CN114540328A - A kind of temperature-sensitive alpha-amylase and its preparation method and application - Google Patents

Abstract

A temperature-sensitive alpha-amylase and a preparation method and application thereof belong to the technical field of bioengineering. The temperature-sensitive α-amylase nucleotide sequence is shown in SEQ ID NO 1, and the amino acid sequence is shown in SEQ ID NO 2. The preparation method of temperature-sensitive α-amylase: 1) Obtain a target gene sequence AAO78808.1 through genome analysis, remove the signal peptide and obtain SEQ ID NO 1 after codon optimization, and SEQ ID NO 1 is handed over to the company Synthesis of α-amylase target gene; 2) Construction of recombinant strain containing α-amylase target gene; 3) Expression and purification of recombinant temperature-sensitive α-amylase. The temperature-sensitive α-amylase has good activity at 30-40°C, and has a sharp drop in activity and poor stability at 50°C. It can be used in textile, paper, detergent, fermentation and food industries.

Description

A kind of temperature-sensitive alpha-amylase and its preparation method and application

(1) Technical field

The invention belongs to the technical field of bioengineering, and in particular relates to a temperature-sensitive alpha-amylase and a preparation method and application thereof.

(2) Background technology

Amylase is a general term for a series of enzymes that can hydrolyze starch molecules to obtain products such as glucose, maltose, and cyclodextrin. Amylases can be divided into α-amylase, β-amylase or γ-amylase according to the isomeric type of enzymatic hydrolysis products; according to their enzymatic properties, amylase can be divided into acid amylase, neutral amylase, alkaline Amylase, low temperature amylase, temperature sensitive amylase, high temperature amylase, etc. Among them, α-amylase, also known as α-1,4-glucan-4-glucanohydrolase, is an endonuclease, which can randomly act on the α-1,4 glycosidic bond inside the starch molecule, thereby releasing glucose, etc. product.

Alpha-amylase has a wide range of commercial applications and is an important class of hydrolytic amylase preparations. In the production process of baked goods such as bread, people generally use alpha-amylase to reduce fermentation time and reduce the viscosity of fermented dough, thereby improving the quality of bread and the production efficiency of products. At the same time, substances such as glucose produced by α-amylase hydrolysis of starch help to improve the coloring of bread, improve the flavor of bread, and further increase the competitiveness of products. However, due to the difficulty in controlling the dosage of enzyme preparations, a slight excess may lead to excessive degradation of starch and increased viscosity of products such as bread. Therefore, people are gradually switching to temperature-sensitive α-amylase, because it can play a role in the early stage of production, gradually inactivate during baking, and finally lose activity completely. And different from low-temperature or high-temperature amylase, the optimum reaction temperature of temperature-sensitive amylase is 30℃～60℃, energy consumption is smaller, and it is more environmentally friendly, thus broadening its application in textile, papermaking, detergent, fermentation and food. applications in industry.

(3) Contents of the invention

The purpose of the present invention is to provide a temperature-sensitive alpha-amylase and a preparation method thereof, which have the advantages of high activity, low energy consumption, safety and environmental protection, and the like.

In order to achieve the above object, the present invention adopts the following technical solutions:

In a first aspect, the present invention provides a temperature-sensitive α-amylase, and the amino acid sequence of the temperature-sensitive α-amylase is shown in SEQ ID NO: 2.

In a second aspect, the present invention provides a gene encoding the above temperature-sensitive α-amylase, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO: 1.

In a third aspect, the present invention provides a recombinant expression plasmid into which the above-mentioned encoding gene is inserted.

Preferably, the recombinant expression plasmid is obtained by inserting the coding gene into the BamHI and XhoI sites of the pET-28a(+) vector.

In a fourth aspect, the present invention provides a recombinant genetically engineered bacterium containing the recombinant expression plasmid.

Specifically, the recombinant genetically engineered bacteria are obtained by transferring the recombinant expression plasmid into a host cell E. coli BL21 (DE3).

In a fifth aspect, the present invention provides an application of the above-mentioned recombinant genetically engineered bacteria in the preparation of temperature-sensitive α-amylase.

Further, the recombinant genetically engineered bacteria are prepared as follows: insert the coding gene shown in SEQ ID NO: 1 into the BamHI and XhoI sites of the pET-28a(+) vector to obtain a recombinant expression plasmid; the recombinant expression The plasmid was transferred into host cell E.coli BL21 (DE3) to obtain the recombinant genetically engineered bacteria;

The application is as follows: inoculating the recombinant genetically engineered bacteria into LB liquid medium containing kanamycin, culturing overnight at 37°C and 120 rpm, adding IPTG with a final concentration of 0.25 mM, and continuing to culture at 16°C and 180 rpm for 6 days. ~8h, the cells were collected by centrifugation, the obtained cell pellets were resuspended with PBS buffer, sonicated under ice bath, centrifuged, and the supernatant was collected as crude enzyme liquid; the crude enzyme liquid was obtained by using Ni-NTA agarose affinity Resin for purification: sequentially equilibrate the Ni-NTA agarose affinity resin with equilibration buffer, wash buffer to remove impurities, elution buffer to separate target protein, collect elution buffer containing target protein, and concentrate by ultrafiltration , to obtain the temperature-sensitive α-amylase.

Further, the composition of the equilibration buffer is: 20 mM Tris, 500 mM NaCl, 10% (v/v) glycerol, and the solvent is water;

The composition of the washing buffer is: 20 mM Tris, 500 mM NaCl, 10% (v/v) glycerol, 20 mM imidazole, and the solvent is water;

The composition of the elution buffer was: 20 mM Tris, 500 mM NaCl, 10% (v/v) glycerol, 250 mM imidazole, and the solvent was water.

Preferably, in the LB liquid medium containing kanamycin, the final concentration of kanamycin is 30 μg/mL.

Compared with the prior art, the beneficial effects of the present invention are:

The temperature-sensitive α-amylase can be used in bakery food industries such as bread production. Adding alpha-amylase during production can help dough fermentation, increase production efficiency, and improve the color and flavor of bread. At the same time, the temperature-sensitive α-amylase will gradually inactivate during bread baking, which can effectively prevent the excessive degradation of starch caused by the continuous fermentation of high-temperature α-amylase during baking.

Porous starch is a modified starch obtained by physical, chemical or biological treatment, and is characterized in that there are pores on the surface of starch granules that extend to the interior of the granules. This porous structure makes it have good adsorption capacity, so it can be used as a microcapsule core material or adsorbent, and is widely used in medicine, chemical industry, food, agriculture and other fields. Since starch will gelatinize at high temperature, temperature-sensitive α-amylase is often used in the production of porous starch.

In the modern textile industry, in order to prevent the fabric from breaking due to excessive tension during the weaving process, it must be protected by adding a slurry during the process. Starch is widely used in industrial production due to its wide range of sources, cheap and easy availability, and easy desizing. The temperature-sensitive α-amylase of the present invention can be used to degrade starch into water-soluble products during fabric desizing, which can be easily washed away with water.

The temperature-sensitive alpha-amylase of the present invention has higher activity and better stability, and can be applied in textile, papermaking, detergent, fermentation and food industries and other fields.

(4) Description of drawings

Fig. 1 is a glucose standard curve;

Figure 2 The effect of different temperatures on enzyme activity;

Fig. 3 Stability experiments of enzymes at different temperatures;

Fig. 4 Effect of different pH on enzyme activity;

Fig. 5 Stability experiments of enzymes at different temperatures;

Figure 6 Effects of metal ions and chelators on enzyme activity;

Fig. 7 is the Lineweaver-Burk diagram of enzyme;

Fig. 8 is the TLC analysis result of enzymolysis product;

Figure 9 shows the results of HPLC analysis of enzymatic hydrolysis products.

(5) Specific implementations

The following embodiments will further illustrate the present invention in conjunction with the accompanying drawings. The embodiments are only used to illustrate the present invention and do not limit the scope of the present invention.

Example 1: Preparation of temperature-sensitive alpha-amylase

Table 1 is the experimental material

Peptone BD Company Yeast extract BD Company Sodium chloride Aladdin Agar BD Company Kanamycin Sulfate (Kan) Shanghai Yuan Ye Isopropylthio-β-D-galactoside (IPTG) Biofroxx BCA Protein Concentration Assay Kit (Enhanced) Biyuntian Ni-NTA agarose affinity resin GenScript Ultrafiltration tube Merck

LB liquid medium: peptone 1%, yeast extract 0.5%, sodium chloride 1%.

LB solid medium: peptone 1%, yeast extract 0.5%, sodium chloride 1%, agar 1%-2%.

IPTG mother liquor: Weigh an appropriate amount of isopropylthio-β-D-galactoside (IPTG) powder, prepare it into 100mM with pure water, filter and sterilize it with a 0.22μm syringe filter, and distribute it into 1.5mL centrifuge tubes for storage. Reserve at -20°C.

Kan mother liquor: Weigh an appropriate amount of kanamycin sulfate (Kan) powder, prepare 30 mg/mL with pure water, filter and sterilize with a 0.22 μm syringe filter, dispense into 1.5 mL centrifuge tubes, and store at -20°C spare.

Equilibration buffer: 20 mM Tris, 500 mM NaCl, 10% (v/v) glycerol.

Washing buffer: 20 mM Tris, 500 mM NaCl, 10% (v/v) glycerol, 20 mM imidazole.

Elution buffer: 20 mM Tris, 500 mM NaCl, 10% (v/v) glycerol, 250 mM imidazole.

Experimental steps:

(1) Construction of recombinant strains containing α-amylase gene

The original sequence of α-amylase AAO78808.1 was obtained from EZbiocloud, the signal peptide was removed and SEQ NO.1 was obtained after codon optimization. SEQ NO.1 was handed over to the bio-company to synthesize the target gene PL2. The designed restriction site is BamHI/XhoI, and the target gene is connected with pET-28a(+) empty vector. The ligation product was mixed with the competent recipient bacteria TOP10, placed on ice for 10-15 minutes, immediately taken out after heat shock at 42°C for 120s, then placed on ice for 2-5 minutes, added 800 μL LB liquid medium, 37°C, 200rpm recovery growth for 45min , transform part or all of it onto LB agar plates containing 100 μg/mL kanamycin, cultivate overnight at 37°C, screen the recombinant plasmid, and verify the recombinant plasmid by electrophoresis and sequencing. The sequencing primer sequences are as follows:

Forward primer:

T7: TAATACGACTCACTATAGGG

Reverse primer:

T7ter:TGCTAGTTATTGCTCAGCGGG

(2) Induction of enzyme expression and preparation of crude enzyme solution

The correct recombinant plasmid was transformed into E. coli BL21 (DE3) to obtain a recombinant strain containing α-amylase gene. The recombinant strain containing the α-amylase gene was inoculated in LB/Kan solid plate (the final concentration of Kan was 30 μg/mL), and cultured at 37°C until a single colony was produced. Pick a single colony and inoculate it in 200mL LB/Kan liquid medium (the final concentration of Kan is 30μg/mL), cultivate at 37°C and 180rpm until the OD ₆₀₀ is 0.6-0.8, add IPTG to make the final concentration 0.25mM, 16°C, The culture was continued at 180 rpm for 6-8 h, and the cells were collected by centrifugation at 8000 rpm for 10 min after induction. The bacterial cells were resuspended with 15 mL of PBS buffer, sonicated in an ice bath, centrifuged at 8000 rpm for 10 min, and the supernatant was collected as the crude enzyme solution.

(3) Purification of enzymes

Take an appropriate amount of Ni-NTA agarose affinity resin and add it to the empty tube of the chromatography tube, drain the storage solution naturally, wash it with pure water, and add 4 times the volume of equilibration buffer to equilibrate the resin. Pipette the crude enzyme solution and mix with the resin, and chelate for 3h at 4°C. After the chelation, the crude enzyme solution was naturally drained, and the resin was washed with 3 times the volume of equilibration buffer, washing buffer and elution buffer, respectively. and the corresponding purity. The target protein was collected according to the results of SDS-PAGE, and the pure α-amylase protein PL2 was obtained after ultrafiltration, concentration and desalting through a 30KDa ultrafiltration tube.

(4) Determination of enzyme concentration

The protein concentrations in this description were all measured using the BCA protein concentration assay kit.

Take an appropriate amount of 25mg/mL protein standard solution and dilute it to 0.5mg/mL with PBS buffer. Add 0, 1, 2, 4, 8, 12, 16, 20 μL of standard to standard wells of 96-well plate, and add standard dilution to make up to 20 μL. Dilute an appropriate amount of pure α-amylase protein with buffer to an appropriate volume, and add 20 μL to the sample well of a 96-well plate. According to the quantity of standard and sample, prepare an appropriate amount of BCA working solution according to the volume ratio of BCA kit A solution: BCA kit B solution 50:1, and mix well. Add 200 μL of BCA working solution to each standard well and sample well, and incubate at 37°C for 30 min. Measure the absorbance of each well at 562 nm with a microplate reader, draw a standard curve, and calculate the protein concentration in the sample according to the standard curve.

Example 2: Enzymatic properties of temperature-sensitive alpha amylase

Glucose standard solution: Accurately weigh 75.0 mg of anhydrous glucose, add an appropriate amount of pure water to dissolve and dilute to a 25 mL volumetric flask.

3,5-Dinitrosalicylic acid (DNS) developer solution: Weigh 18.2g of potassium sodium tartrate and heat it in a small amount of distilled water, add 0.63g of 3,5-dinitrosalicylic acid while still hot, and then add 2mol /L NaOH aqueous solution 26.2mL, weigh 0.5g phenol and 0.5g anhydrous sodium sulfite into the prepared solution, stir to dissolve, dilute to 100mL with distilled water, and store in the dark for one week before use.

Standard curve: Take 0, 20, 40, 60, 80, 100 μL of the above-mentioned glucose standard solution into 1.5 mL plastic centrifuge tubes with corresponding numbers, and then add 100, 80, 60, 40, 20, 0 μL of distilled water, respectively, Add 100 μL of DNS reagent, and heat in a boiling water bath for 5 min after mixing. After cooling in running water, add 800 μL of distilled water to each test tube, mix well, pipette 200 μL from each tube into a 96-well plate, and use a microplate reader to detect the wavelength of 492 nm in each tube. The standard curve was drawn with the concentration of glucose as the abscissa and the absorbance (A492) as the ordinate.

(1) The effect of temperature on the activity of amylase

Take 5μL (2mg/mL) of PL2 enzyme solution, add 95μL PBS, add 100μL 1% soluble starch (dissolved in PBS), at different temperatures (20℃, 30℃, 40℃, 50℃, 60℃, 70℃, 80℃) ) for 30 min. After the incubation, it was inactivated in a boiling water bath for 5 minutes, centrifuged to take 100 μL of the supernatant and mixed with 100 μL of the DNS detection solution, placed in a boiling water bath and heated for 5 minutes. After cooling in running water, 800 μL of pure water was added to the reaction solution, and 200 μL was taken to measure the absorbance at 492 nm. . The enzyme activity at different temperatures was calculated according to the definition of enzyme activity, the maximum enzyme activity was taken as 100%, and the relative enzyme activity at other temperatures was calculated to determine the optimum reaction temperature of amylase. The results showed that the optimum reaction temperature of PL2 in hydrolyzing starch was 40℃, the enzyme had good activity in the range of 20℃～50℃, and the enzyme activity was all above 50%. With the increase of temperature, the enzyme activity gradually decreased, and the enzyme activity decreased sharply when the temperature exceeded 50 °C, and the enzyme activity was almost zero when the temperature exceeded 60 °C.

(2) Thermal stability of amylase

Take 5μL (2mg/mL) of PL2 enzyme solution, add 95μL PBS, and incubate at different temperatures (30°C, 40°C, 50°C) for 1h, 2h, 4h, 8h, then add 100μL of soluble starch, the optimum temperature (40 ℃) for 30 min, the enzyme activity was determined by DNS method, the enzyme activity measured at the optimum temperature without incubation was taken as 100%, and the relative enzyme activity under other conditions was calculated to evaluate its temperature stability. The results showed that the stability of PL2 was better when the incubation temperature was 30 ℃ and 40 ℃, and its enzymatic activity remained basically unchanged within 8 h. But at 50 °C, the activity of PL2 dropped sharply and was completely inactivated within 1 h.

(3) The effect of pH on amylase

Take 5μL (2mg/mL) of PL2 enzyme solution, add 95μL of buffers with different pH, add 100μL of 1% soluble starch (dissolved in different pH buffers), incubate at the optimum temperature (40°C) for 30min, and determine the enzyme activity by DNS method. Taking the maximum enzymatic activity as 100%, the relative enzymatic activity under other conditions was calculated to determine the optimum pH for the reaction. The results showed that the optimum pH of PL2 was 8.6. The enzyme activity showed an upward trend in the range of pH 4 to 7, and there was a certain fluctuation in the range of pH 7 to 8.6. inactive.

(4) pH stability of amylase

Take 5μL (2mg/mL) of PL2 enzyme solution, add 95μL of different pH buffers (pH: 6, 7, 8, 8.6, 9), keep at 4°C for 1h, 2h, and 4h respectively, and then add 1% soluble starch (different). pH buffer solution) 100 μL, incubated at the optimum temperature (40°C) for 30 min, and the enzyme activity was determined by DNS method. relative enzyme activity to evaluate its pH stability. The results showed that in the pH range of 6-9, PL2 had good stability, and it still had a minimum activity of about 50% after being incubated for 8 hours.

(5) Effects of metal ions and chelating agents on the activity of PL2

Take 5μL (2mg/mL) of PL2 enzyme solution, add 93μL of buffer with optimal pH, add 100μL of 1% soluble starch (dissolved in optimal pH buffer), and add 2μL of metal ions and chelating agent respectively to make the final concentration 1mM . Incubate at the optimum temperature (40°C) for 30min, measure the enzyme activity by DNS method, take the measured enzyme activity when no metal ions or chelating agents are added as 100%, and calculate the relative enzyme activity under other conditions to judge different ions and chelators. The effect of mixture on enzyme activity. The results showed that the two metal ions Cu ²⁺ and Co ²⁺ had a strong promoting effect on the enzyme activity, and the four metal ions Mg ²⁺ , Mn ²⁺ , Zn ²⁺ and Ba ²⁺ had a stronger effect on the enzyme activity , while the chelating agent EDTA completely inhibited the enzymatic activity.

(6) Determination of enzyme kinetic parameters

Under the optimum reaction conditions, add 100 μL of soluble starch with concentrations of 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, and 1.0%, respectively, and react for 30 minutes. The inverse of the substrate concentration is the abscissa and the inverse of the reaction rate. is the ordinate, and the _Km and _Vmax values of the enzyme were calculated according to the linear regression equation. According to the calculation of the linear regression equation, K _m is 1.17 mg/mL, V _max is 32.0 μmol/L*min, R ² is equal to 0.9985, and R ² is close to 1, indicating that the experimental results are reliable.

(7) Analysis of amylase enzymatic hydrolysis products

1. TLC product analysis

The enzymatic hydrolysis products were analyzed by thin-layer chromatography. 400 μL (0.2 mg/mL) of PL2 enzyme solution was mixed with 400 μL of 1% soluble starch substrate, reacted at the optimum temperature, and 200 μL were reacted at 0.5 h, 1 h, 2 h and 4 h respectively. The material was inactivated in a 100 °C boiling water bath for 5 min, and the supernatant was collected by centrifugation. Take 2mg/mL maltose (maltobiose) and 2mg/mL glucose as the standard products, respectively take the standard product and the reactant and spot them on the thin layer chromatography plate with capillaries. The composition and volume ratio are: n-butanol: acetic acid: water = 3:1:1. When the liquid level migrates to the top 1cm edge, take out the chromatography plate, dry it with a hair dryer, and then put it into a layer developing cylinder and expand it again. After the layering is finished, soak the layered plate in a 10% H ₂ SO ₄ ethanol solution for about half a minute, and place it in a 110° C. oven to heat for 5 minutes to develop color. After color development, the enzymatic hydrolysis products at different time points were analyzed corresponding to the chromatographic results of the standard. The results showed that the degradation products in different time periods were all glucose, and no other products were produced.

2. High performance liquid chromatography

Sample preparation: Take 200 μL of reaction and blank control respectively, filter with 0.22 μm aqueous needle filter for detection. The detection conditions are as follows:

Liquid phase conditions: the mobile phase was pure water, the chromatographic column was TSK gel G3000Pwxl, the flow rate was 0.5 mL/min, the injection volume was 10 μL, and the collection time was 30 min.

Detector: An evaporative light scattering detector was used.

The results show that: as shown in Figure 9, by comparing the peak time of the glucose and maltose standard (maltobiose) and the enzymatic hydrolysis product, it can be further determined that the enzymatic hydrolysis product of PL2 is glucose.

sequence listing

<110> Zhejiang University of Technology

<120> A temperature-sensitive alpha-amylase and its preparation method and application

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705 710 715

Claims (10)

1. A temperature-sensitive α-amylase, characterized in that: the amino acid sequence of the temperature-sensitive α-amylase is shown in SEQ ID NO: 2. 2 . The encoding gene of temperature-sensitive α-amylase according to claim 1 , wherein the nucleotide sequence of the encoding gene is shown in SEQ ID NO: 1. 3 . 3. The recombinant expression plasmid encoding gene insertion according to claim 2. 4. The recombinant expression plasmid according to claim 3, wherein the recombinant expression plasmid is obtained by inserting the coding gene into the BamHI and XhoI sites of the pET-28a(+) vector. 5. The recombinant genetic engineering bacteria containing the recombinant expression plasmid as claimed in claim 3. 6 . The recombinant genetically engineered bacteria according to claim 5 , wherein the recombinant genetically engineered bacteria is obtained by transferring the recombinant expression plasmid into a host cell E. coli BL21 (DE3). 7 . 7. The application of the recombinant genetically engineered bacteria as claimed in claim 5 in the preparation of temperature-sensitive alpha-amylase. 8. application as claimed in claim 7 is characterized in that: described recombinant genetic engineering bacteria is prepared as follows: the BamHI and XhoI site to obtain a recombinant expression plasmid; transfer the recombinant expression plasmid into a host cell E.coli BL21 (DE3) to obtain the recombinant genetically engineered bacteria; The application is as follows: inoculate the recombinant genetically engineered bacteria into LB liquid medium containing kanamycin, cultivate overnight at 37°C and 120rpm, add IPTG with a final concentration of 0.25mM, and continue to culture at 16°C and 180rpm for 6 days. ~8h, the cells were collected by centrifugation, the obtained cell pellets were resuspended with PBS buffer, sonicated under ice bath, centrifuged, and the supernatant was collected as crude enzyme liquid; the crude enzyme liquid was obtained by using Ni-NTA agarose affinity Resin for purification: sequentially equilibrate the Ni-NTA agarose affinity resin with equilibration buffer, wash buffer to remove impurities, elution buffer to separate target protein, collect elution buffer containing target protein, and concentrate by ultrafiltration , to obtain the temperature-sensitive α-amylase. 9. application as claimed in claim 8 is characterized in that: the composition of described equilibration buffer is: 20mM Tris, 500mMNaCl, 10% (v/v) glycerol, and solvent is water; The composition of described washing buffer is : 20 mM Tris, 500 mM NaCl, 10% (v/v) glycerol, 20 mM imidazole, the solvent is water; the composition of the elution buffer is: 20 mM Tris, 500 mM NaCl, 10% (v/v) glycerol, 250 mM imidazole , the solvent is water. The application according to claim 8, wherein in the LB liquid medium containing kanamycin, the final concentration of kanamycin is 30 μg/mL.

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