CN102533689A - Preparation method for vacuum drying of transglutaminase - Google Patents

Abstract

The invention discloses a preparation method of vacuum drying of transglutaminase, which comprises adding a thermal protection agent to the fermented liquid of transglutaminase, standing at room temperature, obtaining supernatant by high-speed refrigerated centrifugation, and using 95% ethanol Precipitate the fermented liquid, obtain the precipitate by high-speed freezing and centrifugation, add an antioxidant protection agent, mix uniformly, and dry in vacuum to obtain a transglutaminase preparation. The stability of the transglutaminase obtained by the preparation method of the invention is greatly improved.

Description

A kind of preparation method of vacuum drying of transglutaminase

technical field

The invention relates to a production process of an enzyme preparation, in particular to a preparation method for vacuum drying of transglutaminase, adding a heat-resistant and anti-oxidation protective agent to stabilize the transglutaminase enzyme, and applying vacuum drying Method to dry microbial fermentation broth to produce transglutaminase.

Background technique

Transglutaminase is a microbial enzyme that catalyzes the acyl transfer reaction, and catalyzes the cross-linking of the γ-carboxamide group of glutamic acid Gln residue present in the peptide chain and the ε-amino group of lysine Lys residue; Intramolecular or intermolecular ε-(γ-Glu)-Lys bridging and bonding effect is formed to form a cross-linked protein structure, thereby improving the functional properties of the protein. Its safe and super-cross-linking properties have been favored by food at home and abroad. It is widely concerned by scientific and technological workers, and has the reputation of "21st century super adhesive". It is often used as an improver to bind meat, surimi, and bread.

Transglutaminase is a thiolase with cysteine as the active center residue. It has poor oxidation resistance, is easily inactivated under high temperature conditions, and is not easy to store. The current method for producing transglutaminase is mainly freeze-drying, which is costly and expensive, and is not conducive to large-scale industrial production.

The present invention proposes a method for improving the stability of transglutaminase, by adding sugar alcohol protective agents to improve the thermal stability of transglutaminase, by adding active peptides to improve the anti-oxidation of transglutaminase stability. In this way, the microbial fermentation liquid can be dried by using the vacuum drying process, which solves the problem that the transglutaminase can only be produced by freeze-drying in industrial production, improves the stability of the transglutaminase, and saves production costs.

Contents of the invention

The present invention proposes a preparation method of vacuum-drying transglutaminase, adding heat protectant to transglutaminase fermented liquid, standing at room temperature, obtaining supernatant through high-speed refrigerated centrifugation, and using 95% ethanol to The supernatant is precipitated, the precipitate is obtained by high-speed refrigerated centrifugation, an antioxidant protective agent is added, mixed uniformly, and vacuum-dried to obtain a transglutaminase preparation; wherein, the thermal protective agent is sugar alcohol substances, including sorbitol and maltitol, and the antioxidant protection agents include active peptides.

The enzyme activity of transglutaminase in the fermentation broth is 10-50u/ml.

The addition amount of described sorbitol is 0.5%～5%w/v, preferably addition amount is 0.5%～2%w/v; The addition amount of described maltitol is 1%～10%w/v, The preferred addition amount is 2%~4%w/v.

The standing time at room temperature is 0.5h-1h.

The volume ratio of the added amount of ethanol to the supernatant is 0.5:1~1.5:1, preferably 0.5:1.

The antioxidant protection agent is preferably wheat protein peptide.

The mass ratio of the added amount of the antioxidant protection agent to the precipitate is 0.5:1~1:1.5, preferably 0.75:1.

Carry out vacuum drying at 35°C-40°C, preferably for 3h-5h.

In the present invention, two high-speed refrigerated centrifuges are used. After adding a thermal protective agent to the fermentation liquid for the first time, the supernatant is obtained through high-speed refrigerated centrifugation to obtain the supernatant of the fermented liquid. After adding ethanol for precipitation, the second high-speed refrigerated centrifugation A precipitate is obtained and an antioxidant is added. In the present invention, the technical effect of highly purifying transglutaminase is achieved by adopting two high-speed refrigerated centrifuges.

Transglutaminase improves the structure and function of proteins by catalyzing intramolecular and intermolecular covalent crosslinks of protein polypeptides. However, the stability of transglutaminase itself is poor, and it is not suitable for long-term storage, thereby affecting the application of transglutaminase in the food field. Thereby the present invention relates to a kind of technique of applying vacuum drying method to produce transglutaminase, adding thermal protection agent to transglutaminase fermented liquid, standing at room temperature for a period of time, selecting 95% ethanol to carry out precipitation to described fermented liquid , high-speed refrigerated centrifugation, discarding the supernatant, adding an antioxidant protection agent to the obtained precipitate, mixing evenly, and drying in vacuum to obtain a transglutaminase enzyme preparation.

In the present invention, vacuum drying is used instead of freeze-drying to produce transglutaminase, which solves the impact of high temperature on the stability of transglutaminase. The thermal protection agent sugar alcohol in the present invention is the compound that the aldehyde group or ketone group of monosaccharide molecule is reduced to alcohol group, makes sugar change into polyalcohol, has higher stability under thermal environment, and this is because Sugar alcohols contain a large number of hydroxyl groups, which can change the hydration structure of proteins in solution and promote hydrophobic interactions between protein molecules, thereby improving the stability of proteins when heated. Suitable sugar alcohols in the present invention include sorbitol, mannitol, erythritol, maltitol, lactitol, xylitol and the like. Different sugar alcohols have different protective effects on proteins, which may be caused by the difference in the number and position of hydroxyl groups. The present invention proposes that among the sugar alcohols, sorbitol and maltitol have the most ideal thermal protection effect on protein, followed by lactitol and xylitol.

In the present invention, the active peptide is used as the antioxidant protection agent. Active peptides are the general term for different peptides from dipeptides to complex linear and cyclic results formed by 20 natural amino acids in proteins with different compositions and arrangements. According to the classification of raw materials, the active peptides applicable to the method of the present invention include wheat peptides , soybean peptide, corn peptide, protein peptide, silk protein peptide and compound peptide, etc. Active peptides have certain nutritional and physiological functions. The existence form of glutamic acid residue in protein is the glutamine of its amide form, and glutamine is the precursor material of the extremely important anti-oxidant-reduced glutathione in synthetic body, thus the present invention adopts active peptide as Antioxidant protectant. The content of glutamic acid in the wheat protein peptide used in the present invention is the highest among all vegetable proteins, accounting for about 35% of the total protein. Therefore, adding the wheat protein peptide can significantly improve the antioxidant capacity of transglutaminase. In addition to wheat protein peptides, most protein peptides can also improve the antioxidant capacity of transglutaminase, but the effect is not as good as wheat protein peptides. Compared with other protective agents (such as amino acids, monovalent salts, etc.) In long-term storage, the total enzyme activity decreases the least, and the cost of active peptides is low, which is suitable for wide application in industrial production.

The innovative point of the present invention is that in the general process of producing transglutaminase, a thermoprotectant is mainly added to stabilize transglutaminase. The anti-oxidation effect is obvious, and the enzyme activity can be significantly improved, and the enzyme produced by this method has better stability. The present invention selects secondary high-speed freezing and centrifuging, and the obtained enzyme preparation has higher purity than that obtained by one freezing and centrifuging. The beneficial effect of the present invention is that: the thermal protection agent can improve the thermal stability of transglutaminase when heated; the antioxidant protection agent such as wheat protein peptide contains a variety of amino acids, which can improve the oxidation resistance of the enzyme during long-term storage . The invention effectively solves the problem of poor stability of transglutaminase and greatly reduces production cost.

The transglutaminase prepared by the present invention is placed at room temperature, and the enzyme activity is measured every other month. The test results show that the transglutaminase obtained according to the preparation method of the present invention has good enzyme stability.

Description of drawings

FIG. 1 is a schematic diagram showing the stability of the transglutaminase enzyme preparation prepared in Example 1 as a function of time.

Fig. 2 is a schematic diagram showing the stability of the transglutaminase enzyme preparation prepared in Example 2 changing over time.

Fig. 3 is a schematic diagram showing the stability of the transglutaminase enzyme preparation prepared in Example 3 changing over time.

FIG. 4 is a schematic diagram showing the stability of the transglutaminase enzyme preparation prepared in Example 4 changing over time.

Fig. 5 is a schematic diagram showing the stability of the transglutaminase enzyme preparation prepared in Example 5 changing over time.

Fig. 6 is a schematic diagram showing the stability of the transglutaminase enzyme preparation prepared in Example 6 as a function of time.

FIG. 7 is a schematic diagram showing the stability of the transglutaminase preparation prepared in Example 7 over time.

Detailed ways

The present invention will be described in further detail in conjunction with the following specific examples and accompanying drawings, and the protection content of the present invention is not limited to the following examples. Without departing from the spirit and scope of the inventive concept, changes and advantages conceivable by those skilled in the art are all included in the present invention, and the appended claims are the protection scope.

The preparation method of vacuum-drying transglutaminase of the present invention comprises the following steps: adding a thermoprotectant to the fermented liquid of transglutaminase, standing at room temperature, obtaining supernatant through high-speed refrigerated centrifugation, and using 95% ethanol to The supernatant is precipitated, and the precipitate is obtained by high-speed refrigerated centrifugation, and an antioxidant protection agent is added, mixed evenly, and vacuum-dried to obtain a transglutaminase enzyme preparation; wherein, the heat protection agent is a sugar alcohol substance, including sorbic acid Sugar alcohols and maltitol, antioxidant protection including active peptides.

Wherein, the enzyme activity of the transglutaminase in the transglutaminase fermented liquid is 10-50u/ml.

In the present invention, the added amount of sorbitol is 0.5%~5%w/v, preferably 0.5%~2%w/v. The added amount of maltitol is 1%~10%w/v, preferably 2%~4%w/v. The mass ratio of the amount of wheat protein peptide added to the precipitate is preferably 0.75:1 (w/w).

In the present invention, the fermented liquid added with the thermal protection agent is subjected to high-speed freezing and centrifugation, and the sediment is discarded to obtain the supernatant. The volume ratio of the amount of ethanol to the supernatant obtained by high-speed refrigerated centrifugation of the fermented liquid after adding the heat protectant is 0.5:1 (v/v).

After adding the heat protectant to the fermentation broth, it is left at room temperature for 0-2 hours, preferably 0.5-1 hours.

The fermented liquid added with ethanol is subjected to high-speed refrigerated centrifugation, and the supernatant is discarded to obtain a precipitate, and an antioxidant protection agent is added.

Wherein, the vacuum drying is carried out in a vacuum environment of 35°C-45°C, and the drying time is preferably 3-5h.

The thermal protectant in the present invention is sugar alcohols, including maltitol, xylitol, lactitol, sorbitol, mannitol, erythritol and the like. The antioxidant protection agent in the present invention is active peptides, including milk peptides, wheat peptides, soybean peptides, corn peptides, egg white peptides, silk protein peptides and composite peptides.

Example 1

Add ethanol to 100ml of transglutaminase fermentation broth produced by Streptoverticillium mobaraense at a volume ratio of 0.5:1 ethanol:fermentation broth to precipitate the enzyme, high-speed refrigerated centrifuge setting: 6000r/ min, 4°C, and centrifuged for 20 minutes to obtain a precipitate, put the precipitate into a vacuum drying oven, and dry at 40°C for 3 hours to obtain a transglutaminase enzyme preparation. Seal it with a ziplock bag, and measure the enzyme activity regularly.

Example 2

Add 1% (w/v) sorbitol to 100ml of transglutaminase fermentation broth produced by Streptoverticillium mobaraense , stir and place at room temperature for 30min, then use 0.5:1 ethanol: fermentation broth Add ethanol to the volume ratio to precipitate the enzyme, set the high-speed refrigerated centrifuge: 6000r/min, 4°C, centrifuge for 20min to obtain the precipitate, put the precipitate in a vacuum drying oven, dry at 40°C for 3h, and obtain glutamine transfection Aminase enzyme preparation. Seal the bag with a ziplock, and measure the enzyme activity regularly.

Example 3

Add 3% (w/v) maltitol to 100ml of transglutaminase fermented broth produced by Streptoverticillium mobaraense , stir and place at room temperature for 30min, then use 0.5:1 ethanol: fermented broth Add ethanol to the volume ratio to precipitate the enzyme, set the high-speed refrigerated centrifuge: 6000r/min, 4°C, centrifuge for 20min to obtain the precipitate, put the precipitate in a vacuum drying oven, dry at 40°C for 3h, and obtain glutamine transamination Enzyme preparations. Seal the bag with a ziplock, and measure the enzyme activity regularly.

Example 4

Add 3% (w/v) maltitol and 1% sorbitol to 100ml of transglutaminase fermentation broth produced by Streptoverticillium mobaraense , stir and place at room temperature for 30min, then add 0.5:1 Ethanol: the volume ratio of the fermentation broth Add ethanol to precipitate the enzyme, set the high-speed refrigerated centrifuge: 6000r/min, 4°C, centrifuge for 20min to obtain the precipitate, put the precipitate in a vacuum drying oven, dry at 40°C for 3h, A transglutaminase enzyme preparation is obtained. Seal the bag with a ziplock, and measure the enzyme activity regularly.

Example 5

Add ethanol to 100ml of transglutaminase fermentation broth produced by Streptoverticillium mobaraense at a volume ratio of 0.5:1 ethanol:fermentation broth to precipitate the enzyme, high-speed refrigerated centrifuge setting: 6000r/ min, 4°C, and centrifuge for 20 minutes to obtain the precipitate, add wheat protein peptide at a mass ratio of wheat protein peptide: precipitate of 0.75:1, mix well, put it in a vacuum drying oven, and dry for 3 hours to obtain transglutaminase Enzyme. Seal the bag with a ziplock, and measure the enzyme activity regularly.

Example 6

Add 1% (w/v) sorbitol to 100ml of transglutaminase fermentation broth produced by Streptoverticillium mobaraense , stir and place at room temperature for 30min, then mix 0.5:1 ethanol: fermentation broth Add ethanol to precipitate the enzyme, set the high-speed refrigerated centrifuge: 6000r/min, 4°C, centrifuge for 20min, add wheat protein peptide to the obtained precipitate, and the mass ratio of the added amount to the precipitate is 0.75:1 , put into a vacuum drying oven, and dry for 3 hours to obtain a transglutaminase enzyme preparation. Seal the bag with a ziplock, and measure the enzyme activity regularly.

Example 7

Add 1% (w/v) sorbitol and 3% (w/v) maltitol to 100ml of transglutaminase fermentation broth produced by Streptoverticillium mobaraense , stir and place at room temperature for 30min. Add ethanol at a volume ratio of 0.5:1 ethanol:fermentation broth to precipitate the enzyme, set the high-speed refrigerated centrifuge at 6000r/min, 4°C, and centrifuge for 20min, add wheat protein peptide to the obtained precipitate, and the amount of addition is the same as The mass ratio of the precipitate was 0.75:1, put it into a vacuum drying oven, and dry for 3 hours to obtain a transglutaminase preparation. Seal the bag with a ziplock, and measure the enzyme activity regularly.

Example 8

The transglutaminase enzyme preparation that embodiment 1-7 prepares is carried out enzyme activity detection by standard hydroxamate assay method, and its step is: get 2ml reagent A liquid and reagent B liquid respectively and add in the grinding mouth test tube, Incubate at 37°C for 15 minutes, add 0.2ml of enzyme solution, react for 10 minutes, add 2ml of solution B to the test tube added with solution A to terminate the reaction, add 2ml of solution A to the test tube added with solution B, filter, and use the reaction solution of the second test tube as a blank control. OD value was measured at 525nm. A standard curve was made with L-glutamic acid-γ-monohydroxamic acid.

Reagent A (reaction solution): containing 0.2 M Tris-HCl, 0.1 M hydroxylamine hydrochloride, 0.01 M reduced glutathione, 0.03 M Na-CBZ-Gln-Gly, pH 6.0.

Reagent B (stop solution): Mix the following three components in a volume ratio of 1:1:1. 3 mol/L HCl, 12% trichloroacetic acid (W/V), 5% ferric chloride (W/V).

The enzyme activity test results of the transglutaminase preparation prepared in Example 1 are shown in Figure 1. The total enzyme activity of transglutaminase without any protective agent was low, only 2098 (U), and with As time goes by, the total enzyme activity drops more, from 2098U to 1132U, and the decline rate reaches more than 50%, which shows that the stability is poor during long-term storage.

The enzyme activity detection result of the transglutaminase preparation enzyme preparation prepared in embodiment 2 is as shown in Figure 2, compares with Figure 1, after adding sorbitol, the total enzyme activity of the transglutaminase obtained is significantly higher than that without Total enzyme activity of transglutaminase obtained by adding protective agent. The total enzyme activity was 2743U, which increased by 700U, and as time went by, the enzyme activity decreased very little, from 2743U to 2400U.

The enzyme activity detection results of the transglutaminase enzyme preparation prepared in Example 3 are shown in Figure 3. Compared with Figure 1, after adding maltitol, the total enzyme activity of the transglutaminase obtained is significantly higher than that without adding The total enzyme activity of transglutaminase obtained by the protective agent was 2631U, and as time went by, the enzyme activity decreased very little, from 2631U to 2389U.

The enzyme activity detection results of the transglutaminase preparation prepared in Example 4 are shown in Figure 4. Compared with Figure 1, after adding sorbitol and maltitol, the total enzyme activity of the transglutaminase obtained is significantly higher than The total enzyme activity of transglutaminase obtained without adding protective agent is also higher than the total enzyme activity obtained by adding sorbitol and maltitol alone (Example 2-3), the total enzyme activity is 3023U, with time Over time, the enzyme activity decreased slightly, from 3023U to 2632U.

The enzyme activity test results of the transglutaminase preparation prepared in Example 5 are shown in Figure 5. Compared with Figure 1, after adding wheat protein peptide, the total enzyme activity of the transglutaminase obtained is significantly higher than that without The total enzyme activity of transglutaminase obtained by adding the protective agent reached 2754U, and as time went on, the enzyme activity decreased very little, from 2754U to 2471U.

The enzyme activity detection results of the transglutaminase preparation prepared in Example 6 are shown in Figure 6. Compared with Figure 1, after adding sorbitol and wheat protein peptide, the total enzyme activity of the obtained transglutaminase is significantly Higher than the total enzyme activity of the transglutaminase obtained without adding protective agent, also higher than the total enzyme activity of the transglutaminase obtained by embodiment 2-4, the total enzyme activity is 2980U, and with Over time, the enzyme activity decreased very little, from 2980U to 2687U.

The enzyme activity test results of the transglutaminase preparation prepared in Example 7 are shown in Figure 7. Compared with Examples 1-6, according to the preparation method of transglutaminase of the present invention, sorbitol and maltose were added After alcohol and wheat protein peptide, the total enzyme activity of transglutaminase obtained was the highest, which was 3543U, and the enzyme activity decreased the least over time, from 3543U to 3331U. It can be seen that adding 0.5%-2% (w/v) sorbitol, 2%-4% (w/v) maltitol and a certain quality of wheat protein peptide can improve the thermal stability of transglutaminase and antioxidant properties, played a role in long-term preservation.

Example 9

In this embodiment, lactitol and xylitol are used as thermal protectants, and the remaining operating methods are basically the same as in Example 4. After testing, the total enzyme activity of transglutaminase obtained is higher than that of transglutaminase without any protective agent, but not as good as that obtained by adding sorbitol and maltitol total enzyme activity. Therefore, the present invention proposes to use sorbitol and maltitol as heat protectants, which have the most ideal effect on protein heat protection.

Example 10

In this embodiment, soybean protein peptide is used as an antioxidant protection agent, and the remaining operation methods are basically the same as in Embodiment 5. After testing, the obtained transglutaminase is higher than the total enzyme activity of the transglutaminase without adding any protective agent, but not as good as the total enzyme activity of the transglutaminase obtained by adding wheat protein peptide. Therefore, the present invention proposes to use wheaten peptide as an antioxidant protection agent, which has the most ideal effect on protein antioxidant protection.

Claims (8)

1. the vacuum drying preparation method of Transglutaminase EC2.3.2.13 is characterized in that, said method comprises adds thermal protecting agent in glutamine transferred amine enzyme fermentation broth; Room temperature leaves standstill, and obtains supernatant through the high speed frozen centrifugation, with 95% ethanol said supernatant is precipitated; Obtain throw out through the high speed frozen centrifugation; Add anti-oxidant protective agent, through mix, vacuum-drying, obtain the Transglutaminase EC2.3.2.13 zymin; Wherein, said thermal protecting agent is the glycitols material, comprises Sorbitol Powder and maltose alcohol, and said anti-oxidant protective agent comprises bioactive peptide.

2. the vacuum drying preparation method of Transglutaminase EC2.3.2.13 according to claim 1 is characterized in that, the enzyme work of Transglutaminase EC2.3.2.13 is 10 ~ 50u/ml in the described fermented liquid.

3. the vacuum drying preparation method of Transglutaminase EC2.3.2.13 according to claim 1 is characterized in that the addition of said Sorbitol Powder is 0.5% ~ 5%w/v, and preferred addition is 0.5% ~ 2%w/v; The addition of said maltose alcohol is 1% ~ 10%w/v, and preferred addition is 2% ~ 4%w/v.

4. the vacuum drying preparation method of Transglutaminase EC2.3.2.13 according to claim 1 is characterized in that said room temperature time of repose is 0.5h-1h.

5. the vacuum drying preparation method of Transglutaminase EC2.3.2.13 according to claim 1 is characterized in that the volume ratio of said alcoholic acid addition and supernatant is 0.5:1 ~ 1.5:1, and preferred proportion is 0.5:1.

6. the vacuum drying preparation method of Transglutaminase EC2.3.2.13 according to claim 1 is characterized in that said anti-oxidant protective agent is preferably the wheat protein peptide.

7. the vacuum drying preparation method of Transglutaminase EC2.3.2.13 according to claim 1 is characterized in that the addition of said anti-oxidant protective agent and sedimentary mass ratio are 0.5:1 ~ 1:1.5, and preferred proportion is 0.75:1.

8. the vacuum drying preparation method of Transglutaminase EC2.3.2.13 according to claim 1 is characterized in that, under 35 ℃-40 ℃, carries out vacuum-drying, and the preferred time is 3h-5h.

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