CN113801831B - Bacillus subtilis capable of producing neutral protease with high yield and application thereof - Google Patents

Abstract

The invention relates to the technical field of genetic engineering, in particular to a bacillus subtilis strain for high yield of neutral protease and application thereof. The bacillus subtilis is a mutant strain which is obtained by screening through an ultraviolet mutagenesis method and has the yield of neutral protease remarkably improved, and the preservation number is CGMCC No.19499. The strain can be widely applied to fermentation production of neutral protease, is favorable for reducing the production cost of the neutral protease, and has wide application prospect.

Description

Bacillus subtilis capable of producing neutral protease with high yield and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a bacillus subtilis strain for high yield of neutral protease and application thereof.

Background

Protease is added into the detergent in European and American countries at the end of sixty years, so that the great change of the detergent industry is promoted, the new fields of nucleic acid and protein engineering are expanded along with the rise and development of bioengineering in the seventy years, the novel industry of tool enzyme is raised, and the development of enzyme research and enzyme preparation industry is further promoted when biotechnology is used for modifying enzyme-producing bacteria, so that the enzyme becomes a central issue related to international academic conferences. Since the reaction conditions required for alkaline protease and acidic protease limit their wide application, studies on neutral protease have been conducted successively at home and abroad since the beginning of the eighties.

The neutral protease is a kind of protease with the optimum action pH value between 6.0-7.5, and as a biocatalyst, the neutral protease has the advantages of high catalytic reaction speed, no industrial pollution and the like. And the heat resistance is relatively low, and the protein hydrolysate becomes a key for controlling the enzyme activity in the production process of the food protein hydrolysate. Most of microbial neutral proteases contain metal elements, part of the protease protein contains a molecule of zinc, the zinc plays a role of a bridge between the enzyme and a substrate, and the molecules of some enzymes contain a plurality of atoms of calcium, and calcium ions can increase the stability of the neutral proteases.

The protease is widely available, and can be produced by animals, plants and microorganisms. Obtaining protease from animal body requires killing a large number of animals, and the process for purifying enzyme from plant body is difficult, so the application of protease derived from animal and plant is limited. The propagation speed of the growth of the microorganism is high, most of protease derived from the microorganism is exogenous enzyme, and the extraction process is relatively simple, so the research on protease produced by microbial fermentation becomes a hotspot of the research on the protease. Proteases of microbial origin are divided into bacterial neutral proteases, fungal neutral proteases and neutral proteases of other origin, most of the commercially available neutral proteases are mostly produced by the genus Bacillus, such as Bacillus subtilis, B. Neutral proteases are produced by a wide variety of fungi, such as Aspergillus oryzae, rhizopus, mucor, and the like.

At present, neutral protease is widely applied to industries such as food, leather, fur, cosmetics, medicines and the like. Wherein, the neutral protease is added in the formula of the livestock and poultry feed, so that the utilization rate of the protein can be effectively improved, and the feeding cost can be reduced; in addition, the neutral protease has stronger unhairing ability, and can avoid environmental pollution caused by the treatment of sodium sulfate in the traditional leather processing; the neutral protease is applied to the production of beer, and can eliminate the cold turbidity phenomenon generated by protein; the washing effect can be greatly improved by adding neutral protease into the detergent; in the pharmaceutical industry, the neutral protease can also be used as medicines for digestion, inflammation diminishing, phlegm reducing and cough relieving, and the like, and can be used for treating traumatic injury, edema and hematoma, eliminating necrotic tissues and the like.

The production of protease commodities can be traced back to the beginning of the 20 th century at the earliest, and a large number of excellent protease-producing strains, such as bacillus subtilis 1.398, actinomycetes 166 and the like, are successively bred in China since 1956. Currently, most of the neutral proteases on the market are derived from Bacillus. But the problems of strain degeneration, enzyme production capacity reduction, unstable fermentation and the like are faced after long-term passage. How to obtain a new neutral protease producing strain and improve the enzyme producing capability of the strain is still the focus of the current research.

Disclosure of Invention

The invention aims to provide a bacillus subtilis with high yield of neutral protease (bacillus subtilis)Bacillus subtilis) And applications thereof. The applicant firstly constructs and obtains the bacillus subtilis engineering bacteria for recombining and expressing the neutral protease gene, then further performs ultraviolet mutagenesis on the bacillus subtilis engineering bacteria, and screens the bacillus subtilis engineering bacteria to obtain a mutant strain with remarkably improved neutral protease yield, thereby being beneficial to reducing the production cost of the neutral protease and promoting the wide application of the neutral protease.

In one aspect, the invention provides a bacillus subtilis engineering strain, which carries a recombinant plasmid for expressing neutral protease.

The gene sequence of the neutral protease is SEQ ID NO. 3.

In one aspect, the invention provides a mutant strain bacillus subtilis QJNP (B)Bacillus subtilis QJNP), which has been preserved in China general microbiological culture Collection center (CGMCC) at 3 months and 20 days in 2020, with the preservation address of China academy of sciences microbial research institute No. 3 of Beijing province, north Chen Xilu No.1 of Chaoyang, and the preservation number of CGMCC No.19499。

The invention provides an application of the bacillus subtilis in producing neutral protease.

The invention also provides a method for producing neutral protease, which takes the bacillus subtilis as a fermentation strain.

Advantageous effects

The invention firstly expresses neutral protease gene in Bacillus subtilis host, and constructs engineering strain Bacillus subtilis NP for recombinant expression of the neutral protease. The enzyme activity of neutral protease in the supernatant obtained by shake flask fermentation and 20L tank fermentation of the strain respectively reaches 15490U/mL and 37280U/mL.

In order to improve the yield of neutral protease, the applicant uses bacillus subtilis NP as an original strain, and further obtains a mutant bacillus subtilis QJNP by screening through an ultraviolet mutagenesis method. The enzyme activity of neutral protease in the supernatant obtained by shake flask fermentation of the mutant strain is as high as 24410U/mL, which is 57.6% higher than that of the original strain; the enzyme activity of neutral protease in the crude enzyme liquid fermented in the 20L tank is as high as 52750U/mL, which is 41.5% higher than that of the original bacteria, and unexpected technical effects are achieved. The mutant strain can be widely applied to the production of neutral protease, is favorable for reducing the production cost of the neutral protease and promotes the application of the neutral protease.

Drawings

FIG. 1 is a plasmid map of pDG 1662;

FIG. 2 is a pUC19-cat plasmid map;

FIG. 3 is a map of the pPaprE-npr plasmid;

FIG. 4 is a pH-relative enzyme activity curve;

FIG. 5 is a temperature-relative enzyme activity curve.

Detailed Description

The process of the present invention is further illustrated by the following examples, in which experimental procedures not specifically indicated for the conditions may be carried out under conventional conditions, such as those described in molecular cloning, a laboratory Manual written by J. Sambrook et al, or as recommended by the manufacturer. The present invention may be better understood and appreciated by those skilled in the art with reference to the following examples. However, the method of carrying out the present invention should not be limited to the specific method steps described in the examples of the present invention.

The formulation of the culture medium involved in the examples of the present invention is as follows:

the preparation method of the GM I comprises the following steps: 96 mL of 1 × minimum salt solution, 2.5 mL of 20% glucose, 0.4 mL of 5% hydrolyzed casein, and 1 mL of 10% yeast powder juice; the preparation method of the 1 × lowest salt solution comprises the following steps: k ₂ HPO ₄ 14 g/L，KH ₂ PO ₄ 6 g/L，(NH ₄ ) ₂ SO ₄ 2 g/L, trisodium citrate 1 g/L, mgSO ₄ •7H ₂ 0.2 g/L of O, which is dissolved in distilled water in turn;

the preparation method of the GM II comprises the following steps: 97 mL of 1 Xminimum salt solution, 2.5 mL of 20% glucose, 0.08 mL of 5% hydrolyzed casein, 0.04 mL of 10% yeast powder juice, 1M MgCl ₂ 0.25 mL，1 M CaCl ₂ 0.05 mL；

LB plate: tryptone 1%, yeast powder 0.5%, naCl 1%, agar powder 1.5%;

and (3) degreasing milk powder flat plate: 1% of tryptone, 0.5% of yeast powder, 1% of NaCl, 1% of skimmed milk powder and 1.5% of agar powder. (ii) a

Seed culture medium: yeast extract powder 0.5%, tryptone 0.5%, glucose 1%, K ₂ HPO ₄ 1.8 percent and 5 mu g/mL of chloramphenicol;

fermentation medium: 1 to 2 percent of yeast powder, 2 to 5 percent of bean cake powder, 5 to 10 percent of maltodextrin, 0.1 to 0.5 percent of sodium citrate and CaCl ₂ 0.1~0.5%，MgSO ₄ 0.1~0.5%，K ₂ HPO ₄ 0.5~2%；

Example 1 Signal peptide screening for efficient secretory expression of neutral protease

Artificially synthesized gene containing neutral proteasenprThe DNA sequence of (1) contains P43 promoter SEQ ID NO 2 and neutral proteasenprOpen Reading Frame (ORF) of (1) SEQ ID NO 3. WhereinnprThe signal peptide SEQ ID NO. 4 of the gene is a wild-type signal peptide. The sequence SEQ ID NO 1 was cloned into the pUC57 plasmid and named pUC57-npr。

Artificially synthesized DNA sequence SEQ ID NO 5 containing P43 promoter and alkaline protease gene signal peptideThe sequence of the signal peptide of the alkaline protease gene is SEQ ID NO. 6. The sequence SEQ ID NO 5 was cloned into a pUC57 plasmid and named pUC57-P43-SPaprE。

Artificially synthesizing a DNA sequence SEQ ID NO. 7 containing a P43 promoter and a signal peptide of a levansucrase gene of bacillus subtilis, wherein the sequence of the signal peptide of the levansucrase gene is SEQ ID NO. 8. The sequence SEQ ID NO 7 was cloned into a pUC57 plasmid and named pUC57-P43-SPsacB。

1.1 By usingnprNeutral protease integration expression plasmid pP43-SP of signal peptidenpr-nprConstruction of

Designing a cloning primer:

npr-F：5'-CACACAAATTAAAAACTGGTCTGATCGGAATGGGTTTTGTTTTTAAGC-3'

npr-Re：5'-GATGATAAGCTGTCAAACATGAGGCTGTTTGCGTTTTTGCCGTG-3'

pUC57-nprPlasmid as template, usingnpr-F、npr-Re primer, PCR amplified with Phusion Fidelity enzyme from NEB under the following conditions: pre-denaturation at 98 ℃ for 2 min, denaturation at 98 ℃ for 10 s, annealing at 55 ℃ for 20 s, extension at 72 ℃ for 1 min,30 cycles, and extension at 72 ℃ for 5 min. Recovering PCR amplification product named as P43-SP by OMEGA gel recovery kitnpr-nprAnd about 2 kb in size.

Use of pDG1662 plasmidBamHI andEcoperforming double digestion by RI endonuclease, recovering digestion product pDG1662 by OMEGA gel recovery kitBamHI/EcoRI, size about 7 kb.

P43-SPnpr-nprFragment and pDG1662BamHI/EcoMixing RI fragments uniformly according to the molar ratio of 3E. coliDH 5. Alpha. Competent cells were plated with LB plates containing 100. Mu.g/mL ampicillin, transformants were tested by colony PCR, and insert P43-SP was verifiednpr-nprTransformant of fragment plasmid was extracted and sent to Suzhou Jinzhi Biotech limited for sequencing, and the correctly sequenced plasmid was designated as pP43-SPnpr-npr。

1.2 By usingaprENeutral protease integration expression plasmid pP43-SP of signal peptideaprE-nprConstruction of

Designing a cloning primer:

SPaprE-Re：5'-TCGCATCGGCTGCTGAGAATCCTCAGCTTAAAG-3'

SPaprE-F：5'-GGATTCTCAGCAGCCGATGCGATCGATGAACT-3'

pUC57-nprPlasmid as template, usingnpr-F、SPaprERe primer, PCR amplified with Phusion Fidelity enzyme from NEB. Recovering PCR amplification product from OMEGA gel recovery kit, and naming SPaprE-nprPCR1, about 1.67 kb in size.

In the form of pUC57-P43-SPaprEPlasmid as template, SPaprE-F、nprRe primer, PCR amplified with Phusion Fidelity enzyme from NEB. Recovering PCR amplification product named as P43-SP by OMEGA gel recovery kitaprEPCR2, about 0.33 kb in size.

For SPaprE-nprPCR1 and P43-SPaprEAnd performing fusion PCR on the PCR2 fragment, wherein the fusion PCR process is as follows: in the first round, 200 ng of SP were added to each PCR reaction systemaprE-npr PCR1、P43-SPaprEPCR2 fragment was amplified by PCR using Phusion Fidelity enzyme from NEB without primers. In the second round, 10. Mu.l of the PCR product from the first round was used as a template and addednpr-F、nprRe primer, PCR amplified with Phusion Fidelity enzyme from NEB. The OMEGA gel recovery kit recovers about 2 kb of PCR product, which is named as P43-SPaprE-npr。

P43-SPaprE-nprFragments and pDG1662BamHI/EcoMixing RI fragments uniformly according to the molar ratio of 3E. coliDH 5. Alpha. Competent cells, LB plates containing 100. Mu.g/mL ampicillin, transformants were tested by colony PCR and inserts of P43-SPaprE-nprTransformant of fragment plasmid was extracted and sent to Suzhou Jinzhi Biotech limited for sequencing, and the correctly sequenced plasmid was designated as pP43-SPaprE-npr。

1.3 By usingsacBNeutral protease integration expression plasmid pP43-SP of signal peptidesacB-nprConstruction of

Designing a cloning primer:

SPsacB-Re：5'-AAGCTTTTGCCGCTGAGAATCCTCAGCTTAAAG-3'

SPsacB-F：5'-GGATTCTCAGCGGCAAAAGCTTGAGTTGCGCC-3'

pUC57-nprPlasmid as template, usingnpr-F、SPsacBRe primer, PCR amplified with Phusion Fidelity enzyme from NEB. Recovering PCR amplification product from OMEGA gel recovery kit, and naming SPsacB-nprPCR1, size about 1.67 kb.

In the form of pUC57-P43-SPsacBPlasmid as template, SPsacB-F、nprRe primer, PCR amplified with Phusion Fidelity enzyme from NEB. Recovering PCR amplification product named as P43-SP by OMEGA gel recovery kitsacBPCR2, about 0.34 kb in size.

For SPsacB-nprPCR1 and P43-SPsacBAnd performing fusion PCR on the PCR2 fragment, wherein the fusion PCR process is as follows: in the first round, 200 ng of SP was added to each PCR reaction systemsacB-npr PCR1、P43-SPsacBPCR2 fragment was amplified by PCR using Phusion Fidelity enzyme from NEB without primers. In the second round, 10. Mu.l of the first round PCR product was taken as a template and addednpr-F、nprRe primer, PCR amplified with Phusion Fidelity enzyme from NEB. The OMEGA gel recovery kit recovers about 2 kb of PCR product, which is named as P43-SPsacB-npr。

P43-SPsacB-nprFragment and pDG1662BamHI/EcoMixing RI fragments uniformly according to the molar ratio of 3E. coliDH 5. Alpha. Competent cells were plated with LB plates containing 100. Mu.g/mL ampicillin, transformants were tested by colony PCR, and insert P43-SP was verifiedsacB-nprExtracting plasmid from transformant of the fragment, sending the plasmid to Suzhou Jinwei Zhi Biotechnology Limited for sequencing, and designating the correctly sequenced plasmid as pP43-SPsacB-npr。

1.4 Construction of a Single copy integration Strain of Bacillus subtilis Using different Signal peptides

Mixing pP43-SPnpr-nprFor plasmidsBglII andXhoperforming double enzyme digestion by using I endonuclease, recovering enzyme digestion product pP43-SP by using OMEGA gel recovery kitnpr-npr/BglII/XhoI, size about 7.7 kb.

Mixing pP43-SPnpr-npr/BglII/XhoI fragment Bacillus subtilis 1A751 (B.subtilis) transformed by a general competence methodBacillus subtilis 1A751 (apr ^- , his ^- , npr ^- , eglSΔ102, bglT/bglSΔEV)）（Wolf M, et alMicrobiology. 1995 141)nprThe signal peptide neutral protease bacillus subtilis single copy integrated strain is constructed by the following steps:

1) The host Bacillus subtilis 1A751 was streaked on LB plates and cultured at 37 ℃ overnight.

2) The following day, 1 single colony was inoculated into 5 mL of GM I solution and cultured overnight at 30 ℃ with shaking at 125 rpm.

3) The following day, 1 mL of overnight culture was transferred to 9 mL of GM I and cultured at 37 ℃ and 250 rpm for 3.5 h.

4) Then 5 mL of the culture medium from the previous step was transferred to 45 mL of GM II, cultured at 37 ℃ for 90 min at 125 rpm, and centrifuged at 5000 g for 10 min to collect the cells. And (3) lightly suspending the thalli by using 5 mL of original culture solution supernatant, wherein the suspended thalli are competent cells.

5) About 1. Mu.g of pP43-SP was added to 0.2 mL of Bacillus subtilis host competent cellsnpr-npr/BglII/XhoFragment I, 37 ℃, 200 rpm vibration culture 1 h after spreading containing 5 u g/mL chloramphenicol skimmed milk powder plate, 37 ℃ culture overnight.

6) Selecting transformants with remarkable transparent circles on the next day, streaking and purifying the transformants on a skim milk powder plate containing 5 mu g/mL chloramphenicol once, selecting single colonies with remarkable transparent circles, preserving the seeds with 15% -20% glycerol, and naming the strains as BS-SPnpr-npr。

The same method is adopted to mix pP43-SPaprE-npr/BglII/XhoThe I fragment is transformed into a bacillus subtilis host by a common competence method, and the construction adoptsaprESignal peptide-containing single-copy integrated strain of neutral protease bacillus subtilis and named as BS-SPaprE-npr。

The same method is adopted to mix pP43-SPsacB-npr/BglII/XhoThe I fragment is transformed into a bacillus subtilis host by a common competence method, and the construction adoptssacBSignal peptide-containing single-copy integrated strain of neutral protease bacillus subtilis and named as BS-SPsacB-npr。

1.5 Shaking flask fermentation of single copy integrated strain of bacillus subtilis with different signal peptides

BS-SPnpr-npr、BS-SPaprE-npr、BS-SPsacB-nprThe strain is streaked on skim milk powder plates containing 5 mu g/mL chloramphenicol, single colonies with obvious transparent circles are respectively selected from each plate on the next day and inoculated in 20 mL seed culture medium, and the single colonies are subjected to shake culture at 37 ℃ and 220rpm for about 6 h. Then respectively inoculating 2.5 mL of seed culture into 50 mL of fermentation medium, and carrying out shaking culture at 34 ℃ and 220rpm for 60 h;4000 Centrifuging at rpm for 10 min to obtain supernatant; respectively measuring the neutral protease enzyme activity of the fermentation supernatant of the strain by using a method (GB/T23527-2009) for measuring the protease activity of national standard of the people's republic of China (Folin method), wherein BS-SPsacB-nprThe enzyme activity of the recombinant neutral protease is 5828U/mL, which is obviously higher than that of BS-SPnpr-npr(3164U/mL) and BS-SPaprE-npr(4027U/mL) recombinant bacterium, explanationsacBThe signal peptide is more beneficial to the efficient secretory expression of neutral protease.

EXAMPLE 2 expression of plasmid pP for integration of neutral proteaseaprE-nprConstruction of

Selecting newly activated Bacillus subtilis 168: (Bacillus subtilis168 A single colony was inoculated in 5 mL of LB liquid medium, cultured overnight with shaking at 37 ℃ with shaking at 200 rpm in a shaker, and the genome of Bacillus subtilis 168 was extracted according to the instructions of the TIANGEN bacterial genomic DNA extraction kit. The genome containsaprEPromoter (P)aprE) The DNA sequence of (1) is SEQ ID NO 9.

Designing a cloning primer:

cat-F：5'-ACATGCATGCCTGTAATATAAAAACCTTCTTC-3'；

cat-Re：5'-ACGCGTCGACTTTATTCTTCAACTAAAGCAC-3'；

PaprE-F：5'-ACGCGTCGACTGACACAGAAGAAAACGTTGG-3'；

PaprE-Re：5'-TTGATGTTCATTCTTTACCCTCTCCTTTTAAA-3'；

SPsacB-npr-F：5'-GAGGGTAAAGAATGAACATCAAAAAGTTTGCA-3'；

SPsacB-npr-Re：5'-CGCGGATCCGAATGGGTTTTGTTTTTAAGC-3'。

using pDG1662 as templatecat-F、cat-Re primer, PCR amplified with Phusion Fidelity enzyme from NEB under the following conditions: pre-denaturation at 98 ℃ for 2 min, denaturation at 98 ℃ for 10 s, annealing at 55 ℃ for 20 s, extension at 72 ℃ for 30 s, and extension at 72 ℃ for 5 min after 30 cycles. Recovering PCR amplification product by OMEGA gel recovery kit, and namingcatAnd about 0.93 kb in size.

Using pUC19 plasmidSphI andSalperforming double enzyme digestion by using the I endonuclease, and recovering a digestion product pUC19SphI/SalI, size about 2.7 kb. Will be provided withcatFor fragmentsSphI andSalperforming double enzyme digestion by using I endonuclease, and recovering enzyme digestion product by using OMEGA gel recovery kitcat/SphI/SalI, size about 0.93 kb. pUC 19. Dbd.by T4 DNA LigaseSphI/SalI fragment andcat/SphI/Salthe I fragment is subjected to ligation reaction, and the ligation product is transformedE. coliDH 5. Alpha. Competent cells, LB plates containing 100. Mu.g/mL ampicillin, transformants were subjected to colony PCR validation, and inserts were madecatExtracting plasmid from transformant of gene, sending the plasmid to Suzhou Jinzhi Biotechnology limited for sequencing, and naming the plasmid with correct sequencing as pUC19-cat。

Taking Bacillus subtilis 168 genome as template and adopting PaprE-F、PaprERe primer, PCR amplified with Phusion Fidelity enzyme from NEB. Recovering PCR amplification product from OMEGA gel recovery kit, and naming as PaprEAnd the size is about 0.65 kb.

With pP43-SPsacB-nprAs template, use SPsacB-npr-F、SPsacB-nprRe primer, PCR amplified with Phusion Fidelity enzyme from NEB. Recovering PCR amplification product by OMEGA gel recovery kit, and naming asnprAnd about 1.67 kb in size.

To PaprEAndnprthe fragment was subjected to fusion PCR as follows: in the first round, 200 ng of P was added to each PCR reaction systemaprE、nprThe fragments were amplified by PCR using Phusion Fidelity enzyme from NEB without primers. In the second round, 10. Mu.l of the first round PCR product was used as a template, and P was addedaprE-F、SPsacB-nprRe primer, PCR amplified with Phusion Fidelity enzyme from NEB. The OMEGA gel recovery kit recovers about 2.3 kb of PCR product, which is named as PaprE-npr。

pUC19-catFor plasmidsSalI andBamHI endonuclease is subjected to double digestion, and an OMEGA gel recovery kit is used for recovering a digestion product pUC19-cat/SalI/BamHI, size about 3.6 kb. Will PaprE-nprFor fragmentsSalI andBamperforming double enzyme digestion by using HI endonuclease, and recovering enzyme digestion product P by using OMEGA gel recovery kitaprE-npr/SalI/BamHI, size about 2.3 kb. Application of T4 DNA Ligase to pUC19-cat/SalI/BamHI fragment and PaprE-npr/SalI/BamThe HI fragment is subjected to ligation reaction, and the ligation product is transformedE. coliDH 5. Alpha. Competent cells, plated with LB plates containing 100. Mu.g/mL ampicillin, transformants were tested by colony PCR for insert PaprE-nprTransformant of fragment extracting plasmid, sending the plasmid to Suzhou Jinzhi Biotechnology limited for sequencing, and the plasmid with correct sequencing is named as pPaprE-npr. And (5) completing construction of the neutral protease integration expression plasmid.

Example 3 construction and fermentation validation of engineered Bacillus subtilis strains for recombinant expression of neutral protease

3.1 Construction of integrated bacillus subtilis strain and shake flask fermentation

The general competence preparation method of example 1.4 was usedMethod for preparing Bacillus subtilis 1A751 host competent cell, adding about 1 μ g of pP to 0.2 mL of competent cellaprE-nprThe recombinant plasmid was cultured at 37 ℃ for 1 hour with shaking at 200 rpm, then plated with a skim milk powder plate containing 5. Mu.g/mL of chloramphenicol, and cultured overnight at 37 ℃. Selecting 10 single colonies with obvious transparent circles on the next day, streaking and purifying the single colonies on a skim milk powder plate containing 5 mu g/mL chloramphenicol, selecting single colonies with obvious transparent circles on each plate, inoculating the single colonies into 20 mL seed culture medium, and carrying out shake culture at 37 ℃ and 220rpm for 8-9 h. Then respectively inoculating 2.5 mL of seed culture into 50 mL of fermentation medium, and carrying out shaking culture at 34 ℃ and 220rpm for 72 h;4000 Centrifuging at rpm for 10 min, and collecting supernatant; the enzyme activity of neutral protease of the fermentation supernatant of the strain is respectively determined by adopting a method (GB/T23527-2009) (Folin method) for determining the enzyme activity of the national standard protease of the people's republic of China, wherein the enzyme activity of the neutral protease of one recombinant strain is 15490U/mL, which is obviously higher than that of the other 9 recombinant strains. The applicant named this strain Bacillus subtilis NP: (B)Bacillus subtilisNP）。

3.2 Neutral protease strain 20L tank fermentation verification

Bacillus subtilis NP was inoculated into 500 mL of seed medium and cultured with shaking at 220rpm at 37 ℃ for about 12 hours.

Transferring all the seed liquid into 20L fermentation tank (fermentation tank culture medium comprising maltodextrin 3%, glucose 1%, bean cake powder 3%, testa Tritici 3%, and Na) ₂ HPO ₄ 0.78%，KH ₂ PO ₄ 0.05 percent and the volume of the fermentation tank after digestion is 10L). Controlling the temperature at 37 ℃, controlling the pH at the initial fermentation to be 7.2, and controlling the pH to be not lower than 7.0 by ammonia water in the fermentation process. The air volume is 1 to 1.5 vvm, the rotating speed is 300 to 700 rpm, and the DO is not lower than 10% in the fermentation process. And after 3 to 4 hours, beginning to feed 50 percent of glucose at the feeding speed of 3 g/L.h. Fermenting for 30 to 35 hours, and stopping culture after DO and pH rise.

The enzyme activity of neutral protease of the fermentation supernatant is determined, and the result shows that the enzyme activity of the fermentation supernatant of the bacillus subtilis NP strain reaches 37280U/mL. Thus, the recombinant engineering strain of the bacillus subtilis NP constructed by the invention can efficiently over-express the exogenous neutral protease genenpr。

Example 4 analysis of enzymatic Properties

4.1 pH optimum assay

The method comprises the steps of diluting a supernatant obtained by fermenting the recombinant strain bacillus subtilis NP by using disodium hydrogen phosphate-citric acid buffer solutions with pH values of 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0 and borax-boric acid buffer solutions with pH values of 8.5 and 9.0 respectively, preparing substrates by using buffer solutions with corresponding pH values respectively, measuring the activity of protease at 40 ℃, calculating the relative enzyme activity by taking the highest enzyme activity as 100%, and making a pH-relative enzyme activity curve.

As shown in FIG. 4, the protease produced by the recombinant strain NP constructed by the invention has the optimum action pH value of 7.5, is neutral protease, and keeps more than 70% of enzyme activity within the pH value range of 6.5-8.0.

4.2 Optimum action temperature analysis

Respectively carrying out protease enzyme activity determination on the recombinant strain bacillus subtilis NP fermentation supernatant under the conditions of 30 ℃, 35 ℃, 40 ℃, 45 ℃,50 ℃,55 ℃,60 ℃, 65 ℃ and pH 7.5, calculating relative enzyme activity by taking the highest enzyme activity as 100%, and making a temperature-relative enzyme activity curve.

As shown in figure 5, the protease produced by the recombinant strain of the bacillus subtilis NP constructed by the invention has the optimal action temperature of 50 ℃ and can keep more than 90 percent of relative enzyme activity within the range of 45-55 ℃.

EXAMPLE 5 mutagenic screening of strains producing high amounts of neutral protease

The mutation caused by ultraviolet mutagenesis has strong randomness, and the effect generated by mutation is random and difficult to predict. Therefore, in order to obtain effective positive mutations, technicians usually need to perform multiple rounds of ultraviolet mutagenesis, the screening workload is large, and there is a possibility that effective positive mutations cannot be obtained. However, ultraviolet mutagenesis requires simple equipment and low cost, and can obtain a large number of mutants in a short time, so that it is still a common mutagenesis breeding method.

The applicant takes the recombinant strain bacillus subtilis NP obtained in the construction of the embodiment 3 as an original strain, and carries out genetic modification on the original strain by an ultraviolet mutagenesis method, so that the yield of the neutral protease is further improved.

5.1 preparation of bacterial suspension

Streaking and inoculating starting bacillus subtilis NP on an LB inclined plane, and culturing for 24 h at 37 ℃; adding 5 mL of 0.85% sterile physiological saline, washing off all thalli on the inclined plane, transferring into a sterile test tube containing glass beads, and performing vortex oscillation for 10 min to obtain unicellular thalli completely; transferring all the bacterial suspension into a 15 mL centrifuge tube, centrifuging at 6000 rpm for 3 min, collecting thalli, taking supernatant, and suspending the thalli by using 10 mL physiological saline; the cells were washed twice and finally the cell concentration was adjusted to 10 ⁸ one/mL.

5.2 Ultraviolet mutagenesis treatment and mutagenesis dose determination

Opening a 9W ultraviolet lamp switch, and preheating for about 30 min; taking a sterile plate with the diameter of 9 cm, adding the above cell with the concentration of 10 ⁸ Adding 10 mL of bacteria suspension per mL, adding a sterile magnetic stirring rotor, opening a magnetic stirrer, opening a dish cover, and respectively stirring and irradiating for 0.5 min, 1 min, 1.5 min, 2 min, 2.5 min and 3 min at a vertical distance of 15 cm; and (4) covering the dish cover, closing the ultraviolet lamp, and incubating for 30 min in the dark.

Diluting the irradiated bacterial suspension to 10% by gradient with 0.85% normal saline ^-1 ~10 ^-6 (ii) a Get 10 ^-4 、10 ^-5 、10 ^-6 Coating defatted milk powder plate with 100 μ L of each of the three dilutions of bacterial suspension, and coating three plates with each dilution; in the same manner, the bacterial solution which was not subjected to the ultraviolet irradiation treatment was diluted and plated as a control. The plates were spread evenly, wrapped in black cloth or newspaper, and incubated overnight at 37 ℃.

Counting the number of single colonies growing on the plate under each dilution under different irradiation times, and if the number of single colonies growing under a certain dilution is between 30 and 300, determining that the dilution is proper. The number of single colonies growing on the three plates under the dilution was averaged, and the bacterial suspension concentration was calculated according to the following formula:

bacterial suspension concentration (CFU/mL) = average number of colonies at a certain dilution × dilution multiple × 10

The lethality at a certain uv treatment dose was calculated according to the following formula:

lethality (%) = (1-concentration of bacterial suspension after certain dose treatment/concentration of bacterial suspension before treatment) × 100%

The calculated lethality of bacillus subtilis NP at different uv mutagen doses is shown in table 1.

TABLE 1 ultraviolet mutagenesis lethality of Bacillus subtilis NP

Time/min	0.5	1	1.5	2	2.5	3
							Mortality rate/%)	89.0	96.9	99.5	99.9	99.9	99.9

As can be seen from Table 1, the lethality of the bacterial suspension after 1 min of ultraviolet irradiation reaches more than 95%, so that the final mutagenesis time is determined to be 1 min.

5.3 Transparent ring size primary screen for defatted milk powder flat plate

Colonies with obvious transparent circles are picked from a skimmed milk powder flat plate subjected to ultraviolet mutagenesis for 1 min, the colonies are spotted on the skimmed milk powder flat plate, 3 colonies are arranged in parallel in each group, and meanwhile, starting strains are spotted to serve as controls. After culturing for 10 to 12 hours at 37 ℃, selecting single colonies of which the ratio of the diameter of the transparent ring to the diameter of the colonies is more than that of the grown-out bacteria for secondary purification, and selecting 50 single colonies of which the ratio of the diameter of the transparent ring to the diameter of the colonies is more than 30 percent of that of the grown-out bacteria after purification for shake flask secondary screening.

5.4 Shake flask rescreen

Respectively inoculating the screened 50 mutant strains into 50 mL shake flask fermentation culture media, carrying out fermentation culture at 34 ℃ and 220rpm for 60 hours, centrifuging to obtain supernate, respectively measuring the enzyme activity of neutral protease in the fermented supernate, and simultaneously taking the starting strain as a control, selecting the mutant strain with the shake flask fermentation enzyme activity improved by more than 15% compared with the starting strain, and carrying out second round ultraviolet mutagenesis screening.

The applicant continues to carry out 9 rounds of ultraviolet mutagenesis screening according to the method, and finally obtains 1 mutant strain with the yield of neutral protease obviously higher than that of the original strain, namely bacillus subtilis QJNP (B: (B) (B))Bacillus subtilisQJNP). After the strain is fermented and cultured for 60 hours at 34 ℃ and 220rpm in 50 mL of shake flask fermentation medium, the enzyme activity of neutral protease in fermentation supernatant reaches 24410U/mL, which is 57.6 percent higher than that of original strain; the enzyme activity of neutral protease in the crude enzyme liquid fermented in the 20L tank is as high as 52750U/mL, which is improved by 41.5 percent compared with the original bacteria, and unexpected technical effects are obtained.

The applicant has already transformed the above mutant strain Bacillus subtilis QJNP at 20/3/2020: (Bacillus subtilisQJNP) is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is the microorganism research institute of China academy of sciences No. 3 of the Xilu No.1 of Beijing facing the Yangtze district, and the preservation number is CGMCC No.19499.

Sequence listing

<110> Islands Ulva Biometrics Ltd

Weifang kang Di En Biotech Co Ltd

Qingdao blue biological group Co Ltd

<120> bacillus subtilis for high yield of neutral protease and application thereof

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1931

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gctgtttgcg tttttgccgt gatttcgtgt atcattggtt tacttatttt tttgccaaag 60

ctgtaatggc tgaaaattct tacatttatt ttacattttt agaaatgggc gtgaaaaaaa 120

gcgcgcgatt atgtaaaata taaagtgata gcggtaccag gagggctgga agaagcagac 180

cgctaacaca gtacataaaa aaggagacat gaacgatggg tttaggtaag aaattgtctg 240

ttgctgtcgc cgcttccttt atgagtttaa ccatcagtct tccgggtgtt caggccgctg 300

agaatcctca gcttaaagaa aacctgacga actttgtgcc gaagcattct ttggtgcaat 360

ctgaattgcc ttcagtcagt gacaaagcaa tcaagcaata cttgaaacaa aacggcaaag 420

tcttcaaagg caacccttct gagagactga agctaattga ccacacgacc gatgatctcg 480

gctacaagca cttccgttat gtgcctgtcg ttaacggtgt gcctgtgaaa gactcgcaag 540

tcattattca cgtcgataaa tccaacaatg tctatgcgat taacggagaa ttaaacaacg 600

atgcttctgc caaaacggca aacagcaaaa aattatctgc aaatcaagcg ctggatcatg 660

cttttaaagc aatcggcaaa tcacctgaag ccgtctctaa cggcaacgtt gcaaacaaaa 720

acaaagccga gctgaaagca gcggccacaa aagacggtaa ataccgactc gcctatgatg 780

taaccatccg ctacatcgaa ccggaaccag ctaactggga agtaaccgtt gatgcggaaa 840

cagggaaagt cctgaaaaag caaaacaaag tggagcatgc cgctgcaacc ggaacaggta 900

cgactcttaa aggaaaaacg gtctcattaa atatttcttc tgaaagcggc aaatatgtaa 960

tgcgtgatct ttctaaacct accggaacgc aaattattac gtacgatctg caaaaccgac 1020

aatataacct gccgggcacg ctcgtatcaa gcactacaaa ccagttcaca acttcttctc 1080

agcgcgctgc cgttgatgcg cattacaatc tcggcaaagt gtacgattat ttctatcaga 1140

cgtttaaacg caacagctac gacaataaag gcggcaaaat cgtatcttcc gttcattacg 1200

gcagcaaata caacaacgcg gcctggatcg gcgaccaaat gatttacggt gacggtgacg 1260

gctcattctt ctcgcctctt tccggttcaa tggacgtaac ggcccatgaa atgacacacg 1320

gtgttacaca ggaaacagcc aacctgaact atgaaaatca accgggtgct ttaaacgaat 1380

ccttctctga tgtattcgga tacttcaatg atactgagga ctgggatatc ggtgaagata 1440

ttacggtcag ccagccggct ctccgcagtt tatccaatcc gacaaaatac ggacagcccg 1500

accattacaa aaattatcga aaccttccga atactgatgc cggcgactac ggcggcgtgc 1560

atacaaacag cggaattccg aacaaagccg cttacaacac gattacaaaa atcggcgtga 1620

aaaaagcgga gcagatttac taccgtgcac tgacggtata tctcactccg tcatcaagct 1680

ttaaagatgc aaaagcagct ttgattcaat cagcgcggga cctttacggc tctcaagacg 1740

ctgcaagcgt agaagcggcc tggaatgcgg tcggcttgta aacaagaaaa gagaccggag 1800

aaatccggtc tcttttttat atctgaaaca tttcacaatg gcttcaccat gatcatatat 1860

gttttttccc gatcgtcttt ttcaagctta agctgttcaa agccgcattg gcttaaaaac 1920

aaaacccatt c 1931

<210> 2

<211> 215

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gctgtttgcg tttttgccgt gatttcgtgt atcattggtt tacttatttt tttgccaaag 60

ctgtaatggc tgaaaattct tacatttatt ttacattttt agaaatgggc gtgaaaaaaa 120

gcgcgcgatt atgtaaaata taaagtgata gcggtaccag gagggctgga agaagcagac 180

cgctaacaca gtacataaaa aaggagacat gaacg 215

<210> 3

<211> 1566

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgggtttag gtaagaaatt gtctgttgct gtcgccgctt cctttatgag tttaaccatc 60

agtcttccgg gtgttcaggc cgctgagaat cctcagctta aagaaaacct gacgaacttt 120

gtgccgaagc attctttggt gcaatctgaa ttgccttcag tcagtgacaa agcaatcaag 180

caatacttga aacaaaacgg caaagtcttc aaaggcaacc cttctgagag actgaagcta 240

attgaccaca cgaccgatga tctcggctac aagcacttcc gttatgtgcc tgtcgttaac 300

ggtgtgcctg tgaaagactc gcaagtcatt attcacgtcg ataaatccaa caatgtctat 360

gcgattaacg gagaattaaa caacgatgct tctgccaaaa cggcaaacag caaaaaatta 420

tctgcaaatc aagcgctgga tcatgctttt aaagcaatcg gcaaatcacc tgaagccgtc 480

tctaacggca acgttgcaaa caaaaacaaa gccgagctga aagcagcggc cacaaaagac 540

ggtaaatacc gactcgccta tgatgtaacc atccgctaca tcgaaccgga accagctaac 600

tgggaagtaa ccgttgatgc ggaaacaggg aaagtcctga aaaagcaaaa caaagtggag 660

catgccgctg caaccggaac aggtacgact cttaaaggaa aaacggtctc attaaatatt 720

tcttctgaaa gcggcaaata tgtaatgcgt gatctttcta aacctaccgg aacgcaaatt 780

attacgtacg atctgcaaaa ccgacaatat aacctgccgg gcacgctcgt atcaagcact 840

acaaaccagt tcacaacttc ttctcagcgc gctgccgttg atgcgcatta caatctcggc 900

aaagtgtacg attatttcta tcagacgttt aaacgcaaca gctacgacaa taaaggcggc 960

aaaatcgtat cttccgttca ttacggcagc aaatacaaca acgcggcctg gatcggcgac 1020

caaatgattt acggtgacgg tgacggctca ttcttctcgc ctctttccgg ttcaatggac 1080

gtaacggccc atgaaatgac acacggtgtt acacaggaaa cagccaacct gaactatgaa 1140

aatcaaccgg gtgctttaaa cgaatccttc tctgatgtat tcggatactt caatgatact 1200

gaggactggg atatcggtga agatattacg gtcagccagc cggctctccg cagtttatcc 1260

aatccgacaa aatacggaca gcccgaccat tacaaaaatt atcgaaacct tccgaatact 1320

gatgccggcg actacggcgg cgtgcataca aacagcggaa ttccgaacaa agccgcttac 1380

aacacgatta caaaaatcgg cgtgaaaaaa gcggagcaga tttactaccg tgcactgacg 1440

gtatatctca ctccgtcatc aagctttaaa gatgcaaaag cagctttgat tcaatcagcg 1500

cgggaccttt acggctctca agacgctgca agcgtagaag cggcctggaa tgcggtcggc 1560

ttgtaa 1566

<210> 4

<211> 27

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Gly Leu Gly Lys Lys Leu Ser Val Ala Val Ala Ala Ser Phe Met

1 5 10 15

Ser Leu Thr Ile Ser Leu Pro Gly Val Gln Ala

20 25

<210> 5

<211> 296

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gctgtttgcg tttttgccgt gatttcgtgt atcattggtt tacttatttt tttgccaaag 60

ctgtaatggc tgaaaattct tacatttatt ttacattttt agaaatgggc gtgaaaaaaa 120

gcgcgcgatt atgtaaaata taaagtgata gcggtaccag gagggctgga agaagcagac 180

cgctaacaca gtacataaaa aaggagacat gaacgatgaa gaaaccgttg gggaaaattg 240

tcgcaagcac cgcactactc atttctgttg cttttagttc atcgatcgca tcggct 296

<210> 6

<211> 27

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Met Lys Lys Pro Leu Gly Lys Ile Val Ala Ser Thr Ala Leu Leu Ile

1 5 10 15

Ser Val Ala Phe Ser Ser Ser Ile Ala Ser Ala

20 25

<210> 7

<211> 302

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gctgtttgcg tttttgccgt gatttcgtgt atcattggtt tacttatttt tttgccaaag 60

ctgtaatggc tgaaaattct tacatttatt ttacattttt agaaatgggc gtgaaaaaaa 120

gcgcgcgatt atgtaaaata taaagtgata gcggtaccag gagggctgga agaagcagac 180

cgctaacaca gtacataaaa aaggagacat gaacgatgaa catcaaaaag tttgcaaaac 240

aagcaacagt attaaccttt actaccgcac tgctggcagg aggcgcaact caagcttttg 300

cc 302

<210> 8

<211> 29

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Met Asn Ile Lys Lys Phe Ala Lys Gln Ala Thr Val Leu Thr Phe Thr

1 5 10 15

Thr Ala Leu Leu Ala Gly Gly Ala Thr Gln Ala Phe Ala

20 25

<210> 9

<211> 650

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tgacacagaa gaaaacgttg gatagagctg ggtaaagcct atgaattctc cattttcttc 60

tgctatcaaa ataacagact cgtgattttc caaacgagct ttcaaaaaag cctctgcccc 120

ttgcaaatcg gatgcctgtc tataaaattc ccgatattgg ttaaacagcg gcgcaatggc 180

ggccgcatct gatgtctttg cttggcgaat gttcatctta tttcttcctc cctctcaata 240

attttttcat tctatccctt ttctgtaaag tttatttttc agaatacttt tatcatcatg 300

ctttgaaaaa atatcacgat aatatccatt gttctcacgg aagcacacgc aggtcatttg 360

aacgaatttt ttcgacagga atttgccggg actcaggagc atttaaccta aaaaagcatg 420

acatttcagc ataatgaaca tttactcatg tctattttcg ttcttttctg tatgaaaata 480

gttatttcga gtctctacgg aaatagcgag agatgatata cctaaataga gataaaatca 540

tctcaaaaaa atgggtctac taaaatatta ttccatctat tacaataaat tcacagaata 600

gtcttttaag taagtctact ctgaattttt ttaaaaggag agggtaaaga 650

Claims (3)

1. A bacillus subtilis mutant strain is characterized in that the preservation number of the mutant strain is CGMCC No.19499.

2. Use of the bacillus subtilis mutant strain of claim 1 for the production of a neutral protease.

3. A method for producing a neutral protease, comprising using the Bacillus subtilis mutant strain of claim 1 as a fermentation strain.

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