WO2019104761A1 - 一种重组脯氨酸氨肽酶发酵高产及制备脱苦大米肽的方法 - Google Patents

一种重组脯氨酸氨肽酶发酵高产及制备脱苦大米肽的方法 Download PDF

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WO2019104761A1
WO2019104761A1 PCT/CN2017/116188 CN2017116188W WO2019104761A1 WO 2019104761 A1 WO2019104761 A1 WO 2019104761A1 CN 2017116188 W CN2017116188 W CN 2017116188W WO 2019104761 A1 WO2019104761 A1 WO 2019104761A1
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aminopeptidase
fermentation
preparing
appropriate amount
rice
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French (fr)
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田亚平
王开道
李婷婷
张大伟
周楠迪
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江南大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/485—Exopeptidases (3.4.11-3.4.19)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/11—Aminopeptidases (3.4.11)
    • C12Y304/11001—Leucyl aminopeptidase (3.4.11.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/11—Aminopeptidases (3.4.11)
    • C12Y304/11005—Prolyl aminopeptidase (3.4.11.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/11—Aminopeptidases (3.4.11)
    • C12Y304/11009—Xaa-Pro aminopeptidase (3.4.11.9), i.e. aminopeptidase P

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  • the invention relates to a method for high-yield fermentation of recombinant proline aminopeptidase and preparation of debitter rice peptide, belonging to the fields of fermentation technology, enzyme preparation and food additive.
  • the optimization strategy of microbial fermentation culture often starts from the optimization of culture medium and the control of fermentation process. At present, it is not possible to infer and calculate the medium formula and fermentation process control strategy suitable for a certain microorganism from the basic principle of biochemical reaction.
  • the medium and fermentation process control strategies suitable for different microorganisms are different. No culture method can be suitable for fermentation of a variety of microorganisms. Therefore, a small fermentation equipment such as a shake flask or a glass jar is required for a specific microorganism. According to a certain experimental design and test method, the combination of fermentation medium composition and fermentation process control and other fermentation strategies can be selected to select the most suitable culture method.
  • Enzyme preparation refers to a substance with enzymatic properties extracted from living organisms, and has been widely used in various fields such as medicine, chemical industry, food, brewing, etc., and has a wide range of applications.
  • the food processing industry is closely related to people's lives. Enzymes are used more and more in the food processing industry, and their roles are becoming more and more important. They are greatly reflected in meat processing, deep hydrolysis of proteins and as food additives.
  • Non-specific aminopeptidase can broadly hydrolyze the N-terminal amino acid residue of a protein or polypeptide, and can hydrolyze different amino acid residues of the polypeptide or protein N-terminal to release different kinds of amino acids.
  • the highly specific aminopeptidase can hydrolyze the endoprotease and the non-specific exonuclease unhydrolyzable amino acid residues, which can effectively deepen the proteolytic depth and improve the nutritional value of the protein hydrolysate.
  • Rice is one of the most abundant food crops in the world. Rice protein is a product processed from rice.
  • rice protein has a very broad application prospect in the food industry.
  • the hydrolyzate tends to exhibit a strong bitter taste, which affects the field of application. How to improve the degree of hydrolysis of rice protein, remove or reduce the bitterness of hydrolyzate and increase the nutritional value of hydrolysate by synergistic degradation of enzymes has become an urgent problem to be solved.
  • the present invention is based on fermentation kinetics to guide the fermentation high yield and yield of recombinant subtilisin aminopeptidase
  • the proline aminopeptidase was prepared by hydrolyzing rice protein with proline aminopeptidase and alkaline protease and leucine aminopeptidase.
  • the yield of proline aminopeptidase was significantly increased, and the multi-enzyme synergistic hydrolysis improved the hydrolysis degree of rice protein.
  • the bitterness of the hydrolyzate is alleviated and the nutritional value of the hydrolyzate is improved, and the hydrolyzate exhibits a certain ⁇ -glucosidase inhibitory effect.
  • the proline aminopeptidase produced by the invention is an aminopeptidase with strict substrate specificity, and the invention improves the yield of proline aminopeptidase, and reduces the cost and utilization of downstream preparation of proline aminopeptidase.
  • Proline aminopeptidase specifically excise the N-terminal proline residue, which can remove some of the less specific aminopeptidase to further hydrolyze Pro, and can play a better synergistic hydrolysis and debittering effect.
  • the present invention provides a method for efficiently producing proline aminopeptidase using recombinant Bacillus subtilis to increase proline aminopeptidase production and reduce the cost of downstream separation preparation.
  • the method includes the following steps:
  • the activated recombinant Bacillus subtilis is connected to the seed culture medium, and cultured by using a rotary constant temperature shaker, the rotation speed is 220 r/min, the culture temperature is 37 ° C, and the culture is carried out for 20 hours to prepare a seed liquid;
  • the seed medium component in the step (1) is sodium chloride 10 g / L, tryptone 10 g / L, yeast powder 5 g / L, after sterilization, adding filter-sterilized kanamycin to a final concentration of 50 ⁇ g / mL.
  • the fermentation medium component in the step (2) is glucose 20 g / L, yeast extract 60 g / L, fish meal 18.75 g / L, ammonium chloride 3.25 g / L, K 2 HPO 4 12.54 g / L, KH 2 PO 4 2.31 g/L, 0.1% (v/v) phytic acid, pH 7.0.
  • the filter-sterilized kanamycin was added to a final concentration of 50 ⁇ g/mL.
  • the method in particular, activating a recombinant Bacillus subtilis plate preserved at -40 ° C glycerol tube twice to prepare an activated recombinant Bacillus:
  • the volume of the culture medium is 150 mL
  • the shake flask used is a volume of 500 mL
  • the culture is carried out by using a rotary constant temperature shaker
  • the control rotation speed is 220 r/min
  • the culture temperature is 37 ° C
  • the culture is carried out for 20 hours.
  • step (3) fermentation samples were taken every 4 hours to determine the dry weight of the cells, the activity of intracellular proline aminopeptidase and the activity of extracellular proline aminopeptidase.
  • the invention also provides a method for hydrolyzing rice protein, which is followed by hydrolysis with alkaline protease, leucine aminopeptidase and proline aminopeptidase.
  • the method comprises preparing a rice protein solution having a mass concentration of 5-8%,
  • the amount of alkaline protease added in the step (1) is 5000-20000 U/g rice protein.
  • the amount of leucine aminopeptidase added in the step (2) is 1000-4000 U/g rice protein.
  • the amount of proline aminopeptidase added in the step (3) is 50-200 U/g rice protein.
  • the casein solution is prepared by using pure water and adjusting the pH to 9.0 and 90 ° C in a water bath for 30 minutes.
  • the method specifically comprises: preparing a 5% rice protein solution with pure water, adjusting the pH to 9.0, 90 ° C water bath with a base for 30 min, and using the base as a 2 M NaOH solution;
  • the obtained hydrolyzate was centrifuged at 10,000 rpm for 15 min, and the supernatant was taken; the supernatant was diluted with 10% trichloroacetic acid in an equal volume, and allowed to stand for 1 h, and then the needle filter was used. After filtration, the filtrate was centrifuged at 10,000 rpm for 10 min, and the supernatant was again taken. The polypeptide molecular weight distribution and free amino acid analysis were separately performed. At the same time, the hydrolyzate was assayed for the ⁇ -glucosidase inhibitory effect.
  • the invention adopts fermentation kinetic parameters as a guide to obtain a fermentation process condition for high yield proline aminopeptidase; the rice protein is used as a substrate, and the enzyme is hydrolyzed by alkaline protease and leucine aminopeptidase, and the hydrolysis degree is based on To optimize the amount of alkaline protease and leucine aminopeptidase added in rice proteolysis; determine the amount of proline aminopeptidase based on free Pro.
  • the invention also provides for the use of the method in the field of food and beverage and in the processing and utilization of food protein resources.
  • the present invention also provides a method for purifying the ⁇ -glucosidase inhibitory active peptide by centrifuging the hydrolyzate at 10000 rpm for 15 min, taking the supernatant, and lyophilizing.
  • Hi Trip DEAE FF anion exchange layer was dissolved in 20 mM pH 7.5 PB buffer The flow rate was 1 mL/min, and the phase elution was carried out with 150 mM, 350 mM, 550 mM NaCl. Each stage was eluted with 10 column volumes, and the eluate was collected, dialyzed extensively, and concentrated by lyophilization. The lyophilized powder was rehydrated, and the inhibition rate of ⁇ -glucosidase of each peak was measured. The fraction with high inhibition rate was subjected to Sephadex G-15 gel chromatography, collected by peak, lyophilized, and rehydrated to determine the inhibition rate of ⁇ -glucosidase.
  • the invention determines the fermentation process conditions of the high-yield recombinant Bacillus subtilis proline aminopeptidase according to the fermentation kinetic parameters, that is, the rotation speed is adjusted to 0-6h 200rpm, 6-12h is 400rpm, and 12-28h is 500rpm. 28h to the end of fermentation is 400rpm; pH is controlled from 0-12h without pH control, 12-16h is pH7.0, 16-28h pH is not controlled, pH is 7.0 after 28h; temperature is regulated at 40°C 8h before fermentation, 8 -12h is 35 ° C, 12 h to the end of the fermentation is 33 ° C.
  • the optimized yield of proline aminopeptidase reached 174.8 U/mL, which was 1.66 times the highest level before optimization.
  • the enzyme cooperates with alkaline protease and leucine aminopeptidase to hydrolyze rice protein, and determines that the amount of alkaline protease added is 15000 U/g rice protein, hydrolysis is 4 h, and the amount of leucine aminopeptidase is 3000 U/g rice protein. Hydrolyzed for 2h, the free amino acid content of the three enzymes was 3.844mg/mL, which was 27.4 times of the free amino acid content before unhydrolysis.
  • the hydrolyzate mainly existed as small peptides and free amino acids, and the content of small peptides below 180Da reached 44.70%, fully hydrolyzed the exposed N-terminal proline residue, the free proline content was 0.149mg/mL, which was 1064.3 times of the unhydrolyzed free proline content, and the hydrophobic amino acid content in the hydrolyzate was significantly increased.
  • the bitterness of hydrolysis is alleviated, the depth of hydrolysis of rice protein is deepened, the nutritional value of the hydrolyzate is improved, and the ⁇ -glucosidase inhibitory active peptide in the separation and purification of the hydrolyzate has a component with strong inhibitory activity, and functions and health foods. And the beverage has a good application prospect.
  • Figure 2 Analysis of fermentation kinetics under different conditions (a: 1:200 rpm, 2:300 rpm, 3:400 rpm, 4:500 rpm, b: 1:200 rpm, 2:300 rpm, 3:400 rpm, 4:500 rpm; c: 1 : no pH control, 2: pH 7.0, 3: pH 7.5, d: 1: no pH control, 2: pH 7.0, 3: pH 7.5; e: 1:30 ° C, 2: 33 ° C, 3: 35 ° C, 4: 37 ° C, 5: 40 ° C, f: 1: 30 ° C, 2: 33 ° C, 3: 35 ° C, 4: 37 ° C, 5: 40 ° C)
  • Biomaterial sample The recombinant preparation of Bacillus subtilis (Bacillus subtilis WB600 with histidine-tagged proline aminopeptidase, the plasmid used is PMA5) and the purification preparation of proline aminopeptidase are described in Chinese patent CN105925650A. .
  • the enzyme activity unit (U) is defined as the amount of enzyme required to decompose L-valine-p-nitroaniline to produce 1 ⁇ M p-nitroaniline at 50 ° C per minute.
  • Alkaline protease enzyme unit definition The amount of enzyme required to hydrolyze casein to produce 1 ⁇ g of tyrosine per minute at 40 °C.
  • Leucine aminopeptidase enzyme unit definition The amount of enzyme required to decompose L-leucine-p-nitroaniline to produce 1 ⁇ M p-nitroaniline per minute at 50 °C.
  • T&J TypeA 5L fermenter (Shanghai Dibir Bioengineering Co., Ltd.)
  • Polypeptide molecular weight distribution detection Waters600 high performance liquid chromatography (with 2487 UV detector and Empower workstation).
  • ⁇ -glucosidase inhibition rate 1.5U/mL ⁇ -glucosidase solution was prepared with potassium phosphate buffer of pH 6.8, 70 ⁇ L of enzyme solution was mixed with 70 ⁇ L of sample and incubated for 10 min in a 37 ° C water bath, and then added. 70 ⁇ L of potassium phosphate buffer pH 6.8 containing 10 mM pNPG was reacted in a water bath at 37 ° C for 1 h. The reaction was then stopped by the addition of 70 ⁇ L of 1 M Na 2 CO 3 solution. The absorbance A was measured at 405 nm. The ⁇ -glucosidase inhibition rate is calculated as follows:
  • a 0 is the control
  • a i is the sample absorbance
  • a j is the sample control
  • Example 1 Recombinant Bacillus subtilis fermentation culture under different rotation speed conditions
  • the recombinant Bacillus subtilis preserved at -40 °C glycerol tube was inserted into the seed culture medium, and cultured using a rotary constant temperature shaker at a shaking speed of 220 r/min, a culture temperature of 37 ° C, and culture for 20 hours.
  • the seed liquid was inoculated with 5% inoculum, and sterile air was introduced into the fermentation process.
  • the aeration rate was 1.5vvm
  • the fermentation temperature was 37°C, pH7.0, stirring speed. 200 rpm, 300 rpm, 400 rpm, and 500 rpm were set, respectively, and the fermentation time was 36 hours.
  • samples were taken every 2 hours, and the dry weight of the cells, the activity of extracellular proline aminopeptidase and the activity of intracellular proline aminopeptidase were measured.
  • Example 2 Fermentation culture under the guidance of fermentation kinetics
  • the seed solution was introduced into a 5 L fermentor containing 3 L of fermentation medium at a 5% inoculum, and sterilized during the fermentation.
  • samples were taken every 4 hours, and the dry weight of the cells, the extracellular proline aminopeptidase and the activity of intracellular proline aminopeptidase were measured.
  • the content of small peptide below 180Da reached 44.70%, fully hydrolyzed the exposed N-terminal proline residue, free ammonia
  • the acid content was 0.149 mg/mL, which was 1064.3 times the unhydrolyzed free proline content.
  • the inventors also compared the effects of different enzymatic hydrolysis methods on the hydrolysis of rice protein, and the results are shown in Table 1.
  • the results showed that the content of the small peptide below 180 Da was 13.68% when the alkaline protease was hydrolyzed alone, and the content of the small peptide below 180 Da when the alkaline protease and the leucine aminopeptidase were hydrolyzed was 42.21%.
  • 1 rice protein solution
  • 2 alkaline protease single enzyme hydrolyzate
  • 3 alkaline protease and leucine aminopeptidase double enzyme hydrolysate
  • 4 proline aminopeptidase, alkaline protease and leucine Acid aminopeptidase synergistic hydrolyzate.
  • the free amino acid content obtained by the method of the invention is 3.484 mg/mL, which is 27.4 times of the unhydrolyzed free amino acid content, wherein the proline content is 1064.3 times unhydrolyzed, and the hydrophobic amino acid content is significantly improved.
  • the free amino acid content was 1.121 mg/mL
  • the free amino acid content of the alkaline protease and leucine aminopeptidase double enzyme was 3.567 mg/mL.
  • the free proline content was significantly lower than the three enzyme synergistic hydrolysate of 0.083 mg/mL.
  • the invention synergistically hydrolyzes rice protein by proline aminopeptidase and alkaline protease and leucine aminopeptidase, and the content of free amino acid and the content of small peptide in the obtained hydrolysate are greatly increased, and the content of hydrophobic amino acid is also significantly improved.
  • the degree of hydrolysis of rice protein is not greatly improved, and the bitterness of the hydrolyzate is also removed or reduced.
  • the rice protein hydrolysis method of the present invention is to prepare a rice protein solution having a mass concentration of 8%.
  • the amount of alkaline protease added was 20000 U/g rice protein; the amount of leucine aminopeptidase added was 4000 U/g rice protein; the amount of proline aminopeptidase added was 100 U/g rice protein.
  • the polypeptide of 2000 Da or more in the obtained rice protein hydrolyzate was substantially absent, and the small peptide content below 180Da reached 48.26%, the free amino acid content was 5.852 mg/mL, and the free proline content was unhydrolyzed 1136.4. Times.
  • Example 5 Method for separating and purifying ⁇ -glucosidase inhibitory activity peptide
  • the lyophilized powder was dissolved in 1 mL of pure water, and the inhibition rate of ⁇ -glucosidase and the polypeptide concentration were measured.
  • the component F 4 b which gave the most potent ⁇ -glucosidase inhibitory effect had an IC 50 of 132.6 ⁇ g/mL.

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Abstract

一种利用重组枯草芽孢杆菌发酵高产脯氨酸氨肽酶的方法，包括以下步骤：1)将活化后的重组枯草芽孢杆菌接入种子培养基，培养制得种子液；（2）将种子液接入发酵培养基中，通入无菌空气至发酵结束。一种应用脯氨酸氨肽酶制备脱苦大米肽的方法，涉及在大米蛋白溶液中加入适量碱性蛋白酶，反应一定时间后冷却，继续加入适量亮氨酸氨肽酶，反应一定时间后冷却，再加入适量脯氨酸氨肽酶进行酶解。还包括一种制备a-葡萄糖苷酶抑制活性肽的方法，包括制备脱苦大米肽、离心取上清液、以及阴离子交换层析和凝胶层析等步骤。

Description

一种重组脯氨酸氨肽酶发酵高产及制备脱苦大米肽的方法 技术领域
本发明涉及一种重组脯氨酸氨肽酶发酵高产及制备脱苦大米肽的方法，属于发酵技术、酶制剂、食品添加剂领域。
背景技术
微生物的发酵培养优化策略往往从培养基的优化和发酵过程控制两方面入手，目前还不能完全从生化反应的基本原理来推断和计算出适合某种微生物的培养基配方和发酵过程控制策略，且不同的微生物所适合的培养基和发酵过程控制策略都不尽相同，没有一种培养方法能够适合多种微生物的发酵培养，所以对某一特定微生物需要采用摇瓶、玻璃罐等小型发酵设备，按照一定的实验设计和试验方法将培养基组成和发酵过程控制等发酵策略组合优化才能选择出最适合的培养方法。
现阶段脯氨酸氨肽酶的发酵产量还比较低，不能满足工业制备的需求，这也是脯氨酸氨肽酶至今没有商业化产品的关键因素。因此，如何提高通过发酵策略提高脯氨酸氨肽酶发酵产量成为迫切需要解决的问题。
酶制剂是指从生物中提取的具有酶类性质的物质，现已应用在医药、化工、食品、酿造等各领域，应用范围非常的广泛。食品加工业与人们的生活关系紧密，酶在食品加工业中的应用越来越多，作用也越来越重要，在肉类加工、蛋白质的深度水解和作为食品添加剂中有很大体现。
酶制剂伴随着社会的发展其应用也越来越广泛，氨肽酶也在不断的开拓其应用领域。非特异的氨肽酶能广谱的水解蛋白或多肽N-端的氨基酸残基，可以水解多肽或蛋白N-端不同种类的氨基酸残基，游离出不同种类的氨基酸。特异性强的氨肽酶能水解内切蛋白酶及非特异的外切酶不能水解的氨基酸残基，能够有效的加深蛋白水解深度及提高蛋白水解物的营养价值。水稻是全球产量最丰富的粮食作物之一，大米蛋白作为水稻加工成的产品，资源丰富，营养均衡且不易产生过敏。因此，大米蛋白在食品工业中有着非常广阔的应用前景。然而，大米蛋白在深加工过程中，其水解物往往呈现出较强的苦味，影响了其应用的领域。如何通过酶的协同降解提高大米蛋白水解度、去除或减轻水解液苦味、增加水解液的营养价值成为一个亟待解决的问题。
发明内容
为了解决上述问题，本发明基于发酵动力学指导重组枯草脯氨酸氨肽酶的发酵高产及利 用脯氨酸氨肽酶协同碱性蛋白酶和亮氨酸氨肽酶水解大米蛋白制备脱苦大米肽，结果脯氨酸氨肽酶产量显著提高，多酶协同水解提高了大米蛋白的水解度，减轻水解液的苦味及提高了水解物的营养价值，同时水解液表现出一定的α-葡萄糖苷酶抑制作用。本发明生产的脯氨酸氨肽酶是具有严格底物特异性的氨肽酶，本发明提高了脯氨酸氨肽酶的产量，减轻了下游分离制备脯氨酸氨肽酶的成本及利用脯氨酸氨肽酶特异性地切除N末端的脯氨酸残基，可以去除一些特异性不高的氨肽酶进一步水解Pro的障碍，能起较好的协同水解和脱苦作用。
本发明提供了一种利用重组枯草芽孢杆菌高产脯氨酸氨肽酶的方法，以提高脯氨酸氨肽酶产量，降低下游分离制备的成本。
所述方法包括以下步骤：
(1)将活化后的重组枯草芽孢杆菌接入种子培养基，使用回旋式恒温摇床进行培养，控制转速为220r/min，培养温度为37℃，培养20h，制得种子液；
(2)以5％的接种量将种子液接种到5L发酵罐中，发酵罐装液为3L发酵培养基，通入无菌空气，通气量为1.5vvm；转速的调控为0-6h 200rpm，6-12h为400rpm，12-28h为500rpm，28h至发酵结束为400rpm；pH的调控为0-12h不控制pH，12-16h为pH7.0，16-28h pH不控制，28h后pH7.0；温度的调控为40℃发酵前8h，8-12h为35℃，12h至发酵结束为33℃。
所述步骤(1)中种子培养基组分为氯化钠10g/L、胰蛋白胨10g/L、酵母粉5g/L，灭菌后加入过滤除菌的卡那霉素至终浓度为50μg/mL。
所述步骤(2)中发酵培养基组分为葡萄糖20g/L，酵母提取物60g/L，鱼粉18.75g/L，氯化铵3.25g/L，K2HPO412.54g/L，KH2PO42.31g/L，0.1％(v/v)的植酸，pH7.0。灭菌后加入过滤除菌的卡那霉素至终浓度为50μg/mL。
在本发明的一种实施方式中，所述方法，具体是将-40℃甘油管保藏的重组枯草芽孢杆菌平板活化两次，制备活化的重组芽孢杆菌：
(1)接入到种子培养基中，培养基体积为150mL，所用摇瓶为500mL体积，使用回旋式恒温摇床进行培养，控制转速为220r/min，培养温度为37℃，培养20h，制得种子液；
(2)以5％的接种量将种子液接种到装有3L发酵培养基的5L发酵罐中，通入无菌空气，通气量为1.5vvm，25％氨水调pH，有机硅作消泡剂；
(3)转速的调控为0-6h 200rpm，6-12h为400rpm，12-28h为500rpm，28h至发酵结束为400rpm；pH的调控为0-12h不控制pH，12-16h为pH7.0，16-28h pH不控制，28h后pH7.0；温度的调控为40℃发酵前8h，8-12h为35℃，12h至发酵结束为33℃。
所述步骤(3)发酵过程中，每隔4h定时取样，测定菌体干重、胞内脯氨酸氨肽酶酶活力及胞外脯氨酸氨肽酶酶活力。
本发明同时提供了一种大米蛋白的水解方法，是依次用碱性蛋白酶、亮氨酸氨肽酶、脯氨酸氨肽酶进行水解。
所述方法，是配制质量浓度为5-8％的大米蛋白溶液，
(1)加入适量碱性蛋白酶，于40-50℃下反应1-4h，沸水浴10-20min；
(2)冷却后继续加入适量的亮氨酸氨肽酶，45-50℃反应1-4h，沸水浴10-20min；
(3)冷却后再加入适量的脯氨酸氨肽酶，45-50℃反应2-3.5h，沸水浴10-20min。
所述步骤(1)中碱性蛋白酶添加量为5000-20000U/g大米蛋白。
所述步骤(2)中亮氨酸氨肽酶添加量为1000-4000U/g大米蛋白。
所述步骤(3)中脯氨酸氨肽酶添加量为50-200U/g大米蛋白。
在本发明的一种实施方式中，所述酪蛋白溶液是采用纯水配制，并用碱调pH至9.0、90℃水浴30min制得。
在本发明的一种实施方式中，所述方法，具体是：用纯水配制5％的大米蛋白溶液，并用碱调pH至9.0、90℃水浴30min，所用碱为2M NaOH溶液；然后，
(1)加入适量的碱性蛋白酶，50℃反应4h，沸水浴15min；
(2)冷却后继续加入适量的亮氨酸氨肽酶，50℃反应4h，沸水浴15min；
(3)冷却后再加入适量的脯氨酸氨肽酶，50℃反应2h，沸水浴15min。
所述步骤(1)反应中，每隔1h取样，甲醛滴定法测定大米蛋白水解度。
所述步骤(2)反应中，每隔1h取样，甲醛滴定法测定大米蛋白水解度。
所述步骤(3)反应后，冷却后将所得水解液在10000rpm的条件下离心15min，取上清液；将上清液用10％三氯乙酸等体积稀释，放置1h，然后用针头式滤器过滤，滤液于10000rpm离心10min，再次取上清液。然后分别进行多肽分子量分布、游离氨基酸分析。同时，对水解液进行α-葡萄糖苷酶抑制效果的测定。
本发明以发酵动力学参数为指导得到高产脯氨酸氨肽酶的发酵工艺条件；以大米蛋白为底物，该酶与碱性蛋白酶、亮氨酸氨肽酶协同水解，以水解度为依据，优化碱性蛋白酶和亮氨酸氨肽酶在大米蛋白水解中的添加量；以游离的Pro为依据，确定脯氨酸氨肽酶的用量。
本发明还提供所述方法在食品和饮料及在食品蛋白资源加工利用领域的应用。
本发明还提供一种纯化所述α-葡萄糖苷酶抑制活性肽的方法，是将水解液10000rpm离心15min，取上清，冻干。用20mM pH7.5PB缓冲液溶解，进行Hi Trip DEAE FF阴离子交换层 析，流速为1mL/min，用150mM、350mM、550mM浓度NaCl进行阶段洗脱，每个阶段洗脱10个柱体积，收集洗脱液，充分透析，冻干浓缩。将冻干粉复水，测定各峰的α-葡萄糖苷酶的抑制率。将抑制率高的组分，进行Sephadex G-15凝胶层析，按峰收集，冻干浓缩、复水测定α-葡萄糖苷酶的抑制率。
本发明的有益效果：
本发明以发酵动力学参数为指导确定了高产重组枯草芽孢杆菌脯氨酸氨肽酶的发酵工艺条件，即：转速的调控为0-6h 200rpm，6-12h为400rpm，12-28h为500rpm，28h至发酵结束为400rpm；pH的调控为0-12h不控制pH，12-16h为pH7.0,16-28h pH不控制，28h后pH7.0；温度的调控为40℃发酵前8h，8-12h为35℃，12h至发酵结束为33℃。优化后的脯氨酸氨肽酶产量达174.8U/mL是优化前最高水平的1.66倍。
该酶协同碱性蛋白酶和亮氨酸氨肽酶水解大米蛋白，确定了碱性蛋白酶的添加量为15000U/g大米蛋白、水解4h及亮氨酸氨肽酶的添加量为3000U/g大米蛋白、水解2h，三酶协同水解后游离氨基酸含量为3.484mg/mL，为未水解前的游离氨基酸含量的27.4倍，水解液主要以小肽和游离氨基酸形式存在，180Da以下的小肽含量达到了44.70％，充分水解了暴露出的N端脯氨酸残基，游离的脯氨酸含量为0.149mg/mL，是未水解游离脯氨酸含量的1064.3倍，水解液中疏水性氨基酸含量显著增加，减轻了水解的苦味，加深了大米蛋白水解深度，提升了水解液的营养价值，分离纯化水解液中α-葡萄糖苷酶抑制活性肽得到了抑制活性较强的组分，在功能及保健食品和饮料中具有很好的应用前景。
附图说明
图1：转速对重组菌发酵的影响
图2：不同条件下发酵动力学分析(a：1:200rpm，2:300rpm，3:400rpm，4:500rpm，b：1:200rpm，2:300rpm，3:400rpm，4:500rpm；c：1：不控制pH，2：pH 7.0，3：pH 7.5，d：1：不控制pH，2：pH 7.0，3：pH 7.5；e：1:30℃，2:33℃，3:35℃，4:37℃，5:40℃，f：1:30℃，2:33℃，3:35℃，4:37℃，5:40℃)
图3：确定发酵工艺下的发酵过程曲线
图4：水解液疏水氨基酸
具体实施方式
生物材料样品：所用重组枯草芽孢杆菌(带有组氨酸标签的脯氨酸氨肽酶的重组菌株Bacillus subtilis WB600，所用质粒为PMA5)及脯氨酸氨肽酶的纯化制备详见中国专利CN105925650A。
脯氨酸氨肽酶酶活力测定方法：以L-脯氨酸-对硝基苯胺为底物(底物储藏液用Tris-HCl7.5配制，浓度为4.25mM)，反应混合物包括1mL稀释后的酶液，2mLTris-HCl 7.5缓冲液和1mL底物储藏液，50℃水浴反应10min，加入1mL 50％(v/v)的冰醋酸溶液终止反应，在405nm处测定吸光值。
酶活单位(U)定义为50℃每分钟分解L-脯氨酸-对硝基苯胺产生1μM对硝基苯胺所需的酶量。
碱性蛋白酶酶活单位定义：40℃每分钟水解酪蛋白产生1μg酪氨酸所需的酶量。
亮氨酸氨肽酶酶活单位定义：50℃每分钟分解L-亮氨酸-对硝基苯胺产生1μM对硝基苯胺所需的酶量。
所用发酵罐：T&J TypeA 5L发酵罐(上海迪必尔生物工程有限公司)
多肽分子量分布检测：Waters600高效液相色谱仪(配2487紫外检测器和Empower工作站)。
水解液中游离氨基酸的测定：使用氨基酸液相色谱仪(Ag1100)进行测定。
α-葡萄糖苷酶抑制率的测定：用pH6.8的磷酸钾缓冲液配制1.5U/mL的α-葡萄糖苷酶溶液，70μL的酶液混合70μL的样品在37℃水浴温育10min，再加入70μL含有10mM pNPG的pH6.8的磷酸钾缓冲液，37℃水浴反应1h。然后加入70μL 1M Na2CO3溶液终止反应。在405nm下测定吸光度A。α-葡萄糖苷酶抑制率计算公式如下：
Figure PCTCN2017116188-appb-000001
其中A0为对照，Ai为样品吸光度，Aj为样品对照。
实施例1：不同转速条件下重组枯草芽孢杆菌发酵培养
将-40℃甘油管保藏的重组枯草芽孢杆菌接入到种子培养基中，使用回旋式恒温摇床进行培养，摇床转速为220r/min，培养温度37℃，培养20h。在将种子液以5％的接种量接入装有3L发酵培养基的5L发酵罐中，发酵过程中通入无菌空气，通气量1.5vvm，发酵温度为37℃，pH7.0，搅拌转速分别设置200rpm、300rpm、400rpm、500rpm，发酵时间为36h。发酵过程中，每隔2h取样，测定菌体干重、胞外脯氨酸氨肽酶及胞内脯氨酸氨肽酶活力等。
结果表明，将搅拌转速从200rpm提高到500rpm，脯氨酸氨肽酶活力也从38.0U/mL提高到了97.5U/mL。
实施例2：发酵动力学指导下的发酵培养
将种子液以5％的接种量接入装有3L发酵培养基的5L发酵罐中，发酵过程中通入无菌 空气，通气量1.5vvm，发酵条件：转速的调控为0-6h 200rpm，6-12h为400rpm，12-28h为500rpm，28h至发酵结束为400rpm；pH的调控为0-12h不控制pH，12-16h为pH7.0,16-28h pH不控制，28h后pH7.0；温度的调控为40℃发酵前8h，8-12h为35℃,12h至发酵结束为33℃。发酵过程中，每隔4h取样，测定菌体干重、胞外脯氨酸氨肽酶及胞内脯氨酸氨肽酶活力等。
结果表明，动力学分析下的发酵策略能显著提高脯氨酸氨肽酶的产量，脯氨酸氨肽酶活力达到174.8U/mL。
实施例3：重组枯草脯氨酸氨肽酶在大米蛋白水解中的作用
用纯水缓冲液配制5％的大米蛋白溶液，用2M NaOH溶液调pH至9.0,90℃水浴保温30min，冷却后，加入15000U/g大米蛋白的碱性蛋白酶，50℃反应4h，沸水浴15min，冷却后继续加入3000U/g大米蛋白的亮氨酸氨肽酶粉末，50℃反应2h，沸水浴15min，冷却后再加入200U/g大米蛋白的重组脯氨酸氨肽酶酶液，50℃反应2h，沸水浴15min，冷却，水解液主要以小肽和游离氨基酸形式存在，180Da以下的小肽含量达到了44.70％，充分水解了暴露出的N端脯氨酸残基，游离的脯氨酸含量为0.149mg/mL，是未水解游离脯氨酸含量的1064.3倍。
此外，发明人还比较了不同酶解方法对大米蛋白水解效果的影响，结果如表1所示。结果显示，碱性蛋白酶单独水解时180Da以下的小肽含量为13.68％，碱性蛋白酶和亮氨酸氨肽酶双酶水解时180Da以下的小肽含量为42.21％。
表1不同酶解方法对大米蛋白水解效果的影响
Figure PCTCN2017116188-appb-000002
注：1：大米蛋白溶液，2：碱性蛋白酶单酶水解液，3：碱性蛋白酶和亮氨酸氨肽酶双酶水解液，4：脯氨酸氨肽酶、碱性蛋白酶和亮氨酸氨肽酶协同水解液。
另外，本发明方法得到的游离氨基酸含量为3.484mg/mL，为未水解的游离氨基酸含量的27.4倍，其中脯氨酸含量为未水解的1064.3倍，疏水性氨基酸含量显著提升。而碱性蛋白酶单独水解时游离氨基酸含量为1.121mg/mL，碱性蛋白酶和亮氨酸氨肽酶双酶水解时游离氨基酸含量为3.567mg/mL与三酶协同水解时游离氨基酸含量基本平衡，但游离脯氨酸含量明显低于三酶协同水解物为0.083mg/mL。
本发明通过脯氨酸氨肽酶与碱性蛋白酶、亮氨酸氨肽酶协同水解大米蛋白，得到的水解物中游离氨基酸的含量和小肽含量都大幅增加，疏水性氨基酸含量也显著提高，大米蛋白的水解度不仅大大提高，水解液的苦味液也得到了去除或减轻。
实施例4：大米蛋白水解方法
本发明的大米蛋白水解方法，是配制质量浓度为8％的大米蛋白溶液，
(1)加入适量碱性蛋白酶，于40℃下反应3.5h，沸水浴10min；
(2)冷却后继续加入适量的亮氨酸氨肽酶，45℃反应3h，沸水浴10min；
(3)冷却后再加入适量的脯氨酸氨肽酶，45℃反应3h，沸水浴10min；
其中碱性蛋白酶添加量为20000U/g大米蛋白；亮氨酸氨肽酶添加量为4000U/g大米蛋白；脯氨酸氨肽酶添加量为100U/g大米蛋白。
得到的大米蛋白水解液中2000Da以上的多肽基本上已经不存在了，而180Da以下的小肽含量达到了48.26％，游离氨基酸含量为5.852mg/mL，游离的脯氨酸含量为未水解的1136.4倍。
实施例5：α-葡萄糖苷酶抑制活性肽的分离纯化方法
50mL碱性蛋白酶、亮氨酸氨肽酶及脯氨酸氨肽酶协同水解大米蛋白的水解液10000rpm离心15min，取上清，冻干。用20mM pH7.5PB缓冲液溶解冻干粉，用Hi Trip DEAE FF阴离子交换层析进行初步分离，上样量10mL，流速为1mL/min，用150mM、350mM、550mM浓度NaCl溶液进行阶段洗脱，每个阶段洗脱10个柱体积，收集洗脱液，充分透析，冻干浓缩。将冻干粉复水，测定各洗脱峰的α-葡萄糖苷酶的抑制率及多肽浓度。发现洗脱峰F4组分抑制率最高，将该组分进行Sephadex G-15凝胶层析，上样量1mL，流速为0.5mL/min，按峰收集，将各洗脱峰冻干，用1mL纯水溶解冻干粉，测定α-葡萄糖苷酶的抑制率及多肽浓度。得到最强α-葡萄糖苷酶的抑制效果的组分F4b其IC50为132.6μg/mL。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
Figure PCTCN2017116188-appb-000003
Figure PCTCN2017116188-appb-000004
Figure PCTCN2017116188-appb-000005

Claims (9)

  1. 一种利用重组枯草芽孢杆菌发酵高产脯氨酸氨肽酶的方法，其特征在于，所述方法包括以下步骤：
    (1)将活化后的重组枯草芽孢杆菌接入种子培养基，培养制得种子液；
    (2)将种子液接入发酵培养基中，通入无菌空气，通气量为1.2～1.5vvm；转速的调控为0-6h 200rpm，6-12h为400rpm，12-28h为500rpm，28h至发酵结束为400rpm；pH的调控为0-12h不控制pH，12-16h为pH7.0，16-28h pH不控制，28h后pH7.0；温度的调控为40℃发酵前8h，8-12h为35℃，12h至发酵结束为33℃。
  2. 根据权利要求1所述的方法，其特征在于，所述步骤(2)以5％的接种量将种子液接种到5L发酵罐中，发酵罐装液为3L发酵培养基。
  3. 根据权利要求1所述的方法，其特征在于，所述步骤(1)中种子培养基组分为氯化钠10g/L、胰蛋白胨10g/L、酵母粉5g/L，灭菌后加入过滤除菌的卡那霉素至终浓度为50μg/mL。
  4. 根据权利要求1或2所述的方法，其特征在于，所述步骤(2)中发酵培养基组分为葡萄糖20g/L，酵母提取物60g/L，鱼粉18.75g/L，氯化铵3.25g/L，K2HPO412.54g/L，KH2PO42.31g/L，0.1％(v/v)的植酸，pH7.0。
  5. 根据权利要求1所述的方法，其特征在于，所述步骤(2)以5％的接种量将种子液接种到装有3L发酵培养基的5L发酵罐中，通入无菌空气，通气量为1.5vvm，25％氨水调pH，有机硅作消泡剂。
  6. 一种应用脯氨酸氨肽酶制备脱苦大米肽的方法，其特征在于，所述方法，是配制质量浓度为5-8％的大米蛋白溶液，
    (1)加入适量碱性蛋白酶，于40-50℃下反应1-4h，沸水浴10-20min；
    (2)冷却后继续加入适量的亮氨酸氨肽酶，45-50℃反应1-4h，沸水浴10-20min；
    (3)冷却后再加入适量的脯氨酸氨肽酶，45-50℃反应2-3.5h，沸水浴10-20min。
  7. 根据权利要求6所述的一种应用脯氨酸氨肽酶制备脱苦大米肽的方法，其特征在于，所述编码脯氨酸氨肽酶的基因的核苷酸序列如SEQ ID NO.1所示，采用权利要求1～5任一所述方法制备。
  8. 根据权利要求6所述的一种应用脯氨酸氨肽酶制备脱苦大米肽的方法，其特征在于，所述步骤(1)中碱性蛋白酶添加量为5000-20000U/g大米蛋白；所述步骤(2)中亮氨酸氨肽酶添加量为1000-4000U/g大米蛋白；所述步骤(3)中脯氨酸氨肽酶添加量为50-200U/g大米蛋白。
  9. 一种制备α-葡萄糖苷酶抑制活性肽的方法，其特征在于，包括以下步骤，
    (1)配制质量浓度为5-8％的大米蛋白溶液，加入适量碱性蛋白酶，于40-50℃下反应1-4h，沸水浴10-20min；
    (2)冷却后继续加入适量的亮氨酸氨肽酶，45-50℃反应1-4h，沸水浴10-20min；
    (3)冷却后再加入适量的脯氨酸氨肽酶，45-50℃反应2-3.5h，沸水浴10-20min。
    (4)将水解液10000rpm离心15min，取上清，冻干；
    (5)用20mM pH7.5 PB缓冲液溶解，进行Hi Trip DEAE FF阴离子交换层析，流速为1mL/min，用150mM、350mM、550mM浓度NaCl进行阶段洗脱，每个阶段洗脱10个柱体积，收集洗脱液，充分透析，冻干浓缩；将冻干粉复水，测定各峰的α-葡萄糖苷酶的抑制率；将抑制率高的组分，进行Sephadex G-15凝胶层析，按峰收集，冻干浓缩、复水测定α-葡萄糖苷酶的抑制率。
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