Alkaline protease mutant with improved enzyme activity at low temperature and its application

Abstract

The present invention belongs to the fields of microbial genetic engineering and protein engineering, and specifically relates to alkaline protease mutants with enhanced specific enzyme activity at low temperatures and their applications. By performing site-directed mutagenesis on the alkaline protease gene represented by SEQ ID NO. 1, the present invention obtains an alkaline protease mutant with an increased specific enzyme activity at 25°C from 3975 U / mg to 4770 U / mg. This simple and easy method lays the foundation for the efficient expression of alkaline protease in Bacillus licheniformis, thereby promoting the fermentation production of alkaline protease.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial genetic engineering and protein engineering transformation, and particularly relates to an alkaline protease mutant with improved specific enzyme activity at low temperature and its application. Background Art

[0002] Alkaline protease (ALK) refers to a class of enzymes that can hydrolyze protein peptide bonds in the alkaline pH range. It has been widely used in the detergent, pharmaceutical, leather and food industries. Among them, ALK derived from Bacillus Clausii is mainly used in the detergent industry, but its activity at low temperatures is still limited, which limits the effectiveness of detergents under low temperature conditions. Therefore, this patent aims to screen ALK mutants with high activity at low temperatures through molecular modification of ALK, in order to meet the current demand for low-temperature ALK in the detergent industry. This patent determines the enzymatic activity of alkaline protease according to GB / T 23527-2009.

[0003] The principle of alkaline protease activity assay in GB / T 23527-2009 is as follows: under certain temperature and pH conditions, protease hydrolyzes casein substrate to produce phenolic amino acids (such as tryptophan and tyrosine). Under alkaline conditions, the Folin-phenol reagent is reduced to a blue color that can be detected at OD680. The absorbance of the solution at OD680 is measured using a spectrophotometer. The enzyme activity is proportional to the absorbance, and the alkaline protease activity can be calculated. The national standard method is the most widely used method for alkaline protease activity assay, with advantages such as good reproducibility and low testing cost.

[0004] The pH of detergents is generally between 9.0 and 11.0. Alkaline proteases are mainly used in the detergent industry due to their high stability and activity under alkaline conditions. Their use in detergent formulations accounts for 89% of their total sales. According to surveys, the washing temperature in my country is usually concentrated around 25°C. However, most of the proteases added to commercially available detergents are medium-temperature alkaline proteases, which cannot exert their high activity at low temperatures, which has a great impact on the washing performance of detergents. Based on the above background, the applicant has molecularly modified the reported alkaline protease (ALK) and screened ALK mutants with high activity at low temperatures to meet the current needs of the washing industry. Summary of the Invention

[0005] The purpose of the present invention is to provide an alkaline protease mutant with improved enzyme activity at low temperature. The amino acid sequence of the mutant is shown in SEQ ID NO.1.

[0006] Another object of the present invention is to provide the use of the mutant in the preparation of alkaline protease.

[0007] In order to achieve the above object, the present invention adopts the following technical measures:

[0008] The applicant artificially synthesized the starting alkaline protease sequence (Li J, Jiang L, Cao X, et al. Improving the activity and stability of Bacillus clausii alkaline protease using directed evolution and molecular dynamics simulation[J]. Enzyme and Microbial Technology, 2021, 147: 109787-. DOI: 10.1016 / j.enzmictec.2021.109787.), as shown in SEQ ID NO.1, and then modified the alkaline protease enzyme by mutating the glycine at position 113 to proline (i.e., as shown in SEQ ID NO.3). The specific enzyme activity of the alkaline protease at 25°C was increased from the original 3975U / mg to 4770U / mg, which greatly improved the specific activity.

[0009] The protection scope of the present invention also includes:

[0010] A fusion protein obtained by fusing the mutant protein described in SEQ ID NO.3 with a protein tag.

[0011] The gene encoding the mutant or fusion protein described in SEQ ID NO.3.

[0012] An expression cassette, recombinant vector, recombinant microorganism or in vitro recombinant cell having the above-mentioned coding gene.

[0013] The mutant, fusion protein, the mutant or fusion protein encoding gene of SEQ ID NO. 4, the expression cassette, recombinant vector, recombinant microorganism or in vitro recombinant cell having the encoding gene are used in preparing metabolites.

[0014] A method for improving the enzymatic activity of alkaline protease comprises the following steps: performing the following mutation on the alkaline protease: mutating the 113th glycine G in SEQ ID NO.1 to proline P.

[0015] A method for preparing alkaline protease with improved specific enzyme activity comprises the following steps: preparing the alkaline protease with improved specific enzyme activity by culturing the above-mentioned recombinant microorganism.

[0016] The gene encoding the mutant of SEQ ID NO.3 is preferably as shown in SEQ ID NO.4.

[0017] In the above-mentioned applications or methods, preferably, the recombinant microorganism is recombinant Bacillus licheniformis.

[0018] In the above-mentioned use or method, preferably, the Bacillus licheniformis is Bacillus licheniformis DW2.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] The present invention obtains an alkaline protease mutant whose specific enzyme activity at 25°C is increased from the original 3975U / mg to 4770U / mg by performing site-directed mutagenesis on the alkaline protease gene shown in SEQ ID NO.1. This method is simple and easy to implement, laying the foundation for the efficient expression of alkaline protease in Bacillus licheniformis, thereby promoting the fermentation production of alkaline protease. DETAILED DESCRIPTION

[0021] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods well known to those skilled in the art. In addition, the embodiments should be understood to be illustrative rather than limiting the scope of the present invention, and the spirit and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material components and dosages in these embodiments, without departing from the spirit and scope of the present invention, also fall within the scope of protection of the present invention.

[0022] Nickel affinity chromatography filler (Ni Sepharose 6 Fast Flow) and molecular sieve filler (Sephadex G-25) were purchased from GE and packed into columns according to protein purification requirements; the remaining reagents were domestically produced or imported of analytical grade.

[0023] Example 1:

[0024] Construction of alkaline protease mutants from Bacillus clausii

[0025] The artificially synthesized ALK gene is shown in SEQ ID NO.2, and the protein encoded by the gene is shown in SEQ ID NO.1.

[0026] The synthesized ALK gene was ligated into the expression vector pHY-300 via the restriction sites NdeI and NotI to construct the recombinant plasmid pHY-300-ALK. The recombinant plasmid was transformed into Bacillus licheniformis DW2 (CN116656712A) to obtain recombinant Bacillus licheniformis expressing wild-type ALK. Recombinant Bacillus licheniformis pHY-300-ALK expressing the alkaline protease mutant shown in SEQ ID NO.1 G113P The specific preparation steps are as follows:

[0027] Using the plasmid pHY-300-ALK expressing the wild-type ALK gene as a template, the DNA fragment was amplified by PCR reaction with primers G113P-F and G113P-R. The PCR program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, for 30 cycles; and extension at 72°C for 5 min.

[0028] The PCR product was isolated using agarose gel electrophoresis to isolate the target DNA, which was purified using a Winzyme agarose gel recovery kit. The recombinant DNA fragments were circularized using Winzyme's HI-Efficiency Cloning Mix. The recombinant DNA was then transformed into competent Escherichia coli DH5a cells. The cells were plated on Tet-resistant plates for selection and cultured in a 37°C incubator. Transformants were verified by colony PCR using amp-F and amp-R primers. If the target size was correct, sequencing was performed. Vector nucleotide sequence determination was performed by Wuhan Jinkairui Bioengineering Co., Ltd. Sequencing results were analyzed; if the sequence matched the design, the mutant vector was successfully constructed. The recombinant plasmid of the mutant was extracted using a plasmid extraction kit and then transferred into competent Bacillus licheniformis DW2. The bacteria were plated on a culture plate containing Tet resistance for screening and cultured in a 37°C incubator. The transformants were verified by colony PCR using amp-F and amp-R primers. If the target size was correct, the engineered strain was pHY-300-ALK. G113P Build successful.

[0029] The applicant also prepared other mutant proteins as control groups, and the recombinant strains expressing these mutant proteins (as shown in Table 1, including pHY-300-ALK S36A , pHY-300-ALK T51I , pHY-300-ALK H62L , pHY-300-ALK L94C , pHY-300-ALK I105V , pHY-300-ALK G113L , pHY-300-ALK G113T ) The construction process can refer to the recombinant Bacillus licheniformis pHY-300-ALK G113P The corresponding primers used in the construction of the recombinant strain are shown in Table 1.

[0030] Table 1 Primers used for site-directed mutagenesis

[0031]

[0032]

[0033] Example 2:

[0034] Purification and activity determination of the alkaline protease expressed by the recombinant Bacillus licheniformis prepared in Example 1:

[0035] The wild-type ALK recombinant Bacillus licheniformis and the mutant ALK recombinant Bacillus licheniformis prepared in Example 1 were streaked onto solid Tet plates (containing 20 μg / mL kanamycin) and cultured overnight at 37°C to obtain single clones. Single clones were then selected and activated in liquid Tet medium (5 mL) and cultured with shaking at 37°C for 12 hours. One ml of the activated bacterial suspension was then inoculated into 20 mL of liquid Tet medium and cultured with shaking at 37°C overnight. The overnight culture seed solution was inoculated into 20 mL of soybean meal medium at a 1% inoculum, fermented at 37°C and 230 rpm for 48 hours, and the supernatant was collected by centrifugation. The supernatant was applied to a nickel affinity chromatography column, and contaminants were eluted using equilibration buffer (PBS containing 20 mM imidazole). Finally, the target protein was eluted using elution buffer (containing 300 mM imidazole). The eluted protein solution was then desalted with molecular sieves and purified AKL was collected. The enzyme activities of wild-type ALK and six purified ALK mutants were measured at 25°C, and the relative enzyme activities of the wild-type and mutants were calculated. The magnitude of the change was calculated using the formula: (specific enzyme activity of mutants - specific enzyme activity of wild-type) / specific enzyme activity of wild-type.

[0036] The formula of the soybean meal culture medium includes: 45g / L corn starch, 50g / L soybean meal powder, 6g / L calcium carbonate, 5g / L ammonium sulfate, and natural pH.

[0037] Alkaline protease activity was determined using the Folin-phenol method described in Appendix B of GB / T 23527-2009, except that the reaction temperature was adjusted to 25°C. Enzyme activity was defined as the amount of enzyme required to catalyze the production of 1 μmol / min of L-tyrosine at pH 10.5 and 25°C. 1 unit (U) was defined as the amount of enzyme required to catalyze the production of 1 μmol / min of L-tyrosine at pH 10.5 and 25°C.

[0038] The results are shown in the following table:

[0039]

[0040]

[0041] From the table above, we can see that the wild-type AKL has an activity of 3975 U / mg at 25°C, while the AKL mutant G113P has a significantly improved activity of 4770 U / mg at 25°C, an increase of 20%.

Claims

1. An alkaline protease mutant, the amino acid sequence of the protease mutant being shown in SEQ ID NO.

3.

2. A fusion protein obtained by fusing the protease mutant according to claim 1 with a protein tag.

3. A gene encoding the protease mutant according to claim 1 or the fusion protein according to claim 2.

4. An expression cassette, recombinant vector, recombinant microorganism or in vitro recombinant cell comprising the coding gene according to claim 3.

5. Use of the protease mutant according to claim 1, the fusion protein according to claim 2, the encoding gene according to claim 3, the expression cassette, recombinant vector, recombinant microorganism or in vitro recombinant cell according to claim 4 in the preparation of alkaline protease.

6. A method for improving the activity of alkaline protease, comprising the following steps: mutating glycine G at position 113 of SEQ ID NO. 1 to proline P.

7. A method for preparing alkaline protease with improved specific enzyme activity, comprising the steps of: preparing the alkaline protease by culturing the recombinant microorganism according to claim 5.

8. The coding gene according to claim 3, wherein the coding gene of the mutant of SEQ ID NO.3 is shown in SEQ ID NO.

4.

9. The use according to claim 5, wherein the recombinant microorganism is recombinant Bacillus licheniformis.

10. The use according to claim 9, wherein the Bacillus licheniformis is Bacillus licheniformis DW2.

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