ALKALINE PROTEASE MUTANT, AND GENE, ENGINEERED STRAIN, PREPARATION METHOD AND APPLICATION THEREOF

Abstract

An alkaline protease mutant, and a gene, engineered strain, a preparation method and application thereof are provided. The method comprises the following steps of extracting genome DNA of Bacillus clausii, performing PCR amplification to obtain a wild-type alkaline protease gene sequence, mutating the wild-type alkaline protease gene obtained by the amplification through an error-prone PCR, performing high-throughput screening to obtain a plurality of highly active alkaline protease genes, performing DNA shuffling on the highly active alkaline protease genes, and performing screening to obtain eight alkaline protease mutant genes with higher activity.

Claims

1. An alkaline protease mutant, wherein an alkaline protease has a mature peptide obtained by generating any one of the following mutation combinations based on a sequence of a zymogen region of the alkaline protease shown in SEQ ID NO: 6: V11I/G23A/G25P/I35V/G95P/S99H/V145I/N212S/A267G, V11L/G23A/G25P/I35V/G95P/S99H/V145I/N212S/A267G, V11L/G23A/G25A/I35V/G95P/S99H/V145I/N212S/A267G, V11I/G23A/G25A/I35V/G95P/S99H/V145I/N212S/A267G, V11I/G23A/G25A/I35V/G95P/S99H/V145I/N212S/A267P, V11L/G23A/G25A/I35V/G95P/S99H/V145I/N212S/A267P, V11I/G23A/G25P/I35V/G95P/S99H/V145I/N212S/A267P, V11L/G23A/G25P/I35V/G95P/S99H/V145I/N212S/A267P.

2. An encoding gene of the alkaline protease mutant according to claim 1.

3. The encoding gene according to claim 2, wherein the encoding gene is shown in any one of SEQ ID NOS: 7-14.

4. A recombinant vector or a recombinant strain comprising the encoding gene according to claim 2.

5. The recombinant vector or the recombinant strain according to claim 4, wherein an expression vector is pBSA43; and a host cell is a Bacillus subtilis WB600, a Bacillus amyloliquefaciens CGMCC No.11218, or a Bacillus clausii CGMCC No. 12953.

6. A use of the recombinant vector or the recombinant strain according to claim 4 in producing the alkaline protease.

7. A use of the alkaline protease mutant according to claim 1 in fields of detergents and food.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0052] FIG. 1 is an electrophoresis diagram of PCR amplification of a wild-type alkaline protease zymogen gene in an example of the present disclosure, [0053] where, M is DNA Marker and 1 is an alkaline protease zymogen gene apr;

[0054] FIG. 2 is a verification diagram of digestion of a pBAS43-apr plasmid in an example of the present disclosure,

[0055] where, M is DNA Marker and 1 is a double digestion map of pBSA43-apr by BamHI and HindIII;

[0056] FIG. 3 is an electrophoresis diagram of error-prone PCR amplification of an alkaline protease mutant gene in an example of the present disclosure,

[0057] where, M is DNA Marker and 1 and 2 show an electrophoresis diagram of error-prone PCR amplification of an alkaline protease mutant gene aprmx.sub.1;

[0058] FIG. 4 is an electrophoresis diagram of a product of DNA shuffling of an alkaline protease mutant gene in an example of the present disclosure,

[0059] where, M is DNA Marker and 1 and 2 show an electrophoresis diagram of a shuffled gene aprmx.sub.2 of an alkaline protease mutant;

[0060] FIG. 5 is a verification diagram of digestion of a recombinant plasmid of pBSA43-aprmX in an example of the present disclosure,

[0061] where, M is DNA Marker and 1, 2, 3, 4, 5, 6, 7, and 8 show a double digestion map of recombinant plasmids pBSA43 -aprm1, pBSA43 -aprm2, pBSA43-aprm3, pBSA43-aprm4, pBSA43-aprm5, pBSA43-aprm6, pBSA43-aprm7 and pBSA43-aprm8 by BamHI and HindIII; and

[0062] FIGS. 6A-6D show enzymatic properties of a wild-type alkaline protease APR in an example of the present disclosure,

[0063] where, FIG. 6A is an optimal reaction temperature curve of the wild-type alkaline protease APR;

[0064] FIG. 6B is an optimal reaction pH of the wild-type alkaline protease APR;

[0065] FIG. 6C is a temperature stability curve of the wild-type alkaline protease APR at 60° C.; and

[0066] FIG. 6D is a pH stability curve of the wild-type alkaline protease APR at a pH of 11.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0067] To make the objective, technical solutions and advantages of the patent clearer and more comprehensible, the present patent will be further described below in detail in conjunction with specifically examples. It should be understood that the specific examples described herein are merely intended to explain the patent, rather than to limit the present disclosure.

[0068] The Bacillus licheniformis used in the present disclosure is TCCC11965, disclosed in Development and application of a CRISPR/Cas9 system for Bacillus licheniformis genome editing [J]. International Journal of Biological Macromolecules, 2019, 122: 329-337, and currently deposited in Microbial Culture Collection and Management Center of Tianjin University of Science and Technology, and the public can inquire and obtain the strain from the Center.

EXAMPLE 1

Acquisition of Wild-type Alkaline Protease Gene

[0069] 1. A wild-type alkaline protease gene was from a Bacillus clausii CGMCC NO. 12953 strain preserved in the laboratory. Its genomic DNA was extracted by a kit (OMEGA: Bacterial DNA Kit). The genomic DNA of the Bacillus clausii was extracted by the following steps: [0070] (1) strain activation: a little bacterial solution was dipped from a glycerol tube with an inoculation loop and inoculated on an LB solid medium plate in a manner of drawing lines to form three zones, and the bacteria were cultured at 37° C. for 12 h; [0071] (2) strain culturing: single colonies were picked from a plate for culturing bacteria and inoculated into 5 mL of a liquid LB medium, and the bacteria were cultured with shaking at 220 rpm and 37° C. for 12 h; [0072] (3) collection of bacteria: an appropriate amount of a cultured bacterial solution was sub-packaged into sterilized 1.5-mL EP tubes, the bacterial solution was centrifuged at 12,000 rpm for 1 min to collect the bacteria, and a supernatant was discarded; [0073] (4) 100 μL of ddH.sub.2O was added to resuspend the bacteria, 50 μL of 50 mg/mL of lysozyme was added, and water bath was performed at 37° C. for 10 min; [0074] (5) 100 μL of a BTL Buffer and 20 μL of a proteinase K were added, an obtained mixture was vortex-shaken, subjected to water bath at 55° C. for 40-50 min, and shaken and uniformly mixed every other 20-30 min; [0075] (6) 5 μL of RNase was added, and an obtained mixture was uniformly mixed several times in an inverted manner and placed at a room temperature for 5 min; [0076] (7) the obtained mixture was centrifuged at 12,000 rpm for 2 min, an undigested part was removed, a supernatant part was transferred to a new 1.5-mL EP tube, 220 μL of a BDL Buffer was added, and an obtained mixture was shaken and uniformly mixed, and subjected to water bath at 65° C. for 10 min; [0077] (8) 220 μL of absolute ethanol was added and an obtained mixture was pipetted and uniformly mixed; [0078] (9) liquid in the EP tube was transferred to an adsorption column for standing for 2 min and centrifuged at 12,000 rpm for 1 min, a filtrate was poured back into the adsorption column for standing and centrifugation, the operation was repeated twice, and a filtrate was discarded; [0079] (10) 500 μL of a HBC Buffer was added, an obtained mixture stood for 2 min and centrifuged at 12,000 rpm for 1 min, and a filtrate was discarded; [0080] (11) 700 μL of a DNA Wash Buffer was added, an obtained mixture stood for 2 min and centrifuged at 12,000 rpm for 1 min, and a filtrate was discarded; [0081] (12) 500 μL of a DNA Wash Buffer was added, an obtained mixture stood for 2 min and centrifuged at 12,000 rpm for 1 min, and a filtrate was discarded; [0082] (13) centrifugation was performed at 12,000 rpm for 2 min, and the adsorption column was put in a new EP tube, placed in a metal bath at 55° C. for 10 min, and air-dried; and [0083] (14) 50 μL of molecular water at 55° C. was added, and an obtained mixture stood at a room temperature for 3-5 min and was centrifuged at 12,000 rpm for 2 min to collect a genome.

[0084] 2. The extracted genome of the Bacillus clausii was used as a template, a pair of primers was designed at an upstream and a downstream of an ORF according to an alkaline protease sequence registered with a Genbank serial number of FJ940727.1, and restriction sites of BamHI and HindIII were introduced separately. The alkaline protease gene of the present disclosure has amplification primers as follows:

TABLE-US-00001 upstream primer P1 (SEQ ID NO: 1): 5’-CGCGGATCCGCTGAAGAAGCAAAAGAAAAATATTTAAT-3’; and downstream primer P2 (SEQ ID NO: 2): 5’-CCCAAGCTTTTAGCGTGTTGCCGCTTCT-3’.

[0085] P1 and P2 were used as the upstream and downstream primer separately, and the alkaline protease genome of the Bacillus clausii was used as a template for amplification.

[0086] A reaction system for the amplification was as follows:

TABLE-US-00002 10 × PCR Buffer 5.0 μL dNTPs 5.0 μL Upstream primer P1 2.0 μL Downstream primer P2 2.0 μL DNA template 2.0 μL Pyrobest enzyme 0.5 μL ddH.sub.2O 33.5 μL

[0087] An amplification process was as follows: pre-denaturation at 95° C. for 10 min; denaturation at 94° C. for 30 s, annealing at 57° C. for 45 s, and extension at 72° C. for 1 min 20 s, a total of 30 cycles; and extension at 72° C. for 10 min. A PCR amplified product was subjected to 0.8% agarose gel electrophoresis to obtain a band of 1,059 bp (FIG. 1). The PCR product was recovered with a small DNA recovery kit to obtain the wild-type alkaline protein gene apr (SEQ ID NO: 3), the amplified apr was ligated with a vector pBSA43 to obtain a recombinant plasmid pBSA43-apr, a digestion verification was shown in FIG. 2, and the recombinant plasmid was transformed into Escherichia coli JM109 and Bacillus subtilis WB600.

EXAMPLE 2

Screening of Highly Active Alkaline Protease Mutants by Construction of Alkaline Protease Mutant Library by Error-prone PCR

[0088] 1. Random mutation was performed based on error-prone PCR to construct an alkaline protease mutant library. Primers were designed as follows:

TABLE-US-00003 upstream primer P1 (SEQ ID NO: 1): 5’-CGCGGATCCGCTGAAGAAGCAAAAGAAAAATATTTAAT-3’; and downstream primer P2 (SEQ ID NO: 2): 5’-CCCAAGCTTTTAGCGTGTTGCCGCTTCT-3’.

[0089] In an error-prone PCR reaction system, P1 and P2 were used as the upstream and downstream primer separately, a wild-type alkaline protease gene apr was used as a template, and error-prone PCR was performed.

[0090] A reaction system for an amplification was as follows:

TABLE-US-00004 10 × PCR Buffer (free of Mg.sup.2+) 5 μL dATP 0.1 μL dGTP 0.1 μL dCTP 0.5 μL dTTP 0.5 μL Upstream primer P1 2 μL Downstream primer P2 2 μL Wild-type alkaline protease gene 2 μL rTaq DNA polymerase 0.3 μL 25 mM MgCl.sub.2 (10 mM) 20 μL 5 mM MnCl.sub.2 (0.3 mM) 3 μL ddH.sub.2O 14.5 μL

[0091] An amplification process was as follows: pre-denaturation at 95° C. for 10 min; denaturation at 98° C. for 10 s, annealing at 57° C. for 30 s, and extension at 72° C. for 1 min 20 s, a total of 30 cycles; and extension at 72° C. for 10 min. A PCR amplified product was subjected to 0.8% agarose gel electrophoresis (FIG. 3). The PCR product was recovered with a small DNA recovery kit to obtain an alkaline protease gene aprmx.sub.l with random mutation (x.sub.1 represents several different randomly mutated genes).

[0092] 2. The alkaline protease random mutant gene aprmx.sub.1 was ligated with a vector pBSA43 to transform into JM109, the plasmid was extracted to obtain a recombinant plasmid pBSA43-aprmx.sub.1, the recombinant plasmid pBSA43-aprmx.sub.1 was transformed into Bacillus subtilis WB600, transformants were picked into a 48-well plate containing 500 μL of an LB liquid medium, the 48-well plate was placed in a 48-well plate shaker and cultured at 37° C. and 750 r/min for 48 h, a supernatant was taken after the culture to obtain a crude alkaline protease solution, activity of the alkaline protease was determined by a short peptide substrate method, and the transformants with higher enzyme activity than a wild type were picked out. The recombinant plasmids extracted from transformant with high enzyme activity were used as templates, continuous error-prone PCR was performed, screening was performed according to the above method, the operation was repeated for three times, several mutant strains with high alkaline protease activity were finally screened, and the alkaline protease mutant plasmids with high activity were used as templates for DNA shuffling.

[0093] 3. Determination of alkaline protease activity with short peptide substrates

[0094] Short peptide substrates: N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (AAPF, represented by single letter abbreviation of amino acids, similarly hereinafter), AAPY, AAPW, AAPA, AAPR, AAPN, AAPD, AAPC, AAPQ, AAPE, AAPG, AAPH, AAPI, AAPL, AAPK, AAPM, AAPP, AAPS, AAPT and AAPV were mixed and dissolved in dimethyl sulfoxide (DMSO), such that each substrate had a concentration of 6 mmol/L (a method refers to the patent “New method for measuring activity of protease” with an application number of 201910730238.2).

[0095] Determination method: 80 μL of a boric acid buffer at a pH of 10.5 and 20 μL of a short peptide substrate solution were added to a 96-well plate, and incubated in a 40° C. water bath kettle for 1 min, 100 μL of a diluted enzyme solution (100 μL of a boric acid buffer at a pH of 10.5 was added to a negative control) was added, reaction was performed at 40° C. for 10 min, and absorbance was measured at 410 nm with a microplate reader. One unit (U) of enzyme activity was defined as that 1 mL of an enzyme solution hydrolyzes a substrate for 1 min to produce 1 μmol of p-nitroaniline under the above condition.

EXAMPLE 3

Screening of Highly Active Alkaline Protease Mutants by Construction of Alkaline Protease Mutant Library by DNA Shuffling

[0096] The alkaline protease mutant genes obtained by the screening by the error-prone PCR in example 2 were subjected to DNA shuffling and highly active alkaline protease mutants were obtained by high-throughput screening.

[0097] 1. Fragmentation of Alkaline Protease Mutant Genes

[0098] The recombinant plasmids of the alkaline protease mutant strains obtained by the screening by the error-prone PCR were extracted, the recombinant plasmids were digested with restriction enzymes BamHI and HindIII, DNA fragments of the alkaline protease mutant genes were recovered by Gel Extraction, the DNA fragments of the mutant genes were uniformly mixed in an equal amount, 1 μg of the mixed DNA fragments were added to 100 μL of a buffer system (50 mmol/L Tris-HCl at a pH of 7.4 and 1 mmol/L MgCl.sub.2), DNaseI at a final concentration of 0.01 U was added, enzyme digestion was performed at 37° C. for 20 min, and enzyme inactivation was performed at 90° C. for 10 min. A digested product was subjected to 2% agarose gel electrophoresis and fragments of about 50-200 bp were recovered with a small DNA recovery kit.

[0099] 2. Primerless PCR

[0100] The small fragments recovered after the above digestion were used as templates and primerless

[0101] PCR was performed using the small fragments as primers for each other. A reaction system for an amplification was as follows:

TABLE-US-00005 10 × PCR Buffer (free of Mg.sup.2+) 5.0 μL 25 mM MgCl.sub.2 5.0 μL dNTPs 5.0 μL DNA fragment templates 2.0 μL rTaq DNA polymerase 0.5 μL ddH.sub.2O 37.5 μL

[0102] An amplification process was as follows: pre-denaturation at 95° C. for 10 min; denaturation at 98° C. for 10 s, annealing at 50° C. for 30 s, and extension at 72° C. for 1 min 20 s, a total of 30 cycles; and extension at 72° C. for 10 min. A PCR amplified product was subjected to 0.8% agarose gel electrophoresis and a DNA fragment of about 1 kb was recovered with a small DNA recovery kit.

[0103] 2. PCR With Primers

[0104] The primerless PCR product was used as a template to be subjected to a second round of PCR with primers, and the amplification primers were as follows:

TABLE-US-00006 upstream primer P1 (SEQ ID NO: 1): 5’-CGCGGATCCGCTGAAGAAGCAAAAGAAAAATATTTAAT-3’; and downstream primer P2 (SEQ ID NO: 2): 5’-CCCAAGCTTTTAGCGTGTTGCCGCTTCT-3’.

[0105] A reaction system for an amplification was as follows:

TABLE-US-00007 10 × PCR Buffer 5.0 μL dNTPs 5.0 μL Upstream primer P1 2.0 μL Downstream primer P2 2.0 μL Primerless PCR product 2.0 μL Pyrobest enzyme 0.5 μL ddH.sub.2O 33.5 μL

[0106] An amplification process was as follows: pre-denaturation at 95° C. for 10 min; denaturation at 94° C. for 30 s, annealing at 57° C. for 45 s, and extension at 72° C. for 1 min 20 s, a total of 30 cycles; and extension at 72° C. for 10 min. A PCR amplified product was subjected to 0.8% agarose gel electrophoresis (FIG. 4). A DNA fragment of about 1 kb was recovered with a small DNA recovery kit to obtain alkaline protease shuffled genes aprmx.sub.2 (x.sub.2 represents several different shuffled genes).

[0107] 4. The alkaline protease shuffled genes aprmx.sub.2 were separately cloned into an expression vector pBSA43 to obtain several recombinant plasmids pBSA43-aprmx.sub.2 to be transformed into JM109, extraction was performed to obtain the recombinant plasmids pBSA43-aprmx.sub.2, the recombinant plasmids pBSA43-aprmx.sub.2 were transformed into Bacillus subtilis WB600, transformants were picked into a 48-well plate containing 500 μL of an LB liquid medium, the 48-well plate was placed in a 48-well plate shaker and cultured at 37° C. and 750 r/min for 48 h, a supernatant was taken after the culture to obtain a crude alkaline protease solution, activity of the alkaline protease was determined by a short peptide substrate method in example 2, and the transformants with higher enzyme activity than a wild type were picked out. After screening, eight mutant strains WB600/pBSA43-aprmX (X are 1-8 separately and aprmX represents 8 different mutant encoding genes specifically as shown in Table 1) with higher alkaline protease activity were obtained. Plasmids of the obtained highly active alkaline protease mutant strains were extracted and sequenced (Beijing Huada Bioengineering Company). The results showed the obtained 8 highly active alkaline protease mutants in the following table.

TABLE-US-00008 TABLE 1 Information of alkaline protease mutants Gene SEQ Mutants Mutated amino acids residue Name ID NO: APR V11/G23/G25/I35/G95/S99/V145/ apr 3 N212/A267 APRM 1 V11I/G23A/G25P/I35V/G95P/S99H/ aprm1 7 V145I/N212S/A267G APRM 2 V11L/G23A/G25P/I35V/G95P/S99H/ aprm2 8 V145I/N212S/A267G APRM 3 V11L/G23A/G25A/I35V/G95P/S99H/ aprm3 9 V145I/N212S/A267G APRM 4 V11I/G23A/G25A/I35V/G95P/S99H/ aprm4 10 V145I/N212S/A267G APRM 5 V11I/G23A/G25A/I35V/G95P/S99H/ aprm5 11 V145I/N212S/A267P APRM 6 V11L/G23A/G25A/I35V/G95P/S99H/ aprm6 12 V145I/N212S/A267P APRM 7 V11I/G23A/G25P/I35V/G95P/S99H/ aprm7 13 V145I/N212S/A267P APRM 8 V11L/G23A/G25P/I35V/G95P/S99H/ aprm8 14 V145I/N212S/A267P

EXAMPLE 4

Determination of Specific Activity of Highly Active Alkaline Protease Mutants

[0108] The mutant recombinant strains WB600/pBSA43-aprmX (X is 1, 2, 3, 4, 5, 6, 7, and 8, similarly hereinafter) obtained in step 4 of example 3 and a wild-type recombinant strain WB600/pBSA43-apr were inoculated in 5 mL of a LB liquid medium (containing 50 μg/mL of kanamycin) separately and cultured at 37° C. and 220 r/min overnight, the cultured strains were transferred to 50 mL of a fresh LB medium (containing 50 μg/mL of kanamycin) at an inoculum size of 2%, and the strains were continuously cultured at 37° C. and 220 r/min for 48 h.

[0109] A fermentation broth was centrifuged, a supernatant was taken, impure proteins were removed by salting out with ammonium sulfate at a saturation of 25%, and the saturation was increased to 65% to precipitate a target protein. After the precipitate was dissolved, dialysis was performed to remove salt, an active component obtained after salting out to desalt was dissolved with 0.02 mol/L of a Tris-HCl (pH 7.0) buffer, an obtained sample was loaded on a cellulose ion exchange chromatography column, unadsorbed protein was first eluted using the same buffer, and a target protein was collected by gradient elution with 0.02 mol/L of a Tris-HCl (pH 7.0) buffer containing different concentrations of NaCl (0-1 mol/L). The active component obtained by ion exchange was first equilibrated with 0.02 mol/L of a Tris-HCl (pH 7.0) buffer containing 0.15 mol/L of NaCl, and an obtained sample was loaded onto a sephadex g25 gel chromatography column and eluted with the same buffer at a speed of 0.5 mL/min to obtain a purified enzyme solution.

[0110] The alkaline protease activity was determined by the short peptide substrate method in example 2; and a protein concentration was determined by a BCA protein concentration assay kit, the operation was performed according to the instructions, and a specific activity of the alkaline protease was value of a ratio of the enzyme activity (U/ml) to protein concentration (mg/ml). The specific activity of the wild-type recombinant strain was set as 1 and the specific activity of the mutant recombinant strain was expressed as a multiple of the specific activity of the wild-type recombinant strain. The results were as shown in the following table.

[0111] Specific Activity of Alkaline Protease

TABLE-US-00009 Specific activity Specific activity Mutants (U/mg) multiple Wild-type APR 26.6 1 APRM 1 614.5 23.1 APRM 2 670.3 25.2 APRM 3 598.5 22.5 APRM 4 707.6 26.6 APRM 5 696.9 26.2 APRM 6 662.3 24.9 APRM 7 673.0 25.3 APRM 8 686.3 25.8

[0112] Determination of enzymatic properties: enzymatic properties of the wild type and mutants were determined. The results were shown in FIGS. 6A-6D. The wild-type alkaline protease had an optimal reaction temperature of 60° C. and an optimal reaction pH of 10. At a pH of 10 and 60° C., heat preservation was performed for 40 h, and residual enzyme activity was about 6%. At 60° C. and a pH of 11, heat preservation was performed for 70 h, and residual enzyme activity was about 21%. The enzymatic properties of the wild type and mutants were basically the same.

EXAMPLE 5

Construction of Highly Active Alkaline Protease Mutants in Other Bacillus

[0113] 1 μL (50 ng/μL) of pBSA43-aprmX and pBSA43-apr recombinant plasmids were separately added into 50 μL of Bacillus amyloliquefaciens CGMCC No.11218, Bacillus licheniformis TCCC11965, and Bacillus clausii CGMCC No.12953 competent cells to be uniformly mixed, an obtained mixture was transferred to a pre-cooled electroporation cup (1 mm) and subjected to an ice bath for 1-1.5 min, and the treated mixture was electrically shocked once (25 μF, 200 Ω, and 4.5-5.0 ms). Immediately after the electric shock, 1 mL of a resuscitation medium (LB+0.5 mol/L sorbitol+0.38 mol/L mannitol) was added. After cultured in shaking by a shaker at 37° C. for 3 h, a product after the resuscitation was spread on an LB plate containing kanamycin and cultured at 37° C. for 12-24 h. Positive transformants were picked and subjected to a double-enzyme digestion verification (FIG. 5). Recombinant strains of Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus clausii expressing a mutant gene aprmX and a wild-type gene apr was obtained, and named CGMCC No. 11218/pBSA43-aprmX and CGMCC No. 11218/pBSA43-apr; TCCC11965/pB SA43-aprmX and TCCC11965/pB SA43-apr; and CGMCC No. 12953/pBSA43-aprmX and CGMCC No. 12953/pBSA43-apr.

EXAMPLE 6

Expression and Preparation of Alkaline Protease Mutants in Bacillus amyloliquefaciens Recombinant Strain

[0114] The Bacillus amyloliquefaciens mutant recombinant strain CGMCC No. 11218/pBSA43-aprmX and the wild-type recombinant strain CGMCC No. 11218/pBSA43-apr were separately inoculated in 5 mL of a LB liquid medium (containing 50 μg/mL of kanamycin) and cultured at 37° C. and 220 r/min overnight, the cultured strains were transferred to 50 mL of a fresh LB medium (containing 50 μg/mL of kanamycin) at an inoculum size of 2%, and the strains were continuously cultured at 37° C. and 220 r/min for 48 h.

[0115] A fermentation broth was centrifuged, a supernatant was taken, impure proteins were removed by salting out with ammonium sulfate at a saturation of 25%, and the saturation was increased to 65% to precipitate a target protein. After the precipitate was dissolved, dialysis was performed to remove salt, an active component obtained after salting out to desalt was dissolved with 0.02 mol/L of a Tris-HCl (pH 7.0) buffer, an obtained sample was loaded on a cellulose ion exchange chromatography column, unadsorbed protein was first eluted using the same buffer, and a target protein was collected by gradient elution with 0.02 mol/L of a Tris-HCl (pH 7.0) buffer containing different concentrations of NaCl (0-1 mol/L). The active component obtained by ion exchange was first equilibrated with 0.02 mol/L of a Tris-HCl (pH 7.0) buffer containing 0.15 mol/L of NaCl, an obtained sample was loaded onto a sephadex g25 gel chromatography column and eluted with the same buffer at a speed of 0.5 mL/min to obtain a purified enzyme solution, and the enzyme solution was freeze-dried to prepare purified alkaline protease powder. The prepared enzyme powder can be used in detergents, food, leather manufacturing, medicines and other industries.

EXAMPLE 7

Expression and Preparation of Alkaline Protease Mutants in Bacillus licheniformis Recombinant Strain

[0116] The Bacillus licheniformis mutant recombinant strain TCCC11965/pBSA43-aprmX and the wild-type recombinant strain TCCC11965/pBSA43-apr were separately inoculated in 5 mL of a LB liquid medium (containing 50 μg/mL of kanamycin) and cultured at 37° C. and 220 r/min overnight, the cultured strains were transferred to 50 mL of a fresh LB medium (containing 50 μg/mL of kanamycin) at an inoculum size of 2%, and the strains were continuously cultured at 37° C. and 220 r/min for 48 h.

[0117] A fermentation broth was centrifuged, a supernatant was taken, impurity proteins were removed by salting out with ammonium sulfate at a saturation of 25%, and the saturation was increased to 65% to precipitate a target protein. After the precipitate was dissolved, dialysis was performed to remove salt, an active component obtained after salting out to desalt was dissolved with 0.02 mol/L of a Tris-HCl (pH 7.0) buffer, an obtained sample was loaded on a cellulose ion exchange chromatography column, unadsorbed protein was first eluted using the same buffer, and a target protein was collected by gradient elution with 0.02 mol/L of a Tris-HCl (pH 7.0) buffer containing different concentrations of NaCl (0-1 mol/L). The active component obtained by ion exchange was first equilibrated with 0.02 mol/L of a Tris-HCl (pH 7.0) buffer containing 0.15 mol/L of NaCl, an obtained sample was loaded onto a sephadex g25 gel chromatography column and eluted with the same buffer at a speed of 0.5 mL/min to obtain a purified enzyme solution, and the enzyme solution was freeze-dried to prepare purified alkaline protease powder. The prepared enzyme powder can be used in detergents, food, leather manufacturing, medicines and other industries.

EXAMPLE 8

Expression and Preparation of Alkaline Protease Mutants in Bacillus clausii Recombinant Strain

[0118] The Bacillus clausii mutant recombinant strain CGMCC No.12953/pBSA43-aprmX and the wild-type recombinant strain CGMCC No.12953/pBSA43-apr were separately inoculated in 5 mL of a LB liquid medium (containing 50 μg/mL of kanamycin) and cultured at 37° C. and 220 r/min overnight, the cultured strains were transferred to 50 mL of a fresh LB medium (containing 50 μg/mL of kanamycin) at an inoculum size of 2%, and the strains were continuously cultured at 37° C. and 220 r/min for 48 h.

[0119] A fermentation broth was centrifuged, a supernatant was taken, impure proteins were removed by salting out with ammonium sulfate at a saturation of 25%, and the saturation was increased to 65% to precipitate a target protein. After the precipitate was dissolved, dialysis was performed to remove salt, an active component obtained after salting out to desalt was dissolved with 0.02 mol/L of a Tris-HCl (pH 7.0) buffer, an obtained sample was loaded on a cellulose ion exchange chromatography column, unadsorbed protein was first eluted using the same buffer, and a target protein was collected by gradient elution with 0.02 mol/L of a Tris-HCl (pH 7.0) buffer containing different concentrations of NaCl (0-1 mol/L). The active component obtained by ion exchange was first equilibrated with 0.02 mol/L of a Tris-HCl (pH 7.0) buffer containing 0.15 mol/L of NaCl, an obtained sample was loaded onto a sephadex g25 gel chromatography column and eluted with the same buffer at a speed of 0.5 mL/min to obtain a purified enzyme solution, and the enzyme solution was freeze-dried to prepare purified alkaline protease powder. The prepared enzyme powder can be used in detergents, food, leather manufacturing, medicines and other industries.

[0120] The above examples are merely illustrative of several implementations of the present disclosure, and the description thereof is more specific and detailed. However, these examples may not to be construed as a limitation to the scope of the patent. It should be noted that those of ordinary skill in the art can further make several variations, combinations and improvements without departing from the conception of the present disclosure. These variations, combinations and improvements all fall within the protection scope of the patent. Therefore, the protection scope of the patent shall be in accordance with the claims.