阅读说明：本技术 利用枯草芽孢杆菌发酵制备中性蛋白酶的方法 (Method for preparing neutral protease by fermenting bacillus subtilis ) 是由 曹倩荣 吴芳彤 刘春卯 郑翔 康捷 秦艳梅 马清河 于 2019-11-18 设计创作，主要内容包括：本发明属于微生物发酵,特别是指一种利用枯草芽孢杆菌发酵制备中性蛋白酶的方法。本发明通过通过对培养基及发酵条件进行改善和优化,有效解决了现有枯草芽孢杆菌Bacillus subtilis CGMCC No.14837的发酵工艺未经充分优化、距离工业化应用差距较大等技术问题,具有所制备的中性蛋白酶相比西安欧公益制备的中性蛋白酶酶活高、脱毛效果好等优点。(The invention belongs to microbial fermentation, and particularly relates to a method for preparing neutral protease by utilizing bacillus subtilis fermentation. By improving and optimizing the culture medium and the fermentation conditions, the invention effectively solves the technical problems that the existing fermentation process of the Bacillus subtilis CGMCC No.14837 is not fully optimized, the gap from industrial application is large, and the like, and has the advantages that the prepared neutral protease has high enzyme activity and good unhairing effect compared with the neutral protease prepared in the Western-Anoei.)

1. A method for preparing neutral protease by utilizing Bacillus subtilis through fermentation, wherein the Latin name of the Bacillus subtilis is Bacillus subtilis, and the preservation number is CGMCC No. 14837; the method is characterized by comprising the following process steps:

A. preparation of culture medium

Each liter of fermentation medium comprises the following raw materials:

68-72g/L of yeast extract powder; NH (NH)₄Cl 2.5-3.5g/L；K₂HPO₄ 0.8-1.2g/L；MnSO₄·7H₂O 0.01-0.03g/L；CaCl₂·2H₂O2.8-3.2 g/L; 8-12g/L of glucose; MgSO (MgSO)₄·7H₂O1.8-2.2 g/L; the balance of water;

B. inoculation of

B, inoculating the bacillus subtilis into the sterilized culture medium prepared in the step A according to the proportion of 4%, wherein the content of the culture medium in a shake flask is equal to 10% in volume ratio;

C. fermentation of

Fermenting at 37 deg.C and 180 rpm, and adding IPTG to 1 ‰ concentration of glucose when glucose is exhausted; and monitoring the enzyme activity until the enzyme activity is the highest, namely the fermentation end point.

2. The method for preparing neutral protease by fermentation of Bacillus subtilis according to claim 1, wherein the glucose and MgSO in step A are mixed with each other₄·7H₂O is sterilized separately.

3. The method of claim 1, wherein the culture medium of step B further comprises chloramphenicol at a concentration of 10. mu.l/mL.

Technical Field

The invention belongs to microbial fermentation, and particularly relates to a method for preparing neutral protease by utilizing bacillus subtilis fermentation.

Background

In recent years, with the gradual enhancement of environmental awareness of the tanning industry, the traditional extensive development mode is gradually abandoned in the adjustment of the industrial structure, and environmental protection and pollution control become important in the industry. Tanners have made various improvements to the tanning process, and the dehairing process most likely to be industrialized to date is enzymatic dehairing. But because of the reasons of insufficient clean production technology, great pollution treatment difficulty, high investment cost and the like in the leather industry in China, the promotion of clean production in the leather and fur processing industry has always resisted a lot. The protease can digest the protein connected with the epidermis and the dermis around the hair follicle of the raw hide, weaken the close attachment of the hair with the epidermis and the dermis and achieve the aim of hair removal. 1398. 2709 protease preparation was used as unhairing enzyme in leather-making process in the early 20 th century, but collagenase exists in wild strain fermentation protease-producing system, which can hydrolyze collagen in the hide, inevitably damages the structure of dermis, and affects the performance of leather products. The process cost of purifying and removing collagen is far beyond the tolerance of tanneries.

The applicant discloses a neutral protease gene, protein and bacillus subtilis constructed by utilizing molecular chaperone prsA as well as preparation and application thereof in an invention patent application with the publication number of CN 108070606A. Wherein a Bacillus subtilis CGMCC No.14837 for producing neutral protease is constructed for enzyme depilation. The engineering strain does not contain a collagenase gene, but due to the limitation of a protein secretion bottleneck, the fermentation process of the Bacillus is imperfect at the present stage, the used culture medium is the most common LB universal culture medium and is not the culture medium which is most suitable for growth and fermentation of Bacillus subtilis CGMCC No.14837, and the enzyme activity is 1186u/ml after 48-hour fermentation, so that the difference of the Bacillus subtilis CGMCC No.14837 from industrial application is large at present. The optimization of the culture medium and the fermentation conditions plays a significant role in the industrialized production of microorganisms, and not only is a necessary link from a laboratory to industrial production, but also the yield of neutral protease is improved through optimization, so that a brand new direction can be provided for the clean production of the tanning industry.

Disclosure of Invention

The invention aims to provide a method for preparing neutral protease by utilizing bacillus subtilis through fermentation, which achieves the aim of improving the enzyme activity of the neutral protease prepared through fermentation by improving and optimizing a culture medium and fermentation conditions.

The overall technical concept of the invention is as follows:

a method for preparing neutral protease by utilizing Bacillus subtilis through fermentation, wherein the Latin name of the Bacillus subtilis is Bacillus subtilis, and the preservation number is CGMCC No. 14837; the method comprises the following process steps:

A. preparation of culture medium

Each liter of fermentation medium comprises the following raw materials:

68-72g/L of yeast extract powder; NH (NH)₄Cl 2.5-3.5g/L；K₂HPO₄ 0.8-1.2g/L；MnSO₄·7H₂O 0.01-0.03g/L；CaCl₂·2H₂O2.8-3.2 g/L; 8-12g/L of glucose; MgSO (MgSO)₄·7H₂O1.8-2.2 g/L; the balance of water;

B. inoculation of

B, inoculating the bacillus subtilis into the sterilized culture medium prepared in the step A according to the proportion of 4%, wherein the content of the culture medium in a shake flask is equal to 10% in volume ratio;

C. fermentation of

Fermenting at 37 deg.C and 180 rpm, monitoring glucose content, and adding IPTG to 1 ‰ concentration when glucose is exhausted.

The specific technical concept of the invention is as follows:

because the glucose sterilization temperature is too high, furfural is generated, the color of the culture medium is darkened, and the furfural is unfavorable for the growth of thalli; magnesium sulfate is unstable at high temperature and is easy to react with other substances to generate magnesium hydroxide precipitate. Preferably, the glucose and the magnesium sulfate heptahydrate are sterilized separately in the step A.

In order to inhibit the growth of other miscellaneous bacteria in the culture process to influence the fermentation quality and increase the fermentation cost, the preferable technical scheme is that the culture medium in the step B also comprises chloramphenicol with the concentration of 10 mul/mL.

In order to verify the technical effect of the invention, the applicants adopt Folin-phenol method (GB/T23527-2009) to measure the enzyme activity of the neutral protease prepared by the existing process and the method of the invention.

The invention achieves the substantive characteristics and obvious technical progress that:

according to the invention, a fermentation process more suitable for Bacillus subtilis CGMCC No.14837 is obtained by screening and optimally combining a carbon source, a nitrogen source and trace elements in a culture medium, and the enzyme activity of the neutral protease can reach 1653u/ml after the process is used for fermentation for 48 hours, which is detected by an applicant, and is improved by 39.38% compared with the enzyme activity (1186u/ml) of the neutral protease prepared by the existing process; the result of applying a small amount of enzyme solution to the unhairing test is equivalent to that of the neutral protease prepared by the prior art. The invention provides guiding significance for researching the high-density production of the Bacillus subtilis CGMCC No. 14837.

Drawings

FIG. 1 is a graph showing the comparison of the depilation effect of the neutral protease produced by the method of the present invention and the neutral protease produced by the conventional process.

Wherein, FIG. 1A is a schematic surface diagram of a fresh cow leather before depilation treatment by using the neutral protease prepared by the method of the present invention.

FIG. 1B is a schematic representation of the surface of fresh cowhide before dehairing treatment with neutral protease prepared by prior art.

FIG. 1C is a schematic representation of the surface of calfs after 200 minutes of dehairing treatment with 27.2ml of neutral protease prepared by the process of the invention.

FIG. 1D is a schematic representation of the surface of calfskins after 200 minutes of dehairing treatment with 37.9ml of neutral protease prepared by prior art techniques.

As can be seen from the figure, under the same conditions of the unhairing process, the adding amount of the neutral protease liquid is obviously reduced, and the unhairing effect is equivalent to that of the neutral protease liquid prepared by the prior art.

Detailed Description

The present invention is further described with reference to the following examples, but the present invention is not limited thereto. The protection scope of the present invention is subject to the content of the claims, and any equivalent technical means made according to the specification can be substituted without departing from the protection scope of the present invention.