CN116218817A - Beta-mannase mutant beta-ManM, additive prepared from beta-mannase mutant beta-ManM and bile acid and application of additive - Google Patents
Beta-mannase mutant beta-ManM, additive prepared from beta-mannase mutant beta-ManM and bile acid and application of additive Download PDFInfo
- Publication number
- CN116218817A CN116218817A CN202211721662.9A CN202211721662A CN116218817A CN 116218817 A CN116218817 A CN 116218817A CN 202211721662 A CN202211721662 A CN 202211721662A CN 116218817 A CN116218817 A CN 116218817A
- Authority
- CN
- China
- Prior art keywords
- beta
- manm
- mannanase
- mutant
- seq
- 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.)
- Granted
Links
- 239000000654 additive Substances 0.000 title claims abstract description 23
- 230000000996 additive effect Effects 0.000 title claims abstract description 22
- HSINOMROUCMIEA-FGVHQWLLSA-N (2s,4r)-4-[(3r,5s,6r,7r,8s,9s,10s,13r,14s,17r)-6-ethyl-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]-2-methylpentanoic acid Chemical compound C([C@@]12C)C[C@@H](O)C[C@H]1[C@@H](CC)[C@@H](O)[C@@H]1[C@@H]2CC[C@]2(C)[C@@H]([C@H](C)C[C@H](C)C(O)=O)CC[C@H]21 HSINOMROUCMIEA-FGVHQWLLSA-N 0.000 title claims abstract description 14
- 239000003613 bile acid Substances 0.000 title claims abstract description 14
- 108010055059 beta-Mannosidase Proteins 0.000 claims abstract description 59
- 102100032487 Beta-mannosidase Human genes 0.000 claims abstract description 49
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 13
- 241000251468 Actinopterygii Species 0.000 claims abstract description 10
- 238000009360 aquaculture Methods 0.000 claims abstract description 8
- 244000144974 aquaculture Species 0.000 claims abstract description 8
- 238000002156 mixing Methods 0.000 claims abstract description 4
- 238000000855 fermentation Methods 0.000 claims description 18
- 230000004151 fermentation Effects 0.000 claims description 18
- 150000001413 amino acids Chemical group 0.000 claims description 12
- 239000002773 nucleotide Substances 0.000 claims description 7
- 125000003729 nucleotide group Chemical group 0.000 claims description 7
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 6
- 229930182817 methionine Natural products 0.000 claims description 6
- 241001465754 Metazoa Species 0.000 claims description 5
- 229920001184 polypeptide Polymers 0.000 claims description 5
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 5
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 5
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 4
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 4
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 4
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 claims description 2
- 241000238557 Decapoda Species 0.000 claims description 2
- 239000004471 Glycine Substances 0.000 claims description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 claims description 2
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims description 2
- 229960001230 asparagine Drugs 0.000 claims description 2
- 235000009582 asparagine Nutrition 0.000 claims description 2
- 235000018417 cysteine Nutrition 0.000 claims description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 2
- 239000013505 freshwater Substances 0.000 claims description 2
- 241000194108 Bacillus licheniformis Species 0.000 abstract description 5
- 244000063299 Bacillus subtilis Species 0.000 abstract description 5
- 235000014469 Bacillus subtilis Nutrition 0.000 abstract description 5
- 238000012216 screening Methods 0.000 abstract description 5
- 230000004584 weight gain Effects 0.000 abstract description 4
- 235000019786 weight gain Nutrition 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 125000003275 alpha amino acid group Chemical group 0.000 abstract 3
- 230000003248 secreting effect Effects 0.000 abstract 1
- 108090000790 Enzymes Proteins 0.000 description 16
- 102000004190 Enzymes Human genes 0.000 description 16
- 230000035772 mutation Effects 0.000 description 15
- 230000000694 effects Effects 0.000 description 14
- 239000007788 liquid Substances 0.000 description 7
- 229920000057 Mannan Polymers 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 241000196324 Embryophyta Species 0.000 description 4
- 239000003674 animal food additive Substances 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
- LUEWUZLMQUOBSB-GFVSVBBRSA-N mannan Chemical class O[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@H]3[C@H](O[C@@H](O)[C@@H](O)[C@H]3O)CO)[C@@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O LUEWUZLMQUOBSB-GFVSVBBRSA-N 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 241000252210 Cyprinidae Species 0.000 description 3
- 244000046052 Phaseolus vulgaris Species 0.000 description 3
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 3
- 229960000723 ampicillin Drugs 0.000 description 3
- 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 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- DKPMWHFRUGMUKF-UHFFFAOYSA-N (3alpha,5alpha,6alpha,7alpha)-3,6,7-Trihydroxycholan-24-oic acid Natural products OC1C(O)C2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)CC2 DKPMWHFRUGMUKF-UHFFFAOYSA-N 0.000 description 2
- RUDATBOHQWOJDD-UHFFFAOYSA-N (3beta,5beta,7alpha)-3,7-Dihydroxycholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)CC2 RUDATBOHQWOJDD-UHFFFAOYSA-N 0.000 description 2
- JOYGXTIHTHBSOA-UHFFFAOYSA-N 1-(4-chlorophenyl)-3-thiophen-2-ylprop-2-en-1-one Chemical compound C1=CC(Cl)=CC=C1C(=O)C=CC1=CC=CS1 JOYGXTIHTHBSOA-UHFFFAOYSA-N 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 229920002488 Hemicellulose Polymers 0.000 description 2
- DGABKXLVXPYZII-UHFFFAOYSA-N Hyodeoxycholic acid Natural products C1C(O)C2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)CC2 DGABKXLVXPYZII-UHFFFAOYSA-N 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- RUDATBOHQWOJDD-BSWAIDMHSA-N chenodeoxycholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 RUDATBOHQWOJDD-BSWAIDMHSA-N 0.000 description 2
- 229960001091 chenodeoxycholic acid Drugs 0.000 description 2
- 235000005822 corn Nutrition 0.000 description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
- DGABKXLVXPYZII-SIBKNCMHSA-N hyodeoxycholic acid Chemical compound C([C@H]1[C@@H](O)C2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 DGABKXLVXPYZII-SIBKNCMHSA-N 0.000 description 2
- 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 2
- 244000005700 microbiome Species 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 102220000766 rs121434513 Human genes 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 101150084750 1 gene Proteins 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 241000186000 Bifidobacterium Species 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 108090000371 Esterases Proteins 0.000 description 1
- 235000019733 Fish meal Nutrition 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- 238000009004 PCR Kit Methods 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 235000019779 Rapeseed Meal Nutrition 0.000 description 1
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 102000005840 alpha-Galactosidase Human genes 0.000 description 1
- 108010030291 alpha-Galactosidase Proteins 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 230000000433 anti-nutritional effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 102000006995 beta-Glucosidase Human genes 0.000 description 1
- 108010047754 beta-Glucosidase Proteins 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000007799 cork Substances 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000004467 fishmeal Substances 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000001965 increasing effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000007413 intestinal health Effects 0.000 description 1
- 108010059345 keratinase Proteins 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 235000012054 meals Nutrition 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000004456 rapeseed meal Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 235000015099 wheat brans Nutrition 0.000 description 1
- 229920001221 xylan Polymers 0.000 description 1
- 150000004823 xylans Chemical class 0.000 description 1
Images
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)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/2488—Mannanases
- C12N9/2494—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
-
- 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
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
-
- 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
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01078—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
-
- 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)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Polymers & Plastics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Animal Husbandry (AREA)
- Food Science & Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Marine Sciences & Fisheries (AREA)
- Physics & Mathematics (AREA)
- Birds (AREA)
- Insects & Arthropods (AREA)
- Medicinal Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
- Fodder In General (AREA)
Abstract
The invention discloses a beta-mannase mutant beta-ManM, an additive for preparing bile acid and application thereof. The invention mutates mannanase beta-Man from bacillus licheniformis, and the amino acid sequence is obtained by screening and is shown as SEQ ID NO:3, wherein the mannanase mutant beta-ManM 1 has an amino acid sequence shown in SEQ ID NO:5 and the amino acid sequence of the beta-ManM 2 is shown as SEQ ID NO:7, and performing high-efficiency secretory expression in bacillus subtilis. Compared with the original gene, the mannanase mutant has the advantages that the heat stability is respectively improved by 32.5%, 35.0% and 50%, and the additive obtained by mixing the mannanase mutant with bile acid is low in cost, can obviously improve the weight gain rate of fish fries and reduce the bait coefficient of the fish fries, and is very beneficial to aquaculture.
Description
Technical Field
The invention belongs to the field of genetic engineering, and in particular relates to a beta-mannase mutant beta-ManM, an additive for preparing bile acid and application thereof.
Background
Mannans are the main component of plant hemicellulose, mainly existing in cork plants and some special structures such as plant seeds, are the main component of cell walls of various plants and microorganisms, and are hemicellulose with the content inferior to that of xylan in nature. Because of the diversity and structural complexity of mannans, their complete hydrolysis requires the synergistic action of a variety of enzymes to accomplish, including endo- β -mannanase, exo- β -mannosidase, β -glucosidase, acetylmannanase esterase, and α -galactosidase, among others. Among them, endo-beta-mannase can degrade beta-1, 4-glycosidic bond of mannan main chain, and is the enzyme playing the most important role in the degradation process of mannan.
Beta-mannanases (EC 3.2.1.78) are capable of randomly hydrolyzing the beta-1, 4-mannosidic bond of mannans, producing low molecular weight mannooligosaccharides. The beta-mannase can reduce the anti-nutritional factor effect of the mannans, and the degradation product mannooligosaccharide can promote the growth of lactobacillus and bifidobacterium, thereby being beneficial to the intestinal health of animals. However, most mannanases in the current market are not ideal in terms of heat resistance, enzyme activity and the like, and in addition, the beta-mannanases in the domestic and foreign markets have low enzyme activity, low yield and high relative price of products, so that the application range of the beta-mannanases is limited, and the problem needs to be solved urgently.
The microorganism is the most main production source of the enzyme, and the temperature stability of the beta-mannase produced by bacteria is better than that of the fungus, especially the mannase from bacillus licheniformis and the like, but the enzyme has low yield in bacillus licheniformis and is difficult to meet the market demand.
Disclosure of Invention
In order to solve the problems in the background technology, the invention provides a beta-mannase mutant beta-ManM, an additive for preparing the beta-mannase mutant beta-ManM and bile acid and application of the beta-mannase mutant beta-ManM. The beta-mannase mutants beta-ManM 1, beta-ManM 2 and beta-ManM 3 have better thermal stability, and the additive prepared by mixing the beta-mannase mutants beta-ManM 1, beta-ManM 2 and beta-ManM 3 with bile acid has good weight increasing effect in aquaculture.
In order to achieve the aim of the invention, the invention is realized by adopting the following technical scheme:
the invention provides a beta-mannase mutant beta-ManM, the amino acid sequence of which is shown in SEQ ID NO:3, or as set forth in SEQ ID NO:5, or as set forth in SEQ ID NO: shown at 7.
Further, the beta-mannase mutant beta-ManM specifically comprises a polypeptide sequence with an amino acid sequence of SEQ ID NO:1 into methionine at the 68 th position of mannanase.
Furthermore, the beta-mannase mutant beta-ManM specifically comprises a sequence represented by SEQ ID NO:1, changing tyrosine at 68 th site of mannanase shown in the formula 1 into methionine and changing glycine at 97 th site into cysteine to obtain mannanase mutant beta-ManM 2; consists of a polypeptide with an amino acid sequence of SEQ ID NO:1, wherein tyrosine at the 68 th position of mannanase shown in the formula 1 is changed into methionine, asparagine at the 273 th position of mannanase is changed into proline, and the mannanase mutant beta-ManM 3 is obtained.
The invention provides a gene for encoding the beta-mannase mutant beta-ManM, and the nucleotide sequence of the gene is shown in SEQ ID NO:4, or as set forth in SEQ ID NO:6, or as set forth in SEQ ID NO: shown at 8.
The invention provides a recombinant engineering strain containing the gene.
The invention provides an additive containing the beta-mannase mutant beta-ManM, which also contains bile acid.
Further, the additive is prepared by mixing fermentation liquor prepared by fermenting recombinant engineering strains containing beta-mannase mutants beta-ManM with bile acid in a volume ratio of 3-5:1.
The invention provides an application of the beta-mannase mutant beta-ManM or the additive in aquaculture.
Furthermore, the dosage of the beta-mannase mutant beta-ManM or the additive is 0.02-0.1% of the weight of the basic ration of the aquatic animal.
Further, the aquatic animals are freshwater fish, shrimp and crab.
Compared with the prior art, the invention has the advantages and technical effects that:
the invention takes mannanase beta-Man (GenBank: GQ 859482.1) from bacillus licheniformis as a basis for mutation improvement, and mannanase mutant beta-ManM 1 with mutation sites of single-point mutation Y68M, mannanase mutant beta-ManM 2 with double-point mutation Y68M/G97C and mannanase mutant beta-ManM 3 with mutation sites of Y68M/N273P are obtained through screening. Compared with the original gene, the heat stability of the mannanase mutant obtained by the invention is respectively improved by 32.5%, 35.0% and 50%, and the enzyme production level reaches 12800 ten thousand U/mL. The invention also utilizes the fermentation liquor obtained by fermenting the engineering bacteria containing mannanase beta-Man to compound with bile acid, and the invention can not only obviously improve the weight gain rate of fish fries, but also reduce the bait coefficient thereof when used in aquaculture, and the additive has low cost and is very beneficial to aquaculture.
Drawings
FIG. 1 shows the amplification results of mannanase gene.
FIG. 2 is a comparison of the thermal stability of mannanase mutations.
FIG. 3 shows the loss of enzyme activity of mannanase mutant beta-ManM 3 at 80℃incubation.
FIG. 4 is data of the fermentation of mannanase mutant beta-ManM 3 in a 30L fermenter.
Detailed Description
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.
The invention adopts a determination method in a spectrophotometry for determining the activity of beta-mannase of a feed additive GB/T36861-2018.
Example 1: construction and screening of mannanase mutant genes
Referring to the amino acid sequence of mannanase beta-Man (GenBank: GQ 859482.1) of bacillus licheniformis, translating the mannanase beta-Man into a corresponding nucleotide sequence, and after gene sequence optimization, artificially synthesizing the mannanase beta-Man by the synthetic nucleotide sequence shown in SEQ ID NO:2, the amino acid sequence is shown as SEQ ID NO: 1. Primers were designed as follows, ecoR I restriction sites were designed at the 5 'end and Not I restriction sites were designed at the 3' end.
β-Man-F1：CCGGAATTCCACACCGTTTCTCCGGTGAA(SEQ ID NO：9)；
β-Man-R1：ATAAGCGGCCGCTTATTCCACGACAGGCGTCA(SEQ ID NO：10)。
Random mutation is carried out by using a GeneMorph II random mutation PCR kit and using an artificially synthesized gene as a template, and the PCR amplification result is shown in figure 1 by using the primer pair designed as above.
The amplified random mutation PCR product is digested with EcoR I and Not I, purified and recovered, and then connected to a pET-21a (+) vector, and escherichia coli BL21-DE3 is transformed, and ampicillin resistance LB plates are used for screening positive clones to obtain pET-beta-ManMx. The synthesized original gene is connected to a pET-21a (+) vector and transformed into escherichia coli BL21-DE3 by the same method to obtain pET-beta-Man 0.
The screened single colonies were inoculated into 96-well deep well plates. 2 single colonies expressing β -Man0 were inoculated per plate as controls. 300uL of LB liquid medium (containing 100 mug/mL of ampicillin) is filled into each hole, after shaking culture is carried out for 4 hours at 37 ℃ and 200rpm, 50uL of bacterial liquid is transferred to a new 96-hole flat plate for seed preservation, 200uL of LB-Amp medium containing IPTG is added into the remaining bacterial liquid of the flat plate, the final concentration of the IPTG is 1mM, the final concentration of the ampicillin is 100 mug/mL, and the mannanase is induced to be expressed by shaking culture for 10 hours at 200rpm at 37 ℃.
Repeatedly freezing and thawing the induced bacterial liquid for crushing, centrifuging the crushed cell liquid to obtain supernatant, and detecting the enzyme activity and heat resistance of mannanase. Mutant genes with higher thermostability than the control were sequenced. After sequencing, the mannanase mutant beta-ManM 1 is obtained, and the amino acid sequence of the mannanase mutant beta-ManM 1 is shown as SEQ ID NO:3, the corresponding nucleotide sequence is shown as SEQ ID NO:4 is shown in the figure; the mutation pattern of beta-ManM 1 is Y68M.
Example 2: random mutation by taking mannanase mutant beta-ManM 1 gene as template
The mutant beta-ManM 1 obtained in example 1 was used as a template, the same random mutation method as in example 1 was used to carry out the second round of mutation and screening, and beta-ManM 1 was used as a control, and heat resistance of mannanase was detected to obtain two mutants beta-ManM 2 and beta-ManM 3 with improved heat stability. Through sequencing, the amino acid sequence of the beta-ManM 2 is shown as SEQ ID NO:5, the nucleotide sequence is shown as SEQ ID NO:6, the mutation mode is Y68M/G97C; the amino acid sequence of the beta-ManM 3 is shown as SEQ ID NO:7, the nucleotide sequence is shown as SEQ ID NO:8, the mutation mode is Y68M/N273P.
Example 3: expression verification of mannanase mutant in bacillus subtilis
The mannanase mutant is amplified by adopting primers beta-Man-F2 and beta-Man-R2 respectively, cloned into Xho I and BamH I sites of plasmid pWB980, and transformed into bacillus subtilis WB600 by referring to a bacillus subtilis transformation method established by Spizizezen to obtain recombinant bacteria. The recombinant bacteria are subjected to shaking flask fermentation for 78 hours in a fermentation medium (the formula is that bean pulp powder is 50-80g/L, corn starch is 60-100g/L, disodium hydrogen phosphate is 2-4g/L, sodium carbonate is 1-2g/L and pH is natural), and the culture solution is centrifuged to obtain a supernatant for later use. The primer sequences were as follows:
β-Man-F2：CCGCTCGAGCCACACCGTTTCTCCGGTGAA(SEQ ID NO：11)；
β-Man-R2：CGCGGATCCTTATTCCACGACAGGCGTCA(SEQ ID NO：12)。
according to the method for determining mannanase in national standard, taking supernatant after fermentation broth centrifugation, determining average enzyme activity of supernatant of each mutant fermentation broth, taking supernatant of fermentation of transformant with highest enzyme activity in each mutant, and comparing enzyme activity retention rate after water bath treatment at 80 ℃ for 3 min.
As shown in FIG. 2, the heat stability of the mannanase mutant is improved by 32.5%, 35.0% and 50% respectively compared with that of the control group, and the enzyme activity residual rate of the beta-ManM 3 is obviously higher than that of the control group (FIG. 3) under the water bath heat preservation at 80 ℃.
Example 4: mannanase mutant beta-ManM 3 fermentation and preparation in 30L fermenter
The genetically engineered bacterium expressing the mannanase mutant beta-ManM 3 of example 3 was streaked on LB plate containing kanamycin resistance (final concentration: 20. Mu.g/mL), cultured at 37℃until single colony developed, single colony with good growth was picked up and continued streaking on LB plate containing kanamycin resistance (final concentration: 20. Mu.g/mL), so-activated for three generations, and the obtained recombinant Bacillus subtilis colony was inoculated in 50mL LB medium containing kanamycin final concentration: 20. Mu.g/mL, and cultured at 37℃and 200rpm for 24 hours. The resulting culture was inoculated into 1L of LB medium containing kanamycin at a final concentration of 20. Mu.g/mL at 2% of the inoculum size, and cultured at 37℃and 200rpm until the OD600 became about 5, and used as a seed liquid 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 keratinase, 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 value in the fermentation process is natural, the enzyme activity is measured after fermentation is carried out for 24 hours, and the fermentation liquid is obtained after the fermentation is finished (generally 48 hours).
The fermentation process curve is shown in fig. 4: sampling every 8h, measuring enzyme production level, fermenting for 48h, and reaching the highest enzyme activity level.
Example 5: application experiment of mannanase mutant-containing additive in fish fry culture
The fermentation broth containing the mannanase mutant in example 4 was uniformly mixed with bile acid (the active ingredient composition includes hyocholic acid, hyodeoxycholic acid and chenodeoxycholic acid, wherein the sum of the hyocholic acid and the hyodeoxycholic acid is 78.0% by weight, the chenodeoxycholic acid is 20.0% by weight, and the balance is water and ash) in a volume ratio of 3:1 (the units of the fermentation broth and the bile acid are ml), to obtain a feed additive which can be used for aquaculture.
400 fish fries with weight close to that of the carps are taken, the weight of the carps is 9.0-10.0g, and the carps are randomly divided into 4 groups. Each group had 5 replicates, 20 replicates each. Basic ration (10% of fish meal, 25% of bean pulp, 20% of rapeseed meal, 5% of cotton pulp, 25% of corn germ cake and 15% of wheat bran) is fed, pre-feeding is carried out for 5 days, and proper adjustment is carried out on fish fries, so that the cultivation experiment is carried out for 42 days.
The control group is fed with normal basic ration, and 0.02% of feed additives containing mannanase mutants beta-ManM 1, beta-ManM 2 or beta-ManM 3 are added into the daily ration of the test group, wherein the feed additives comprise the test group 1, the test group 2 and the test group 3. After 42 days of cultivation, all fries of the control group and the experimental group are hungry cultivated for 24 hours, and are weighed after being respectively fished out and absorbed with the surface moisture of the fish body, and the average weight, the weight gain rate and the bait coefficient of each group are counted.
The results are shown in Table 1, it can be seen that the additive containing the mannanase mutant can obviously improve the weight gain rate of fish fries and reduce the bait coefficient to a certain extent, so that the mannanase mutant has important application value in the field of aquaculture.
TABLE 1 Effect of mutant-containing additives on fry growth Properties
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. The beta-mannase mutant beta-ManM is characterized in that the amino acid sequence of the beta-mannase mutant beta-ManM is shown in SEQ ID NO:3, or as set forth in SEQ ID NO:5, or as set forth in SEQ ID NO: shown at 7.
2. The beta-mannanase mutant beta-ManM according to claim 1, characterized in that it consists in particular of a polypeptide having the amino acid sequence of SEQ ID NO:1 into methionine at the 68 th position of mannanase.
3. The β -mannanase mutant β -ManM according to claim 1, characterized in that it is further specifically a polypeptide consisting of the amino acid sequence of SEQ ID NO:1, changing tyrosine at 68 th site of mannanase shown in the formula 1 into methionine and changing glycine at 97 th site into cysteine to obtain mannanase mutant beta-ManM 2; consists of a polypeptide with an amino acid sequence of SEQ ID NO:1, wherein tyrosine at the 68 th position of mannanase shown in the formula 1 is changed into methionine, asparagine at the 273 th position of mannanase is changed into proline, and the mannanase mutant beta-ManM 3 is obtained.
4. A gene encoding the beta-mannanase mutant beta-ManM of any one of claims 1-3, wherein the nucleotide sequence of the gene is as set forth in SEQ ID NO:4, or as set forth in SEQ ID NO:6, or as set forth in SEQ ID NO: shown at 8.
5. A recombinant engineering strain comprising the gene of claim 4.
6. An additive comprising the β -mannanase mutant β -ManM of claim 1, wherein the additive further comprises a bile acid.
7. The additive according to claim 6, wherein the additive is prepared by mixing fermentation liquor prepared by fermenting a recombinant engineering strain containing beta-mannase mutant beta-ManM with bile acid in a volume ratio of 3-5:1.
8. Use of the β -mannanase mutant β -ManM of claim 1 or the additive of claim 6 in aquaculture.
9. The use according to claim 8, wherein the beta-mannase mutant beta-ManM or additive is used in an amount of 0.02% to 0.1% by weight of the aquatic animal base ration.
10. The use according to claim 8, wherein the aquatic animals are freshwater fish, shrimp and crab.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211721662.9A CN116218817B (en) | 2022-12-30 | 2022-12-30 | Beta-mannase mutant beta-ManM, additive prepared from beta-mannase mutant beta-ManM and bile acid and application of additive |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211721662.9A CN116218817B (en) | 2022-12-30 | 2022-12-30 | Beta-mannase mutant beta-ManM, additive prepared from beta-mannase mutant beta-ManM and bile acid and application of additive |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116218817A true CN116218817A (en) | 2023-06-06 |
| CN116218817B CN116218817B (en) | 2024-06-21 |
Family
ID=86583538
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211721662.9A Active CN116218817B (en) | 2022-12-30 | 2022-12-30 | Beta-mannase mutant beta-ManM, additive prepared from beta-mannase mutant beta-ManM and bile acid and application of additive |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116218817B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102277342A (en) * | 2011-08-16 | 2011-12-14 | 青岛康地恩生物科技有限公司 | Mannose and mutants thereof |
| CN112410322A (en) * | 2020-11-30 | 2021-02-26 | 河北科技大学 | Bacillus licheniformis beta-mannase mutant and application thereof |
-
2022
- 2022-12-30 CN CN202211721662.9A patent/CN116218817B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102277342A (en) * | 2011-08-16 | 2011-12-14 | 青岛康地恩生物科技有限公司 | Mannose and mutants thereof |
| CN112410322A (en) * | 2020-11-30 | 2021-02-26 | 河北科技大学 | Bacillus licheniformis beta-mannase mutant and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| YAOZHONG CHEN ET AL.: "Enhanced production of heterologous proteins by Bacillus licheniformis with defective d-alanylation of lipoteichoic acid", WORLD JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY, vol. 34, 31 December 2018 (2018-12-31), pages 1 - 9 * |
| 张蕊: "β-甘露聚糖酶分子生物学研究进展", 中国农业科技导报, 31 December 2018 (2018-12-31), pages 34 - 36 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116218817B (en) | 2024-06-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106520642B (en) | Bacillus amyloliquefaciens and application thereof | |
| CN104686838A (en) | Composite microecological feed additive capable of increasing freshwater fish growth rate and application of composite microecological feed additive | |
| CN103773746B (en) | Lipase and mutant thereof | |
| CN116218817B (en) | Beta-mannase mutant beta-ManM, additive prepared from beta-mannase mutant beta-ManM and bile acid and application of additive | |
| CN100491533C (en) | Improved xylanase with high specific activity, its gene expression vector and recombinant yeast cell, and expression method | |
| CN110734867B (en) | Deep-sea-derived yeast Q7-7 and application thereof | |
| CN102220304A (en) | Low-temperature xylanase XynAHJ2 and gene thereof | |
| CN101701214B (en) | Xylanase XYNA4 with wide pH applicability and gene and application thereof | |
| Turaeva et al. | Isolation and identification of bacteria with cellulolytic activity from the microflora of animal juice | |
| CN101849618A (en) | Method for producing complex enzyme for feed | |
| Fadel | High-level xylanase production from sorghum flour by a newly isolate of Trichoderma harzianum cultivated under solid state fermentation | |
| CN108118006A (en) | A kind of heatproof zytase kluyveromyces marxianus engineered strain and its application | |
| CN114921440A (en) | Xylanase mutant and application thereof | |
| CN116004585B (en) | Eucalyptus essential oil mutant enzyme preparation and application thereof in livestock cultivation | |
| CN116042581B (en) | Co-expression mutant enzyme recombinant bacterium, preparation thereof and application thereof in preparation of feed additive for aquatic animals | |
| CN115851670B (en) | Xylanase mutant, compound preparation of xylanase mutant and eucommia ulmoides leaf extract and application of xylanase mutant and eucommia ulmoides leaf extract in feed additive | |
| CN116064479B (en) | Compound preparation containing xylanase mutant and bile acid and application thereof | |
| CN115975992B (en) | Lysozyme mutant with improved enzyme activity, bile acid enzyme preparation and application | |
| CN112641012A (en) | Method for preparing large yellow croaker biological fermentation feed | |
| CN115851671B (en) | Xylanase mutant xynH, enzyme preparation compounded with bile acid and application thereof | |
| RU2747782C1 (en) | Recombinant yeast strain ogataea haglerorum producing beta-mannanase bacillus subtilis | |
| RU2764793C1 (en) | Yeast transformant ogataea haglerorum producing beta-mannanase, containing synthetic mans gene in chromosome | |
| CN114634942B (en) | Cold-resistant xylanase and gene thereof, engineering bacteria containing gene and application of engineering bacteria | |
| CN115820612B (en) | Keratinase, bile acid enzyme preparation containing keratinase and application of bile acid enzyme preparation | |
| CN118562772A (en) | Crayfish intestinal microecological additive containing low-temperature protease and metazoan preparation, and preparation method 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 | ||
| CB02 | Change of applicant information |
Address after: No. 9 Yanying North Road, Yanbei Street Office, Qihe County, Dezhou City, Shandong Province, 253000 Applicant after: Shandong Longchang Animal Health Products Co.,Ltd. Address before: 251100 No.1, North Park Road, Qihe Economic Development Zone, Dezhou City, Shandong Province Applicant before: SHANDONG LONGCHANG ANIMAL HEALTH PRODUCT Co.,Ltd. |
|
| CB02 | Change of applicant information | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |