阅读说明：本技术 一种提高胞内普鲁兰酶提取效率的方法 (Method for improving extraction efficiency of intracellular pullulanase ) 是由 祝冬君 王施岚 德青美朵 杨梦莲 沈微 陈献忠 杨海泉 夏媛媛 于 2020-05-08 设计创作，主要内容包括：一种提高胞内普鲁兰酶提取效率的方法,属于微生物和酶工程技术领域。本发明采用两种特殊试剂对巴斯德毕赤酵母突变株处理,首先将重组巴斯德毕赤酵母突变体GS115/pPICK-BdP5 WB138与10×试剂D反应,随后在冰水中放置,再加入10×试剂E反应。离心收集细胞后用柠檬酸缓冲液悬浮细胞进行超声波破碎。经过两种试剂处理的细胞经超声波处理6 min就将细胞全部破碎,破碎液中酶活保留在85%以上。使用本发明所提供的方法及专用处理液可以大幅度减少细胞破碎所需要消耗的电力等成本,减少设备占用时间。(The invention relates to a method for improving extraction efficiency of intracellular pullulanase, which belongs to the technical field of microorganism and enzyme engineering, and adopts two special reagents to treat a pichia pastoris mutant strain, wherein a recombinant pichia pastoris mutant GS115/pPICK-BdP5 WB138 is firstly reacted with a reagent D10 ×, then placed in ice water, and then a reagent E10 × is added for reaction, after cells are centrifugally collected, the cells are suspended by a citric acid buffer solution for ultrasonic crushing, the cells treated by the two reagents are completely crushed after being treated by ultrasonic for 6min, and the enzyme activity in the crushing solution is kept above 85 percent.)

1. A method for improving extraction efficiency of intracellular pullulanase is characterized by comprising the following steps:

(1) fermenting the recombinant Pichia pastoris GS115/pPICK-BdP5 WB138, centrifuging at 5000r/min at the fermentation end point for 2min, collecting cells, suspending the obtained cells with water, and controlling the volume of the suspension to be 0.8 times of the volume of the original fermentation liquid;

(2) adding 10 × reagent D in the volume of original fermentation liquid 1/10 into the cell suspension in the step (1), mixing, reacting at 45 ℃ for 15min, then putting the cell suspension into an ice-water mixture to quickly cool the suspension, maintaining for 10min, and standing at 4 ℃ for 3 h;

(3) continuously adding 10 × reagent E of the volume of the original fermentation liquor 1/10 obtained in the step (1) into the reaction liquid obtained in the step (2), mixing, and then reacting for 20min at 60 ℃;

(4) centrifuging at 5000r/min for 2min to collect cells, suspending the cells with citric acid-sodium citrate buffer solution of 20 mmol/L and pH5.0, controlling the volume to be the same as that of the original fermentation liquid, and performing ultrasonic disruption at an interval of 1s of 250W for 1s, wherein the complete disruption time of the cell suspension is 20m L and is not more than 6 min.

2. The method for increasing the extraction efficiency of intracellular pullulanase according to claim 1, wherein the 10 × reagent D of step (2) is formulated with 12.5mM Tris-HCl, alkaline protease 50U/m L, bromelain 50U/m L₄2 mmol/L, 20 g/L of glycerol, 20 g/L of ethanol and 7.4-7.6 of pH.

3. The method for increasing extraction efficiency of intracellular pullulanase according to claim 2, wherein the 10 × reagent D in the step (2) is prepared by adding 800m L deionized water to a 1L container, adding 1.51g Tris Tris hydroxymethyl aminomethane and 0.1 mol/L hydrochloric acid solution 50m L, mixing, and adding FeSO₄·H₂0.56g of O and 20g of glycerol20g of ethanol, adding 50000U of each of alkaline protease and bromelain, stirring and mixing, adding water to 1L, and finally adjusting the pH to 7.4-7.6 by adopting a pH regulator.

4. The method for improving extraction efficiency of intracellular pullulanase according to claim 3, wherein: the pH regulator is HCl or NaOH solution.

5. The method for improving extraction efficiency of intracellular pullulanase of claim 1, wherein the 10 × reagent E in step (3) is 200mM citric acid-sodium citrate, 80 mmol/L Na₂SO₄，70mmol/L KCl，10mmol/LNa₂SO₃，5mmol/L KH₂PO₄，pH3.4-3.6。

6. The method for improving extraction efficiency of intracellular pullulanase of claim 5, wherein the 10 × reagent E of step (3) is prepared by adding L m deionized water to a 1L container, adding 33.6g citric acid monohydrate and 11.8g trisodium citrate dihydrate, mixing and dissolving, adding 11.4g anhydrous sodium sulfate, 5.2g potassium chloride, 2.5g sodium sulfite heptahydrate and 0.7g potassium dihydrogen phosphate, stirring and dissolving, and adjusting pH to 3.4-3.6 with pH regulator.

7. The method for improving extraction efficiency of intracellular pullulanase according to claim 6, wherein: the pH regulator is citric acid or sodium citrate.

Technical Field

The invention relates to a method for improving extraction efficiency of intracellular pullulanase, in particular to a method for treating a pichia pastoris mutant strain by adopting two special reagents, and belongs to the technical field of microorganism and enzyme engineering.

Background

Pullulanase (pullulanase) specifically hydrolyzes 1, 6-glycosidic bond of starch molecules, and has wide application in saccharification process of starch sugar production. In the subject group, a recombinant pichia pastoris capable of producing pullulanase at high yield is constructed in earlier stage work, and a mutant strain capable of producing pullulanase at high yield under acidic conditions is further obtained through mutation breeding: pichia pastoris GS115/pPICK-BdP5 WB138[ see details: high-efficiency expression of a codon-optimized acidic pullulanase gene in pichia pastoris, food and fermentation industries, 2016, 42(7): 9-15; de qingmei, zhuixin, jiu chang, mao bank, sheng wei, chen donated faithful, fan you and so on "a recombinant pichia pastoris mutant of high-yield pullulanase with improved fermentation speed under acidic condition", patent application no: 201710825655.6].

When the pichia pastoris GS115/pPICK-BdP5 WB138 (hereinafter referred to as WB 138) is used for fermentation and preparation of the pullulanase, fermentation can be carried out under the acidic condition of pH4.0, the problem of bacterial contamination is not easy to occur, and the fermentation level is higher. However, pullulanase obtained by fermenting the strain is intracellular enzyme, and finally, a usable product can be obtained by cell disruption. Pichia pastoris has firm cell walls, cells are difficult to break, and the ultrasonic method is generally used for breaking the cells for about 30min before the cells can be completely broken. The enzyme capable of cracking the yeast cell wall can reduce the cell wall strength and is beneficial to breaking the wall of the yeast. Snailase, yeast lywallzyme (Zymolyase), etc. are often used for breaking yeast cell walls to extract yeast intracellular proteins, but these enzymes are expensive and generally can only be used for experimental studies and are difficult to be used for the production of industrial enzyme preparations.

The invention discloses a method for pretreating Pichia pastoris GS115/pPICK-BdP5 WB138 cells by adopting a low-cost enzyme preparation for food industry, wherein the treated cells are easier to break, and the obtained enzyme solution is basically stable.

Disclosure of Invention

The invention aims to overcome the defects and provide the method for improving the extraction efficiency of the intracellular pullulanase, the treated cells are easier to break, and the extracted pullulanase is more stable.

According to the technical scheme, the method for improving the extraction efficiency of intracellular pullulanase comprises the steps of centrifugally collecting cells at the fermentation end point of a recombinant Pichia pastoris mutant GS115/pPICK-BdP5 WB138, suspending the cells with water, pretreating the cells with two reagents in sequence, and then crushing the cells.

The recombinant Pichia pastoris mutant GS115/pPICK-BdP5 WB138 is disclosed in application No. 201710825655.6, the name of which is: a recombinant Pichia pastoris mutant with high pullulanase yield and improved fermentation speed under acidic conditions, (Deqingmei, Zhuxin, Caishi, Maolin, Shenwei, Chengxi, Chenxiang, Yanyou) is obtained.

The method comprises the following specific steps:

(1) fermenting the recombinant Pichia pastoris GS115/pPICK-BdP5 WB138, centrifuging at 5000r/min at the fermentation end point for 2min, collecting cells, suspending the obtained cells with water, and controlling the volume of the suspension to be 0.8 times of the volume of the original fermentation liquid;

(2) adding 10 × reagent D in the volume of original fermentation liquid 1/10 into the cell suspension in the step (1), mixing, reacting at 45 ℃ for 15min, then putting the cell suspension into an ice-water mixture to quickly cool the suspension, maintaining for 10min, and standing at 4 ℃ for 3 h;

(3) continuously adding 10 × reagent E of the volume of the original fermentation liquor 1/10 obtained in the step (1) into the reaction liquid obtained in the step (2), mixing, and then reacting for 20min at 60 ℃;

(4) centrifuging at 5000r/min for 2min to collect cells, suspending the cells with citric acid-sodium citrate buffer solution of 20 mmol/L and pH5.0, controlling the volume to be the same as that of the original fermentation liquid, and performing ultrasonic disruption at an interval of 1s of 250W for 1s, wherein the complete disruption time of the cell suspension is 20m L and is not more than 6 min.

Further, the 10 × reagent D of step (2) is formulated as follows, 12.5mM Tris-HCl, alcalase 50U/m L, bromelain 50U/m L₄2 mmol/L, 20 g/L of glycerol, 20 g/L of ethanol and 7.4-7.6 of pH.

The preparation method of the 10 × reagent D in the step (2) comprises the steps of adding 800m L deionized water into a 1L container, adding 1.51g Tris Tris (hydroxymethyl) aminomethane and 0.1 mol/L hydrochloric acid solution 50m L, mixing, and adding FeSO₄·H₂0.56g of O, 20g of glycerol and 20g of ethanol, mixing, adding 50000U of each of alkaline protease and bromelain, stirring and mixing, adding water to 1L, and finally adjusting the pH to 7.4-7.6 by using a pH regulator.

The pH regulator is HCl or NaOH solution.

The 10 × reagent E in the step (3) is 200mM citric acid-sodium citrate, 80 mmol/L Na₂SO₄，70mmol/L KCl，10mmol/L Na₂SO₃，5mmol/L KH₂PO₄，pH3.4-3.6。

The preparation method of the 10 × reagent E in the step (3) comprises the following steps of adding 800m L deionized water into a 1L container, adding 33.6g of citric acid monohydrate and 11.8g of trisodium citrate dihydrate, mixing and dissolving, adding 11.4g of anhydrous sodium sulfate, 5.2g of potassium chloride, 2.5g of sodium sulfite heptahydrate and 0.7g of potassium dihydrogen phosphate, stirring and dissolving, and finally adjusting the pH to 3.4-3.6 by adopting a pH regulator.

The pH regulator is citric acid or sodium citrate.

The invention has the beneficial effects that: when the pichia pastoris GS115/pPICK-BdP5 WB138 is fermented to prepare the recombinant pullulanase, the method and the special treatment liquid provided by the invention are used for treating cells in the fermentation liquid, so that the ultrasonic disruption time can be greatly shortened, the equipment occupation time is reduced, and the cost of electric power consumption and the like required by cell disruption is saved.

The ultrasonic treatment time required by the cells treated by the method is shortened to be within 6min, the enzyme activity loss is within 15 percent, and the cell breakage without the treatment of the method needs more than 30 min.

Detailed Description

In the following examples the alkaline protease is a dupont jenenaceae P100 detergent specific liquid alkaline protease. Bromelain and papain are both products of Panningpobo bioengineering, Inc. Other enzymes referred to in the examples: the glucanase is a product of Shandong Su Koehan bioengineering limited company, and the chitinase is purchased from Jiangsu Ruiyang biotechnology limited company. The other reagents are domestic analytical pure reagents.

The fermentation tank is B L BIO-5GJ, a product of Shanghai Bailun Biotechnology GmbH.

The strains and the enzyme activity detection method of the pullulanase are the same as the literature (Deqingmei, Zhuxinwen, Jiefang, Mao bank, Shenwei, Chengxiaozhi, Yongwu, a recombinant Pichia pastoris mutant of high-yield pullulanase with improved fermentation speed under acidic condition, application No. 201710825655.6)

The ultrasonic disruption method comprises taking cell suspension 20m L, immersing metal head of ultrasonic cell disruptor in the yeast cell suspension, starting the cell disruptor, controlling power to 250W, and initiating disruption under 1 second interval until cell suspension is clarified, wherein the ultrasonic disruptor is Scientiz-II D type ultrasonic cell disruptor Ningbo New Ganoderma Biotech Co Ltd, and detecting OD (OD) at 600 nm with common visible light spectrophotometer after ultrasonic disruption treatment, i.e. OD₆₀₀. Criteria for complete cell disruption (alternatively referred to as cell suspension clarification) according to the invention: when the crushing liquid OD₆₀₀When 1/10 before ultrasonication was reached, the cells were considered to be completely disrupted.

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention in any way.