ethanol, 90% acetone, 90% tert-butanol and 90% acetonitrile as precipitant respectively, take 100 μL Mix the multi-enzyme solution at a ratio of 1:0.25:1 (U:U:U), add 900 μL of the above precipitant respectively, precipitate at 25°C for 30 minutes, centrifuge at 8000 rpm for 5 minutes, wash, and wash with 1 mL of disodium hydrogen phosphate pH7.0/ Resuspend the precipitate in sodium dihydrogen phosphate buffer, measure the enzyme activity, and calculate the recovery rate of the enzyme activity.

Determination of the saturation of the precipitating agent: take 100 μL of the mixed enzyme solution, and slowly add 900 μL of pH7. Ammonium sulfate solution, precipitate at 25°C for 30 minutes, measure the activity of each enzyme, and calculate the recovery rate of the enzyme activity.

Determination of precipitation pH: Take 100 μL of mixed enzyme solution, slowly add 900 μL of saturated ammonium sulfate solutions with pHs of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0, precipitate at 25°C for 30 minutes, measure the activity of each enzyme, and calculate Enzyme activity recovery.

Determination of multi-enzyme ratio: Referring to Example 1, fix the enzyme amount of the first enzyme ARO8, and according to the enzyme activity, ARO8, ARO10 and ADH1 are respectively 1:1:1, 1:0.5:1, 1:0.25:2 , 1:0.25:1, 1:0.25:0.5, 1:0.15:0.5 and 1:0.15:0.25 to prepare multi-enzyme mixed solution, take 100 μL mixed enzyme solution, slowly add 900 μL saturated ammonium sulfate solution to it, 25 Precipitate at ℃ for 30 minutes, measure the activity of each enzyme, and calculate the recovery rate of the enzyme activity.

Determination of cross-linking agent concentration: prepare the multi-enzyme aggregate suspension according to the aforementioned optimal conditions, add glutaraldehyde solutions with final concentrations of 0.05%, 0.10%, 0.15%, 0.25%, 0.50% and 0.75% respectively and Stir slightly, after cross-linking at 25°C for 120 min, centrifuge at 4000 rpm for 10 min, wash the precipitate three times with 10 mL of pH 7.0 phosphate buffer, and finally resuspend the precipitate with 1 mL of this buffer, measure the enzyme activity, and calculate the recovery rate of the enzyme activity.

Determination of cross-linking agent temperature: prepare multi-enzyme aggregate suspension, add glutaraldehyde solution of appropriate concentration, cross-link at 0°C, 10°C, 25°C, 35°C and 45°C for 120min, centrifuge, wash, Resuspend the pellet with 1 mL of buffer, measure the enzyme activity, and calculate the recovery rate of the enzyme activity.

Determination of cross-linking time: prepare multi-enzyme aggregate suspension, add glutaraldehyde solution, cross-link at the optimum temperature for 30, 60, 90, 120 and 150 min respectively, centrifuge, wash, and resuspend the precipitate with 1 mL buffer , measure the activity of each enzyme, and calculate the recovery rate of the enzyme activity.

3. Using the response surface analysis method to verify the multi-enzyme co-immobilization conditions of the present invention

According to the results of the single factor experiment, the Box-Behnken test design was adopted, and the significant factors affecting the activity of Combi-CLEAs were designed with 3 factors and 3 levels. The codes and levels of each factor are shown in Table 1.

After carrying out the test according to the corresponding test table, carry out quadratic regression fitting on the data, obtain the quadratic equation including the first-order term, the square term and the interaction term, analyze the main effect and the interaction effect of each factor, and finally calculate within a certain level range Take the best value.

Table 1 Box-Behnken response surface design experiment factor levels and codes

4. From co-immobilized multi-enzymes (Combi-CLEAs) enzymatic properties research to determine the process parameters of the present invention

(1) The optimum temperature of Combi-CLEAs: the enzyme activities of Combi-CLEAs were measured at 30°C, 33°C, 35°C, 37°C, 40°C, 45°C and 50°C respectively, and the results were expressed as relative enzyme activities, The maximum enzyme activity determined is 100%. The optimal ratio of free multienzyme ARO8:ARO10:ADH1 was 1:0.25:1 (U:U:U) as the control.

(2) Temperature stability of Combi-CLEAs: The Combi-CLEAs were incubated at 10°C, 30°C, 50°C, 70°C and 90°C for 5 hours, and the remaining enzyme activity was determined. The measurement results are expressed by relative enzyme activity, taking the unincubated enzyme activity as 100%. The optimal ratio of free multienzyme ARO8:ARO10:ADH1 was 1:0.25:1 (U:U:U) as the control.

(3) Optimum pH of Combi-CLEAs: Add Combi-CLEAs into buffer solutions with different pHs, mix well, place at 4°C for 30 min, react under optimum temperature conditions, and measure enzyme activity. The pH systems are 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0, of which pH3.0~5.0 is 0.1M sodium acetate buffer, pH6.0~7.0 is 0.1M sodium phosphate buffer, and pH8.0 is 0.1 M Tris-HCl buffer; pH 9.0 is 0.05M Gly-NaOH buffer. The results are expressed by relative enzyme activity, with the maximum enzyme activity determined as 100%. The optimal ratio of free multienzyme ARO8:ARO10:ADH1 was 1:0.25:1 (U:U:U) as the control.

(4) Effect of substrate concentration on the activity of Combi-CLEAs: The molar ratios of Combi-CLEAs in L-phenylalanine and oxaloacetate were determined to be 0.2:1, 0.5:1, 0.75:1, 1:1, Enzyme activity under the conditions of 1.5:1 and 2:1, the result is expressed by relative enzyme activity, and the maximum enzyme activity determined is 100%. The optimal ratio of free multienzyme ARO8:ARO10:ADH1 was 1:0.25:1 (U:U:U) as the control.

(5) Storage stability of Combi-CLEAs: Take Combi-CLEAs and store them in a refrigerator at 4°C for 40 days, take out an equal amount of enzyme solution at regular intervals to measure the enzyme activity, and take the maximum enzyme activity as 100%. Enzyme activity indicated. The optimal ratio of free multienzyme ARO8:ARO10:ADH1 was 1:0.25:1 (U:U:U) as the control.

(6) Operational stability of Combi-CLEAs: Take equal amounts of free multi-enzymes and Combi-CLEAs to act on L-phenylalanine respectively, after terminating the reaction, filter out the enzyme solution to continue the next batch of reactions, and measure the enzyme in each batch of reactions The maximum enzyme activity measured is 100%, and the result is expressed by relative enzyme activity.

2. Test results

1. Research results of multi-enzyme co-immobilization conditions

(1) Selection of the type of precipitant

Precipitating free enzymes using neutral salts, water-soluble organic solvents, and non-ionic polymers is the first step in the preparation of CLEAs. Different types of precipitating agents induce different enzyme conformations, and the subsequent cross-linking reaction freeze-frame methods are also different. The activity of the final cross-linked enzyme aggregates varies greatly. Therefore, the type of precipitant has a great influence on the enzyme activity. After targeted research, screening and comparative tests, in this example, 8 different precipitants ((NH4) ₂ SO ₄ , tert-butanol, PEG6000, isopropanol, n-butanol, ethanol , acetone, acetonitrile), the enzyme activities of various aggregates were measured as shown in Figure 13, in Figure 13, CK: free enzyme; A: 90% (NH4) ₂ SO ₄ ; B: 90% tert-butanol; C : 60% PEG6000; D: 90% isopropanol; E: 90% n-butanol; F: 90% ethanol; G: 90% acetone; H: 90% acetonitrile). It can be seen from Figure 13 that various precipitants have different precipitation effects on mixed multi-enzymes. Ammonium sulfate and tert-butanol are used as precipitants to finally obtain enzyme polymers with higher activity, and the recovery rate of enzyme activity reaches 68%. Ammonium sulfate is a neutral salt, which can destroy the hydration layer of protein in solution and cause protein coagulation and precipitation. Tert-butanol is a water-soluble organic solvent-like protein precipitant. The principle of precipitation is mainly that organic solvents can reduce the dielectric constant of aqueous solution, reduce the polarity of the solvent, weaken the interaction between solvent molecules and protein molecules, and increase The interaction between charged particles increases the mutual attraction between protein molecules, resulting in easy aggregation between protein particles; secondly, this polar organic solvent is miscible with water, removes the hydration layer around protein molecules, and destroys the hydration layer of protein molecules. Instead of a water film, the protein precipitates. PEG 6000 is an uncharged linear polymer, which mainly destroys the hydration layer on the surface of protein molecules through steric position repulsion and strong hydrophilicity, causing enzyme aggregation or dehydration to precipitate. In this paper, the relative enzymatic activity of aggregates prepared with 60% PEG 6000 is only 43%, which may be due to the low concentration of PEG and the weak steric repulsion to enzymes. In addition, the high viscosity of PEG solution affects the operation and the test repeatability is not good. it is good. Other organic solvents such as acetone and acetonitrile have better precipitation effects, but the enzyme activity is not high. This is because these organic solvents have a great impact on the enzyme activity during the precipitation process, resulting in partial inactivation of the enzyme. Therefore, after analysis and test verification, the present invention selects ammonium sulfate as the precipitant for preparing Combi-CLEAs.

(2) Determination of saturation of precipitant

In this example, the effect of the concentration of the precipitating agent on the activity of multi-enzyme aggregates was studied. The multi-enzyme mixture was precipitated with different saturations of ammonium sulfate for 30 minutes, and the activity of each aggregate was measured after centrifugation. The recovery rate of the enzyme activity was 100% with the same amount of free multi-enzyme, and the results are shown in FIG. 14 . With the increase of precipitant concentration, the amount of precipitated protein also increased, so the activity of multi-enzyme aggregates showed an upward trend; the greater the concentration of precipitant, the more complete the precipitation of enzyme protein, and the higher the enzyme activity in the aggregate. When the saturation of ammonium sulfate increased from 20% to 40%, the precipitation of multi-enzymes was the fastest, and the relative activity of aggregates increased significantly from 18% to 58%. As the saturation of ammonium sulfate continued to increase, the relative enzyme activity gradually increased. High; when its saturation is 90%, most of the multi-enzymes are precipitated, and the relative enzyme activity reaches the maximum, but the relative enzyme activities of the three saturation degrees of 70%, 80% and 90% are not significantly different, from the preparation cost Consider, the final saturation of the precipitant is 70%.

(3) Determination of precipitation pH

The multi-enzyme aggregates were prepared with different pH ammonium sulfate precipitants, and the enzyme activity was measured. The recovery rate of the enzyme activity of the same amount of free multi-enzymes was 100%. The results are shown in FIG. 15 . It can be seen from Figure 15 that with the increase of pH value, the enzyme activity showed a trend of first increasing and then decreasing; the relative enzyme activity in the range of pH 4.0-6.0 was higher, between 67% and 77%; At 0, the activity of the aggregates was the greatest, because the isoelectric points of the recombinases ARO8, ARO10 and ADH1 were 5.81, 4.91 and 5.06, respectively, so the precipitation was most complete near pH5.0. These enzyme aggregates precipitated by ammonium sulfate in aqueous solution, the enzyme molecules are bound together by non-covalent bonds, when the enzyme aggregates are placed in the aqueous phase reaction system, the aggregates are decomposed due to instability, the enzyme Molecules are re-dissolved in water, and the spatial conformation of enzyme molecules is destroyed, thereby affecting enzyme activity. It can be seen from the foregoing test results that even under the condition of complete precipitation, the maximum relative enzyme activity of aggregates is less than 80%. Therefore, in order to avoid The re-dissolution of the aggregates needs to be cross-linked with a certain concentration of cross-linking agent after the multi-enzyme is precipitated.

(4) Determination of multi-enzyme ratio

When determining the multi-enzyme ratio, fix the enzyme amount of the first enzyme ARO8, match the three enzymes in different proportions according to the enzyme activity, and precipitate with ammonium sulfate with pH 5.0 and 70% saturation at 25°C for 30 minutes, and centrifuge Afterwards, an appropriate amount of glutaraldehyde was added to each aggregate for cross-linking to prepare Combi-CLEAs, and its enzyme activity was measured. The recovery rate of the enzyme activity of the same amount of free multienzyme was 100%. The results are shown in FIG. 16 . Different enzyme ratios lead to different enzyme activities; when ARO8:ARO10:ADH1 is 1:0.5:1 (U/U/U), the relative enzyme activity is the largest, which is the same as the optimal ratio of the aforementioned free three enzymes. A certain deviation may be due to the fact that when the ratio of the three enzymes is 1:0.5:1, the particle size of the cross-linked aggregates formed by the three recombinant enzymes with different molecular diameters and spatial conformations is suitable, and the substrate molecules are easy to diffuse into the aggregates, and the enzyme The intermolecular distance is closer than that of free enzymes. The phenylpyruvate produced by ARO8 is easily decarboxylated by ARO10 in situ, and the phenylacetaldehyde is easily dehydrogenated by ADH1 in situ. Therefore, Combi- CLEAs obtained the highest vigor.

(5) Determination of crosslinking agent concentration

Glutaraldehyde is suitable for the immobilization system of the present invention, and its cross-linking of enzymes is mainly through the reaction of aldehyde groups and amine groups on the surface of enzyme molecules to obtain insoluble enzyme cross-linking aggregates. However, the amount of glutaraldehyde had different effects on the prepared enzyme-crosslinked aggregates. After ARO8, ARO10 and ADH1 were mixed in a ratio of 1:0.5:1 according to the enzyme activity, after 70% saturated ammonium sulfate precipitation, different volumes of glutaraldehyde were added to cross-link the multi-enzyme aggregates, so that the glutaraldehyde in the solution The final concentrations were: 0.05%, 0.10%, 0.15%, 0.25%, 0.50% and 0.75% (V/V). Various Combi-CLEAs were prepared and their enzyme activities were measured. The results are shown in FIG. 17 . It can be seen from Figure 17 that with the increase of the concentration of glutaraldehyde, the enzyme activity showed a trend of a small increase and then a continuous decline. When the concentration of glutaraldehyde was 0.10%, the relative enzyme activity reached the highest; When it was increased to 0.75%, the relative enzyme activity dropped sharply by 30%. It shows that within a certain range, the amount of glutaraldehyde increases, the amount of Combi-CLEAs obtained also increases, and the enzyme activity increases. However, if the amount of glutaraldehyde is too large, it will cause excessive cross-linking of the enzyme, resulting in serious enzyme activity. loss. This is because glutaraldehyde mainly reduces the loss of enzyme through cross-linking. At low concentrations, only a small amount of enzyme molecules can be cross-linked, and the amount of insoluble cross-linked enzyme aggregates obtained is very small, and there are a large amount of cross-linked enzyme aggregates. The enzyme is not cross-linked, so it leaks and loses during the washing process, so the enzyme activity is low; but when the concentration is too high, glutaraldehyde binds to the amino acids of the enzyme molecule itself at multiple sites, causing the higher-order structure of the protein to occur to a certain extent. Destruction not only causes the loss of enzyme activity, but also forms tightly structured aggregates that make it difficult for substrate molecules to approach the active center of the enzyme, thereby affecting the effectiveness of the active center of the enzyme molecule, thus showing lower enzyme activity.

(6) Determination of crosslinking temperature

Temperature is also an important factor affecting the activity of multi-enzyme cross-linked aggregates. Add glutaraldehyde at a final concentration of 0.10% to the multi-enzyme aggregates for cross-linking, and place them at 0, 10, 20, 25, 30, 35, and 45°C for 120 minutes to determine the activity of each Combi-CLEAs , to calculate the relative enzyme activity. It can be seen from Figure 18 that when the temperature rises from 0°C to 30°C, the enzyme activity increases continuously; in the range of 20-30°C, the enzyme activity increases significantly; at 30°C, the relative enzyme activity reaches the maximum, reaching 74 %. As the temperature continued to rise, the loss of enzyme activity increased, and the relative activity of Combi-CLEAs decreased to 43% at 45°C. Therefore, the optimum temperature for the preparation of multi-enzyme cross-linked aggregates was selected as 30°C.

(7) Determination of crosslinking time

Cross-linking time affects the activity of multi-enzyme aggregates. Glutaraldehyde with a final concentration of 0.10% cross-linked multi-enzyme aggregates at 30°C for 30, 60, 90, 120 and 150 min respectively. The activity of each Combi-CLEAs was measured and the relative enzyme activity was calculated. The results are shown in Figure 19, with the increase of cross-linking time, the relative enzymatic activity of Combi-CLEAs also increases steadily, this is because the longer the cross-linking time, the more thorough the cross-linking, and the greater the amount of multi-enzyme cross-linking aggregates. more, the greater the activity of the enzyme; when it exceeds 120min, the enzyme activity begins to decline, and when the crosslinking time is 150min, the relative enzyme activity drops to 41%. This is because the crosslinking time is too long, and too much glutaraldehyde covers part of the activity of the enzyme In addition, if the cross-linking time is too long, glutaraldehyde is prone to reactions such as polymerization and oxidation, thereby affecting the enzyme activity and cross-linking effect. Therefore, the cross-linking time is very important to the stability and activity of Combi-CLEAs, so 120min was determined as the appropriate cross-linking time.

3. Response surface research verification experiment of multi-enzyme co-immobilization conditions

1. Box-Benhnken test results and analysis

The present invention adopts Box-Behnken test design, with (NH4)2SO4 is precipitating agent, selects multi-enzyme ratio as 1:0.5:1, precipitation pH is 5.0, cross-linking time is 120min, to the significant factor that influences multi-enzyme CLEAs activity The design of 3 factors and 3 levels was carried out, and the average value of the enzyme activity recovery rate obtained in 3 experiments was the response value (Y). The specific experimental design and results are shown in Table 2.

Table 2 Box-Behnken experimental design and its results

(2) Quadratic regression fitting and analysis of variance

Statistical software SAS 8.0 (Statistics Analysis System) was used to perform quadratic multinomial regression fitting on the data in Table 2. The statistical significance was determined by T test, and the coefficient of determination and coefficient of variation were determined by F test. The quadratic regression equation between the response value (Y) and each factor (A, B, C) obtained by fitting the Box-Behnken test data is:

Y＝-1024.29+17.18A+519.85B+31.61C+8.63AB-0.079AC+11.78BC-0.11A ² -7488.50B ² -0.47C ²

Significance analysis and variance analysis were carried out on the above binary regression equation, and the results are shown in Table 3.

Table 3 Analysis of variance of regression equation of Box-Behnken experimental design

The significance of the linear relationship between each factor and the response value was judged by the F value test, and the smaller the value of the probability P (F>F _α ), the higher the significance of the variable. Lack of fit is an important data used to evaluate the reliability of the equation. If it is significant, it indicates that the equation simulation is not good; The regression equation is used to replace the real test points to analyze and predict the experimental results. As can be seen from Table 3, the significant level of the model is 0.0001 (<0.01), indicating that the model has a very significant impact on the response value (enzyme activity recovery rate), and the model is statistically significant. The influence of cross-linking temperature is extremely significant, and the significance of the influence of the three factors on the recovery rate of enzyme activity is cross-linking temperature > ammonium sulfate saturation > glutaraldehyde concentration; interaction terms A (ammonium sulfate concentration) and B (glutaraldehyde concentration), The interaction between A (ammonium sulfate concentration) and C (crosslinking temperature) is significant, indicating that these three factors are the key control factors in the preparation process of Combi-CLEAs; the quadratic items A ² , B ² , and C ² have very significant effects. The lack of fit item P=0.6311 (>0.05), has no significant meaning, indicating that there is no abnormal point in the data, and there is no need to introduce a higher order item, and the model is appropriate.

Credibility analysis of the model in Table 4

Std. Dev. 2.58 R-Squared 0.9864 mean 46.63 Adj R-Squared 0.9688 C.V.% 5.54 Pred R-Squared 0.9153 PRESS 290.63 Adeq Precision 19.485

It can be seen from Table 4 that the correlation coefficient R ² =0.9864, indicating that 98.64% of the change in the response value comes from the three selected variables. The correction coefficient of determination R ² (Adj) = 0.9688 (>0.80), indicating that 96.88% of the variability of the experimental data can be explained by this model; while the lower coefficient of variation CV% (5.54%) shows that only 5.54% of the variability in the model Cannot be explained by the model, that is, 94.45% of the variance can be explained by the model, indicating that the model is highly significant. It is more reasonable for the signal-to-noise ratio Adeq Precision to be greater than 4. The signal-to-noise ratio of the model is 19.485, which shows that the model can well reflect the real experimental value. In summary, the regression equation can better describe the true relationship between each factor and the response value, and can be used to determine the optimal preparation conditions of Combi-CLEAs.

3. Verification test

According to the analysis of the Box-Behnken design model, the optimized process parameters of multi-enzyme co-immobilization conditions were: (NH ₄ ) ₂ SO ₄ saturation was 71.57%, glutaraldehyde concentration was 0.10%, and crosslinking temperature was 28.55℃. Under these conditions, the predicted recovery rate of enzyme activity was 67.29%. In order to confirm whether the established model is consistent with the experimental results, according to the actual operation, the immobilization process parameters were corrected as (NH ₄ ) ₂ SO ₄ saturation was 72%, the crosslinking temperature was 29°C, and the concentration of glutaraldehyde was still 0.10%. ; Under this parameter, three groups of experiments were carried out, and the recovery rate of enzyme activity was 65.76±3.27%, which was close to the predicted value of the model. The fitted data model is well consistent with the actual scheme of the present invention.

4. Study on the enzymatic properties of co-immobilized multienzymes

(1) Effect of temperature on co-immobilized multienzyme activity

The dissociation of the active conformation of the enzyme at high temperature is the root cause of the reduction or loss of enzyme activity, and the immobilization technology can effectively reduce the dissociation of this conformation through the connection of various covalent bonds between enzyme molecules or between the enzyme and the carrier. Dissociate. Therefore, after most enzymes are immobilized, the optimum temperature will be higher than that of free enzymes. In this experiment, the activities of Combi-CLEAs and free enzymes were measured at different temperatures, and the results are shown in Figure 20. It can be seen from Figure 20 that the optimum temperature of the free enzyme is 37°C, and the optimum temperature of Combi-CLEAs has a slight increase, which is 40°C; and the optimum temperature range of Combi-CLEAs is wider than that of free enzymes, at 37-50°C Within the range, its relative enzyme activity remains above 86% of the highest enzyme activity. It shows that the cross-linking immobilization technology improves the catalytic temperature of the enzyme. The reason is that the cross-linked enzyme aggregates formed by the cross-linking agent glutaraldehyde have a relatively dense structure and have a certain protective effect on the active groups of the enzyme. It can also be seen from the figure that with the further increase of temperature, the activities of immobilized enzymes and free enzymes are gradually reduced. Therefore, in addition to determining the optimum temperature, the thermal stability of immobilized enzymes should also be considered.

(2) Temperature stability of co-immobilized multienzymes

Thermostability is an important indicator for examining the performance of immobilized enzymes. The immobilized enzyme and the free enzyme were treated at different temperatures for 5 hours, and the remaining enzyme activity was measured. The results are shown in FIG. 21 . It can be seen from the figure that as the temperature increases, the activities of Combi-CLEAs and free multienzymes both show a downward trend, but the activity of free enzymes decreases more; after incubation at 50°C for 5 hours, the relative enzyme activity of Combi-CLEAs remains the same. It remained at 80%, while the free enzyme was only 50%. After being treated at 90°C for 5 hours, the remaining activity of the former was 25%, while that of the latter was less than 10%. It can be seen that the immobilization method of co-crosslinked enzyme polymers can effectively improve the thermal stability of enzymes and significantly reduce the degree of thermal inactivation. The reason is that after the free enzyme forms CLEAs, the spatial structure changes, and some active groups are embedded in the aggregate, which is protected, so the heat resistance is enhanced; in addition, the higher temperature is conducive to the substrate molecules Diffuse into the interior of the aggregate to react with the enzyme.

(3) Effect of pH on co-immobilized multienzyme activity

Enzyme activity is easily affected by the pH of the environment, and only at a certain pH can the enzyme exhibit maximum activity. Due to the impact of the carrier on the ionic strength in the microenvironment, the optimum pH of the immobilized enzyme tends to drift. In this example, the activities of Combi-CLEAs and free multienzyme in the buffer solution with pH 3.0-9.0 were measured, and the results are shown in FIG. 22 . It can be seen from the figure that the optimum pH of Combi-CLEAs is 5.0, and the optimum pH of free multienzyme is 6.0. Combi-CLEAs shifts to acidity by one unit, and its suitable pH range also shifts to acid. It may be because during the cross-linking process, the aldehyde group in the glutaraldehyde molecule reacts with the amino group in the enzyme molecule, which increases the negative charge of the side chain group of the protein molecule, and the pH in the external solution must be shifted towards acidity. neutralize these negative charges.

(4) Effect of substrate concentration on co-immobilized multienzyme activity

When the concentration ratio of substrates affects the activity of co-immobilized multi-enzymes (Combi-CLEAs), when the concentration of substrate oxaloacetate is kept constant at 10mM, the substrates L-phenylalanine and oxaloacetate are used Different molar ratios were kept under the optimal conditions of Combi-CLEAs for 5 minutes, and the enzyme activity was measured, and the results are shown in Figure 23. It can be seen from the figure that the substrate ratio has the same effect on co-immobilized multienzyme and free multienzyme. The increase of L-phenylalanine concentration increases, but the relative enzymatic activity of immobilized multienzyme is slightly higher; There is a decline, and the immobilized multi-enzyme decline rate is slightly faster. It can be seen that when the molar ratio of L-phenylalanine to oxaloacetate is 0.75:1, the relative enzyme activity of immobilized multienzyme and free multienzyme is the largest.

(5) Storage stability of co-immobilized multienzymes

The storage stability of an enzyme is very important for its commercial application. The free multi-enzyme and co-immobilized multi-enzyme CLEAs were stored in a refrigerator at 4°C for 40 days, and the enzyme activity was measured at regular intervals. The results are shown in Figure 24. In the first 10 days of storage, the activities of both did not change significantly; after 20 days, the activity of the immobilized enzyme maintained 98%, and the activity of the free enzyme was 95%; The decline trend was slower than that of the free enzyme, and the activities of the two maintained 87% and 70% of the initial enzyme activity, respectively. It shows that the storage stability of the immobilized enzyme is better, which is mainly because the precipitation and cross-linking steps in the preparation process of Combi-CLEAs can purify the enzyme and avoid the hydrolysis of the enzyme caused by impurities. In addition, the enzyme molecules are firmly locked in a region in the form of multi-point covalent cross-linking, which prevents the enzyme from leaking into the solution in a free state.

(6) Operational stability of co-immobilized multienzymes

Reusability is also an important indicator to measure the performance of immobilized enzymes. After the end of each batch of reaction, the Combi-CLEAs were filtered out to continue the next batch of reaction, the results are shown in Figure 25. After the immobilized multi-enzyme was reused 4 times, the conversion rate of the substrate was 23%, maintaining 66% of the initial enzyme activity, while the free enzyme retained only 4% of the enzyme activity after 1 use because it was difficult to recover. -CLEAs have good reproducibility; after 8 consecutive uses, the activity of immobilized multi-enzyme dropped greatly, and only 19% of the activity remained, the main reason was that repeated repetitions affected its stability, followed by Combi-CLEAs particles Small, affecting recycling.

Example 4 Multi-enzyme co-immobilization system applied to co-reaction to prepare 2-phenylethanol

According to the optimal process parameters obtained by response surface analysis, multi-enzyme co-immobilization was carried out, and 2-phenylethanol was synthesized according to the optimal conditions. The amount of Combi-CLEAs added was 100 μL, and after pH 5.0, 40°C water bath for 5 min, the reaction was terminated with an equal volume of acetonitrile, and the content of 2-phenylethanol was measured by HPLC as 28.61 μM. The enzyme activity of co-immobilized multienzyme was 0.057μmol/(min·mL).

The construction method of three kinds of recombinant enzymes optimized by embodiment 5

The following construction methods that are not specifically described refer to conventional techniques in the art, and the biological materials that are not described are all commercially available or conventionally used biological materials in the field.

1. From different strains of Saccharomyces cerevisiae, 11 genes of 3 key enzymes for the synthesis of 2-phenylethanol by Ehrlich pathway were cloned.

(1) Three aromatic aminotransferase I genes. The DNA sequence length is 1503bp, the relative molecular mass is 56.2kD, and the theoretical isoelectric point is 5.81. The Genbank accession numbers of the three genes are KC422721.1, KC422722.1 and KC422723.1, respectively.

(2) Three phenylpyruvate decarboxylase genes. The DNA sequence length is 1908bp, the relative molecular mass is 71.4kD, and the theoretical isoelectric point is 6.10. Two of the genes were registered in Genbank with accession numbers KC422719.1 and KC422720.1, respectively.

(3) Five alcohol dehydrogenase genes. They are: 2 alcohol dehydrogenase Adh1 genes, 1 alcohol dehydrogenase Adh2 gene, 1 alcohol dehydrogenase Adh3 gene and 1 alcohol dehydrogenase Sfa1 gene. Among them, the DNA sequence length of Adh1 and Adh2 genes is 1047bp, the relative molecular mass is about 36.8kD, the theoretical isoelectric point of DV10-Adh1 gene is 6.07, and the theoretical isoelectric point of EC1118-Adh1 and DV10-Adh2 is 6.21. The Genbank accession numbers of EC1118-Adh1 gene and DV10-Adh2 gene are KC491732.1 and KC491733.1, respectively. The DNA sequence length of YT0801-Adh3 gene is 1128bp, the relative molecular mass is 40.4kD, the theoretical isoelectric point is 8.65, and its Genbank accession number is KC422718.1. The DNA sequence length of YT0801-Sfa1 gene is 1161bp, the relative molecular mass is 41kD, the theoretical isoelectric point is 6.51, and its Genbank accession number is KC491734.1.

Materials: Saccharomyces cerevisiae strain and Escherichia coli strain DH5ɑ, conventional strains, strains used in routine experiments in other laboratories can be used, and the strains used in this example are experimental strains routinely preserved in this laboratory. Cloning vector pGEM-T Easy Vector, product of Promega Company.

TakaRaEx Taq enzyme was purchased from Dalian Bao Biological Engineering Co., Ltd.; ordinary agar gel DNA recovery kit and plasmid mini-extraction kit were purchased from Tiangen Biochemical Technology Co., Ltd.; conventional reagents were domestic analytical grade.

According to the reported target gene sequence information, primer 5 and oligo 6.0 were used to design specific primers, which were synthesized by Shanghai Jierui Bioengineering Co., Ltd. The primer sequence information is shown in Table 5.

Table 5 Primer sequence information table

Cultivation of Saccharomyces cerevisiae and extraction of total DNA:

Genomic DNA was extracted from different Saccharomyces cerevisiae strains, referring to the overnight treatment with helicase (Wang Ping et al., 2008; Zhao Hongyu et al., 2011). Using the total DNA of Saccharomyces cerevisiae as a template, use the above-mentioned specific primers to perform PCR reaction to amplify the full-length DNA fragment of the target gene. The PCR amplification reaction system is:

PCR reaction parameters: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 45 s, annealing at 46°C for 45 s, extension at 72°C for 90 s; a total of 33 cycles; extension at 72°C for 10 min, and storage of PCR products at 4°C.

Recovery of the target fragment PCR amplification product: After the amplified PCR product was subjected to 1.0% agarose gel electrophoresis, the target band was excised and recovered with TIANGEN ordinary agarose gel DNA recovery kit.

Connection of PCR products: The recovered PCR products were connected with the carrier T-easy Vector, and the recombinant plasmid was transformed into E. coli DH5α. The connection reaction system is as follows:

The ligation mixture was ligated overnight at 4°C, and transformed into Escherichia coli competent DH5α (CaCl ₂ method).

The transformation of the ligation product and the preparation of Escherichia coli competent cells refer to existing conventional techniques.

The transformation of the ligation product includes the following steps:

Step 1: Preparation of Escherichia coli Competent Cells

(1) Pick a single colony from a fresh plate cultured at 37°C for 16h, transfer it to a 250mL Erlenmeyer flask containing 100mL LB medium, and shake vigorously at 37°C for 3h (220rpm).

(2) Transfer the E. coli to a sterile, disposable pre-cooled 50mL centrifuge tube under aseptic conditions, and place it on ice for 10 minutes.

(3) The cells were collected by centrifugation at 4°C at 4000 rpm for 10 min with a GS3 rotor.

(4) Pour out the culture solution and invert the tube for 1 min to drain the last remaining trace of the culture solution.

(5) Resuspend each pellet in 10 mL of 0.1 M CaCl ₂ pre-cooled with ice, and after 30 min in ice bath, centrifuge at 4000 rpm for 10 min to collect the cells.

(6) Discard the supernatant, resuspend the pellet with 2 mL of ice-cold 0.1M CaCl ₂ , store at 4°C, and use within 72 hours; or add glycerol to the final concentration of 20% on the basis of 0.1M CaCl ₂ Store at -70°C for long-term use.

Step 2: Identification of E. coli Competent Cells

(1) Aspirate 100 μL of the competent cell suspension, add 2 μL of a plasmid (anti-Amp) without the target gene, mix well, and bathe in ice for 30 minutes. At the same time, make a blank control, that is, draw another 100 μL of competent cell suspension without adding plasmid.

(2) Place in a water bath at 42°C for 90 seconds, and quickly transfer to ice to rapidly cool the cells for 1-2 minutes.

(3) Add 400 μL of LB liquid, recover in a 37° C. water bath for 5 minutes, transfer to a 37° C. shaker at 200 rpm and shake for 45 minutes.

(4) Add 200 μL of Amp (50 μg/μL) to 100 mL of LB solid medium cooled to 46° C., mix well, and pour into a plate.

(5) Add 100 μL of (3) and spread evenly.

(6) After drying, invert the plate, culture overnight at 37°C, and observe the results.

(7) No colonies were produced in the control without plasmids; uniform and dense colonies were produced in the control with plasmids. It shows that the competent cells have good transformation ability, and there is no anti-Amp bacteria.

Step 3: Conversion of Ligation Products

(1) Take 200 μL of competent cell suspension from the -70°C refrigerator, thaw it at room temperature, and place it on ice immediately after thawing;

(2) Add 10 μL of the connection mixture, shake gently, and place on ice for 30 minutes;

(3) Heat shock in a water bath at 42°C for 90 seconds, and immediately place on ice to cool for 3 to 5 minutes after the heat shock;

(4) Add 1 mL of LB liquid medium without Amp, mix well, and culture with shaking at 37°C for 1 hour to restore the bacteria to normal growth state and express the antibiotic resistance gene (Amp ⁺ ) encoded by the plasmid;

(5) Shake the above-mentioned bacterial solution, take 100 μL and spread it evenly on the screening plate containing Amp ⁺ , and place it face up for 0.5 h to allow the liquid to be absorbed;

(6) After the bacterial solution is completely absorbed by the medium, invert the plate and incubate at 37°C for 16-24 hours;

(7) After the colony is fully colored, pick the clone (white spot) with the recombinant plasmid for culture, pick the transformed white single colony in the LB solid medium with a sterile toothpick, and inoculate it into 2 mL of LB containing the antibiotic ampicillin In liquid culture medium, shake culture at 37°C for 8-12 hours.

Extraction of recombinant plasmids: Extract recombinant plasmids from transformed strains with the TIANGEN plasmid mini-extraction kit according to the instructions.

Enzyme digestion identification of recombinant plasmids: The enzyme digestion reaction system is:

Incubate at 37°C for 3h, and perform 1.0% agarose gel electrophoresis.

The clones carrying the strains identified by PCR and enzyme digestion were sequenced by Beijing Liuhe Huada Gene Technology Co., Ltd. Guangzhou Branch.

Nucleic acid sequence alignment was performed on NCBI BLAST (http://blast.ncbi.nlm.nih.gov), amino acid sequence alignment was performed on EBI-BLAST (http://www.ebi.ac.uk), nucleic acid Sequence analysis was completed with DNassist 2.0 and NCBI Spidey, nucleic acid and amino acid sequence drawing was completed with DNAMAN, and protein structure and function prediction was performed on the platform linked on the Swissprot (http://www.expasy.ch/sprot/) website.

In this example, the total DNA of 17 different Saccharomyces cerevisiae strains were respectively extracted, and PCR amplification was performed with ARO8F and ARO8R as primers. As a result, the PCR products obtained from the three yeast strains were all about 1500bp in size, which was in line with expectations. Transform E.coli DH5α, extract the positive recombinant plasmid, and identify it by EcoRI digestion. The size of the inserted fragment is about 1500bp, which is close to the expected target gene, as shown in Figure 26. Among them, in Fig. 26, the left (a) is the electrophoresis analysis result of the Aro8 gene PCR product; the right (b) is the enzyme digestion identification result of the recombinant plasmid. M: MakerⅢ; ₁ ～ ₃ : PCR products of _YT0801 -Aro8, EC1118-Aro8 and DV10-Aro8; ₁₀ ～ ₃₀ : recombinant plasmids; 11～33: enzyme-digested products of plasmids ₁₀ ～30.

In this example, the total DNA of 17 different Saccharomyces cerevisiae strains was extracted, and ARO10F and ARO10R were used as primers for PCR amplification. Results The PCR products obtained from the three yeast strains were all about 1900bp in size, which was in line with expectations. The PCR products were recovered, transformed into DH5α competent cells, and screened by blue and white spots on LB plates containing Amp ⁺ . The positive recombinant plasmids were extracted and identified by EcoRI digestion. The results of enzyme digestion showed that the electrophoresis bands were clear, and the size of the inserted fragment was about 1900bp, which was close to the expected target gene, as shown in Figure 27. In the figure, the left picture (a) is the electrophoresis analysis of the Aro10 gene PCR product; the right picture (b): Enzyme digestion identification of recombinant plasmids. M: MakerⅢ; ₁ ～ ₃ : PCR products of _YT0801 -Aro10, EC1118-Aro10 and DV10-Aro10; ₁₀ ～ ₃₀ : recombinant plasmids; 11～33: enzyme-digested products of plasmids ₁₀ ～30.

In this example, the total DNA of 17 different strains of Saccharomyces cerevisiae was extracted for PCR amplification. Get a PCR product (about 1050bp) that matches the size of the Adh1 gene from 2 strains of yeast, a PCR product (about 1050bp) that matches the size of the Adh2 gene from 1 strain of yeast, and a PCR product that matches the size of the Adh3 gene from 1 strain of yeast (about 1150bp), a PCR product (about 1150bp) corresponding to the size of the Sfa1 gene was obtained from a yeast strain. The PCR product was recovered, transformed into E.coli DH5α, screened by blue and white spots, and positive recombinant plasmids were extracted, identified by EcoRI digestion, and the size of the inserted fragment was close to the expected target gene, as shown in Figure 28. In Figure 28, the left panel (a) is the electrophoresis analysis of the PCR product; the right panel (b) is the enzyme digestion identification of the recombinant plasmid, M: MakerⅢ; 1～5: DV10-Adh1, EC1118-Adh1, DV10-Adh2, YT0801 -PCR product of Adh3 and YT0801-Sfa1; 1 ₀ to 5 ₀ : recombinant plasmid; 1 ₁ to 5 ₅ : restriction product of 1 ₀ to 5 ₀ recombinant plasmid.

2. Escherichia coli BL21(DE3) was used to prokaryotically express the proteins encoded by the three key enzyme genes, and the molecular weights of the three proteins were basically consistent with those predicted by bioinformatics methods as detected by SDS-PAGE.

(1) The YT0801-Aro8 gene was cloned into the expression vector pET-32a(+), and the aromatic aminotransaminase Ⅰ genetic engineering strain was constructed. At 30°C, induced by IPTG for 8 hours, the engineered strain can express the recombinase ARO8. After purification, the concentration of fusion protein ARO8 was 462.3mg/L. As detected by SDS-PAGE, its molecular size is about 70kDa.

(2) The YT0801-Aro10 gene was cloned into the expression vector pET-32a(+), and a phenylpyruvate decarboxylase genetically engineered strain was constructed. At 28°C, induced by IPTG for 3 hours, the engineered strain can express the recombinase ARO10. After purification, the concentration of fusion protein ARO10 was 202.2mg/L. As detected by SDS-PAGE, its molecular size is about 90kDa.

(3) The DV10-Adh1 gene was cloned into the expression vector pET-32a(+), and an alcohol dehydrogenase genetically engineered strain was constructed. At 28°C, induced by IPTG for 3 hours, the engineered strain can express the recombinase ADH1. After purification, the concentration of fusion protein ADH1 was 252.6mg/L. As detected by SDS-PAGE, its molecular size is 61kDa.

Materials: E.coli DH5α and E.coli BL21(DE3) were donated by Professor Liu Yuhuan, School of Life Sciences, Sun Yat-sen University, and the recipient bacteria routinely used in this field can also be used. The pMD18-T vector was purchased from Promega, and the pET-32a(+) vector was donated by Professor Liu Yuhuan, School of Life Sciences, Sun Yat-sen University, and the vectors routinely used in the field can also be used.

Ampicillin, Isopropyl-β-D-thiogalactoside (IPTG), 5-Bromo-4-chloro-3-indole-β-D-galactoside (X-gal), Trimethylol Aminomethane (Tris), acrylamide (Acrylamide), methylene bisacrylamide (BIS), β-mercaptoethanol, bromophenol blue (BPB), agarose, and agar powder were all imported and purchased from Beijing Puboxin Biological Technology Co., Ltd. Agarose gel DNA recovery kit and plasmid small extraction kit were purchased from Tiangen Biochemical Technology Co., Ltd.; protein purification resin His·bind resin and purification column were purchased from Merck (Germany); various restriction enzymes, T4DNA Ligase and low molecular weight protein standards were purchased from Bao Biological (Dalian) Engineering Co., Ltd. DNAMarkerⅢ、1kb plus DNA Ladder(MD113)

, Protein Marker, purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. Chemical standards such as L-phenylalanine, oxaloacetate, aspartic acid, phenylpyruvate, phenylacetaldehyde, and 2-phenylethanol were purchased from Sigma-Aldrich, USA. All other chemical reagents were domestic analytically pure or chromatographically pure.

Medium: LB (Luria-Bertani) medium (1000ml): 10g tryptone, 5g yeast extract, 10g NaCl, natural pH (an additional 20g agar powder is needed for solid medium), sterilized at 121°C for 15min. Cool to room temperature and add ampicillin to the culture medium at a final concentration of 1 g/L. LB medium-X-gal-IPTG (1000ml): 10g tryptone, 5g yeast extract, 10g NaCl, natural pH, 20g agar powder, sterilized at 121°C for 15min. After cooling to room temperature, 100 μL of ampicillin (100 mg/mL) was added, 10 μL of IPTG (24 mg/mL) and 200 μL of X-gal (20 mg/mL) were added to the culture medium.

(1) Primer design

According to the known sequence information at both ends of the target sequence, design PCR amplification primers, as shown in Table 6.

Table 6 Primer sequence information table

Primers were synthesized by Shanghai Jierui Bioengineering Co., Ltd. DNA sequencing was performed by Huada Genomics Co., Ltd.

(2) PCR amplification of cloned gene

Using the extracted cloning plasmid as a template, carry out PCR amplification reaction, the reaction system is as follows:

YT0801-Aro8 gene PCR reaction parameters: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 45 s, annealing at 59°C for 45 s, extension at 72°C for 90 s; a total of 33 cycles; extension at 72°C for 10 min, and storage of PCR products at 4°C.

YT0801-Aro10 gene PCR reaction parameters: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 45 s, annealing at 60°C for 45 s, extension at 72°C for 120 s; a total of 35 cycles; extension at 72°C for 10 min, and storage of PCR products at 4°C.

DV10-Adh1 gene PCR reaction parameters: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 45 s, annealing at 58°C for 45 s, extension at 72°C for 60 s; a total of 33 cycles; extension at 72°C for 10 min, and storage of PCR products at 4°C.

(3) Recovery, connection and transformation of PCR amplification products

After the PCR amplification product was recovered, the pMD18-T vector was connected overnight at 4°C to transform E.coli DH5α. The connection reaction system is as follows:

(4) Double enzyme digestion identification and sequence determination of the recombinant plasmid

Extract the recombinant plasmid and carry out double enzyme digestion identification. The enzyme digestion system is as follows:

The recombinant plasmid containing the Aro8 gene involved in this example was identified by digestion with EcoR I and Xho I, and the recombinant plasmid containing the Aro10 and Adh1 gene was identified by digestion with BamH I and Xho I. The product should be two fragments in size. Sequencing of recombinant plasmids that were identified correctly.

(5) Ligation and transformation of vector pET-32a(+) and recombinant plasmid

Digest the vector pET-32a(+) and the recombinant plasmid with double restriction enzymes, and conduct electrophoresis in a water bath at 37°C for 3 hours. The enzyme digestion system is as follows:

Use agarose gel to recover small and large DNA fragments respectively, and establish a connection system. The amount of inserted fragments and the amount of vectors depend on the size of the fragments and their DNA concentration, generally at a ratio of 3:1. Add T4 ligase (350U/μL, 1.0 μL) and ligase buffer (1.0 μL), replenish the system to 10 μL with deionized water, flick and mix well, connect at 16°C overnight, transform E.coli DH5α, and extract the recombinant plasmid , identified by double enzyme digestion and sequence determination. According to the source of the gene, the expression vector plasmids were named p32-YT0801-Aro8, p32-YT0801-Aro10 and p32-DV10-Adh1, respectively. Electrophoretic analysis is shown in Figure 29, Figure 30 and Figure 31. The recombinant plasmid was sequenced and identified, and the sequence result was completely consistent with the target gene sequence. It indicated that the expression vectors p32-YT0801-Aro8, p32-YT0801-Aro10 and p32-DV10-Adh1 had been constructed successfully. In Fig. 29, M: Maker III; 1: plasmid; 2: digested product. In FIG. 30 , M: MD113; 1-6: plasmids; 1 ₀ , 5 ₀ : digestion products of plasmids No. 1 and No. 5. In Fig. 31, 1-5: plasmids; 1 ₀₂ , 2 ₀ , 4 ₀ : digestion products of plasmids 1, 2 and 4.

(6) Transformation of the expression vector: the expression vector plasmid was transformed into the expression strain E. coli BL21 (DE3). The recombinant plasmid was extracted and identified by double enzyme digestion.

Induced expression and purification of recombinant proteins:

(1) Induced expression of recombinant protein mainly includes the following steps: (1) Streak the preserved recombinant positive clones on an LB plate containing 100 μg/mL Amp and culture overnight at 37°C. (2) Pick a single colony, inoculate into 15 mL liquid LB medium containing 100 μg/mL Amp, and culture overnight at 37° C. and 200 r/m. (3) Take 500 μL of the bacterial liquid and transfer it to 50 mL of LB medium containing Amp (1:100), expand the culture at 37 °C and 200 rpm until the OD is 0.8 (about 2.5 h), and add 100 mM IPTG to a final concentration of 1 mM. The expression is induced at a certain temperature for a certain period of time. (4) Centrifuge at 8000rpm for 15min, collect bacteria and supernatant respectively, and store at -20°C for later use. (5) Ultrasonic disruption and protein electrophoresis to observe protein expression.

(2) Extraction of recombinant protein: Wash the bacteria 2 to 3 times with PBS, add lysate at a ratio of 1/5 to 1/10 of the volume of the original bacteria solution, and resuspend the bacteria. Bacteria were disrupted by ultrasonication in an ice bath, and the ultrasonic conditions were 400W power, 3s ultrasonication, 2s interval, and 60 times of disruption. Centrifuge at 12000rpm for 5min, collect the supernatant with a centrifuge tube, and perform SDS-PAGE analysis.

(3) Purification of recombinant protein mainly includes the following steps: (1) Add 1 mL of 50% Ni-NTA His·bind resin suspension into 4 mL of 1×Ni-NTA binding buffer, and mix gently. (2) After the resin settles naturally, absorb 4 mL of the supernatant with a pipette tip, add 4 mL of the prepared supernatant, mix gently, and combine at 4°C for 60 min.

(3) Add the lysate Ni-NTAHis·Bind resin mixture into the empty chromatographic column with the lower end closed, remove the lower end closed cap, collect the effluent (through the peak), save it, and use it for SDS-PAGE electrophoresis analysis. (4) Rinse twice with 4 mL of 1×Ni-NTA washing buffer, collect the rinsing components, and use for SDS-PAGE electrophoresis analysis. (5) The target protein was eluted 4 times with 0.5mL 1×Ni-NTA washing buffer, and the eluted fraction was divided into 4 parts to collect, and SDS-PAGE electrophoresis was performed to analyze each preserved fraction, and finally the target protein was determined. (4) SDS-PAGE electrophoresis analysis of the recombinant protein: for the electrophoresis analysis of the recombinant protein in the present invention, an electrophoresis scheme of 8% SDS-PAGE (>10kDa) is selected according to its molecular weight. The formulation of the electrophoresis gel, the electrophoresis parameters and the staining and decolorization methods of the gel are all carried out according to the standard experimental method.

(5) Determination of the concentration of the recombinant protein with reference to Osper's method (1998):

Determination of the concentration of recombinant protein ARO8: make a standard curve of bovine serum, the equation is Y=0.0009X-0.0086, the equation correction coefficient is 0.9967, and the linear fitting is better. The concentrations of the supernatant total protein and the purified target protein were determined to be 1149.2 mg/L and 462.3 mg/L respectively, and the target protein accounted for 40.2% of the total protein.

Determination of the concentration of recombinant protein ARO10: measure the absorbance value of the supernatant total protein and the purified ARO10 protein, and calculate the concentration of the total protein and the purified ARO10 according to the BSA standard curve, which are 789.9mg/L and 202.2mg/L respectively , the target protein accounted for 25.6% of the total protein.

Determination of the concentration of recombinant protein ADH1: measure the absorbance value of the supernatant total protein and the purified ADH1 protein, and calculate the concentration of the total protein and the purified ADH1 according to the BSA standard curve, which are 880.1mg/L and 252.6mg/L respectively , the target protein accounted for 28.7% of the total protein.

3. The biological activities of three recombinant enzymes were identified, and the enzymatic properties of the recombinant enzymes were studied respectively.

(1) As detected by HPLC and LC-MS, the recombinant enzyme ARO8 has the normal biological activity of aromatic transaminase I, and can catalyze L-phenylalanine to generate phenylpyruvate. The optimum temperature and optimum pH of ARO8 are 30℃ and 7.0 respectively, and the thermal stability is good. Its catalytic activity requires the participation of Mg ²⁺ , and Fe ³⁺ , Fe ²⁺ and Cu ²⁺ inhibit its activity. Low concentrations of organic solvents such as methanol and DMSO can promote the enzyme activity. At 30°C and pH 7.0, the Michaelis constant K _m of recombinant enzyme ARO8 acting on L-phe is 6.577mM, and the maximum reaction rate V _max is 10.352μM/min; the Michaelis constant K _m acting on oxaloacetate It is 15.779mM, and the maximum reaction rate V _max is 16.863μM/min. The enzyme activity under the optimum condition was 0.87μmol/(min·mL).

(2) As detected by GC-FID and GC-MS, the recombinant enzyme ARO10 has the normal biological activity of phenylpyruvate decarboxylase and can catalyze phenylpyruvate to generate phenylacetaldehyde. The optimum temperature and optimum pH of ARO10 are 37°C and 6.0, respectively, and the thermal stability is good. ARO10 needs ThPP and Mg ²⁺ as cofactors, Cu ²⁺ and Mn ²⁺ can promote the enzyme activity, and metal ions such as Fe ³⁺ can inhibit the enzyme activity. Organic solvents such as methanol inhibit the enzyme activity. At 37°C and pH 6.0, the K _m of recombinant ARO10 acting on phenylpyruvate was 8.239 mM, and the maximum reaction rate V _max was 2.994 μM/min. The enzyme activity under the optimum condition was 0.22μmol/(min·mL).

(3) As detected by HPLC and LC-MS, the recombinant alcohol dehydrogenase ADH1 has the normal biological activity of the enzyme and can reduce phenylacetaldehyde to 2-phenylethanol. The optimum temperature and optimum pH of ADH1 are 35°C and 6.0, respectively, and are sensitive to heat. Zn ²⁺ and Mg ²⁺ promote its activity, while metal ions such as Fe ³⁺ inhibit its activity. Organic solvents such as methanol and low concentration of ethanol can also promote enzyme activity. At pH6.0 and 35℃, the Michaelis constant K _m of ADH1 acting on phenylacetaldehyde was 3.612mM, and the maximum reaction rate V _max was 18.587μM/min. The enzyme activity under the optimum condition was 1.75μmol/(min·mL).

SEQUENCE LISTING

<110> South China Agricultural University

<120> Preparation method and application of 2-phenylethanol by non-cell synthetic biology

<130>

<160> 18

<170> PatentIn version 3.3

<210> 1

<211> 25

<212>DNA

<213> Primer ARO8F

<400> 1

atgactttac ctgaatcaaa agact 25

<210> 2

<211> 25

<212>DNA

<213> Primer ARO8R

<400> 2

ctatttggaa ataccaaatt cttcg 25

<210> 3

<211> 23

<212>DNA

<213> Primer ARO10F

<400> 3

ttaagcatgg cacctgttac aat 23

<210> 4

<211> 25

<212>DNA

<213> Primer ARO10R

<400> 4

gcgcccacaa gtttctattt tttat 25

<210> 5

<211> 25

<212>DNA

<213> Primer ADH1F

<400> 5

gcttatttag aagtgtcaac aacgt 25

<210> 6

<211> 25

<212>DNA

<213> Primer ADH1R

<400> 6

atgtctatcc cagaaactca aaaag 25

<210> 7

<211> 25

<212>DNA

<213> Primer ADH2F

<400> 7

atgtctattc cagaaactca aaaag 25

<210> 8

<211> 26

<212>DNA

<213> Primer ADH2R

<400> 8

ttattattagaa gtgtcaacaa cgtatc 26

<210> 9

<211> 21

<212>DNA

<213> Primer ADH3F

<400> 9

atgttgagaa cgtcaacatt g 21

<210> 10

<211> 24

<212>DNA

<213> Primer ADH3R

<400> 10

ttattacta gtatcgacga cgta 24

<210> 11

<211> 25

<212>DNA

<213> Primer SFA1F

<400> 11

atgtccgccg ctactgttgg taaac 25

<210> 12

<211> 32

<212>DNA

<213> Primer SFA1R

<400> 12

ctattttatt tcatcagact tcaagacggt tc 32

<210> 13

<211> 30

<212>DNA

<213> Primer p32-ARO8F

<400> 13

cggcggaatt catgacttta cctgaatcaa 30

<210> 14

<211> 28

<212>DNA

<213> Primer p32-ARO8R

<400> 14

ggcctcgagc tatttggaaa taccaaat 28

<210> 15

<211> 31

<212>DNA

<213> Primer p32-ARO10F

<400> 15

tggatccatg gcacctgtta caattgaaaa g 31

<210> 16

<211> 34

<212>DNA

<213> Primer p32-ARO10R

<400> 16

gcgctcgagc tattttttatttcttttaag tgcc 34

<210> 17

<211> 32

<212>DNA

<213> Primer p32-ADH1F

<400> 17

cggatccatg tctatcccag aaactcaaaa ag 32

<210> 18

<211> 31

<212>DNA

<213> Primer p32-ADH1R

<400> 18

cgctcgagtt atttagaagt gtcaacaacgt 31

Claims (10)

1. the acellular synthetic biology preparation method of a 2 phenylethyl alcohol, it is characterised in that be logical in same reaction system Cross the common catalyst system and catalyzing catalyzed conversion of restructuring transaminase I ARO8, phenylpyruvate decarboxylase ARO10 and alcoholdehydrogenase ADH1 composition L-phenylalanine, L-phenylalanine is converted into 2 phenylethyl alcohol via phenylpyruvic acid, hyacinthin；Wherein, in described reaction system, it is With L-phenylalanine and oxaloacetic acid as substrate, recombinase ARO8, ARO10 and ADH1 press enzyme activity with 1:0.25:1,1:1:1, 1:0.5:1 or 1:0.25:2 (U/U/U) proportioning determines, pH value be 5.5～7.5, Mg²⁺Concentration is the reaction system of 1.0～5mM In, under the conditions of bath temperature is 30～40 DEG C, after reaction converts 4～7min, terminate reaction with isopyknic acetonitrile, i.e. prepare 2- Phenethanol.

The acellular synthetic biology preparation method of 2 phenylethyl alcohol the most according to claim 1, it is characterised in that described L-benzene The mol ratio of alanine and oxaloacetic acid is 0.75:l.

The acellular synthetic biology preparation method of 2 phenylethyl alcohol the most according to claim 1, it is characterised in that described The proportioning of ARO8, ARO10 and ADH1 is 1:0.25:1.

The acellular synthetic biology preparation method of 2 phenylethyl alcohol the most according to claim 1, it is characterised in that described Mg²⁺ Concentration is 1.0～2.5mM；It is preferably 1.5mM.

The acellular synthetic biology preparation method of 2 phenylethyl alcohol the most according to claim 1, it is characterised in that described water-bath Temperature is 33～40 DEG C；Response time is 5min.

The acellular synthetic biology preparation method of 2 phenylethyl alcohol the most according to claim 1, it is characterised in that described pH value It is 5.5～6.5；The most described pH value is 6.

The acellular synthetic biology preparation method of 2 phenylethyl alcohol the most according to claim 1, it is characterised in that be first by institute State restructuring transaminase I ARO8, phenylpyruvate decarboxylase ARO10 and alcoholdehydrogenase ADH1 and prepare catalysis altogether by common fixing means System Combi-CLEAs, then utilizes Combi-CLEAs that L-phenylalanine is carried out catalyzed conversion, via phenylpyruvic acid, benzene second Aldehyde is converted into 2 phenylethyl alcohol.

The acellular synthetic biology preparation method of 2 phenylethyl alcohol the most according to claim 7, it is characterised in that described reaction In system, pH value is 5.0, Mg²⁺Concentration is 1.0～5mM, and bath temperature is 40 DEG C, and reaction transformation time is 5min.

The acellular synthetic biology preparation method of 2 phenylethyl alcohol the most according to claim 7, it is characterised in that described urge altogether The preparation method of change system Combi-CLEAs is: ARO8, ARO10 and ADH1 are joined according to 1:1:1,1:0.5:1 or 1:0.25:1 After Bi, after precipitant ammonium sulfate precipitation, form many enzyme aggregates, add glutaraldehyde and many enzyme aggregates are cross-linked, prepare many Enzyme fixed system altogether；Wherein, the saturation of ammonium sulfate is 60～90%, and pH value is 4～6, and the addition of glutaraldehyde is according to it eventually Concentration is 0.05～0.25% to determine.

The acellular synthetic biology preparation method of 2 phenylethyl alcohol the most according to claim 9, it is characterised in that described heavy The saturation 70% of shallow lake agent ammonium sulfate；The pH value of precipitant ammonium sulfate is 5；The enzyme proportioning of ARO8, ARO10 and ADH1 is 1:0.5: 1；The addition of described glutaraldehyde final concentration of 0.1% determines according to it；The temperature of described crosslinking is 20～35 DEG C；Described crosslinking Time be 30～150min.