阅读说明：本技术 一种提高普鲁兰酶终浓度的发酵液处理方法 (Fermentation liquor treatment method for improving final concentration of pullulanase ) 是由 祝冬君 德青美朵 王施岚 孙娟娟 沈微 陈献忠 杨海泉 夏媛媛 于 2019-11-21 设计创作，主要内容包括：一种提高普鲁兰酶终浓度的发酵液处理方法,属于酶工程和发酵工程技术领域。本发明将处理溶液C与普鲁兰酶发酵上清液混合后保温,超滤浓缩,即得高酶活的普鲁兰酶发酵液。采用本发明方法,在以重组菌解淀粉芽孢杆菌BF7658/pUC980-<I>BdP</I>发酵制备普鲁兰酶的后提取阶段,可以改善普鲁兰酶发酵液的超滤性能。经本发明处理的发酵液上清液经超滤浓缩后得到的浓缩液的酶活是未经溶液C处理的发酵液经同样超滤浓缩后得到的浓缩液的酶活的1.75倍以上。(A fermentation liquor treatment method for improving the final concentration of pullulanase belongs to the technical field of enzyme engineering and fermentation engineering. The treated solution C and the pullulanase fermentation supernatant are mixed, then the mixture is subjected to heat preservation and ultrafiltration concentration, and the high-enzyme-activity pullulanase fermentation liquor is obtained. By adopting the method of the invention, the recombinant bacillus amyloliquefaciens BF7658/pUC980- BdP The post-extraction stage of the pullulanase prepared by fermentation can improve the ultrafiltration performance of the pullulanase fermentation liquor. The enzyme activity of the concentrated solution obtained by ultrafiltration and concentration of the supernatant of the fermentation liquor treated by the method is more than 1.75 times of that of the concentrated solution obtained by ultrafiltration and concentration of the fermentation liquor which is not treated by the solution C.)

1. A fermentation liquor treatment method for improving the final concentration of pullulanase is characterized by comprising the following steps: and mixing the treated solution C with the pullulanase fermentation supernatant, preserving heat, and performing ultrafiltration concentration to obtain the high-enzyme-activity pullulanase fermentation concentrated solution.

2. The method for treating fermentation liquor for increasing the final concentration of pullulanase according to claim 1, wherein: mixing the treated solution C with 9 times of pullulanase fermentation supernatant; keeping the temperature of the mixed solution at 60 ℃ for 30 min; concentrating the treated supernatant of the fermentation liquor for 20min by adopting an ultrafiltration method at 20 ℃ and 0.09-0.11MPa to obtain a pullulanase fermentation concentrated solution with high enzyme activity.

3. The method for treating fermentation liquor for increasing the final concentration of pullulanase according to claim 1, wherein the treatment solution C is specifically: contains 200mM citric acid-sodium citrate, 100mM CaCl₂50mmol/L KCl, 20mmol/L NaCl and 1mmol/L FeSO₄The pH of the aqueous solution of (1) is 5.4-5.6.

4. The method as claimed in claim 1, wherein the supernatant of pullulanase fermentation is Bacillus amyloliquefaciens BF7658/pUC980-BdPFermentation supernatant obtained in the post-extraction process of preparing pullulanase by fermentation.

5. The method for treating fermentation liquor capable of increasing the final concentration of pullulanase according to claim 1, wherein the preparation method of pullulanase fermentation supernatant is as follows: the recombinant bacterium Bacillus amyloliquefaciens/pUC 980-stored in a glycerol tube at the temperature of-70 DEG CBdPStreaking on LB plate containing 20mg/L kanamycin, and picking out single colony; inoculating LB liquid culture medium containing 20mg/L kanamycin; the liquid loading amount in a 500mL triangular flask is 100mL, the flask is shaken at 34 ℃ and 200r/min for fermentation for 24 h; inoculating 10% of the inoculum size into a 5L fermentation tank filled with sterilized 2.7L fermentation liquid in advance, and controlling pH to 6.8-7.5 with ammonia water during fermentation; and after the fermentation is finished, putting the fermentation liquor into a centrifugal tube, and centrifuging at 4000rpm for 10min to obtain the pullulanase fermentation supernatant.

6. The method for treating fermentation liquor for increasing the final concentration of pullulanase according to claim 1, wherein: the enzyme activity of the concentrated solution obtained by ultrafiltration and concentration of the treated supernatant of the pullulanase fermentation liquor is more than 1.75 times of that of the concentrated solution obtained by ultrafiltration and concentration of the fermentation liquor which is not treated by the solution C.

Technical Field

The invention relates to a fermentation liquor treatment method for improving the final concentration of pullulanase, belonging to the technical field of enzyme engineering and fermentation engineering.

Background

Pullulanase (EC 3.2.1.41) has the function of hydrolyzing alpha-1, 6 glycosidic bond in starch, and can improve the yield of glucose by the synergistic action with saccharifying enzyme in the production of glucose. Recombinant Bacillus amyloliquefaciens/pUC 980-BdPThe secretory expression of pullulanase gene in Bacillus amyloliquefaciens BF7658 strain, industrial microbe 2011, 41(6): 18-22]Has better capability of producing pullulanase.

In the production of industrial enzyme preparations, in order to obtain a product with a high enzyme concentration, ultrafiltration concentration of a fermentation broth is often required to remove a part of water, reduce the volume of an enzyme solution, and realize concentration of the enzyme. Bacillus amyloliquefaciens/pUC 980-BdPThe prepared pullulanase fermentation liquor has higher viscosity, and when the fermentation liquor is concentrated by an ultrafiltration method after solid-liquid separation operation, generally after the final enzyme liquid is concentrated to 1/5 with the original volume, if the ultrafiltration operation is continued, the volume of the fermentation liquor cannot be reduced continuously, and the enzyme concentration cannot be further improved.

Disclosure of Invention

The invention aims to overcome the defects and provide a fermentation liquor treatment method for improving the final concentration of pullulanase.

According to the technical scheme, the fermentation liquor treatment method for improving the final concentration of the pullulanase comprises the steps of mixing the treatment solution C with the pullulanase fermentation supernatant, preserving heat, and carrying out ultrafiltration concentration to obtain the high-enzyme-activity pullulanase fermentation liquor concentrated solution.

Further, mixing the treatment solution C with 9 times of pullulanase fermentation supernatant; keeping the temperature of the mixed solution at 60 ℃ for 30 min; concentrating the treated supernatant of the fermentation liquor at 20 ℃ and 0.09-0.11MPa by adopting an ultrafiltration method until the ultrafiltration end point to obtain the pullulanase fermentation concentrated solution with high enzyme activity.

The preparation method of the treatment solution C comprises the following steps: contains 200mM citric acid-sodium citrate, 100mM CaCl₂50mmol/LKCl, 20mmol/L NaCl and 1mmol/L FeSO₄The pH of the aqueous solution of (1) is 5.4-5.6.

The configuration method comprises the following steps: in a 1L volumetric flask, 800mL of deionized water was added, and citric acid monohydrate ((C)₆H₈O₇）·H₂O) 12.6 g, sodium citrate monohydrate ((Na)₃C₆H₅O₇）·H₂O) 41.2g, anhydrous calcium chloride 11.1g, potassium chloride 3.7g, sodium chloride 1.17g, ferrous sulfate heptahydrate 278.2mg, adjusting pH to 5.5 + -0.1 with NaOH or HCl if pH deviates, and adding water to 1L.

The pullulanase fermentation supernatant is BF7658/pUC980-BdPFermentation supernatant obtained in the post-extraction process of preparing pullulanase by fermentation.

Further, the preparation method of the recombinant pullulanase fermentation supernatant comprises the following steps: the recombinant bacterium Bacillus amyloliquefaciens/pUC 980-stored in a glycerol tube at the temperature of-70 DEG CBdPStreaking on LB plate containing 20mg/L kanamycin, selecting single colony, inoculating LB liquid culture medium containing 20mg/L kanamycin, filling 100mL liquid into 500mL triangular flask, generally making 10 flasks at the same time, shaking flask for 24h at 34 ℃, 200r/min, inoculating 5L fermentation tank containing sterilized 2.7L fermentation liquor in advance according to 10% inoculation amount, controlling pH to be 6.8-7.5 by using ammonia water in the fermentation process, sampling and detecting enzyme activity at intervals of 4h after fermenting for 24h, ending fermentation when the enzyme activity reaches about 25U/mL, taking fermentation liquor after the fermentation is ended, centrifuging for 10min at 4000rpm, generally making 2 ~ 3 jars for fermentation at the same time in actual work, centrifuging the fermentation liquor, removing precipitate, mixing supernatant, carrying out subsequent experiment, and carrying out centrifugal operation to ensure that the enzyme activity of pullulanase is generally 20-30U/mLA Hunan apparatus CL5R high-capacity refrigerated centrifuge is used.

And (3) pullulanase enzyme activity determination: according to the literature [ Bibel M, Brett C, Gosslar U, et al, isolationd analysis of amylolytic enzyme of the hyperthermophilic bacterial thermatology maritime, FEMS Microbiol Lett, 1998, 158:9-15 ].

The ultrafiltration concentration method of the fermentation supernatant comprises the following steps: performing enzyme liquid ultrafiltration performance experiment in UF101 ultrafilter with PES30-1512, collecting 1L fermentation supernatant in ultrafilter tank, and ultrafiltering according to the instruction; wherein the ultrafiltration pressure is controlled at 0.1 + -0.01 MPa.

The ultrafiltration membrane has a molecular weight cut-off of 30kDa, and water, salts and other relatively small molecules smaller than this molecular weight flow out of the waste pipe in the form of waste liquid. In the ultrafiltration process, along with the outflow of small molecules such as water and the like, the volume of enzyme liquid in the storage tank is reduced, meanwhile, large molecules such as pullulanase and the like are concentrated, the filtration resistance is increased, and the outflow speed of waste liquid is mainly reduced. Control criteria for ultrafiltration endpoint: and when the effluent velocity of the waste liquid is less than 1mL/min, determining the ultrafiltration endpoint. And measuring enzyme liquid remained in the storage tank and the ultrafiltration membrane column after the end point is reached, recording the enzyme liquid and the volume of the concentrated solution, calculating the ultrafiltration time, and detecting the enzyme activity of the concentrated solution.

Model number of the ultrafilter is UF101, and model number of the ultrafiltration membrane is PES30-1512, which are all products of Shanghai Furit industries, Ltd.

Culture medium: LB is described in Sambrook et al 1989 handbook of molecular cloning experiments. Fermentation medium: 50g/L of lactose, 30g/L of bean cake powder, 0.5g/L of yeast extract, 10g/L of ammonium sulfate and 0.5g/L of anhydrous calcium chloride. The fermentation medium and the fermentation method can be referred to documents [ Shenwei, Shoibang, Korea ice, Liting Lin, Zhujincun, Chengxing, Yanxiang.

The pullulanase is prepared from recombinant bacillus amyloliquefaciens BF7658/pUC980-BdPThe preparation method can be found in the literature [ secret expression of Prochloraceae gene in Bacillus amyloliquefaciens BF7658 strain, industrial microorganism 2011, 41(6): 18-22%]。

The enzyme activity of the concentrated solution obtained by ultrafiltration and concentration of the supernatant of the pullulanase fermentation liquor treated by the solution C is more than 1.75 times of that of the concentrated solution obtained by ultrafiltration and concentration of the fermentation liquor not treated by the solution C.

Uses Bacillus amyloliquefaciens BF7658/pUC980-BdPThe method for preparing the pullulanase is beneficial to improving the concentration of the pullulanase final product and reducing the storage space of the product.

The invention has the beneficial effects that: by adopting the method of the invention, the recombinant bacillus amyloliquefaciens BF7658/pUC980-BdPThe post-extraction stage of the pullulanase prepared by fermentation can improve the filtration performance of fermentation liquor; the enzyme activity of the concentrated solution obtained by ultrafiltration and concentration of the supernatant of the pullulanase fermentation liquor treated by the solution C is more than 1.75 times of that of the concentrated solution obtained by ultrafiltration and concentration of the fermentation liquor not treated by the solution C.

Detailed Description

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