CN111500561B - Method for improving extraction efficiency of intracellular pullulanase - Google Patents
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Abstract
A method for improving extraction efficiency of intracellular pullulanase belongs to the technical field of microorganism and enzyme engineering. The invention adopts two special reagents to treat the pichia pastoris mutant strain, firstly, the recombinant pichia pastoris mutant GS115/pPICK-BdP5 WB138 reacts with 10 times of reagent D, then the recombinant pichia pastoris GS115/pPICK-BdP5 WB138 is placed in ice water, and then 10 times of reagent E is added for reaction. After the cells were collected by centrifugation, the cells were suspended in a citrate buffer and disrupted by sonication. The cells treated by the two reagents are treated by ultrasonic waves for 6min to completely break the cells, and the enzyme activity in the broken liquid is kept above 85 percent. The method and the special treatment liquid provided by the invention can greatly reduce the cost of electric power and the like required by cell disruption and reduce the occupied time of equipment.
Description
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 for details: high-efficiency expression of a codon-optimized acidic pullulanase gene in pichia pastoris, food and fermentation industry, 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, and cells are difficult to break, and the cells can be completely broken by adopting an ultrasonic method generally within about 30 min. 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) and the like are often used for breaking the wall of yeast cells to extract yeast intracellular proteins, but the enzymes are high in price and generally can only be used for experimental research and are difficult to be used for producing 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 easy 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, so that the treated cells are easier to break, and the pullulanase obtained by extraction 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 the application number 201710825655.6, and the invention name is as follows: 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 multiplied by reagent D with the volume of 1/10 of the original fermentation liquid into the cell suspension in the step (1), mixing and reacting for 15min at 45 ℃; then putting the cell suspension into an ice water mixture to rapidly cool the suspension, maintaining for 10min, and then standing for 3h at 4 ℃;
(3) Continuously adding 10 times of reagent E with the volume of 1/10 of the original fermentation liquor 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 cells by using 20mmol/L citric acid-sodium citrate buffer solution with pH of 5.0, and controlling the volume to be the same as that of the original fermentation liquid; the ultrasonic disruption is carried out under the condition of 250W and 1s interval of 1s, and the complete disruption time of 20mL of the cell suspension is not more than 6 min.
Further, the formulation of 10 × reagent D described in step (2) is as follows: 12.5mM Tris-HCl, alkaline protease 50U/mL, bromelain 50U/mL, feSO 4 2mmol/L, 20g/L of glycerol, 20g/L of ethanol and pH 7.4-7.6.
The method for preparing the 10 × reagent D in the step (2) is as follows: at 1LAdding 800mL of deionized water into a container, adding 1.51g of Tris Tris (hydroxymethyl) aminomethane and 50mL of 0.1mol/L hydrochloric acid solution, and mixing; then adding FeSO 4 ·H 2 0.56 g of O, 20g of glycerol and 20g of ethanol, mixing, adding 50000U of each of alkaline protease and bromelain, stirring and mixing, and adding water to 1L; finally, pH is adjusted to 7.4-7.6 by a pH regulator.
The pH regulator is HCl or NaOH solution.
The 10 × reagent E in the step (3) is specifically: 200mM citric acid-sodium citrate, 80mmol/L Na 2 SO 4 ，70mmol/L KCl，10mmol/L Na 2 SO 3 ，5mmol/L KH 2 PO 4 ，pH3.4-3.6。
The preparation method of the 10X reagent E in the step (3) is as follows: adding 800mL of deionized water into a 1L container, adding 33.6g of citric acid monohydrate and 11.8g of trisodium citrate dihydrate, mixing and dissolving; then 11.4g of anhydrous sodium sulfate, 5.2g of potassium chloride, 2.5g of sodium sulfite heptahydrate and 0.7g of potassium dihydrogen phosphate are added and stirred for dissolution; finally, pH is adjusted to 3.4-3.6 by 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 fermentation liquor, 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 Nanning Pompe bioengineering, inc. Other enzymes referred to in the examples: the glucanase is a product of Shandong Su Kehan bioengineering limited company, and the chitinase is purchased from Jiangsu Runyang biotechnology limited company. The other reagents are domestic analytical pure reagents.
The fermentation tank is BLBIO-5GJ, a product of Bailun Biotechnology, inc. of Shanghai.
The used strains and the enzyme activity detection method of the pullulanase are the same as the literature (Deqingmei, zhuxin, jiefu, mao bank, shenwei, chengxiao and Youxianfan. A recombinant Pichia pastoris mutant with high pullulanase yield and improved fermentation speed under the acidic condition, application number: 201715608255.6)
The ultrasonic crushing method comprises the following steps: taking 20mL of cell suspension, immersing a metal head of an ultrasonic cell crusher into the yeast cell suspension, starting the cell crusher, controlling the power to be 250W, and starting crushing under the crushing condition of 1 second at intervals of 1 second until the cell suspension is clear. The ultrasonic disruptor is Scientiz-II D type ultrasonic cell disruptor Ningbo Xin Zhi Biotech GmbH. The cell suspension treated by ultrasonication is subjected to common visible spectrophotometer to detect OD (optical density) at wavelength of 600 nm, i.e. OD 600 . Criteria for complete cell disruption (alternatively referred to as cell suspension clarification) according to the invention: when the crushing liquid OD 600 When 1/10 of the time 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.
Example 1
Fermentation was carried out as described in literature (Deqingmei, zhu Xinweng, jiu Shing, mao bank, shenwei, chen faithful, yanfan. A recombinant Pichia pastoris mutant with high pullulanase yield increased in fermentation rate under acidic conditions application No. 201710825655.6). At the end of fermentation, cells were collected by centrifugation at 5,000 r/min for 2min, and the cells were suspended in tap water, and the volume of the suspension was controlled to be 0.8 times the volume of the original fermentation broth. Then adding a 10 Xreagent D into the cell suspension according to the volume of 1/10 of the original fermentation liquor, mixing and reacting for 15min at 45 ℃. The cell suspension was then placed in an ice-water mixture to allow the suspension to cool rapidly for 10 min. The mixture was left at 4 ℃ for 3 hours. Then according to the volume of 1/10 of the original fermentation liquor, adding 10 times of reagent E, mixing and reacting for 20min at 60 ℃. After the cells are collected by centrifugation, the cells are suspended by using a citric acid-sodium citrate buffer solution with the pH value of 5.0 and the volume of the buffer solution is controlled to be the same as that of the original fermentation liquor, and then the cells are crushed by ultrasonic waves. And (5) carrying out enzyme activity detection on the crushing liquid. The cells treated by the two reagents can be completely crushed by ultrasonic treatment for 6min, and the enzyme activity in the crushing liquid is kept above 85%.
The formulation of the above 10 × reagent D is as follows: 12.5mM Tris-HCl, alkaline protease 50U/mL, bromelain 50U/mL, feSO 4 2mmol/L, 20g/L of glycerol, 20g/L of ethanol and pH 7.5 +/-0.1.
The above 10 × reagent E specifically is: 200mM citric acid-sodium citrate, 80mmol/L Na 2 SO 4 ，70 mmol/L KCl，10 mmol/L Na 2 SO 3 ，5 mmol/L KH 2 PO 4 ，pH3.5±0.1。
The preparation method of the 10 × reagent D is as follows: in a 1L vessel, 800mL of deionized water was charged, and 1.51g of Tris (Tris-hydroxymethyl-aminomethane) and 50mL of 0.1mol/L hydrochloric acid solution were added and mixed. Then adding FeSO 4 .H 2 O0.56 g, glycerol (glycerin) 20g, ethanol 20g, mixing, adding alkaline protease and bromelain 50000U each, stirring, mixing, adding water to 1L, and adjusting pH to 7.5 + -0.1 with HCl or NaOH if there is a pH deviation. After the preparation is finished, the mixture is generally subpackaged into 20mL portions for freezing preservation.
The preparation method of the 10 × reagent E is as follows: in a 1L container, 800mL of deionized water was added, and 33.6g of citric acid monohydrate and 11.8g of trisodium citrate dihydrate were added, mixed and dissolved. Then adding: 11.4g of anhydrous sodium sulfate, 5.2g of potassium chloride, sodium sulfite heptahydrate: 2.5g, potassium dihydrogen phosphate 0.7g, dissolved by stirring. Citric acid or sodium citrate is added to adjust the pH to 3.5 ± 0.1 if the pH deviates.
Application example 1 comparison of the present invention with a general ultrasonic cell disruption method
The results of treating the cells by the general ultrasonic cell disruption method and comparing with example 1 of the present invention are shown in Table 1.
TABLE 1
Note: the untreated cells refer to cells suspended by tap water after the cells are collected by centrifugation of the fermentation liquor, the cells suspended by citric acid-sodium citrate buffer solution with the pH value of 5.0 and the volume of the cell suspension liquid is controlled to be the same as that of the original fermentation liquor after the cells are collected by centrifugation again, and then the cells are used for an ultrasonic disruption experiment.
As can be seen from Table 1, the cells without treatment, which were disrupted by ultrasonication, took about 34 min to achieve the target OD of the cell suspension of 1/10. The cells treated by the method can be broken only by 5 min. Of course, the method of the invention has defects, mainly including certain loss of enzyme activity, about 13 percent loss.
Example 2 comparison of the Process of the invention with other Processes
The comparison of the enzyme activity and enzyme solution retention rate obtained in example 1 of the present invention with the single enzyme treatment method is shown in Table 2.
The experimental method for different pH conditions is as follows: the fermentation broth was centrifuged to collect the cells, the cells were suspended in tap water, and 10 Xbuffer solutions of different pH were added at 1/10 volume. All solid enzymes were dissolved in tap water, and the enzyme solution was prepared to be 100 times as active as the enzyme used and added in 1/100 volume. Wherein the final concentration of chitinase and glucanase is 15U/mL, and the final concentration of bromelain and alkaline protease are both 5U/mL. 0.04 mol/L barbiturate buffer solution is used for pH9.0, 0.1mol/L citric acid buffer solution is used for pH6.5, pH5.5 and pH3.5, and preparation method references [ ZhuGejian king Zhengxiang Industrial microorganism experiment technical Manual, beijing, light industry Press, 1994:683-688.]. The pH 7.5 was 12.5mM Tris-HCl buffer, prepared in the same manner as the 10 Xreagent D of the present invention, except that Tris and hydrochloric acid were added and other components such as metal salts were not added.
TABLE 2
As can be seen from Table 2, the chitinase, glucanase, alkaline protease or chitinase in combination with glucanase did not effectively reduce the cell wall strength of yeast WB138, and the time required for the disruption of the enzyme-treated cells was not much different from that of the untreated cells. The bromelain can effectively destroy the strength of WB138 cell walls, the time required for cell disruption after enzyme treatment is reduced from 30min to 5-6 min, and a plurality of industrial enzyme preparations are tested in the research process of the invention, and the result shows that only bromelain and papain in the industrial enzyme preparation with low price can effectively destroy the strength of the WB138 cell walls, thereby achieving the purpose of greatly reducing the ultrasonic disruption time.
The biggest problem when bromelain is used for crushing WB138 is that the loss of pullulanase in a crushing liquid is large, the loss rate is close to 90 percent, and the practical value is obviously difficult to have. Under the same conditions, the wall breaking effect of the papain is better than that of the bromelain, but the enzyme activity loss of the pullulanase in the obtained cell disruption solution is larger. The invention further changes the temperature within the range of 20 to 60 ℃ to carry out the experiment, and the result is basically consistent with the result in table 2, namely only the pH is below 7.5, when bromelain is used, the cell crushing time is greatly reduced, but simultaneously the enzyme activity of pullulanase in the obtained crushing liquid is greatly reduced, the enzyme activity retention rate is below 20 percent, and the industrial application value is basically not existed. The results obtained with papain instead of bromelain were substantially similar, except that the enzymatic activity of the enzyme solution obtained was lower. When the method is adopted, if the bromelain is replaced by the papain, the time required for ultrasonically breaking the cells of the treated cells is generally shortened to be within 3 min, but the preservation rate of the enzyme activity of the obtained enzyme solution is not more than 30 percent, and the loss of the enzyme activity cannot be obviously reduced by increasing or reducing the dosage of the papain. It can be seen that although papain is less expensive than bromelain, it is essential to use bromelain in the methods of the invention.
Example 3 Combined use of Bromelain with other enzymes
Bromelain was used in combination with other enzymes, treated according to the method of the present invention in example 2, and the resulting broths were tested for enzyme activity and enzyme activity retention, with the results shown in table 3.
TABLE 3
As can be seen from Table 3, in the samples without bromelain, the required time is over 25 min after cell treatment by using ultrasonic wave disruption, and the reduction range of the required time is small. When the pH was 7.5 or less, the time required for cell disruption was reduced to 7 min or less after the yeast cells were treated with the solution containing bromelain, and it was found that it had a significant effect on weakening the cell wall strength. However, in the samples using bromelain, the enzyme activity of the pullulanase in the obtained crushing liquid is greatly reduced, and the enzyme activity is kept below 15 percent. The experiment is further carried out at a temperature of 20-60 ℃, and the result is basically consistent with that in the table 3, namely, only the pH is below 7.5, when bromelain is used, the cell disruption time is greatly reduced, but the enzyme activity of the pullulanase in the obtained disruption solution is greatly reduced, the enzyme activity retention rate is below 20 percent, and the industrial application value is basically absent. The results obtained by using papain to replace bromelain are basically similar, but the enzyme activity of the obtained enzyme solution is lower, and the condition that the enzyme activity is seriously reduced cannot be improved by increasing or reducing the using amount of the papain.
Therefore, in the research process of the invention, the bromelain and the alkaline protease are combined and treated by the special method of the invention, so that the cell disruption efficiency can be effectively improved, and the higher enzyme activity yield of the pullulanase can be kept.
Claims (3)
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 multiplied by reagent D with the volume of 1/10 of the original fermentation liquid into the cell suspension in the step (1), mixing and reacting for 15min at 45 ℃; then putting the cell suspension into an ice water mixture to rapidly cool the suspension, maintaining for 10min, and then standing for 3h at 4 ℃;
(3) Continuously adding 10 times of reagent E with the volume of 1/10 of the original fermentation liquid 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 cells by using a citric acid-sodium citrate buffer solution with the concentration of 20mmol/L and the pH value of 5.0, wherein the control volume of the buffer solution is the same as that of the original fermentation liquor; carrying out ultrasonic disruption under the conditions of 250W and 1s interval of 1s, wherein the complete disruption time of 20mL of the cell suspension is not more than 6 min;
the formula of the 10X reagent D in the step (2) is as follows: 12.5mM Tris-HCl, alkaline protease 50U/mL, bromelain 50U/mL, feSO 4 2mmol/L, 20g/L of glycerol, 20g/L of ethanol and 7.4-7.6 of pH;
the method for disposing the 10 × reagent D is as follows: adding 800mL of deionized water into a 1L container, adding 1.51g of Tris Tris (hydroxymethyl) aminomethane and 50mL of 0.1mol/L hydrochloric acid solution, and mixing; then adding FeSO 4 ·H 2 0.56 g of O, 20g of glycerol and 20g of ethanol, mixing, adding 50000U of each of alkaline protease and bromelain, stirring and mixing, and adding water to 1L; finally, regulating the pH value to 7.4-7.6 by adopting a pH regulator;
the 10 × reagent E in the step (3) is specifically: 200mM citric acid-sodium citrate, 80mmol/L Na 2 SO 4 ，70mmol/L KCl，10mmol/L Na 2 SO 3 ，5mmol/L KH 2 PO 4 ，pH3.4-3.6；
The preparation method of the 10X reagent E is as follows: adding 800mL of deionized water into a 1L container, adding 33.6g of citric acid monohydrate and 11.8g of trisodium citrate dihydrate, mixing and dissolving; then 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; finally, pH is adjusted to 3.4-3.6 by a pH regulator.
2. The method for improving extraction efficiency of intracellular pullulanase according to claim 1, wherein: the pH regulator adopted by the reagent D is specifically HCl or NaOH solution.
3. The method for improving the extraction efficiency of intracellular pullulanase according to claim 1, wherein the method comprises the following steps: the pH regulator adopted by the reagent E is citric acid or sodium citrate.
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