阅读说明：本技术 一种角蛋白酶高效异源表达的方法 (Method for high-efficiency heterologous expression of keratinase ) 是由 龚劲松 史劲松 许正宏 苏畅 于 2018-08-16 设计创作，主要内容包括：本发明公开了一种角蛋白酶高效异源表达的方法,属于工业生物技术领域。本发明成功构建了携带16种不同启动子序列的角蛋白酶重组菌,其中7种能够提高角蛋白酶表达量,PaprE启动子改造重组菌的酶活水平最高,达到2605U/mL,相比对照菌提高20倍。在5L发酵罐上发酵液中角蛋白酶活力高达7176U/mL,是目前文献报道重组角蛋白酶表达的最高水平,能更好地服务于实际应用。本发明为角蛋白酶的高效表达及产酶研究提供了一种有效策略。传统基因工程改造通常需构建高通量筛选方法,进行大量文库筛选,工作量大、周期长、成本高,该改造方法相比传统方法极大的减小了工作量,提高了高表达效率。(The invention discloses a method for high-efficiency heterologous expression of keratinase, belonging to the technical field of industrial biology. According to the invention, keratinase recombinant bacteria carrying 16 different promoter sequences are successfully constructed, wherein 7 keratinase expression levels can be improved, the highest enzyme activity level of the recombinant bacteria modified by the PaprE promoter reaches 2605U/mL, and the enzyme activity level is improved by 20 times compared with that of a control bacterium. The activity of the keratinase in fermentation liquor on a 5L fermentation tank is as high as 7176U/mL, which is the highest level of recombinant keratinase expression reported in the literature at present and can better serve the practical application. The invention provides an effective strategy for the research of the high-efficiency expression and the enzyme production of the keratinase. The traditional genetic engineering modification usually needs to construct a high-throughput screening method for screening a large amount of libraries, and has the advantages of large workload, long period and high cost.)

1. A promoter is characterized in that the promoter is a promoter of a keratinase gene, and the nucleotide sequence of the promoter is shown as SEQ ID No. 13.

2. The promoter according to claim 1, wherein the nucleotide sequence of the keratinase gene is a nucleotide sequence shown as SEQ ID No.17, SEQ ID No.18 or SEQ ID No.19, or a nucleotide sequence having homology of 95% or more with SEQ ID No.17 to 19.

3. An expression vector comprising the promoter of claim 1 or 2.

4. The expression vector of claim 3, wherein the vector is a vector suitable for expression in Bacillus.

5. A recombinant bacterium which expresses keratinase and which comprises the promoter according to claim 1.

6. The recombinant bacterium according to claim 5, wherein the host of the recombinant bacterium is Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, Bacillus megaterium, Bacillus fragilis, Bacillus clausii, Bacillus alkalophilus, or Bacillus thuringiensis.

7. A method for producing a recombinant bacterium according to claim 5 or 6, comprising the steps of:

(1) amplifying a promoter sequence by taking a bacillus subtilis genome as a template, and carrying out mutation to obtain a promoter sequence with a nucleotide sequence shown as SEQ ID No. 13;

(2) recovering the promoter fragment in the step (1), and constructing the promoter fragment into a plasmid carrying a keratinase gene to obtain a recombinant plasmid;

(3) and (3) transforming the recombinant plasmid in the step (2) into host bacteria to obtain recombinant bacteria.

8. The method for producing keratinase by using the recombinant strain as claimed in claim 5 or 6, wherein the recombinant strain is inoculated into a fermentation tank at an inoculation amount of 1-10%, and the keratinase is obtained by fermentation production under the conditions of a fermentation temperature of 28-40 ℃, a rotation speed of 100-1000 rpm, an air flow of 0.5-3 vvm and a pH value of 5.5-8.5.

9. Use of a promoter according to claim 1 or 2 to promote expression of keratinase.

Technical Field

The invention relates to a method for high-efficiency heterologous expression of keratinase, belonging to the technical field of industrial biology.

Background

Keratin is an insoluble structural protein with high stability due to high cross-linking of disulfide bonds and hydrogen bonds. As a byproduct in the development process of animal husbandry, leather industry and textile industry, a large amount of hair is abandoned in the nature every year, and huge pollution is caused to the environment. At present, the hair is treated by mainly physical and chemical methods such as high temperature, high pressure, acid, alkali, oxidation and the like, but the methods can consume useful amino acids in keratin, and have low efficiency, high energy consumption and large pollution. Keratinase opens complex disulfide bonds in keratin by disulfide bond reduction, and then realizes degradation of keratin by proteolysis. The keratin degradation by adopting the keratinase is a green degradation process, and the environment-friendly biological treatment mode has wide prospect from the aspects of economy and environmental protection.

However, in summary, it has been found that the enzymatic activity and thermal stability of keratinase are generally poor. Although many documents report that the keratinase genes from different microorganisms successfully realize heterologous expression, the keratinase expression level of most genetic engineering bacteria is not broken through, so that the realization of the wide application of the keratinase and the satisfaction of the industrial large-scale use requirement become difficulties. In recent years, researchers gradually shift the research focus to the heterogenous high-efficiency expression of keratinase, and a genetic engineering modification means is a key method for improving the expression level of recombinant keratinase. Radha utilizes inducible promoters PxylA and Pamyl to realize the high-efficiency expression of keratinase in Bacillus megaterium, the expression level of recombinase is about 3 times higher than that of control bacteria, and the enzyme activity reaches 168.6U/mL (Radha S, et al. Bioresource Technol,2008,99: 5528-. Wang et al integrated multiple copies of keratinase genes into the Bacillus licheniformis genome to construct a recombinant strain with stable high keratinase yield (Wang J, et al, Biotechnol Bioeng,2004,87: 459-464). However, the high-efficiency expression of the keratinase through the transformation of a promoter is not reported in any literature at present, which is also significant for realizing the deep and wide application of the keratinase.

Disclosure of Invention

In order to solve the problems, the invention provides a promoter for high-efficiency heterologous expression of keratinase, which improves the expression quantity and enzyme activity of the keratinase.

The first purpose of the invention is to provide a promoter, the promoter is a promoter of a keratinase gene, and the nucleotide sequence of the promoter is shown as SEQ ID NO.2, SEQ ID NO.6, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.15 or SEQ ID NO. 16.

In one embodiment of the present invention, the nucleotide sequence of the keratinase gene is a nucleotide sequence shown as SEQ ID NO.17, SEQ ID NO.18 or SEQ ID NO.19, or a nucleotide sequence having a homology of 95% or more with SEQ ID NO.17 to 19.

The second object of the present invention is to provide an expression vector containing the above promoter.

In one embodiment of the invention, the vector is a vector suitable for expression by bacillus.

The third purpose of the invention is to provide a recombinant bacterium for expressing keratinase, wherein the recombinant bacterium contains the promoter.

In one embodiment of the present invention, the host of the recombinant bacterium includes, but is not limited to, bacillus subtilis, bacillus cereus, bacillus licheniformis, bacillus megaterium, bacillus fragilis, bacillus clausii, bacillus alcalophilus, or bacillus thuringiensis.

The fourth purpose of the invention is to provide a preparation method of the recombinant bacterium, which comprises the following steps:

(1) amplifying a promoter sequence by taking a bacillus subtilis genome as a template, and carrying out mutation to obtain the promoter sequence with a nucleotide sequence shown as SEQ ID NO.2, SEQ ID NO.6, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.15 or SEQ ID NO. 16;

(2) recovering the promoter fragment in the step (1), and constructing the promoter fragment into a plasmid carrying a keratinase gene to obtain a recombinant plasmid;

(3) and (3) transforming the recombinant plasmid in the step (2) into host bacteria to obtain recombinant bacteria.

The fifth purpose of the invention is to provide a method for producing keratinase by using the recombinant bacteria, wherein the recombinant bacteria are inoculated into a fermentation tank in an inoculation amount of 1-10%, and fermentation production is carried out under the conditions of a fermentation temperature of 28-40 ℃, a rotation speed of 100-1000 rpm, an air flow of 0.5-3 vvm and a pH value of 5.5-8.5, so as to obtain the keratinase.

The sixth purpose of the invention is to provide the application of the promoter or the recombinant bacterium in animal husbandry, leather making industry or textile industry.

In one embodiment of the invention, the application is to construct a recombinant bacterium by using the promoter, and produce the keratinase degradation keratin by fermentation.

The invention has the beneficial effects that: the keratinase recombinant strain carrying 16 different promoter sequences is successfully constructed, wherein the enzyme activity level of the recombinant strain modified by the PaprE promoter is the highest and reaches 2605U/mL, and is improved by 20 times compared with a control strain. The fermentation research strategy of culture medium nutrient component optimization is adopted, the yield of the mutant transformed recombinant bacterium keratinase is improved, the activity of the keratinase in fermentation liquor on a 5L fermentation tank is as high as 7176U/mL, the activity is the highest level of the expression of the recombinant keratinase reported in the current literature, and the recombinant keratinase can be better served for practical application. The invention provides an effective strategy for the research of the high-efficiency expression and the enzyme production of the keratinase. The traditional genetic engineering modification usually needs to construct a high-throughput screening method for screening a large amount of libraries, and has the advantages of large workload, long period and high cost.

Drawings

FIG. 1 is a double restriction enzyme-digestion-verified electrophoresis of recombinant plasmid; m: DL 10000DNA Marker; CT: pMA5-kerBv initial control plasmid; 1-16: transforming recombinant plasmids by using each promoter;

FIG. 2 shows the keratinase enzyme activity of the promoter-modified recombinant bacteria; CT: initial control strain containing pMA5-kerBv plasmid; 1-16: recombinant bacteria containing modified recombinant plasmids of each promoter;

FIG. 3 is SDS-PAGE analysis of recombinant bacteria enzyme production by promoter modification; m: protein molecular weight standards; CT 1: b.subtilis WB600 containing pMA5 plasmid; CT 2: an initial enzyme-producing strain containing the pMA5-kerBv plasmid; 1-16 recombinant bacteria containing various promoter modified recombinant plasmids;

FIG. 4 is a graph of the effect of different nutrient components on enzyme production by fermentation;

FIG. 5 shows the amplified enzyme production results in a 5L fermenter.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

The enzyme activity detection method of the keratinase comprises the following steps:

the enzyme activity of the keratinase is detected by taking soluble keratin which is sold in the market as a substrate.

Preparation of 1% keratin substrate solution: first, 0.1M Tris-HCl (pH 9.0) solution was prepared, then 5% soluble keratin stock solution was added, and deionized water was added to dilute the Tris-HCl solution to 0.05M and the keratin solution to 1%.

Enzyme reaction: taking 100 mu L of moderately diluted enzyme solution, adding 100 mu L of substrate solution, placing in a water bath at 50 ℃ for accurate reaction for 15min, and immediately adding 200 mu L of 5% (w/v) TCA to terminate the reaction after the reaction is finished. In the control group, 200. mu.L of TCA was added, and 100. mu.L of substrate solution was added after the completion of the enzyme reaction. After the above reaction, all samples were centrifuged at 12000rpm for 5min, and then 200. mu.L of the supernatant was aspirated into a new centrifuge tube, and 1mL of 0.4M Na was added₂CO₃Adding 200 mu L of Folin phenol solutionAnd carrying out color reaction in a water bath at 40 ℃ for 20min, and then detecting the light absorption value at 680 nm.

Definition of enzyme activity: under the above reaction conditions, the difference in absorbance at 680nm of the substrate hydrolyzed by the enzyme solution was defined as one unit of enzyme activity U.

The enzyme activity calculation formula is as follows: u ═ a-B) × 100 × N

A: represents the absorbance value of the experimental group at 680 nm; b: represents the absorbance value of the control group at 680 nm; 100: the expression that the difference in absorbance is 1 is multiplied by 100; n: the dilution factor is indicated.