CN116790564A - Heat-resistant protease mutant and encoding gene and application thereof - Google Patents
Heat-resistant protease mutant and encoding gene and application thereof Download PDFInfo
- Publication number
- CN116790564A CN116790564A CN202310770625.5A CN202310770625A CN116790564A CN 116790564 A CN116790564 A CN 116790564A CN 202310770625 A CN202310770625 A CN 202310770625A CN 116790564 A CN116790564 A CN 116790564A
- Authority
- CN
- China
- Prior art keywords
- seq
- protease
- set forth
- amino acid
- changed
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108091005804 Peptidases Proteins 0.000 title claims abstract description 92
- 239000004365 Protease Substances 0.000 title claims abstract description 88
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 title claims abstract description 81
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 claims abstract description 15
- 230000008569 process Effects 0.000 claims abstract description 7
- 238000012216 screening Methods 0.000 claims abstract description 5
- 150000001413 amino acids Chemical group 0.000 claims description 56
- 239000002773 nucleotide Substances 0.000 claims description 36
- 125000003729 nucleotide group Chemical group 0.000 claims description 36
- CKLJMWTZIZZHCS-REOHCLBHSA-N aspartic acid group Chemical group N[C@@H](CC(=O)O)C(=O)O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 35
- 239000004475 Arginine Substances 0.000 claims description 32
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 32
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 32
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 31
- 239000004472 Lysine Substances 0.000 claims description 30
- 235000003704 aspartic acid Nutrition 0.000 claims description 27
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims description 27
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 claims description 24
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 24
- 235000009582 asparagine Nutrition 0.000 claims description 24
- 229960001230 asparagine Drugs 0.000 claims description 24
- 238000000855 fermentation Methods 0.000 claims description 24
- 230000004151 fermentation Effects 0.000 claims description 24
- 235000013922 glutamic acid Nutrition 0.000 claims description 24
- 239000004220 glutamic acid Substances 0.000 claims description 24
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 claims description 19
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 claims description 18
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 16
- 241000894006 Bacteria Species 0.000 claims description 14
- 125000000613 asparagine group Chemical group N[C@@H](CC(N)=O)C(=O)* 0.000 claims description 14
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical group OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims description 12
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical group NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 claims description 12
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 claims description 10
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 claims description 10
- 229920001184 polypeptide Polymers 0.000 claims description 8
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 8
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 8
- 239000004474 valine Substances 0.000 claims description 8
- 239000004471 Glycine Substances 0.000 claims description 7
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims description 6
- 238000010353 genetic engineering Methods 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 5
- 125000002987 valine group Chemical group [H]N([H])C([H])(C(*)=O)C([H])(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 5
- 239000013598 vector Substances 0.000 claims description 5
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 claims description 4
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 claims description 4
- 239000003674 animal food additive Substances 0.000 claims description 4
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 4
- 239000013604 expression vector Substances 0.000 claims description 4
- 235000013373 food additive Nutrition 0.000 claims description 4
- 239000002778 food additive Substances 0.000 claims description 4
- 238000003259 recombinant expression Methods 0.000 claims description 4
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 claims description 4
- 239000004382 Amylase Substances 0.000 claims description 3
- 102000013142 Amylases Human genes 0.000 claims description 3
- 108010065511 Amylases Proteins 0.000 claims description 3
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 3
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 claims description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 3
- 235000019764 Soybean Meal Nutrition 0.000 claims description 3
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Chemical group C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 3
- 240000008042 Zea mays Species 0.000 claims description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 3
- 235000004279 alanine Nutrition 0.000 claims description 3
- 235000019418 amylase Nutrition 0.000 claims description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 3
- 235000005822 corn Nutrition 0.000 claims description 3
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 claims description 3
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 235000012054 meals Nutrition 0.000 claims description 3
- 229930182817 methionine Natural products 0.000 claims description 3
- 239000001301 oxygen Substances 0.000 claims description 3
- 229910052760 oxygen Inorganic materials 0.000 claims description 3
- 239000004455 soybean meal Substances 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Chemical group OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 3
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 claims description 3
- 238000009423 ventilation Methods 0.000 claims description 3
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 claims description 2
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- 239000003550 marker Substances 0.000 claims description 2
- 125000001500 prolyl group Chemical group [H]N1C([H])(C(=O)[*])C([H])([H])C([H])([H])C1([H])[H] 0.000 claims description 2
- 230000001131 transforming effect Effects 0.000 claims description 2
- 125000000430 tryptophan group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12 0.000 claims description 2
- 230000035772 mutation Effects 0.000 abstract description 16
- 230000009286 beneficial effect Effects 0.000 abstract description 5
- 235000013305 food Nutrition 0.000 abstract description 5
- 238000012545 processing Methods 0.000 abstract description 2
- 230000000694 effects Effects 0.000 description 16
- 102000004190 Enzymes Human genes 0.000 description 15
- 108090000790 Enzymes Proteins 0.000 description 15
- 229940088598 enzyme Drugs 0.000 description 15
- 244000063299 Bacillus subtilis Species 0.000 description 7
- 235000014469 Bacillus subtilis Nutrition 0.000 description 7
- 102000035195 Peptidases Human genes 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 239000007788 liquid Substances 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 229960000723 ampicillin Drugs 0.000 description 4
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 229930027917 kanamycin Natural products 0.000 description 4
- 229960000318 kanamycin Drugs 0.000 description 4
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 4
- 229930182823 kanamycin A Natural products 0.000 description 4
- 230000014759 maintenance of location Effects 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- 238000009004 PCR Kit Methods 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000012474 protein marker Substances 0.000 description 2
- 238000002708 random mutagenesis Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 241000024188 Andala Species 0.000 description 1
- 241000193744 Bacillus amyloliquefaciens Species 0.000 description 1
- 241000194108 Bacillus licheniformis Species 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229940079919 digestives enzyme preparation Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 239000010985 leather Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 230000036438 mutation frequency Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000009145 protein modification Effects 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
-
- 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
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/189—Enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/06—Enzymes
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Polymers & Plastics (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Animal Husbandry (AREA)
- Nutrition Science (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention provides a heat-resistant protease mutant, and a coding gene and application thereof. The invention constructs mutant library by utilizing error-prone PCR method, and the method is used for preparing the mutant libraryBacillus licheniformis Mutation of WX-02-derived protease gene, and directional screening to obtain multiple heat-resistant protease mutants, specifically BLAPR 01-BLAPR 12, and wildCompared with the raw protease, the heat resistance of the raw protease is respectively improved by 31.0%, 35.5%, 35.6%, 45.5%, 39.0%, 54.0%, 49%, 51.0%, 56.5%, 58%, 55.5% and 60.0%, so that the heat resistance is obviously improved, the productivity of the raw protease in the high-temperature processing process is improved, and the raw protease is more beneficial to industrialization and application in various fields such as feed, food and the like.
Description
Technical Field
The invention belongs to the fields of genetic engineering and enzyme engineering, and in particular relates to a heat-resistant protease mutant, and a coding gene and application thereof.
Background
Proteases are a class of enzymes that catalyze the hydrolysis of peptide bonds in proteins, which are widely found in animals, plants and microorganisms and have many different physiological functions. Proteases are one of the most widely used enzyme preparations, such as the food industry, brewing, detergent industry, feed industry, leather industry, silk industry and pharmaceutical industry, and so forth, and therefore, there is a need for improved yields and properties of proteases in such environments.
Most proteases in the current market only have less than 20% of enzyme activity after being treated at 70 ℃, and the total temperature resistance is poor, so that the wide use of the proteases is limited. For example, in the production of feed, the enzyme preparation is granulated at high temperature after being mixed with the feed, and the enzyme is inactivated during this process. Therefore, it is important to improve the thermostability of the protease.
The error-prone PCR technology is that when DNA polymerase is adopted to carry out PCR reaction amplification of target fragments, mutation frequency in the amplification process is increased by adjusting reaction conditions, so that mutation is randomly introduced into target genes at a certain frequency, a mutant library is constructed, and therefore required forward mutants are screened. The error-prone PCR technique can be well applied to the molecular modification of proteins.
Disclosure of Invention
The invention provides a heat-resistant protease mutant, and a coding gene and application thereof. The invention constructs mutant library and directionally screens by error-prone PCR method, and the method is used forBacillus licheniformis The protease gene from WX-02 is improved, and a plurality of mutants with improved heat resistance are obtained through screening, so that the mutants are beneficial to improving the application of the mutants in the fields of feed, food and the like.
In order to achieve the aim of the invention, the invention is realized by adopting the following technical scheme:
the invention provides a heat-resistant protease mutant, which has one of the following amino acid sequences:
(1) As set forth in SEQ ID NO:3, an amino acid sequence shown in 3;
(2) As set forth in SEQ ID NO:5, and a polypeptide sequence shown in the figure;
(3) As set forth in SEQ ID NO: 7;
(4) As set forth in SEQ ID NO: 9;
(5) As set forth in SEQ ID NO:11, and a polypeptide comprising the amino acid sequence shown in seq id no;
(6) As set forth in SEQ ID NO:13, an amino acid sequence shown in seq id no;
(7) As set forth in SEQ ID NO:15, and a polypeptide comprising the amino acid sequence shown in seq id no;
(8) As set forth in SEQ ID NO:17, an amino acid sequence shown in seq id no;
(9) As set forth in SEQ ID NO:19, an amino acid sequence shown in seq id no;
(10) As set forth in SEQ ID NO:21, an amino acid sequence shown in seq id no;
(11) As set forth in SEQ ID NO:23, an amino acid sequence shown in seq id no;
(12) As set forth in SEQ ID NO:25, and a polypeptide comprising the amino acid sequence shown in seq id no.
Further, the thermostable protease mutant specifically comprises a sequence represented by SEQ ID NO:1 to valine at position 127 of a protease represented by formula (i); the amino acid sequence is shown as SEQ ID NO:1 to arginine; the amino acid sequence is shown as SEQ ID NO:1 to arginine from lysine at position 127 and glycine from aspartic acid at position 180 of the protease shown in FIG. 1; the amino acid sequence is shown as SEQ ID NO:1, and a blast 04 obtained by converting lysine at position 127 of the protease shown in fig. 1 into arginine and converting asparagine at position 352 into histidine; the amino acid sequence is shown as SEQ ID NO:1 to arginine for lysine at position 127 and proline for serine at position 363 of the protease shown in FIG. 1; the amino acid sequence is shown as SEQ ID NO:1, from lysine to arginine at position 127, glutamic acid to aspartic acid at position 216, and asparagine to serine at position 344; the amino acid sequence is shown as SEQ ID NO:1, a protease having a sequence of blast 07 obtained by converting lysine at position 127 to arginine, glutamic acid at position 216 to aspartic acid, asparagine at position 344 to serine, and tyrosine at position 247 to aspartic acid; the amino acid sequence is shown as SEQ ID NO:1, a protease of which lysine at position 127 is changed to arginine, glutamic acid at position 216 is changed to aspartic acid, asparagine at position 344 is changed to serine, and valine at position 149 is changed to glycine; the amino acid sequence is shown as SEQ ID NO:1, a protease having a sequence of blast 09 obtained by converting lysine at position 127 of the protease shown in fig. 1 into arginine, glutamic acid at position 216 into aspartic acid, asparagine at position 344 into serine, and tryptophan at position 217 into valine; the amino acid sequence is shown as SEQ ID NO:1, a protease having a sequence of a blast r10 wherein lysine at position 127 of the protease is changed to arginine, glutamic acid at position 216 is changed to aspartic acid, asparagine at position 344 is changed to serine, and proline at position 272 is changed to glutamic acid; the amino acid sequence is shown as SEQ ID NO:1, a protease of which lysine at position 127 is changed to arginine, glutamic acid at position 216 is changed to aspartic acid, asparagine at position 344 is changed to serine, and glycine at position 214 is changed to leucine; the amino acid sequence is shown as SEQ ID NO:1, a protease of which lysine at position 127 is changed to arginine, glutamic acid at position 216 is changed to aspartic acid, asparagine at position 344 is changed to serine, and alanine at position 118 is changed to methionine.
The invention also provides a coding gene of the heat-resistant protease mutant, which has one of the following nucleotide sequences:
(1) As set forth in SEQ ID NO:4, a nucleotide sequence shown in seq id no;
(2) As set forth in SEQ ID NO:6, a nucleotide sequence shown in seq id no;
(3) As set forth in SEQ ID NO:8, a nucleotide sequence shown in seq id no;
(4) As set forth in SEQ ID NO:10, a nucleotide sequence shown in seq id no;
(5) As set forth in SEQ ID NO:12, a nucleotide sequence shown in seq id no;
(6) As set forth in SEQ ID NO:14, a nucleotide sequence shown in seq id no;
(7) As set forth in SEQ ID NO:16, a nucleotide sequence shown in seq id no;
(8) As set forth in SEQ ID NO:18, a nucleotide sequence shown in seq id no;
(9) As set forth in SEQ ID NO:20, a nucleotide sequence shown in seq id no;
(10) As set forth in SEQ ID NO:22, a nucleotide sequence shown in seq id no;
(11) As set forth in SEQ ID NO:24, a nucleotide sequence shown in seq id no;
(12) As set forth in SEQ ID NO:26, and a nucleotide sequence shown in seq id no.
The invention also provides a recombinant expression vector containing the coding gene.
The invention also provides recombinant genetic engineering bacteria containing the coding gene.
Further, the genetically engineered bacteria are bacillus subtilis, bacillus licheniformis or bacillus amyloliquefaciens.
The invention also provides a preparation method of the heat-resistant protease mutant, which comprises the following steps:
(1) Connecting the encoding gene of the heat-resistant protease mutant to a pUB110 vector, transforming the obtained recombinant expression vector into competent cells, and screening positive clones by using a resistance marker to obtain recombinant genetic engineering bacteria;
(2) Carrying out shake flask fermentation on the recombinant genetically engineered bacteria, shake culturing, and fermenting to generate protease mutants;
(3) Inoculating the recombinant genetic engineering strain subjected to shaking fermentation into a fermentation tank for expansion fermentation culture to obtain heat-resistant protease mutants BLAPR01, BLAPR02, BLAPR03, BLAPR04, BLAPR05, BLAPR06, BLAPR07, BLAPR08, BLAPR09, BLAPR10, BLAPR11 or BLAPR12.
Further, the process of the expansion fermentation culture comprises the following steps: the pH is natural, the temperature is 37-40 ℃, the stirring speed is 600-800 rpm, the ventilation rate is 1.5 (v/v), and the dissolved oxygen is controlled to be more than 20%.
Further, the culture medium for the enlarged fermentation culture comprises the following components in percentage by weight: 5-10% of soybean meal, 1-5% of corn meal, 0.1-1.0% of PPG-20000.1-1.0% of protease, 0.1-1.0% of amylase and 0.2-0.5% of disodium hydrogen phosphate 12.
The invention also provides application of the heat-resistant protease mutant in preparing feed additives and/or food additives.
Further, the feed additive or the food additive contains at least one of heat-resistant protease mutants BLAPR01, BLAPR02, BLAPR03, BLAPR04, BLAPR05, BLAPR06, BLAPR07, BLAPR08, BLAPR09, BLAPR10, BLAPR11 and BLAPR12.
Compared with the prior art, the invention has the advantages and beneficial technical effects that:
the invention usesBacillus licheniformis The protease genes derived from WX-02 are provided as single-point mutants BLAPR01 and BLAPR02 comprising K127V, K127R, and as double-point mutants BLAPR 03-BLAPR 05 comprising K127R/D180G, K R/N352H, K R/S363P, as triple-point mutants BLAPR06 comprising K127R/E216D/N344S, as triple-point mutants BLAPR 07-BLAPR 12 comprising K127R/E216D/N344S/247D, K R/E216D/N344S/149G, K R/E216D/N344S/217V, K R/E216D/N344S/272E, K R/E216D/N344S/214L, K R/E216D/N344S/118M, respectively.
The modified mutant BLAPR01 to BLAPR12 has the heat resistance improved by 31.0%, 35.5%, 35.6%, 45.5%, 39.0%, 54.0%, 49%, 51.0%, 56.5%, 58%, 55.5% and 60.0% respectively when being treated for 3 minutes at 75 ℃, thus the heat resistance of the modified mutant BLAPR01 to BLAPR12 is obviously improved compared with that of wild protease, the activity loss is less in the processing, and the modified mutant BLAPR is more beneficial to industrialization and wide application in various fields such as feed, food and the like.
Drawings
FIG. 1 is a diagram showing construction and verification of recombinant bacteria of protease mutants in the present invention; wherein the left side A and B are recombinant bacteria for colony PCR verification, and the left side C is a control group; the recombinant bacteria of protease mutants BLAPR 01-BLAPR 06 are arranged on the right side 1-6, and the unmutated protease recombinant bacteria are arranged on the 0 side.
FIG. 2 is a comparison of thermostability of protease mutants of the present invention in a water bath at 75℃for 3 min.
FIG. 3 is fermentation data of protease mutants of the present invention in a 30L fermenter.
FIG. 4 shows the results of the activity retention of protease mutants of the present invention after 3min treatment at 80 ℃.
Description of the embodiments
The present invention will be described more fully hereinafter with reference to the accompanying drawings and examples, in which the invention is shown, but the scope of the invention is not limited to the specific examples.
The molecular biology experimental methods not specifically described in the following examples can be performed with reference to the specific methods listed in the "molecular cloning Experimental guidelines (third edition) J.Sam Brookfield, or according to the kit and product instructions. Reagents and biological materials used in the specific examples are commercially available unless otherwise specified.
1. Strain and vector
Bacillus subtilis WB600, plasmid pUB110, E.coli BL21, plasmid pET-21a (+) were purchased from Invitrogen corporation.
2. Reagent and culture medium
Plasmid extraction kits, fragment purification recovery kits, restriction enzymes, and the like are purchased from Takara bioengineering (Dalian) limited; geneMorph II random mutagenesis PCR kit was purchased from Stratagene Inc.; ampicillin, IPTG, etc. were purchased from the division of bioengineering (Shanghai); protein Marker: blue Plus II Protein Marker (14-120 kDa) was purchased from Beijing full gold Biotechnology Co.
LB medium formula: 1% tryptone, 0.5% yeast extract, 1% NaCl.
Fermentation medium: 50-80g/L of soybean meal, 60-100g/L of corn starch, 2-4g/L of disodium hydrogen phosphate, 1-2g/L of sodium carbonate and natural pH.
Example 1: error-prone PCR construction of protease mutant libraries
Reference toBacillus licheniformis The primers were designed for the amino acid sequence (SEQ ID NO: 1) and the DNA sequence (SEQ ID NO: 2) of WX-02-derived protease, the Xba I restriction enzyme site was designed at the 5 'end, and the 3' endBamH I restriction sites were counted.
Random mutation is carried out by using a GeneMorph II random mutation PCR kit and using a gene of SEQ ID NO:2 as a template, wherein the sequence of the used primer is as follows:
BLAPR-F：GCTCTAGAATGATGAGGAAGAAATC（SEQ ID NO:27）；
BLAPR-R：CGCGGATCCTTACTGTGCAGCTGCTTCGA（SEQ ID NO:28）。
the reaction conditions are as follows: pre-denaturation at 94℃for 3min, denaturation at 94℃for 30s, annealing at 58℃for 30s and elongation at 72℃for 1min for 30 cycles.
The amplified random mutation PCR product is digested with Xba I and BamH I, purified and recovered, and then connected to pET-21a (+) vector, and E.coli BL21-DE3 is transformed, ampicillin resistance LB plate is used for screening positive clone, thus obtaining pET-BLAPR0x. The synthesized original gene was ligated to pET-21a (+) vector in the same manner and transformed into E.coli BL21-DE3 to obtain pET-BLAPR0.
The screened single colonies were inoculated into 96-well deep well plates. 2 single colonies expressing BLAPR0 were inoculated per plate as controls. After 300. Mu.L of LB liquid medium (containing 100. Mu.g/mL ampicillin) was added to each well and shake-cultured at 37℃and 200rpm for 4 hours, 50. Mu.L of the bacterial liquid was transferred to a new 96-well plate for seed preservation, 200. Mu.L of LB-Amp medium containing IPTG was added to the remaining bacterial liquid of the plate, and the resulting mixture was subjected to shaking culture at 37℃and 200rpm for 10 hours to give a final concentration of 1mM of IPTG and a final concentration of 100. Mu.g/mL of ampicillin, thereby inducing protease expression.
Repeatedly freezing and thawing the induced bacterial liquid for crushing, centrifuging the crushed cell liquid, taking the supernatant, performing heat treatment (water bath at 75 ℃ for 3 min), and then detecting the residual activity of the protease. Mutant genes with residual enzyme activities higher than the control were sequenced.
Mutants K127V and K127R (named BLAPR01 and BLAPR 02) with improved heat resistance and taking BLAPR0 as a starting template are selected, and after sequencing, the amino acid sequence of the BLAPR01 is shown as SEQ ID NO. 3, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 4; the amino acid sequence of BLAPR02 is shown as SEQ ID NO. 5, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 6.
Example 2: construction of mutant library of protease BLAPR02 by second round error-prone PCR
A second round of random mutagenesis was performed using the protease gene BLAPR02 selected in example 1 as a template (BLAPR 02 has a higher heat resistance than BLAPR 01), and the construction process of the mutant library and the material reagents, primers and operating conditions used were the same as those used in example 1. The mutant was cultured and screened using BLAPR02 as a control, and the residual activity of the protease mutant was measured after heat treatment (water bath at 75℃for 3 min). Mutant genes with residual enzyme activities higher than BLAPR02 were sequenced.
The following mutants with improved heat resistance were finally selected:
blast 03: the mutation mode is K127R/D180G (the 127 th lysine is changed into arginine, the 180 th aspartic acid is changed into glycine), the amino acid sequence is shown as SEQ ID NO. 7, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 8;
blast 04: the mutation mode is K127R/N352H (the 127 th lysine is changed into arginine and the 352 th asparagine is changed into histidine), the amino acid sequence is shown as SEQ ID NO. 9, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 10;
blast 05: the mutation mode is K127R/S363P (the 127 th lysine is changed into arginine, the 363 rd serine is changed into proline), the amino acid sequence is shown as SEQ ID NO. 11, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 12;
blast 06: the mutation mode is K127R/E216D/N344S (the 127 th lysine is changed into arginine, the 216 th glutamic acid is changed into aspartic acid and the 344 th asparagine is changed into serine), the amino acid sequence is shown as SEQ ID NO. 13, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 14;
blast 07: the mutation mode is K127R/E216D/N344S/247D (the lysine at 127 th is changed into arginine, the glutamic acid at 216 th is changed into aspartic acid, the asparagine at 344 th is changed into serine and the tyrosine at 247 th is changed into aspartic acid), the amino acid sequence is shown as SEQ ID NO. 15, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 16;
blast 08: the mutation mode is K127R/E216D/N344S/149G (the lysine at 127 th is changed into arginine, the glutamic acid at 216 th is changed into aspartic acid, the asparagine at 344 th is changed into serine and the valine at 149 th is changed into glycine), the amino acid sequence is shown as SEQ ID NO. 17, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 18;
blast 09: the mutation mode is K127R/E216D/N344S/217V (the 127 th lysine is changed into arginine, the 216 th glutamic acid is changed into aspartic acid, the 344 th asparagine is changed into serine and the 217 th tryptophan is changed into valine), the amino acid sequence is shown as SEQ ID NO. 19, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 20;
blast 10: the mutation mode is K127R/E216D/N344S/272E (the 127 th lysine is changed into arginine, the 216 th glutamic acid is changed into aspartic acid, the 344 th asparagine is changed into serine and the 272 th proline is changed into glutamic acid), the amino acid sequence is shown as SEQ ID NO. 21, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 22;
blast 11: the mutation mode is K127R/E216D/N344S/214L (the 127 th lysine is changed into arginine, the 216 th glutamic acid is changed into aspartic acid, the 344 th asparagine is changed into serine and the 214 th glycine is changed into leucine), the amino acid sequence is shown as SEQ ID NO. 23, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 24;
blast 12: the mutation mode is K127R/E216D/N344S/118M (the 127 th lysine is changed into arginine, the 216 th glutamic acid is changed into aspartic acid, the 344 th asparagine is changed into serine and the 118 th alanine is changed into methionine), the amino acid sequence is shown as SEQ ID NO. 25, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 26.
Example 3: verification of expression of thermostable protease mutants in Bacillus subtilis
The mutants of examples 1 and 2, respectively, and BLAPR0 were cloned into the Xba I and BamH I sites of plasmid pUB110, respectively; referring to the Bacillus subtilis transformation method created by Spizizzen, the recombinant plasmid is transformed into Bacillus subtilis WB600 to obtain recombinant bacteria of BLAPR0 and BLAPR 01-BLAPR 06 mutants (shown in figure 1). After shaking flask fermentation of recombinant bacteria in a fermentation medium for 78 hours, centrifuging the culture solution to obtain a supernatant, measuring the average enzyme activity of the supernatant of each mutant fermentation broth, taking the fermentation supernatant of the transformant with the highest enzyme activity in each mutant, and comparing the enzyme activity retention rate after water bath treatment at 75 ℃ for 3 minutes.
The mutants BLAPR07 to BLAPR12 were transformed into Bacillus subtilis WB600 according to the same method as described above, and the enzyme activity retention after treatment in a water bath at 75℃for 3min was fermented and measured.
As a result, as shown in FIG. 2, the heat resistance of the mutant proteases BLAPR01 to BLAPR12 was improved by 31.0%, 35.5%, 35.6%, 45.5%, 39.0%, 54.0%, 49%, 51.0%, 56.5%, 58%, 55.5% and 60.0% when they were treated at 75℃for 3 minutes, respectively, as compared with the original proteases.
The above results indicate that mutation of Lys at position 127 of BLAPR0 to Val or Arg can improve the thermostability of the enzyme while maintaining the original enzyme activity. Mutants obtained by mutating Asp at position 180 of the mutant into Gly or mutants obtained by mutating Asn at position 352 of the mutant into His on the basis of BLAPR02; or a mutant obtained by mutating Ser at position 363 to Pro; or a mutant obtained by mutating Glu at position 216 to Asp and simultaneously mutating Asn at position 344 to Ser; or a mutant obtained by mutating Glu at position 216 thereof to Asp, mutating Asn at position 344 thereof to Ser and simultaneously mutating Tyr at position 247 thereof to Asp; or a mutant obtained by mutating Glu at position 216 to Asp, mutating Asn at position 344 to Ser and simultaneously mutating Val at position 149 to Gly; or a mutant obtained by mutating Glu at position 216 to Asp, mutating Asn at position 344 to Ser and simultaneously mutating Trp at position 217 to Val; or a mutant in which Glu at position 216 is changed to Asp, asn at position 344 is changed to Ser, and Pro at position 272 is changed to Glu; or a mutant obtained by mutating Glu at position 216 to Asp, mutating Asn at position 344 to Ser and simultaneously mutating Gly at position 214 to Leu; or a mutant in which Glu at position 216 is mutated to Asp, asn at position 344 is mutated to Ser and Ala at position 118 is mutated to Met, and the heat resistance is further improved. Therefore, compared with the wild type, the heat resistance of the mutated protease is greatly improved, and the mutated protease is more beneficial to the application in the fields of feed, food or washing.
Example 4: fermentation and preparation of protease mutants in a 30L fermenter
Recombinant bacteria expressing protease BLAPR0 and protease mutants BLAPR01, BLAPR02, BLAPR03, BLAPR04, BLAPR05 and BLAPR06 in example 3 were streaked on LB plates containing kanamycin (final concentration: 20. Mu.g/mL) resistance, cultured at 37℃until single colonies were grown, single colonies excellent in growth were picked up and streaked on LB plates containing kanamycin (final concentration: 20. Mu.g/mL) resistance, and thus three generations of the obtained recombinant Bacillus subtilis colonies were activated, inoculated in 50mL of LB medium containing kanamycin (final concentration: 20. Mu.g/mL), and cultured at 37℃and 200rpm for 24 hours. The seed solution was inoculated into 1L of LB medium containing kanamycin (final concentration: 20. Mu.g/mL) at an inoculum size of 2%, and cultured at 37℃and 200rpm until the OD600 became about 5, and used as a seed solution inoculation fermenter.
The fermentation production process comprises the following steps: 5-10% of bean pulp, 1-5% of corn meal, 0.1-1.0% of PPG-20000, 0.1-1.0% of protease, 0.1-1.0% of amylase, 0.2-0.5% of disodium hydrogen phosphate (12 water), natural pH, 37 ℃ of temperature, 600rpm of stirring speed, 1.5 (v/v) of ventilation quantity and more than 20% of dissolved oxygen. The pH of the fermentation process is natural, and the enzyme activity is measured after 24 hours of fermentation, and the measurement is carried out every 4 hours until the fermentation is finished (48 hours).
As shown in FIG. 3, the enzyme activity of the protease mutant was comparable to that of the wild-type protease BLAPR0, but the heat resistance of the protease mutant was better than that of the wild-type protease, as the fermentation time increased.
The blast process treatment was performed on blast 0 and the mutants blast 05 and blast 06, respectively, and the heat resistance of the blast powder, that is, the activity retention rate after the treatment at 80 ℃ for 3min was measured, and the results are shown in fig. 4, the blast 05 and blast 06 mutants were significantly improved in heat resistance compared with blast 0.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be apparent to one skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Claims (10)
1. A thermostable protease mutant, characterized in that the thermostable protease mutant has one of the following amino acid sequences:
(1) As set forth in SEQ ID NO:3, an amino acid sequence shown in 3;
(2) As set forth in SEQ ID NO:5, and a polypeptide sequence shown in the figure;
(3) As set forth in SEQ ID NO: 7;
(4) As set forth in SEQ ID NO: 9;
(5) As set forth in SEQ ID NO:11, and a polypeptide comprising the amino acid sequence shown in seq id no;
(6) As set forth in SEQ ID NO:13, an amino acid sequence shown in seq id no;
(7) As set forth in SEQ ID NO:15, and a polypeptide comprising the amino acid sequence shown in seq id no;
(8) As set forth in SEQ ID NO:17, an amino acid sequence shown in seq id no;
(9) As set forth in SEQ ID NO:19, an amino acid sequence shown in seq id no;
(10) As set forth in SEQ ID NO:21, an amino acid sequence shown in seq id no;
(11) As set forth in SEQ ID NO:23, an amino acid sequence shown in seq id no;
(12) As set forth in SEQ ID NO:25, and a polypeptide comprising the amino acid sequence shown in seq id no.
2. The thermostable protease mutant according to claim 1, characterized in that the thermostable protease mutant consists of the amino acid sequence as set forth in SEQ ID NO:1 to valine at position 127 of a protease represented by formula (i); the amino acid sequence is shown as SEQ ID NO:1 to arginine; the amino acid sequence is shown as SEQ ID NO:1 to arginine from lysine at position 127 and glycine from aspartic acid at position 180 of the protease shown in FIG. 1; the amino acid sequence is shown as SEQ ID NO:1, and a blast 04 obtained by converting lysine at position 127 of the protease shown in fig. 1 into arginine and converting asparagine at position 352 into histidine; the amino acid sequence is shown as SEQ ID NO:1 to arginine for lysine at position 127 and proline for serine at position 363 of the protease shown in FIG. 1; the amino acid sequence is shown as SEQ ID NO:1, from lysine to arginine at position 127, glutamic acid to aspartic acid at position 216, and asparagine to serine at position 344; the amino acid sequence is shown as SEQ ID NO:1, a protease having a sequence of blast 07 obtained by converting lysine at position 127 to arginine, glutamic acid at position 216 to aspartic acid, asparagine at position 344 to serine, and tyrosine at position 247 to aspartic acid; the amino acid sequence is shown as SEQ ID NO:1, a protease of which lysine at position 127 is changed to arginine, glutamic acid at position 216 is changed to aspartic acid, asparagine at position 344 is changed to serine, and valine at position 149 is changed to glycine; the amino acid sequence is shown as SEQ ID NO:1, a protease having a sequence of blast 09 obtained by converting lysine at position 127 of the protease shown in fig. 1 into arginine, glutamic acid at position 216 into aspartic acid, asparagine at position 344 into serine, and tryptophan at position 217 into valine; the amino acid sequence is shown as SEQ ID NO:1, a protease having a sequence of a blast r10 wherein lysine at position 127 of the protease is changed to arginine, glutamic acid at position 216 is changed to aspartic acid, asparagine at position 344 is changed to serine, and proline at position 272 is changed to glutamic acid; the amino acid sequence is shown as SEQ ID NO:1, a protease of which lysine at position 127 is changed to arginine, glutamic acid at position 216 is changed to aspartic acid, asparagine at position 344 is changed to serine, and glycine at position 214 is changed to leucine; the amino acid sequence is shown as SEQ ID NO:1, a protease of which lysine at position 127 is changed to arginine, glutamic acid at position 216 is changed to aspartic acid, asparagine at position 344 is changed to serine, and alanine at position 118 is changed to methionine.
3. The thermostable protease mutant encoding gene according to claim 1, wherein the encoding gene has one of the following nucleotide sequences:
(1) As set forth in SEQ ID NO:4, a nucleotide sequence shown in seq id no;
(2) As set forth in SEQ ID NO:6, a nucleotide sequence shown in seq id no;
(3) As set forth in SEQ ID NO:8, a nucleotide sequence shown in seq id no;
(4) As set forth in SEQ ID NO:10, a nucleotide sequence shown in seq id no;
(5) As set forth in SEQ ID NO:12, a nucleotide sequence shown in seq id no;
(6) As set forth in SEQ ID NO:14, a nucleotide sequence shown in seq id no;
(7) As set forth in SEQ ID NO:16, a nucleotide sequence shown in seq id no;
(8) As set forth in SEQ ID NO:18, a nucleotide sequence shown in seq id no;
(9) As set forth in SEQ ID NO:20, a nucleotide sequence shown in seq id no;
(10) As set forth in SEQ ID NO:22, a nucleotide sequence shown in seq id no;
(11) As set forth in SEQ ID NO:24, a nucleotide sequence shown in seq id no;
(12) As set forth in SEQ ID NO:26, and a nucleotide sequence shown in seq id no.
4. A recombinant expression vector comprising the coding gene of claim 3.
5. A recombinant genetically engineered bacterium comprising the coding gene of claim 3.
6. The method for producing a thermostable protease mutant according to claim 2, comprising the steps of:
(1) Connecting the encoding gene of the heat-resistant protease mutant to a pUB110 vector, transforming the obtained recombinant expression vector into competent cells, and screening positive clones by using a resistance marker to obtain recombinant genetic engineering bacteria;
(2) Carrying out shake flask fermentation on the recombinant genetically engineered bacteria, shake culturing, and fermenting to generate protease mutants;
(3) Inoculating the recombinant genetic engineering strain subjected to shaking fermentation into a fermentation tank for expansion fermentation culture to obtain heat-resistant protease mutants BLAPR01, BLAPR02, BLAPR03, BLAPR04, BLAPR05, BLAPR06, BLAPR07, BLAPR08, BLAPR09, BLAPR10, BLAPR11 or BLAPR12.
7. The method according to claim 6, wherein the process of the enlarged fermentation culture is as follows: the pH is natural, the temperature is 37-40 ℃, the stirring speed is 600-800 rpm, the ventilation rate is 1.5 (v/v), and the dissolved oxygen is controlled to be more than 20%.
8. The preparation method of claim 6, wherein the medium for the expanded fermentation culture comprises the following components in percentage by weight: 5-10% of soybean meal, 1-5% of corn meal, 0.1-1.0% of PPG-20000.1-1.0% of protease, 0.1-1.0% of amylase and 0.2-0.5% of disodium hydrogen phosphate 12.
9. Use of the thermostable protease mutant according to claim 1 or 2 for the preparation of feed additives and/or food additives.
10. The use according to claim 9, wherein the feed additive or food additive comprises at least one of the thermostable protease mutants BLAPR01, BLAPR02, BLAPR03, BLAPR04, BLAPR05, BLAPR06, BLAPR07, BLAPR08, BLAPR09, BLAPR10, BLAPR11 and BLAPR12.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310770625.5A CN116790564A (en) | 2023-06-28 | 2023-06-28 | Heat-resistant protease mutant and encoding gene and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310770625.5A CN116790564A (en) | 2023-06-28 | 2023-06-28 | Heat-resistant protease mutant and encoding gene and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116790564A true CN116790564A (en) | 2023-09-22 |
Family
ID=88044743
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310770625.5A Pending CN116790564A (en) | 2023-06-28 | 2023-06-28 | Heat-resistant protease mutant and encoding gene and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116790564A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118516332A (en) * | 2024-07-22 | 2024-08-20 | 上海先赛生物科技有限公司 | Cpf1 mutants and uses thereof |
| CN118516332B (en) * | 2024-07-22 | 2024-10-29 | 上海先赛生物科技有限公司 | Cpf1 mutants and uses thereof |
-
2023
- 2023-06-28 CN CN202310770625.5A patent/CN116790564A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118516332A (en) * | 2024-07-22 | 2024-08-20 | 上海先赛生物科技有限公司 | Cpf1 mutants and uses thereof |
| CN118516332B (en) * | 2024-07-22 | 2024-10-29 | 上海先赛生物科技有限公司 | Cpf1 mutants and uses thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114561375B (en) | Protease mutant BLAPR2 with improved thermal stability, and encoding gene and application thereof | |
| WO2022134236A1 (en) | Alkaline protease mutant, gene thereof, engineering bacteria thereof, preparation method therefor and use thereof | |
| CN114107266B (en) | Protease mutant with improved heat resistance, encoding gene and application thereof | |
| KR102183558B1 (en) | Gene encoding L-alanyl-L-glutamine biosynthetic enzyme and use thereof | |
| CN114480352B (en) | Alkaline protease mutant and application thereof | |
| CN110982807B (en) | High-efficiency stable cellulase mutant | |
| CN107586764B (en) | Glutamine transaminase mutant, gene, engineering bacteria and preparation method thereof | |
| CN110042092B (en) | Xylanase mutant with improved stability and coding gene and application thereof | |
| CN109943546B (en) | Glutamine transaminase mutant and preparation method and application thereof | |
| CN112522173A (en) | Engineering bacterium for producing heterologous alkaline protease and construction method thereof | |
| CN108004220B (en) | Alkaline protease BmP mutant for improving thermal stability and gene and application thereof | |
| CN113151270A (en) | Promoter for efficiently expressing alkaline protease and application thereof | |
| CN111153968A (en) | Signal peptide mutant for improving expression quantity of exogenous alkaline protease and construction method and application thereof | |
| CN111944790B (en) | Neutral protease gene, neutral protease, preparation method and application thereof | |
| CN113699138A (en) | Alkaline protease gene, hybrid promoter, recombinant expression vector, recombinant expression engineering bacterium, alkaline protease, method and application | |
| CN116790564A (en) | Heat-resistant protease mutant and encoding gene and application thereof | |
| CN110878293B (en) | Application of bacillus licheniformis with deletion of yceD gene in production of heterologous protein | |
| CN117904082A (en) | Protease with improved acid resistance, mutant and application thereof | |
| CN115927267B (en) | Bile acid complex enzyme preparation and application thereof | |
| CN117535273B (en) | Temperature-sensitive alkaline protease variants and uses thereof | |
| CN115927264B (en) | Keratinase, compound enzyme preparation of keratinase and eucommia ulmoides leaf extract and bile acid and application of compound enzyme preparation | |
| CN114350643B (en) | Recombinant strain for producing aminopeptidase and application of recombinant strain in efficient proteolysis | |
| CN115125247B (en) | Combined promoter palpha 2-alpha 2 and application thereof | |
| CN113801831B (en) | Bacillus subtilis capable of producing neutral protease with high yield and application thereof | |
| CN115125248B (en) | Combined promoter pctsR-alpha 2 and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |