WO2022134236A1 - 一种碱性蛋白酶突变体及其基因、工程菌、制备方法和应用 - Google Patents

一种碱性蛋白酶突变体及其基因、工程菌、制备方法和应用 Download PDF

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WO2022134236A1
WO2022134236A1 PCT/CN2021/071281 CN2021071281W WO2022134236A1 WO 2022134236 A1 WO2022134236 A1 WO 2022134236A1 CN 2021071281 W CN2021071281 W CN 2021071281W WO 2022134236 A1 WO2022134236 A1 WO 2022134236A1
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alkaline protease
gene
mutant
bacillus
wild
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PCT/CN2021/071281
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French (fr)
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路福平
刘逸寒
李玉
王兴吉
王克芬
刘文龙
刘夫锋
张会图
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天津科技大学
山东隆科特酶制剂有限公司
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Priority to US17/878,040 priority Critical patent/US11655464B2/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C12N15/1031—Mutagenizing nucleic acids mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus

Definitions

  • the invention belongs to the technical field of bioengineering, in particular to an alkaline protease mutant and its gene, engineering bacteria, preparation method and application.
  • Protease is a type of hydrolase that catalyzes the cleavage of protein peptide bonds and can degrade protein molecules and polypeptides into small peptide chains and amino acids.
  • Protease wherein the optimal pH value of alkaline protease is generally greater than 9.
  • alkaline protease has stronger enzymatic activity, heat resistance and alkali resistance, and has esterase properties.
  • Adding alkaline protease to the detergent can maintain the original color of the laundry, improve the decontamination effect of the product, effectively reduce the amount of surfactants and certain auxiliaries in the detergent, and increase water saving, energy saving and environmental protection. protection benefits. It is mainly used to hydrolyze vegetable protein in food. After hydrolysis, vegetable protein is converted into peptides and amino acids with smaller molecular weight, which is more conducive to digestion and absorption. The nutritional value of the product is higher, and the product quality and safety are relatively better. In the tanning process, the skin and hair mainly composed of protein and protein analogs are usually difficult to deal with. The traditional method is to use toxic chemical substances for treatment, which not only endangers people's own safety, but also harms the environment. There is also a lot of pollution, and protease can replace these chemicals to degrade non-colloidal components and non-fibrin in the tanning process, and can also reduce environmental pollution.
  • Microorganisms are an important source of proteases. Compared with plant proteases and animal proteases, microorganisms, because of their rapid growth and ease of artificial genetic modification, produce protease-rich resources that can be cultured in large quantities in a relatively short period of time. Enzymes to meet production needs.
  • Alkaline protease-producing microorganisms are mainly isolated from alkaline environments such as saline-alkali lakes, deep seas, and sandy land. At present, Bacillus, Actinomycetes and fungi have been reported to produce alkaline proteases, but in industrial production, they are mainly Bacillus sp.
  • Protein engineering is a new technology based on genetic engineering. It mainly relies on computer software and other assisted design and basic knowledge of protein chemistry and other disciplines. Technology for novel proteins that meet human needs.
  • the directed evolution of enzymes also known as in vitro molecular directed evolution of enzymes, belongs to the irrational design of proteins and is a new development direction of protein engineering.
  • the evolutionary process artificially creating special evolutionary conditions, starting from one or more existing parent enzymes (natural or artificially obtained enzyme precursors), randomly mutating genes or in vitro gene recombination in vitro or in vivo, constructing artificial The mutant enzyme library, further through certain screening or selection methods, finally obtains the evolutionary enzymes with certain characteristics expected in advance.
  • Error-prone PCR is to reduce the fidelity of DNA replication by using low-fidelity TaqDNA polymerase and changing PCR reaction conditions, such as adding Mn, changing the number of cycles and dNTP concentration, etc., and increasing base errors during the synthesis of new DNA strands.
  • a method for inducing DNA sequence variation in vitro which is a method for inducing DNA sequence variation in the amplified product by matching with more point mutations.
  • DNA shuffling is to cut one or a group of closely related gene sequences into a series of small DNA fragments of random size under the action of DNaseI. Due to the homology of genes, some of the base sequences overlap between these small fragments. Self-guided, random recombination, and finally through PCR with specific primers, the full-length gene is generated. In this process, the exchange of related sequences occurs due to the transformation of the template, thus generating a variety of gene recombination libraries. The products of gene expression are screened to achieve the directed evolution of the target gene.
  • the invention provides an alkaline protease mutant and its gene, engineering bacteria, preparation method and application, and specifically provides a highly active alkaline protease mutant and its gene, engineering bacteria and the like.
  • the Bacillus expression system has the following advantages: 1. Bacillus has a set of efficient secretion signal peptide and molecular chaperone systems, which is conducive to the efficient expression of the target protein; 2. Most Bacillus are non-pathogenic This is in line with the general safety requirements in industrial production; 3. The composition of the cell wall of Bacillus should be relatively simple, which is conducive to the extracellular secretion of expressed proteins, will not lead to the accumulation of secreted proteins in cells, and is conducive to the downstream recovery of proteins and Purification; 4. As a single-celled organism, Bacillus can achieve a high cell density in a short time, and the required medium composition is relatively simple, and the cost is low, which meets the requirements of industrial production.
  • the alkaline protease gene derived from Bacillus clausii is molecularly modified to obtain a highly active alkaline protease gene , and successfully expressed in Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus clausii systems.
  • One of the technical solutions provided by the present invention is: using the genome of Bacillus clausii CGMCC NO.12953 as a template, after cloning the sequence of the wild-type alkaline protease zymogen region gene apr (shown in SEQ ID NO.3) (amino acid sequence As shown in SEQ ID NO.4), random mutation was performed on the wild-type alkaline protease gene by continuous error-prone PCR, and then several high-activity mutant genes of alkaline protease were obtained by high-throughput screening using the Bacillus subtilis expression system. DNA shuffling was performed on these high-activity alkaline protease mutant genes, and high-activity alkaline protease mutant genes were obtained after screening.
  • the second technical solution provided by the present invention is: constructing the above mutant gene into a recombinant vector and successfully expressing it in Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus clausii to obtain a recombinant strain with improved enzyme production activity, and further fermenting Process optimization to obtain a new type of alkaline protease, which can be used in detergents, food, leather, medicine and other fields.
  • amino acid replaced at the original amino acid position is used to refer to the mutated amino acid in the alkaline protease high activity mutant.
  • the position of the mutation point of alkaline protease is numbered according to the amino acid sequence of its mature peptide, and the numbering of the position corresponds to the amino acid sequence number of the mature peptide of wild-type alkaline protease in SEQ ID NO.
  • the 212th amino acid in the amino acid sequence of the mature peptide of alkaline protease is Asn, and Asn212Ser means that the amino acid at position 212 is replaced by Asn of wild-type alkaline protease to Ser, and it can also be represented by amino acid single-letter abbreviations, such as N212S, and multiple sites occur at the same time.
  • Mutations are indicated by "/" connecting each mutation site, such as V11I/G95V/V145I/N212S, which means that the amino acids at positions 11, 95, 145 and 212 are sequentially replaced by V of wild-type alkaline protease with I, and by G is replaced by V, V is replaced by I, and N is replaced by S;
  • the nucleotide representation method is similar to the amino acid representation method, and the numbering of the positions corresponds to the nucleotide sequence numbering of the wild-type alkaline protease in SEQ ID NO.5 , such as C425, the 425th base of the alkaline protease nucleotide sequence is C.
  • APR represents wild-type alkaline protease, that is, the original sequence is shown in SEQ ID NO.4, and its encoding gene is shown as apr (shown in SEQ ID NO.3).
  • Each alkaline protease mutant is represented by APRM plus a number X, and the coding gene of each mutant is represented in lowercase italics in its amino acid representation.
  • the alkaline protease mutant has proteolytic activity, and its mature peptide is:
  • the invention uses error-prone PCR technology and DNA shuffling technology to mutate the wild-type alkaline protease gene, and obtains eight alkaline protease high activity mutants by screening.
  • the present invention also provides genes encoding the above mutants.
  • the gene encoding the mutant is shown in any of SEQ ID NO. 7-14.
  • the present invention also provides recombinant vectors or recombinant bacteria comprising the above mutants or their encoding genes.
  • the expression vector of the recombinant vector is pBSA43;
  • the host cell expressing the gene encoding the mutant is Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, or Bacillus clausii.
  • the high-activity mutant gene of alkaline protease in the present invention is expressed in Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus clausii expression system, and purified to obtain high-activity alkaline protease enzyme powder.
  • the Bacillus subtilis is WB600.
  • the Bacillus amyloliquefaciens is CGMCC No. 11218.
  • the Bacillus licheniformis is TCCC11965.
  • the Bacillus clausii is CGMCC No. 12953.
  • pBSA43 is obtained by using the Escherichia coli-Bacillus shuttle cloning vector pBE2 as the backbone, cloned into a strong Bacillus constitutive promoter P43, and the signal sequence sacB that can directly secrete the recombinant protein into the medium. It carries the Amp r gene, which can be used as a selection marker for ampicillin resistance in Escherichia coli; it also has a Km r gene, which can use kanamycin resistance as a selection marker in Bacillus subtilis and Bacillus licheniformis.
  • the present invention uses error-prone PCR technology and DNA shuffling technology to mutate the wild-type alkaline protease gene, and obtains eight alkaline protease high-activity mutants by screening.
  • the high-activity mutant gene of alkaline protease in the present invention is expressed in the expression system of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus clausii, and purified to obtain high activity alkaline protease enzyme pink.
  • FIG. 1 is an electrophoresis diagram of PCR amplification of the wild-type alkaline protease zymogen gene in the example of the present invention.
  • M is DNA Marker
  • 1 is alkaline protease zymogen gene apr.
  • FIG. 2 is a verification diagram of the digestion of pBAS43-apr plasmid in the embodiment of the present invention.
  • M is DNA Marker
  • 1 is the double digestion map of pBSA43-apr by BamHI and HindIII.
  • FIG. 3 is an electropherogram of the error-prone PCR amplification of the alkaline protease mutant gene in the example of the present invention.
  • M is DNA Marker
  • 1 and 2 are the error-prone PCR amplification electrophoresis images of alkaline protease mutant gene aprmx 1 .
  • Figure 4 is an electrophoresis diagram of the DNA shuffling product of the alkaline protease mutant gene in the example of the present invention.
  • M is DNA Marker
  • 1 and 2 are electrophoresis images of alkaline protease mutant shuffling gene aprmx 2 .
  • Fig. 5 is the verification diagram of the recombinant plasmid pBSA43-aprmX of the present invention in the embodiment of the present invention.
  • M is DNA Marker
  • 1, 2, 3, 4, 5, 6, 7, and 8 are the recombinant plasmids pBSA43-aprm1, pBSA43-aprm2, pBSA43-aprm3, pBSA43-aprm4, pBSA43-aprm5, pBSA43-aprm6, PBSA43-aprm7 and pBSA43-aprm8 were digested by BamHI and HindIII.
  • FIG. 6 is a graph showing the enzymatic properties of wild-type alkaline protease APR in the embodiment of the present invention.
  • A is the optimal reaction temperature curve of wild-type alkaline protease APR
  • B is the pH curve of the optimal reaction of wild-type alkaline protease APR
  • C is the temperature stability curve of wild-type alkaline protease APR at 60°C;
  • the Bacillus licheniformis used in the present invention is TCCC11965, disclosed in: Development and application of a CRISPR/Cas9system for Bacillus licheniformis genome editing[J].International Journal of Biological Macromolecules, 2019,122:329-337, currently preserved in Tianjin Science and Technology The University's Microbial Culture Collection and Management Center, where the public can inquire and obtain bacterial cultures.
  • the wild-type alkaline protease gene comes from the Bacillus clausii CGMCC NO.12953 strain preserved in the laboratory, and its genomic DNA is extracted with a kit (OMEGA: Bacterial DNA Kit). Among them, the Bacillus clausii genome The steps of DNA extraction are as follows:
  • the amplification primer of the alkaline protease gene of the present invention is as follows:
  • Upstream primer P1 (SEQ ID NO.1):
  • Downstream primer P2 (SEQ ID NO.2):
  • P1 and P2 were used as upstream and downstream primers, and Bacillus clausii alkaline protease genome was used as a template for amplification.
  • the amplification reaction system is:
  • the amplification program 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, extension at 72 °C for 1 min 20 s, reaction for 30 cycles; extension at 72 °C for 10 min.
  • the PCR amplification product was subjected to 0.8% agarose gel electrophoresis to obtain a band of 1059 bp (Fig. 1), and the PCR product was recovered with a small amount of DNA recovery kit to obtain the wild-type basic protein gene apr (SEQ ID NO.
  • Upstream primer P1 (SEQ ID NO.1):
  • Downstream primer P2 (SEQ ID NO.2):
  • the amplification reaction system is:
  • the amplification program 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, extension at 72 °C for 1 min and 20 s for 30 cycles; extension at 72 °C for 10 min.
  • the PCR amplification product was subjected to 0.8% agarose gel electrophoresis ( Figure 3), and the PCR product was recovered with a small amount of DNA recovery kit to obtain the gene aprmx 1 with random mutation of alkaline protease (x 1 represents several different random mutations Gene).
  • alkaline protease The enzyme activity of alkaline protease was determined by the short peptide substrate method, and the transformants with higher enzyme activity than the wild type were picked out. Then use the high enzyme activity transformant plasmid as the template to carry out continuous error-prone PCR, screen according to the above method, repeat three times, and finally screen to obtain several mutant strains with high alkaline protease activity. Plasmids are used as templates for DNA shuffling.
  • Short peptide substrate N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (AAPF, represented by amino acid single letter abbreviation, the same below), AAPY, AAPW, AAPA, AAPR, AAPN, AAPD, AAPC, AAPQ , AAPE, AAPG, AAPH, AAPI, AAPL, AAPK, AAPM, AAPP, AAPS, AAPT, AAPV were mixed and dissolved in dimethyl sulfoxide (DMSO) so that the concentration of each substrate was 6 mmol/L (method reference Patent "A New Method for Determining Protease Activity", application number: 201910730238.2).
  • DMSO dimethyl sulfoxide
  • Determination method Add 80 ⁇ L of boric acid buffer at pH 10.5 and 20 ⁇ L of short peptide substrate solution to 96-well plate, incubate in a water bath at 40°C for 1 min, and then add 100 ⁇ L of diluted enzyme solution (add 100 ⁇ L of pH 10.5 to the negative control). Boric acid buffer), react at 40 °C for 10 min, and measure the absorbance at 410 nm with a microplate reader. Under the above conditions, 1 mL of enzyme solution hydrolyzed the substrate for 1 min to produce 1 ⁇ mol of p-nitroaniline, which was defined as one enzyme activity unit U.
  • DNA shuffling was performed on the alkaline protease mutant gene screened by error-prone PCR in Example 2, and then high-activity alkaline protease mutant was obtained through high-throughput screening.
  • the plasmids of the alkaline protease mutant strains obtained by the error-prone PCR screening were extracted, the recombinant plasmids were digested with restriction enzymes BamHI and HindIII, and the DNA fragments of the alkaline protease mutant genes were recovered by cutting the gel.
  • the fragments were mixed in equal amounts, and 1 ⁇ g was added to 100 ⁇ L buffer system (50 mmol/L Tris-HCl pH 7.4, 1 mmol/L MgCl 2 ), and then added with a final concentration of 0.01 U DNaseI, digested at 37 °C for 20 min, and inactivated at 90 °C for 10 min .
  • the digested products were subjected to 2% agarose gel electrophoresis, and fragments of about 50-200 bp were recovered with a small amount of DNA recovery kit.
  • the small fragments recovered after the above digestion were used as templates, and primerless PCR was performed as primers for each other.
  • the amplification reaction system is:
  • the amplification procedure 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, extension at 72 °C for 1 min 20 s, and the reaction was performed for 30 cycles; extension at 72 °C for 10 min.
  • the PCR amplification products were subjected to 0.8% agarose gel electrophoresis, and DNA fragments of about 1 kb were recovered with a small amount of DNA recovery kit.
  • Upstream primer P1 (SEQ ID NO.1):
  • Downstream primer P2 (SEQ ID NO.2):
  • the amplification reaction system is:
  • the amplification program 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, extension at 72 °C for 1 min 20 s, 30 cycles of reaction; extension at 72 °C for 10 min.
  • the PCR amplification product was subjected to 0.8% agarose gel electrophoresis ( Figure 4), and a DNA fragment of about 1 kb was recovered with a small amount of DNA recovery kit, which is the shuffled gene aprmx 2 of alkaline protease (x 2 represents several different shuffled genes) .
  • the alkaline protease shuffling gene aprmx 2 was cloned into the expression vector pBSA43 respectively to obtain several recombinant plasmids pBSA43-aprmx 2 , which were transformed into JM109 and extracted to obtain the recombinant plasmid pBSA43-aprmx 2 , and then the recombinant plasmid pBSA43 -aprmx 2 was transformed into Bacillus subtilis WB600, the transformants were picked into a 48-well plate containing 500 ⁇ L of LB liquid medium, placed in a 48-well plate shaker and incubated at 37°C, 750r/min for 48h, After culturing, the supernatant was collected by centrifugation to obtain crude alkaline protease enzyme solution.
  • the enzyme activity of alkaline protease was measured by the short peptide substrate method in Example 2, and transformants with higher specific enzyme activity than wild-type were picked out. After screening, eight mutant strains WB600/pBSA43-aprmX with high alkaline protease activity were obtained (X are 1-8 respectively, and aprmX represents 8 different mutant encoding genes, as shown in Table 1). The obtained high-activity alkaline protease mutant strain was extracted with plasmids and sequenced (Beijing Huada Bioengineering Company). The results show that the obtained information of 8 high-activity alkaline protease mutants is as follows:
  • the mutant recombinant strain WB600/pBSA43-aprmX (X is 1, 2, 3, 4, 5, 6, 7, 8, the same below) obtained in step 4 of Example 3 above and the wild-type recombinant strain WB600/pBSA43 -apr was inoculated in 5mL of LB liquid medium (containing kanamycin, 50 ⁇ g/mL), cultured at 37°C at 220r/min overnight, and transferred to 50mL of fresh LB medium (containing kanamycin according to 2% inoculum volume) element, 50 ⁇ g/mL), continue to culture at 37 °C, 220 r/min for 48 h.
  • LB liquid medium containing kanamycin, 50 ⁇ g/mL
  • the fermentation broth was centrifuged to take the supernatant, and firstly, the impurity protein was removed by salting out with ammonium sulfate with a saturation of 25%, and then the saturation was increased to 65% to precipitate the target protein. After dissolving, dialysis to remove salt, and then the active component obtained after salting out and desalting is dissolved with 0.02mol/L Tris-HCl (pH 7.0) buffer solution, and the same buffer solution is used after loading on cellulose ion exchange chromatography column.
  • Tris-HCl (pH 7.0) buffer solution 0.02mol/L Tris-HCl (pH 7.0) buffer solution
  • the unadsorbed protein was first eluted, and then the target protein was collected by gradient elution with 0.02 mol/L Tris-HCl (pH 7.0) buffer containing different concentrations of NaCl (0-1 mol/L).
  • the active components obtained by ion exchange were first equilibrated with 0.02mol/L Tris-HCl (pH 7.0) buffer containing 0.15mol/L NaCl, and then loaded onto a sephadex g25 gel chromatography column with 0.5mL of the same buffer. Elution at a speed of /min to obtain purified enzyme solution.
  • the alkaline protease activity was determined by the short peptide substrate method in Example 2; the protein concentration was determined by the BCA protein concentration assay kit, and the operation was performed according to its instructions.
  • the specific enzyme activity of alkaline protease was the enzyme activity (U/ml) and Ratio of protein concentration (mg/ml).
  • the specific enzyme activity of the wild-type recombinant strain was set as 1, and the specific enzyme activity of the mutant recombinant strain was expressed as a multiple of the specific enzyme activity of the wild-type recombinant strain. The results are shown in the following table.
  • the enzymatic properties of wild-type and mutants were determined. The results are shown in Figure 6.
  • the optimal reaction temperature of wild-type alkaline protease is 60 °C, and the optimal reaction pH is 10. At pH 10, 60
  • the enzymatic properties of the mutant were basically the same as the wild type.
  • the recombinant strains of Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus clausii that express the mutant gene aprmX and the wild-type gene apr were determined to be obtained, named CGMCC No.11218/pBSA43-aprmX, CGMCC No.11218 respectively /pBSA43-apr; TCCC11965/pBSA43-aprmX, TCCC11965/pBSA43-apr; CGMCC No. 12953/pBSA43-aprmX, CGMCC No. 12953/pBSA43-aprmX, CGMCC No. 12953/pBSA43-aprmX.
  • Bacillus amyloliquefaciens mutant recombinant strain CGMCC No.11218/pBSA43-aprmX and wild-type recombinant strain CGMCC No.11218/pBSA43-apr were inoculated in 5 mL of LB liquid medium (containing kanamycin, 50 ⁇ g/mL) medium, 37°C, 220r/min overnight culture, transfer to 50mL fresh LB medium (containing kanamycin, 50 ⁇ g/mL) according to 2% inoculum, and continue to culture at 37°C, 220r/min for 48h.
  • the fermentation broth was centrifuged to take the supernatant, and firstly, the impurity protein was removed by salting out with ammonium sulfate with a saturation of 25%, and then the saturation was increased to 65% to precipitate the target protein. After dissolving, dialysis to remove salt, and then the active component obtained after salting out and desalting is dissolved with 0.02mol/L Tris-HCl (pH 7.0) buffer solution, and the same buffer solution is used after loading on cellulose ion exchange chromatography column.
  • Tris-HCl (pH 7.0) buffer solution 0.02mol/L Tris-HCl (pH 7.0) buffer solution
  • the unadsorbed protein was first eluted, and then the target protein was collected by gradient elution with 0.02 mol/L Tris-HCl (pH 7.0) buffer containing different concentrations of NaCl (0-1 mol/L).
  • the active components obtained by ion exchange were first equilibrated with 0.02mol/L Tris-HCl (pH 7.0) buffer containing 0.15mol/L NaCl, and then loaded onto a sephadex g25 gel chromatography column with 0.5mL of the same buffer. Elution at a speed of /min to obtain a purified enzyme liquid, which is freeze-dried to obtain pure alkaline protease enzyme powder.
  • the prepared enzyme powder can be used in detergent, food, leather, medicine and other industries.
  • Bacillus licheniformis mutant recombinant strain TCCC11965/pBSA43-aprmX and wild-type recombinant strain TCCC11965/pBSA43-apr were inoculated in 5 mL of LB liquid medium (containing kanamycin, 50 ⁇ g/mL), 37 ° C, 220r/ Min culture overnight, transfer to 50 mL of fresh LB medium (containing kanamycin, 50 ⁇ g/mL) according to 2% of the inoculum, and continue to culture at 37° C., 220 r/min for 48 h.
  • the fermentation broth was centrifuged to take the supernatant, and firstly, the impurity protein was removed by salting out with ammonium sulfate with a saturation of 25%, and then the saturation was increased to 65% to precipitate the target protein. After dissolving, dialysis to remove salt, and then the active component obtained after salting out and desalting is dissolved with 0.02mol/L Tris-HCl (pH 7.0) buffer solution, and the same buffer solution is used after loading on cellulose ion exchange chromatography column.
  • Tris-HCl (pH 7.0) buffer solution 0.02mol/L Tris-HCl (pH 7.0) buffer solution
  • the unadsorbed protein was first eluted, and then the target protein was collected by gradient elution with 0.02 mol/L Tris-HCl (pH 7.0) buffer containing different concentrations of NaCl (0-1 mol/L).
  • the active components obtained by ion exchange were first equilibrated with 0.02mol/L Tris-HCl (pH 7.0) buffer containing 0.15mol/L NaCl, and then loaded onto a sephadex g25 gel chromatography column with 0.5mL of the same buffer. Elution at a speed of /min to obtain a purified enzyme liquid, which is freeze-dried to obtain pure alkaline protease enzyme powder.
  • the prepared enzyme powder can be used in detergent, food, leather, medicine and other industries.
  • the Bacillus clausii mutant recombinant strain CGMCC No.12953/pBSA43-aprmX and the wild-type recombinant strain CGMCC No.12953/pBSA43-apr were inoculated in 5 mL of LB liquid medium (containing kanamycin, 50 ⁇ g/mL), respectively. ), cultured overnight at 37°C, 220r/min, transferred to 50mL of fresh LB medium (containing kanamycin, 50 ⁇ g/mL) according to 2% of the inoculum, and continued to culture at 37°C, 220r/min for 48h.
  • the fermentation broth was centrifuged to take the supernatant, and the impurity protein was removed by salting out with ammonium sulfate with a saturation of 25%, and then the saturation was increased to 65% to precipitate the target protein.
  • dialysis to remove salt, and then the active component obtained after salting out and desalting is dissolved with 0.02mol/L Tris-HCl (pH 7.0) buffer solution, and the same buffer solution is used after loading the sample onto cellulose ion exchange chromatography column.
  • the unadsorbed protein was first eluted, and then the target protein was collected by gradient elution with 0.02 mol/L Tris-HCl (pH 7.0) buffer containing different concentrations of NaCl (0-1 mol/L).
  • the active components obtained by ion exchange were first equilibrated with 0.02mol/L Tris-HCl (pH 7.0) buffer containing 0.15mol/L NaCl, and then loaded onto a sephadex g25 gel chromatography column with the same buffer solution to 0.5mL Elution at a speed of /min to obtain a purified enzyme solution, which is freeze-dried to obtain pure alkaline protease enzyme powder.
  • the prepared enzyme powder can be used in detergent, food, leather, medicine and other industries.

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Abstract

本发明涉及一种碱性蛋白酶突变体及其基因、工程菌、制备方法和应用，属于生物工程技术领域。本发明通过提取克劳氏芽孢杆菌基因组DNA，经PCR扩增得到野生型碱性蛋白酶基因序列，将扩增得到的野生型碱性蛋白酶基因通过易错PCR进行突变，通过高通量筛选后获得了若干个高活力碱性蛋白酶基因，再将这些高活力碱性蛋白酶基因进行DNA改组，通过筛选后获得八个更高活力的碱性蛋白酶突变体基因。

Description

一种碱性蛋白酶突变体及其基因、工程菌、制备方法和应用
本申请要求于2020年12月21日提交中国专利局、申请号为CN2020115133251、发明名称为一种碱性蛋白酶突变体及其基因、工程菌、制备方法和应用的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于生物工程技术领域，具体涉及一种碱性蛋白酶突变体及其基因、工程菌、制备方法和应用。
背景技术
蛋白酶是催化蛋白肽键裂解，可将蛋白分子、多肽降解为小的肽链、氨基酸的一类水解酶，根据蛋白酶作用时适宜pH值的不同，可分为中性蛋白酶、酸性蛋白酶和碱性蛋白酶，其中碱性蛋白酶的最适反应pH值一般大于9，较其它蛋白酶而言，碱性蛋白酶的酶活力、耐热、耐碱能力更强，并且具有酯酶特性。这些优势使得碱性蛋白酶在工业上的应用更为广泛，其在洗涤剂、食品加工、饲料、环境保护、皮革制造和丝绸制造等行业都有着十分重要的作用。在洗涤剂中加入碱性蛋白酶能保持被洗衣物的原有色彩，提高产品的去污效果，有效减少洗涤剂中表面活性剂和某些助剂的用量，还能增加节水、节能和环境保护的效益。在食品中主要用来水解植物蛋白质，植物蛋白水解后转化成分子量更小的肽和氨基酸，更利于消化吸收，产品营养价值更高，产品质量和安全性也相对更好。在制革工艺中以蛋白质和蛋白质类似物为主要成分的皮与毛，通常情况下处理较为困难，传统的方法是利用有毒的化学物质进行处理，这种方法不但危害人们自身安全，而且对环境也有着很大的污染，而蛋白酶可以代替这些化学物质来降解制革过程中非胶质组分和非纤维蛋白，同时也可以减少环境的污染。
微生物是蛋白酶的重要来源，相比于植物蛋白酶和动物蛋白酶，微生物由于其生长迅速、易于人工遗传改造，产生的蛋白酶类资源丰富微生物可以在相对较短的时间内大量培养，因此可以生产大量的满足生产需要的酶类。产碱性蛋白酶的微生物主要从盐碱湖、深海、沙地等碱性环境中分离得到，目前，芽孢杆菌、放线菌以及真菌均有报道可以产碱性蛋白酶，但在工业生产中主要是芽孢杆菌属。然而，由于菌株自身产酶能力有 限，导致了发酵酶活水平不高，芽孢杆菌产碱性蛋白酶的产品成本较高，限制了其大规模的应用。因此，提高碱性蛋白酶活力对其在工业生产及应用具有重要的意义。
蛋白质工程是建立在基因工程基础上的新技术，主要依靠计算机软件等辅助设计和蛋白质化学等多学科的基础知识，通过对蛋白编码基因的人工定向改造，对蛋白质进行修饰、改造和拼接以获得能满足人类需要的新型蛋白质的技术。酶的定向进化又称为酶的体外分子定向进化，属于蛋白质的非理性设计，是蛋白质工程新的发展方向，它不用事先了解蛋白质的结构、活性位点、催化机制等因素，而是模拟自然进化过程，人为地创造特殊的进化条件，从一个或多个已经存在的亲本酶(天然的或者人为获得的酶前体)出发，在体外或体内对基因进行随机突变或体外基因重组，构建人工突变酶库，进一步通过一定的筛选或选择方法，最终获得预先所期望的具有某些特性的进化酶。
体外定向进化常用方法主要是易错PCR(Error-prone PCR)和DNA改组(DNA shuffling)。易错PCR是通过利用低保真度TaqDNA聚合酶和改变PCR反应条件，如加入Mn、改变循环次数和dNTP浓度等，降低DNA复制的保真度，在新DNA链合成过程中增加碱基错配，从而使扩增产物出现较多点突变的一种体外诱导DNA序列变异的方法。
DNA改组是将一个或一组密切相关的基因序列，在DNaseI作用下切割成一系列随机大小的DNA小片段，由于基因的同源性，这些小片段之间有部分的碱基序列重叠，它们通过自身引导，随机重组，最后通过特定引物的PCR，生成全长基因，在这一过程中，由于模板的变换产生了相关序列间的交换，从而产生了多样的基因重组文库，再进一步对改组的基因表达的产物进行筛选，从而达到目的基因的定向进化。
发明内容
本发明提供了一种碱性蛋白酶突变体及其基因、工程菌、制备方法和应用，具体提供了高活力碱性蛋白酶突变体及其基因、工程菌等。
本发明中，芽孢杆菌表达***具有以下优点：1、芽孢杆菌拥有一套高效的分泌信号肽和分子伴侣***，这有利于实现目的蛋白的高效表达；2、大多数芽孢杆菌都是没有致病性的，这符合工业生产中的一般安全需求；3、芽孢杆菌的细胞壁组成要相对简单，这有利于表达蛋白的胞外分泌，不会导致分泌蛋白在细胞内积累，有利于蛋白的下游回收和纯化；4、芽孢杆菌作为单细胞生物，可以较短时间内达到较高的菌体密度，且所需培养基组成相对较简单，成本较低符合工业生产的要求。
本发明中，基于碱性蛋白酶在枯草芽孢杆菌中的表达平台，利用易错PCR及DNA改组技术，对来源于克劳氏芽孢杆菌的碱性蛋白酶基因进行分子改造，获得高活力碱性蛋白酶基因，并成功在解淀粉芽孢杆菌、地衣芽孢杆菌、克劳氏芽孢杆菌体系中进行表达。
本发明提供的技术方案之一为：以克劳氏芽孢杆菌CGMCC NO.12953基因组为模板，克隆出野生型碱性蛋白酶酶原区基因apr(SEQ ID NO.3所示)序列后(氨基酸序列如SEQ ID NO.4所示)，通过连续易错PCR对野生型碱性蛋白酶基因进行随机突变，再利用枯草芽孢杆菌表达***进行高通量筛选获得若干碱性蛋白酶高活力突变体基因，再将这些高活力碱性蛋白酶突变体基因进行DNA改组，经过筛选后获得高活力的碱性蛋白酶突变体基因。
本发明提供的技术方案之二为：将上述突变体基因构建重组载体并在解淀粉芽孢杆菌、地衣芽孢杆菌、克劳氏芽孢杆菌中成功表达，得到产酶活力提高的重组菌株，进一步通过发酵工艺优化获得新型碱性蛋白酶，可应用在洗涤剂、食品、制革、医药等领域。
在本发明中采用如下定义：
1、氨基酸和DNA核酸序列的命名法
使用氨基酸残基的公认IUPAC命名法，采用三字母代码形式。DNA核酸序列采用公认IUPAC命名法。
2、碱性蛋白酶高活力突变体的标识
采用“原始氨基酸位置替换的氨基酸”来表示碱性蛋白酶高活力突变体中突变的氨基酸。在本发明中，碱性蛋白酶的突变点位置按其成熟肽的氨基酸序列进行编号，位置的编号对应于SEQ ID NO.6中野生型碱性蛋白酶成熟肽的氨基酸序列编号，如Asn212表示野生型碱性蛋白酶成熟肽氨基酸序列的第212位氨基酸为Asn，Asn212Ser表示位置212的氨基酸由野生型碱性蛋白酶的Asn替换成Ser，也可用氨基酸单字母简称进行表示，如N212S，同时发生多位点突变的采用“/”连接各突变位点的方式表示，如V11I/G95V/V145I/N212S，表示位置11、95、145和212位的氨基酸依次由野生型碱性蛋白酶的V替换成I、由G替换为V、由V替换为I、由N替换为S；核苷酸表示方法与氨基酸表示方法类似，位置的编号对应于SEQ ID NO.5中野生型碱性蛋白酶的核苷酸序列编号，如C425，表示碱性蛋白酶核苷酸序列的第425位碱基为C。
在本发明中，APR表示野生型碱性蛋白酶，即原始序列如SEQ ID NO.4所示，其编码基因表示为apr(如SEQ ID NO.3所示)。用APRM加数字X的方式表示各个碱性蛋白酶突变体，各突变体的编码基因则以其氨基酸表示形式的小写斜体表示。
本发明中，所述碱性蛋白酶突变体具有蛋白水解活性，其成熟肽是：
(1)在SEQ ID NO.6所示的野生型碱性蛋白酶成熟肽基础上发生包含以下突变中的任意一种获得的：
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；或
(2)与(1)同源性75％以上的氨基酸序列；或
(3)在(1)的基础上进行一个或多个氨基酸替换，和/或缺失，和/或添加后获得的具有(1)相同功能的氨基酸序列。
本发明使用易错PCR技术及DNA改组技术，对野生型碱性蛋白酶基因进行突变，筛选获得了八个碱性蛋白酶高活力突变体。
本发明还提供上述突变体的编码基因。
在一些实施例中，所述突变体的编码基因如SEQ ID NO.7-14任一所示。
本发明还提供包含上述突变体或其编码基因的重组载体或重组菌。
在一些实施例中，所述重组载体的表达载体为pBSA43；
在一些实施例中，表达所述突变体编码基因的宿主细胞为枯草芽孢杆菌、解淀粉芽孢杆菌、地衣芽孢杆菌或克劳氏芽孢杆菌。
本发明中碱性蛋白酶高活力突变体基因在枯草芽孢杆菌、解淀粉芽孢杆菌、地衣芽孢杆菌、克劳氏芽孢杆菌表达***中进行了表达，并经过纯化制得了高活力碱性蛋白酶酶粉。
在一些实施例中，所述枯草芽孢杆菌为WB600。
在一些实施例中，所述解淀粉芽孢杆菌为CGMCC No.11218。
在一些实施例中，所述地衣芽孢杆菌为TCCC11965。
在一些实施例中，所述克劳氏芽孢杆菌为CGMCC No.12953。
pBSA43是以大肠杆菌-芽孢杆菌穿梭克隆载体pBE2为骨架，克隆入一个强的芽孢杆菌组成型启动子P43，以及能够使重组蛋白直接分泌到培养基中果聚糖蔗糖酶信号序 列sacB而获得。它带有Amp r基因，可以在大肠杆菌中利用氨苄青霉素抗性作为筛选标记；同时也具有Km r基因，可以在枯草芽孢杆菌、地衣芽孢杆菌中利用卡那霉素抗性作为筛选标记。
本发明实验步骤具体如下：
(1)将来自克劳氏芽孢杆菌的碱性蛋白酶基因通过易错PCR进行随机突变，获得随机突变的基因aprmx 1，将其连接到表达载体后转化至枯草芽孢杆菌WB600中进行筛选，将获得的高活力突变体基因作为模板再次进行易错PCR，重复三轮，最终获得若干碱性蛋白酶高活力突变体基因；
(2)将经易错PCR筛选获得的高活力突变体基因进行DNA改组，再将改组后的突变基因aprmx 2连接到表达载体后转化至枯草芽孢杆菌中，通过高通量筛选后获得了八个碱性蛋白酶高活力突变体基因；
(3)将获得的碱性蛋白酶高活力突变体基因连接到表达载体，将其转至解淀粉芽孢杆菌、地衣芽孢杆菌及克劳氏芽孢杆菌中，获得各重组菌株；
(4)表达所述的重组菌株，纯化后获得碱性蛋白酶高活力突变体APRMX。
有益效果：
1、本发明使用易错PCR技术及DNA改组技术，对野生型碱性蛋白酶基因进行突变，筛选获得了八个碱性蛋白酶高活力突变体。
2、本发明中碱性蛋白酶高活力突变体基因在枯草芽孢杆菌、解淀粉芽孢杆菌、地衣芽孢杆菌、克劳氏芽孢杆菌表达***中进行了表达，并经过纯化制得了高活力碱性蛋白酶酶粉。
附图说明
图1为本发明实施例中野生型碱性蛋白酶酶原基因的PCR扩增电泳图。
其中：M为DNA Marker，1为碱性蛋白酶酶原基因apr。
图2为本发明实施例中pBAS43-apr质粒酶切验证图。
其中：M为DNA Marker，1为pBSA43-apr经BamHI和HindIII双酶切图。
图3为本发明实施例中碱性蛋白酶突变体基因易错PCR扩增电泳图。
其中：M为DNA Marker，1、2为碱性蛋白酶突变体基因aprmx 1易错PCR扩增电泳图。
图4为本发明实施例中碱性蛋白酶突变体基因DNA改组产物电泳图。
其中：M为DNA Marker，1、2为碱性蛋白酶突变体改组基因aprmx 2电泳图。
图5为本发明实施例中本发明重组质粒pBSA43-aprmX酶切验证图。
其中：M为DNA Marker，1、2、3、4、5、6、7、8分别为重组质粒pBSA43-aprm1、pBSA43-aprm2、pBSA43-aprm3、pBSA43-aprm4、pBSA43-aprm5、pBSA43-aprm6、pBSA43-aprm7、pBSA43-aprm8经BamHI和HindIII双酶切图。
图6为本发明实施例中野生型碱性蛋白酶APR酶学性质曲线图。
其中：A为野生型碱性蛋白酶APR最适反应温度曲线图；
B为野生型碱性蛋白酶APR最适反应pH曲线图；
C为野生型碱性蛋白酶APR在60℃条件下的温度稳定性曲线图；
D为野生型碱性蛋白酶APR在pH＝11条件下的pH稳定性曲线图。
具体实施方式
为了使本专利的目的、技术方案及优点更加清楚明白，以下结合具体实施例，对本专利进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本专利，并不用于限定本发明。
本发明所使用的地衣芽孢杆菌为TCCC11965，公开于：Development and application of a CRISPR/Cas9system for Bacillus licheniformis genome editing[J].International Journal of Biological Macromolecules,2019,122:329-337，目前保存于天津科技大学微生物菌种保藏管理中心，公众可查询并可从该中心获取菌种。
实施例1 野生型碱性蛋白酶基因的获得
1、野生型碱性蛋白酶基因来自实验室保存的克劳氏芽孢杆菌(Bacillus clausii)CGMCC NO.12953菌株，利用试剂盒(OMEGA:Bacterial DNA Kit)提取其基因组DNA，其中克劳氏芽孢杆菌基因组DNA的提取步骤如下：
(1)菌株活化：用接种环从甘油管中蘸取少许菌液接种于LB固体培养基平板上，三区划线，37℃恒温培养12h；
(2)转接：从培养菌体的平板上挑取单菌落接种于含5mL液体LB培养基中，于220rpm，37℃条件下振荡培养12h；
(3)收集菌体：取适量培养菌液分装于已灭菌的1.5mL EP管中，12000rpm离心1min收集菌体，弃上清；
(4)加入100μL ddH 2O重悬菌体，并加入50μL的50mg/mL溶菌酶，37℃水浴10min；
(5)加入100μL BTL Buffer和20μL蛋白酶K，旋涡振荡，55℃水浴40-50min，每隔20-30min，振荡混匀；
(6)加入5μL RNA酶，颠倒混匀数次，室温放置5min；
(7)12000rpm离心2min，去掉未消化部分，将上清部分转移至新的1.5mL EP管中，加入220μL BDL Buffer，振荡混匀，65℃水浴10min；
(8)加入220μL无水乙醇，吹吸混匀；
(9)将EP管中的液体转移至吸附柱中静止2min，12000rpm离心1min，将滤液重新倒入吸附柱中静置、离心，重复两次，弃滤液；
(10)加入500μL HBC Buffer，静置2min，12000rpm离心1min，弃滤液；
(11)加入700μL DNA Wash Buffer，静置2min，12000rpm离心1min，弃滤液；
(12)加入500μL DNA Wash Buffer，静置2min，12000rpm离心1min，弃滤液；
(13)12000rpm空离2min，将吸附柱放到一个新的EP管上，置于55℃金属浴10min，晾干；
(14)加入50μL的55℃分子水，室温静置3-5min，12000rpm离心2min收集基因组。
2、以提取的克劳氏芽孢杆菌的基因组为模板，根据Genbank序列号FJ940727.1登录的碱性蛋白酶序列，在ORF框上下游设计一对引物，分别引入限制性酶切位点BamHI、HindIII，本发明的碱性蛋白酶基因的扩增引物如下：
上游引物P1(SEQ ID NO.1)：
5’-CGCGGATCCGCTGAAGAAGCAAAAGAAAAATATTTAAT-3’
下游引物P2(SEQ ID NO.2)：
5’-CCCAAGCTTTTAGCGTGTTGCCGCTTCT-3’
以P1和P2作为上、下游引物，以克劳氏芽孢杆菌碱性蛋白酶基因组为模板进行扩增。
其扩增的反应体系为：
10×PCR Buffer 5.0μL
dNTPs 5.0μL
上游引物P1 2.0μL
下游引物P2 2.0μL
DNA模板 2.0μL
Pyrobest酶 0.5μL
ddH 2O 33.5μL
扩增程序为：95℃预变性10min；94℃变性30s，57℃退火45s，72℃延伸1min20s，反应30个循环；72℃延伸10min。PCR扩增产物经0.8％琼脂糖凝胶电泳，得到1059bp 的条带(图1)，用小量DNA回收试剂盒回收PCR产物，得到本发明的野生型碱性蛋白基因apr(SEQ ID NO.3)，将扩增获得的apr与载体pBSA43载体连接，得到重组质粒pBSA43-apr，酶切验证如图2所示，并将其转化至大肠杆菌JM109及枯草芽孢杆菌WB600中。
实施例2 易错PCR构建碱性蛋白酶突变体文库筛选高活力碱性蛋白酶突变体
1、基于易错PCR技术进行随机突变，构建碱性蛋白酶突变体文库，设计引物如下：
上游引物P1(SEQ ID NO.1)：
5’-CGCGGATCCGCTGAAGAAGCAAAAGAAAAATATTTAAT-3’
下游引物P2(SEQ ID NO.2)：
5’-CCCAAGCTTTTAGCGTGTTGCCGCTTCT-3’
在易错PCR反应体系中，以P1和P2作为上、下游引物，以野生型碱性蛋白酶基因apr为模板，进行易错PCR。
其扩增的反应体系为：
10×PCR缓冲液(无Mg 2+) 5μL
dATP 0.1μL
dGTP 0.1μL
dCTP 0.5μL
dTTP 0.5μL
上游引物P1 2μL
下游引物P2 2μL
野生型碱性蛋白酶基因 2μL
rTaq DNA聚合酶 0.3μL
25mM MgCl 2(10mM) 20μL
5mM MnCl 2(0.3mM) 3μL
ddH 2O 14.5μL
扩增程序为：95℃预变性10min；98℃变性10s，57℃退火30s，72℃延伸1min20s反应30个循环；72℃延伸10min。PCR扩增产物经0.8％琼脂糖凝胶电泳(图3)，用小量DNA回收试剂盒回收PCR产物，得到带有随机突变的碱性蛋白酶的基因aprmx 1(x 1表示若干不同的随机突变基因)。
2、将碱性蛋白酶随机突变体基因aprmx 1与表达载体pBSA43连接后转化至JM109中并 提取其质粒即得重组质粒pBSA43-aprmx 1，再将重组质粒pBSA43-aprmx 1转化至枯草芽孢杆菌WB600中，将转化子挑接至装有500μL的LB液体培养基的48孔板中，放入四十八孔板摇床在37℃，750r/min条件下培养48h，培养完成后离心取上清即得碱性蛋白酶粗酶液，用短肽底物法测定碱性蛋白酶酶活力，从中挑出比酶活力比野生型高的转化子。再以高酶活力转化子质粒为模板进行连续易错PCR，按上述方式进行筛选，重复三次，最终筛选获得若干株碱性蛋白酶活力较高的突变体菌株，以这些高活力碱性蛋白酶突变体质粒为模板进行DNA改组。
3、短肽底物测定碱性蛋白酶酶活力
短肽底物：将N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide(AAPF，用氨基酸单字母简称表示，下同)、AAPY、AAPW、AAPA、AAPR、AAPN、AAPD、AAPC、AAPQ、AAPE、AAPG、AAPH、AAPI、AAPL、AAPK、AAPM、AAPP、AAPS、AAPT、AAPV混合，用二甲基亚砜(DMSO)溶解，使得每种底物的浓度均为6mmol/L(方法参考专利《一种测定蛋白酶活力的新方法》，申请号：201910730238.2)。
测定方法：在96孔板中加入80μL pH 10.5的硼酸缓冲液和20μL短肽底物溶液，在40℃水浴锅中保温1min，再加入100μL稀释后的酶液(阴性对照中加100μL pH 10.5的硼酸缓冲液)，40℃反应10min，用酶标仪在410nm测定其吸光值。在上述条件下，1mL酶液1min水解底物产生1μmol对硝基苯胺定义为一个酶活力单位U。
实施例3 DNA改组构建碱性蛋白酶突变体文库筛选高活力碱性蛋白酶突变体
将实施例2中经易错PCR筛选得到的碱性蛋白酶突变体基因进行DNA改组，再通过高通量筛选得到高活力碱性蛋白酶突变体。
1、碱性蛋白酶突变体基因的片段化
提取易错PCR筛选获得的碱性蛋白酶突变体菌株的质粒，使用限制性内切酶BamHI和HindIII酶切重组质粒，切胶回收碱性蛋白酶突变体基因的DNA片段，将这些突变体基因的DNA片段等量混匀，取1μg加入100μL缓冲体系(50mmol/L Tris-HCl pH 7.4，1mmol/L MgCl 2)中，再加入终浓度为0.01U DNaseI，37℃酶切20min，90℃灭活10min。酶切产物经2％琼脂糖凝胶电泳，用小量DNA回收试剂盒回收50-200bp左右的片段。
2、无引物PCR
将上述消化后回收的小片段作为模板，互为引物进行无引物PCR。其扩增的反应体系为：
10×PCR Buffer(无Mg 2+) 5.0μL
25mM MgCl 2 5.0μL
dNTPs 5.0μL
DNA片段模板 2.0μL
rTaq DNA聚合酶 0.5μL
ddH 2O 37.5μL
扩增程序为：95℃预变性10min；98℃变性10s，50℃退火30s，72℃延伸1min20s，反应30个循环；72℃延伸10min。PCR扩增产物经0.8％琼脂糖凝胶电泳，用小量DNA回收试剂盒回收1kb左右的DNA片段。
3、有引物PCR
取无引物PCR产物为模板进行第二轮有引物PCR，其扩增引物如下：
上游引物P1(SEQ ID NO.1)：
5’-CGCGGATCCGCTGAAGAAGCAAAAGAAAAATATTTAAT-3’
下游引物P2(SEQ ID NO.2)：
5’-CCCAAGCTTTTAGCGTGTTGCCGCTTCT-3’
其扩增的反应体系为：
10×PCR Buffer 5.0μL
dNTPs 5.0μL
上游引物P1 2.0μL
下游引物P2 2.0μL
无引物PCR产物 2.0μL
Pyrobest酶 0.5μL
ddH 2O 33.5μL
扩增程序为：95℃预变性10min；94℃变性30s，57℃退火45s，72℃延伸1min20s，反应30个循环；72℃延伸10min。PCR扩增产物经0.8％琼脂糖凝胶电泳(图4)，用小量DNA回收试剂盒回收1kb左右的DNA片段即为碱性蛋白酶的改组基因aprmx 2(x 2表示若干不同的改组基因)。
4、将碱性蛋白酶改组基因aprmx 2分别克隆入表达载体pBSA43中，得到若干重组质粒pBSA43-aprmx 2，并转化至JM109中并提取其质粒即得重组质粒pBSA43-aprmx 2，再将重组质粒pBSA43-aprmx 2转化至枯草芽孢杆菌WB600中，将转化子挑接至装有500μL的LB液体培养基的48孔板中，放入四十八孔板摇床在37℃，750r/min条件下培养48h，培养完成后离心取上清即得碱性蛋白酶粗酶液，用实施例2中的短肽底物法测定碱 性蛋白酶酶活力，从中挑出比酶活力比野生型高的转化子。经过筛选，得到了八株碱性蛋白酶活力较高的突变体菌株WB600/pBSA43-aprmX(X分别为1-8，aprmX表示表示8个不同的突变体编码基因，具体如表1)，将所获得的高活力碱性蛋白酶突变体菌株提取质粒并进行测序(北京华大生物工程公司)。结果表明，获得的8个高活力碱性蛋白酶突变体信息如下表：
表1 碱性蛋白酶突变体信息
Figure PCTCN2021071281-appb-000001
实施例4 碱性蛋白酶高活力突变体比酶活力评估
将上述实施例3步骤4所获得的突变体重组菌株WB600/pBSA43-aprmX(X分别为1、2、3、4、5、6、7、8，下同)和野生型重组菌株WB600/pBSA43-apr接种于5mL的LB液体培养基(含卡那霉素，50μg/mL)中，37℃，220r/min培养过夜，按照2％接种量转接于50mL新鲜LB培养基(含卡那霉素，50μg/mL)中，继续以37℃，220r/min培养48h。
发酵液离心取上清，先以25％饱和度的硫酸铵盐析除去杂蛋白，再把饱和度加大到65％，沉淀目的蛋白。溶解后，透析除盐，再将盐析脱盐后得到的活性组分用0.02mol/L Tris-HCl(pH 7.0)缓冲液溶解，上样至纤维素离子交换层析柱后用同样的缓冲液先洗脱未吸附的蛋白，再用含不同浓度NaCl(0～1mol/L)的0.02mol/L Tris-HCl(pH 7.0)缓冲液梯度洗脱，收集目的蛋白。离子交换得到的活性组分先用含0.15mol/L NaCl的0.02mol/L Tris-HCl(pH 7.0)缓冲液平衡，上样至sephadex g25凝胶层析柱后用相同的缓冲液以0.5mL/min的速度洗脱，获得纯化的酶液。
使用实施例2中的短肽底物法测定其碱性蛋白酶活力；蛋白质浓度由BCA蛋白浓 度测定试剂盒测定，按照其说明书进行操作，碱性蛋白酶比酶活力为酶活力(U/ml)与蛋白质浓度(mg/ml)的比值。将野生型重组菌株比酶活力定为1，突变体重组菌株比酶活力以野生型重组菌株比酶活力倍数进行表示，其结果如下表所示。
碱性蛋白酶比活力
突变体 比酶活(U/mg) 比酶活力倍数
野生型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
酶学性质测定：对野生型及突变体的酶学性质进行测定，结果如图6所示，野生型碱性蛋白酶最适反应温度为60℃，最适反应pH为10，在pH 10，60℃条件下保温40h其残余酶活在6％左右，在60℃，pH＝11条件下保温70h其残余酶活在21％左右，突变体酶学性质与野生型基本一致。
实施例5 碱性蛋白酶高活力突变体在其它芽孢杆菌中的构建
将1μL(50ng/μL)的pBSA43-aprmX和pBSA43-apr重组质粒分别加入到50μL的解淀粉芽孢杆菌CGMCC No.11218、地衣芽孢杆菌TCCC11965、克劳氏芽孢杆菌CGMCC No.12953感受态细胞中并混匀，之后转移到预冷的电转杯(1mm)中，冰浴1-1.5min后，电击一次(25μF，200Ω，4.5-5.0ms)。电击完毕之后，立即加入1mL复苏培养基(LB+0.5mol/L山梨醇+0.38mol/L甘露醇)。37℃摇床，震荡培养3h之后，将复苏物涂布于含有卡那霉素的LB平板上，37℃培养12-24h，挑取阳性转化子，并进行双酶切验证(图5)，确定获得表达突变体基因aprmX和野生型基因apr的解淀粉芽孢杆菌重组菌株、地衣芽孢杆菌重组菌株、克劳氏芽孢杆菌重组菌株，分别命名为CGMCC No.11218/pBSA43-aprmX、CGMCC No.11218/pBSA43-apr；TCCC11965/pBSA43-aprmX、TCCC11965/pBSA43-apr；CGMCC No.12953/pBSA43-aprmX、CGMCC No.12953/pBSA43-apr。
实施例6 碱性蛋白酶突变体在解淀粉芽孢杆菌重组菌株中的表达及制备
分别将解淀粉芽孢杆菌突变体重组菌株CGMCC No.11218/pBSA43-aprmX和野生型重组菌CGMCC No.11218/pBSA43-apr接种于5mL的LB液体培养基(含卡那霉素，50μg/mL)中，37℃，220r/min培养过夜，按照2％接种量转接于50mL新鲜LB培养基(含卡那霉素，50μg/mL)中，继续以37℃，220r/min培养48h。
发酵液离心取上清，先以25％饱和度的硫酸铵盐析除去杂蛋白，再把饱和度加大到65％，沉淀目的蛋白。溶解后，透析除盐，再将盐析脱盐后得到的活性组分用0.02mol/L Tris-HCl(pH 7.0)缓冲液溶解，上样至纤维素离子交换层析柱后用同样的缓冲液先洗脱未吸附的蛋白，再用含不同浓度NaCl(0～1mol/L)的0.02mol/L Tris-HCl(pH 7.0)缓冲液梯度洗脱，收集目的蛋白。离子交换得到的活性组分先用含0.15mol/L NaCl的0.02mol/L Tris-HCl(pH 7.0)缓冲液平衡，上样至sephadex g25凝胶层析柱后用相同的缓冲液以0.5mL/min的速度洗脱，获得纯化的酶液，经冷冻干燥制得碱性蛋白酶纯酶酶粉。制得的酶粉可以应用于洗涤剂、食品、制革、医药等行业领域。
实施例7 碱性蛋白酶突变体在地衣芽孢杆菌重组菌株中的表达及制备
分别将地衣芽孢杆菌突变体重组菌株TCCC11965/pBSA43-aprmX和野生型重组菌TCCC11965/pBSA43-apr接种于5mL的LB液体培养基(含卡那霉素，50μg/mL)中，37℃，220r/min培养过夜，按照2％接种量转接于50mL新鲜LB培养基(含卡那霉素，50μg/mL)中，继续以37℃，220r/min培养48h。
发酵液离心取上清，先以25％饱和度的硫酸铵盐析除去杂蛋白，再把饱和度加大到65％，沉淀目的蛋白。溶解后，透析除盐，再将盐析脱盐后得到的活性组分用0.02mol/L Tris-HCl(pH 7.0)缓冲液溶解，上样至纤维素离子交换层析柱后用同样的缓冲液先洗脱未吸附的蛋白，再用含不同浓度NaCl(0～1mol/L)的0.02mol/L Tris-HCl(pH 7.0)缓冲液梯度洗脱，收集目的蛋白。离子交换得到的活性组分先用含0.15mol/L NaCl的0.02mol/L Tris-HCl(pH 7.0)缓冲液平衡，上样至sephadex g25凝胶层析柱后用相同的缓冲液以0.5mL/min的速度洗脱，获得纯化的酶液，经冷冻干燥制得碱性蛋白酶纯酶酶粉。制得的酶粉可以应用于洗涤剂、食品、制革、医药等行业领域。
实施例8 碱性蛋白酶突变体在克劳氏芽孢杆菌重组菌株中的表达及制备
分别将克劳氏芽孢杆菌突变体重组菌株CGMCC No.12953/pBSA43-aprmX和野生型重组菌CGMCC No.12953/pBSA43-apr接种于5mL的LB液体培养基(含卡那霉素，50μg/mL)中，37℃，220r/min培养过夜，按照2％接种量转接于50mL新鲜LB培养 基(含卡那霉素，50μg/mL)中，继续以37℃，220r/min培养48h。
发酵液离心取上清，先以25％饱和度的硫酸铵盐析除去杂蛋白，再把饱和度加大到65％，沉淀目的蛋白。溶解后，透析除盐，再将盐析脱盐后得到的活性组分用0.02mol/L Tris-HCl(pH 7.0)缓冲液溶解，上样至纤维素离子交换层析柱后用同样的缓冲液先洗脱未吸附的蛋白，再用含不同浓度NaCl(0～1mol/L)的0.02mol/L Tris-HCl(pH 7.0)缓冲液梯度洗脱，收集目的蛋白。离子交换得到的活性组分先用含0.15mol/L NaCl的0.02mol/L Tris-HCl(pH 7.0)缓冲液平衡，上样至sephadex g25凝胶层析柱后用相同的缓冲液以0.5mL/min的速度洗脱，获得纯化的酶液，经冷冻干燥制得碱性蛋白酶纯酶酶粉。制得的酶粉可以应用于洗涤剂、食品、制革、医药等行业领域。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本专利构思的前提下，上述各实施方式还可以做出若干变形、组合和改进，这些都属于本专利的保护范围。因此，本专利的保护范围应以权利要求为准。

Claims (8)

  1. 一种碱性蛋白酶突变体，其特征在于，所述碱性蛋白酶的成熟肽是：
    (1)在SEQ ID NO.6所示的碱性蛋白酶酶原区序列的基础上，发生包含以下突变组合中的任意一种获得的：
    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、
    或V11L/G23A/G25P/I35V/G95P/S99H/V145I/N212S/A267P；或者
    (2)与(1)同源性75％以上的氨基酸序列；或者
    (3)在(1)的基础上进行一个或多个氨基酸替换，和/或缺失，和/或添加后获得的具有(1)相同功能的氨基酸序列。
  2. 权利要求1所述碱性蛋白酶突变体的编码基因。
  3. 如权利要求2所述的编码基因，其特征在于，如序列表SEQ ID NO.7-14任一所示。
  4. 包含权利要求2所述基因的重组载体或重组菌株。
  5. 权利要求4所述的重组载体或重组菌株，其特征在于，表达载体为pBSA43；宿主细胞为枯草芽孢杆菌WB600、解淀粉芽孢杆菌CGMCC No.11218、地衣芽孢杆菌TCCC11965、或者克劳氏芽孢杆菌CGMCC No.12953。
  6. 权利要求4所述重组载体或重组菌株在生产碱性蛋白酶中的应用。
  7. 权利要求1所述碱性蛋白酶突变体的应用。
  8. 如权利要求7所述的应用，其特征在于，应用于洗涤剂、制革、食品或饲料领域。
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