CN115975991A - Medium temperature amylase mutant and application thereof - Google Patents
Medium temperature amylase mutant and application thereof Download PDFInfo
- Publication number
- CN115975991A CN115975991A CN202210839096.5A CN202210839096A CN115975991A CN 115975991 A CN115975991 A CN 115975991A CN 202210839096 A CN202210839096 A CN 202210839096A CN 115975991 A CN115975991 A CN 115975991A
- Authority
- CN
- China
- Prior art keywords
- amylase
- mutant
- enzyme
- amino acid
- specific activity
- 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
- 108010065511 Amylases Proteins 0.000 title claims abstract description 70
- 102000013142 Amylases Human genes 0.000 title claims abstract description 64
- 235000019418 amylase Nutrition 0.000 title claims abstract description 64
- 239000004382 Amylase Substances 0.000 title claims abstract description 62
- 238000004519 manufacturing process Methods 0.000 claims abstract description 10
- 235000013305 food Nutrition 0.000 claims abstract description 6
- 238000012545 processing Methods 0.000 claims abstract description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 14
- 241000235058 Komagataella pastoris Species 0.000 claims description 9
- 239000013604 expression vector Substances 0.000 claims description 7
- 150000001413 amino acids Chemical class 0.000 claims description 5
- 108020004414 DNA Proteins 0.000 claims description 4
- 102000053602 DNA Human genes 0.000 claims description 4
- 238000003259 recombinant expression Methods 0.000 claims description 4
- 230000000694 effects Effects 0.000 abstract description 33
- 102000004190 Enzymes Human genes 0.000 abstract description 23
- 108090000790 Enzymes Proteins 0.000 abstract description 23
- 230000035772 mutation Effects 0.000 abstract description 15
- 230000009286 beneficial effect Effects 0.000 abstract description 4
- 238000010353 genetic engineering Methods 0.000 abstract description 3
- 230000001737 promoting effect Effects 0.000 abstract description 3
- 230000009145 protein modification Effects 0.000 abstract description 2
- 229940088598 enzyme Drugs 0.000 description 21
- 239000002609 medium Substances 0.000 description 15
- 108090000637 alpha-Amylases Proteins 0.000 description 13
- 102000004139 alpha-Amylases Human genes 0.000 description 12
- 229940024171 alpha-amylase Drugs 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 10
- 239000000047 product Substances 0.000 description 9
- 239000001963 growth medium Substances 0.000 description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 229920002472 Starch Polymers 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 239000008107 starch Substances 0.000 description 6
- 235000019698 starch Nutrition 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 230000029087 digestion Effects 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108091008146 restriction endonucleases Proteins 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 239000001888 Peptone Substances 0.000 description 4
- 108010080698 Peptones Proteins 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 229940041514 candida albicans extract Drugs 0.000 description 4
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 4
- 238000001976 enzyme digestion Methods 0.000 description 4
- 238000000855 fermentation Methods 0.000 description 4
- 230000004151 fermentation Effects 0.000 description 4
- BRZYSWJRSDMWLG-CAXSIQPQSA-N geneticin Natural products O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](C(C)O)O2)N)[C@@H](N)C[C@H]1N BRZYSWJRSDMWLG-CAXSIQPQSA-N 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 235000019319 peptone Nutrition 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000012138 yeast extract Substances 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 102000003960 Ligases Human genes 0.000 description 3
- 108090000364 Ligases Proteins 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 3
- 108010084455 Zeocin Proteins 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 229960002685 biotin Drugs 0.000 description 3
- 235000020958 biotin Nutrition 0.000 description 3
- 239000011616 biotin Substances 0.000 description 3
- 229940098773 bovine serum albumin Drugs 0.000 description 3
- 238000004364 calculation method Methods 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- CWCMIVBLVUHDHK-ZSNHEYEWSA-N phleomycin D1 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC[C@@H](N=1)C=1SC=C(N=1)C(=O)NCCCCNC(N)=N)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C CWCMIVBLVUHDHK-ZSNHEYEWSA-N 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 239000013598 vector Substances 0.000 description 3
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 2
- 241001506991 Komagataella phaffii GS115 Species 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- YVNQAIFQFWTPLQ-UHFFFAOYSA-O [4-[[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfophenyl)methyl]amino]-2-methylphenyl]methylidene]-3-methylcyclohexa-2,5-dien-1-ylidene]-ethyl-[(3-sulfophenyl)methyl]azanium Chemical compound C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=C1 YVNQAIFQFWTPLQ-UHFFFAOYSA-O 0.000 description 2
- 229940025131 amylases Drugs 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 230000031700 light absorption Effects 0.000 description 2
- 244000144972 livestock Species 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 239000008057 potassium phosphate buffer Substances 0.000 description 2
- 244000144977 poultry Species 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 108010011619 6-Phytase Proteins 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 244000153158 Ammi visnaga Species 0.000 description 1
- 235000010585 Ammi visnaga Nutrition 0.000 description 1
- 229920000945 Amylopectin Polymers 0.000 description 1
- 229920000856 Amylose Polymers 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 108700010070 Codon Usage Proteins 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
- 229920001353 Dextrin Polymers 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 229920001503 Glucan Polymers 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 238000007696 Kjeldahl method Methods 0.000 description 1
- 229920000881 Modified starch Polymers 0.000 description 1
- 239000004368 Modified starch Substances 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 238000009004 PCR Kit Methods 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 102100026367 Pancreatic alpha-amylase Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 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 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 239000006030 antibiotic growth promoter Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 235000013405 beer Nutrition 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 229940079919 digestives enzyme preparation Drugs 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000012215 gene cloning Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000003262 industrial enzyme Substances 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 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 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 235000019426 modified starch Nutrition 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229940085127 phytase Drugs 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 239000010453 quartz Substances 0.000 description 1
- 238000002708 random mutagenesis Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 239000007222 ypd medium Substances 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Abstract
The invention belongs to the technical field of genetic engineering and protein modification, and particularly provides a medium-temperature amylase mutant and application thereof. The invention provides a mutant containing a single mutation site of N455A, D K or G573V based on a wild type moderate temperature amylase ZD 5. Compared with the wild type, the specific activity of the amylase single-point mutant provided by the invention is generally improved by 87.8% -146.7%; the mutant ZD5-1 containing the N455A single-point mutation has the highest specific activity which reaches 415U/mg, thereby being beneficial to reducing the production cost of the enzyme and promoting the wide application of the enzyme in the fields of feed production or food processing.
Description
Technical Field
The invention relates to the technical field of genetic engineering and protein modification, in particular to a medium temperature amylase mutant and application thereof.
Background
Amylases (Amyalase, E.C. 3.2.1.1) are a generic term for a class of enzymes that hydrolyze amylopectin, amylose or other glucan molecules in a random manner to produce linear and branched oligosaccharides of unequal length. The medium-temperature alpha-amylase is an alpha-amylase with the optimal reaction temperature of 50-70 ℃, is also the most widely applied amylase at present, and is widely applied to various fields of modified starch, starch sugar, baking industry, beer brewing, alcohol industry, feed, light industry, food, paper making, medicine, oil exploitation and the like. Alpha-amylases are all referred to as alpha-1,4-glucanohydrolase (EC 3.2.1.1), and when they act on starch, they cleave the alpha-1,4-glycosidic bond from the inside of the molecule to produce dextrins and reducing sugars, and they are called alpha-amylases since the C1 carbon atom of the terminal glucose residue of the product is in the alpha-configuration. Alpha-amylase is the most important enzyme, and is the enzyme preparation variety which is the first to realize industrial production and has the widest application and the largest yield so far. Statistically, amylase accounts for about 25% of the market for enzyme preparations and is a very important industrial enzyme. The medium-temperature alpha-amylase is used as a food additive, a feed additive or an auxiliary additive to be added into food, feed, medicines and daily chemical products, and has wide application value.
Starch is the main component of carbohydrates in grain feeds and is also an important energy source for energy required by livestock and poultry. The immature animal has immature digestive system and insufficient amylase secretion, so that the digestion and absorption of starch are limited. Research reports that exogenous alpha-amylase is added into feed to supplement the shortage of endogenous enzyme of animals, help young animals digest and utilize starch, and the growth performance and the feed conversion rate of the feed are very beneficial. The addition of the culture of the living escherichia coli producing the alpha-amylase in the daily ration of the broiler chicken can play a role of an antibiotic growth promoter, improve the intestinal form and improve the production performance. At present, in the feed production, alpha-amylase is mainly mixed with enzymes such as cellulase, protease, phytase and the like and added into the feed to play a role in intestinal tracts of livestock and poultry, decompose starch substances in the feed, promote digestion and improve the utilization rate.
The development of the medium-temperature amylase has very important significance for realizing domestic production as soon as possible, meeting market demands, adjusting the industrial structure of the enzyme preparation industry in China, saving foreign exchange expenditure and the like. However, the enzyme activity and yield of the existing mesophilic amylase are difficult to satisfy, so that mesophilic alpha-amylase with high enzyme activity, high yield, low production cost and wide pH range is urgently needed to be developed so as to meet the increasing industrial production requirements. The early research work of the medium-temperature alpha-amylase mainly comprises the aspects of breeding of enzyme-producing microbial strains, optimization of fermentation conditions, purification and physicochemical properties of enzyme, an enzyme hydrolysis action mechanism and the like. With the wide application of gene and protein engineering technologies, the research on mesophilic alpha-amylase is turning to the research on the aspects of gene cloning expression, active sites and the like so as to meet the development requirements of the feed industry.
Disclosure of Invention
The invention aims to provide a medium-temperature amylase mutant and application thereof. The specific activity of the mutant is obviously higher than that of a wild type mutant, and the mutant can be widely applied to the fields of feed, food processing and the like.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides amylase, and the amino acid sequence of the amylase is SEQ ID NO. 1.
In one aspect, the invention provides an amylase mutant, wherein the 455 th amino acid of the amylase with the amino acid sequence of SEQ ID NO. 1 is changed from Asn to Ala.
The invention also provides an amylase mutant, wherein the 487 th amino acid of the amylase with the amino acid sequence of SEQ ID NO. 1 is changed from Asp to Lys.
The invention also provides an amylase mutant, wherein the 573 th amino acid of the amylase with the amino acid sequence of SEQ ID NO. 1 is changed from Gly to Val.
The invention also relates to a DNA molecule encoding the amylase mutant.
The invention also relates to a recombinant expression vector containing the DNA molecule.
The invention also relates to a host cell comprising the recombinant expression vector.
The host cell is pichia pastoris (Pichia pastoris）。
The plasmid is transferred into a host cell, and the specific activity of the recombinant amylase mutant is obviously improved.
The invention also provides application of the amylase mutant in the field of feed production or food processing.
The invention provides a mutant containing a single mutation site of N455A, D K or G573V based on a wild type moderate temperature amylase ZD 5. Compared with the wild type, the specific activity of the amylase single-point mutant provided by the invention is generally improved by 87.8% -146.7%; the mutant ZD5-1 containing the N455A single-point mutation has the highest specific activity, and has unexpected technical effects.
The three mutation sites of N455A, D K and G573V provided by the invention can obviously improve the specific activity of the mesothermal amylase ZD5, thereby being beneficial to reducing the production cost of the enzyme and promoting the wide application of the enzyme in the industrial field.
Detailed Description
The present invention uses conventional techniques and methods used in the fields of genetic engineering and molecular biology, such as MOLEC m LAR CLONING: a Laboratory Manual,3nd Ed. (Sambrook, 2001) and CURRENT PROTOCOLS IN MOLEC m.Lan BIOLOGY (Ausubel, 2003). These general references provide definitions and methods known to those skilled in the art. However, those skilled in the art can adopt other conventional methods, experimental schemes and reagents in the field on the basis of the technical scheme described in the invention, and the invention is not limited to the specific embodiment of the invention. For example, the following experimental materials and reagents may be selected for use in the present invention:
the experimental reagent: DNA polymerase was purchased from Takara, T4 ligase, restriction enzyme from Fermentas, plasmid extraction kit and gel purification recovery kit from Omega, geneMorph II random mutagenesis kit from Beijing Bomais Biotech Ltd.
Bacterial strain and carrier: coli DH 5. Alpha. Deposited from Invitrogen, pichia pastoris GS115, vectors pPIC9k, pPICZA, amp, G418, zeocin were purchased from Invitrogen.
Culture medium:
LB medium (e.coli medium): 0.5% yeast extract, 1% peptone, 1% NaCl, pH7.0;
LB + Amp medium: adding 100 mu g/ml ampicillin in LB culture medium;
YPD medium (yeast medium): 1% yeast extract, 2% peptone, 2% glucose;
YPD + Zeocin medium: adding 100 mu g/ml Zeocin into YPD culture medium;
MD medium (yeast screening medium): 1.34% YNB, 4X 10 -5 Biotin, 1% glycerol, 2% agarose;
BMGY medium: 2% peptone, 1% yeast extract, 100 mM potassium phosphate buffer (pH 6.0), 1.34% YNB, 4X 10 -5 % biotin, 1% glycerol;
BMMY medium: 2% peptone, 1% yeast extract, 100 mM potassium phosphate buffer (pH6.0), 1.34% YNB, 4X 10 -5 % biotin, 0.5% methanol;
the enzyme activity detection method of the amylase in the embodiment of the invention refers to GB/T24401-2009.
The present invention will be further described with reference to the following detailed description.
EXAMPLE 1 mesophilic amylase gene synthesis
The amino acid sequence of the mesophilic amylase gene (GenBank number is AFI 62032.1) is taken as a basis, the amino acid sequence of the mesophilic amylase is analyzed, the signal peptide of the mesophilic amylase gene is removed, codon optimization is carried out on the mesophilic amylase according to the codon preference of pichia pastoris, and the Huada gene company carries out whole-gene synthesis. The applicant names the moderate temperature amylase ZD5, the amino acid sequence of the moderate temperature amylase is SEQ ID NO. 1, and the coding nucleotide sequence of the moderate temperature amylase is SEQ ID NO. 2.
Example 2 screening of high specific Activity mesophilic amylase mutants
In order to improve the specific activity of the mesophilic amylase ZD5, the applicant has screened a large number of mutations by directed evolution techniques.
Design of PCR primer 1 (F) and primer 1 (R):
primer 1 (F): GCGCGAATTCGAAACTGCTAATAAATCTAATGAAT (restriction enzymes underlined)EcoR I recognition site);
primer 1 (R): TAAAGCGGCCGCTTAATGTGGCAAAGAACCAGACAAA (restriction enzymes underlined)Not I recognition site).
Using a gene fragment of the medium-temperature amylase ZD5 as a template, and performing PCR amplification by using the primer and a GeneMorph II random mutation PCR kit (Bomeis); the PCR product is recovered by glue and then,EcoRI、Noti, performing enzyme digestion treatment, connecting the enzyme digested product with a pET21a vector, transforming the enzyme digested product into escherichia coli BL21 (DE 3), coating the escherichia coli BL21 (DE 3) in an LB + Amp plate, and performing inverted culture at 37 ℃; after the transformants appear, picking the transformants to a 96-well plate one by using toothpicks, adding 150 ul LB + Amp culture medium containing 0.1mM IPTG into each well, culturing the cells at 37 ℃ and 220rpm for about 6 h, centrifuging, discarding the supernatant, resuspending the thalli by using a buffer solution, and repeatedly freezing and thawing to break the wall to obtain an escherichia coli cell lysate containing medium temperature amylase; and centrifuging the cell lysate, taking supernatant, respectively carrying out amylase activity determination and protein content determination, and calculating the specific activity levels of different mutants.
The experimental results show that some mutations have no influence on the specific activity of the mesophilic amylase ZD5, and some mutations even make the specific activity lower. Finally, the applicant screened a single point mutation that could significantly improve the specific activity of the mesophilic amylase ZD 5: N455A, D487K, G573V.
The moderate temperature amylase mutant containing N455A single point mutation is named ZD5-1;
the medium temperature amylase mutant containing D487K single point mutation is named as ZD5-2;
the intermediate temperature amylase mutant containing the G573V single point mutation is named as ZD5-3.
The amino acid sequences of the mutants ZD5-1, ZD5-2 and ZD5-3 are SEQ ID NO 3, SEQ ID NO 4 and SEQ ID NO 5 respectively.
The nucleotide sequences encoding the mutants were each synthesized with reference to the above amino acid sequences.
Example 3 expression of mesophilic amylases in Pichia pastoris
1. Construction of recombinant plasmid
Respectively using restriction enzyme to make intermediate temperature amylase gene ZD5 and its mutant gene (ZD 5-1, ZD5-2, ZD 5-3)EcoR I andNoti, double digestion is carried out, the expression vector pPIC9K is subjected to double digestion by using the same restriction enzyme, and the digestion system of 100 mu l is as follows: 40. Mu.l of gene fragment/vector, 10 XH buffer 10. Mu.l, 10 XBSA 10. Mu.l,EcoR I 5 μl、Not I 5 μl、ddH 2 O30. Mu.l. After digesting 4h at 37 ℃, the product was recovered by agarose gel electrophoresis.
The medium temperature amylase gene fragments after double enzyme digestion are respectively connected with an expression vector pPIC9K, and the connection system is as follows: 5 mul of expression vector pPIC9K double enzyme digestion product, 3 mul of moderate temperature amylase gene double enzyme digestion product and 10 XT 4 ligase buffer 1 μl、T 4 1 μ l of ligase. 22. The mixture is connected at the temperature of overnight, transformed into escherichia coli DH5 alpha, and transformants are picked and sequenced for verification. And transferring the transformant which is verified to be correct by sequencing into an LB + Amp liquid culture medium, carrying out overnight culture at 37 ℃, and extracting the plasmid, namely the recombinant yeast expression plasmid.
Transformation and screening
The recombinant yeast expression plasmids constructed above were used separatelySalI, linearization is carried out, a linearization product is purified by a column purification kit, and then pichia pastoris GS115 is transformed by an electroporation method and coated on an MD plate. The colony growing on the MD plate is a pichia pastoris engineering strain for recombining and expressing the mesophilic amylase ZD5 or a mutant thereof, and then YPD plates containing different concentrations of geneticin G418 are coated to screen multi-copy transformants.
Shake flask fermentation verification
Single multi-copy transformants are picked and respectively inoculated into a BMGY culture medium, and after shaking culture is carried out at 30 ℃ and 220rpm for 24 hours, the single multi-copy transformants are transferred into a BMMY culture medium and are subjected to shaking culture at 30 ℃ and 220rpm, and 0.5% methanol is added every 24 hours. After the induction expression is carried out for 4 days, the thalli are removed by centrifugation, the supernatant is respectively subjected to medium temperature amylase activity determination and protein content determination, and the specific activity is calculated.
The result shows that the specific activity of the fermentation supernatant in a transformant expressing the wild type moderate temperature amylase ZD5 reaches 168.2U/mg at the highest level under the shake flask level; the specific activity of the fermentation supernatant in the transformants expressing the amylase single-point mutants ZD5-1, ZD5-1 and ZD5-3 is respectively 415U/mg, 342.9U/mg and 315.8U/mg which is increased by 87.8-146.7% compared with the wild type; the mutant ZD5-1 containing the N455A single-point mutation has the highest specific activity, and has unexpected technical effects.
Method for detecting enzyme activity of amylase
Reference is made to the GB/T24401-2009 method.
Detection of protein content by Coomassie brilliant blue method
1. Reagent
(1) Coomassie brilliant blue G-250 staining solution: dissolving Coomassie brilliant blue G-250 100mg in 50ml 95% ethanol, adding 100ml 85% phosphoric acid, diluting with water to 1L, and using at normal temperature for 1 month;
(2) Standard protein solution: measuring the protein content by using bovine serum albumin through a micro Kjeldahl method in advance, and preparing a protein standard solution of 1 mg/ml according to the purity of the protein;
(3) Preparing a standard stock solution: accurately weighing 0.05g of crystallized bovine serum albumin on an analytical balance, adding a small amount of distilled water into a small beaker, dissolving, transferring into a 50ml volumetric flask, washing residual liquid in the beaker with a small amount of distilled water for several times, pouring the washing liquid into the volumetric flask together, and finally fixing the volume to the scale with the distilled water. A standard stock solution was prepared in which the concentration of bovine serum albumin was 1000. Mu.g/ml.
2. And (5) drawing a standard curve.
(1) The 6 test tubes are respectively numbered, the reagents are added according to the following table, and the mixture is uniformly mixed.
| Pipe number | 1 | 2 | 3 | 4 | 5 | 6 |
| Sample (ml) | 0 | 0.1 | 0.2 | 0.3 | 0.4 | 0.5 |
| Water (ml) | 2.0 | 1.9 | 1.8 | 1.7 | 1.6 | 1.5 |
| Protein content (mg/ml) | 0 | 0.05 | 0.1 | 0.15 | 0.2 | 0.25 |
Accurately sucking 2.5ml of Coomassie brilliant blue solution into 6 dry test tubes, accurately sucking 0.1ml of the above solutions, placing the solutions into test tubes with respective numbers, mixing uniformly by vortex, standing at room temperature for 5min, zeroing with test tube No. 1, measuring at 595nm, comparing color, and recording light absorption value.
(2) Drawing a standard curve: the absorbance values read by the 1-6 tubes were recorded, and a standard curve was drawn with the protein content (μ g) as the abscissa and the absorbance as the ordinate. Note that the cuvette had to be cleaned due to the strong staining ability of coomassie brilliant blue. Cannot be measured with a quartz cup.
3. Determination of samples
Preparation of samples:
(1) Liquid sample: diluting a sample to be detected to the protein content of 0.1-0.3mg/ml, and controlling the light absorption value (after blank is subtracted) after blank is removed to be 0.2-0.4;
(2) Solid sample: accurately weighing 1.0000g of sample in a 100ml triangular flask, adding 20ml of deionized water by using a pipette gun, magnetically stirring for 10min, centrifuging at 4000rpm for 10min, taking supernatant, and further diluting to determine protein content, wherein the dilution method refers to liquid samples.
Sample detection:
adding a clean test tube into a solution containing 2.5ml of Coomassie brilliant blue, adding a sample to be tested, uniformly shaking in a vortex manner, standing at room temperature for 5min, taking a blank of a standard curve as a control, measuring absorbance at 595nm by using a microcuvette with an optical diameter of 1cm, and obtaining the protein content according to the standard curve.
4. Protein content calculation
Protein content = X × dilution factor × standard conversion factor.
X: protein content (mg/ml) determined from standard;
reduced value of standard sample: the standard sample is 47mg/ml, and a coefficient is converted according to an actual measurement value.
(III) calculation of specific Activity
"Specific Activity" means: the number of units of enzyme activity per weight of protein is generally expressed as U/mg protein. In general, the higher the specific activity of the enzyme, the purer the enzyme.
The specific activity calculation formula is as follows: specific activity (U/mg) = enzyme activity (U/mL)/protein content (mg/mL).
In conclusion, the three mutation sites of N455A, D487K and G573V provided by the invention can obviously improve the specific activity of the mesophilic amylase, thereby being beneficial to reducing the production cost of the mesophilic amylase and promoting the wide application of the mesophilic amylase in the industrial field.
Claims (10)
1. An amylase, wherein the amino acid sequence of the amylase is SEQ ID NO. 1.
2. An amylase mutant, wherein the mutant has the amino acid sequence of SEQ ID NO. 1, wherein the amino acid at position 455 of the amylase is changed from Asn to Ala.
3. An amylase mutant wherein the amino acid 487 of the amylase having the amino acid sequence of SEQ ID NO. 1 is changed from Asp to Lys.
4. An amylase mutant comprising an amylase having an amino acid sequence of SEQ ID NO. 1 wherein amino acid 573 of the amylase is changed from Gly to Val.
5. A DNA molecule encoding an amylase mutant according to any of claims 2-4.
6. A recombinant expression vector comprising the DNA molecule of claim 5.
7. A host cell comprising the recombinant expression vector of claim 6.
8. The host cell of claim 7, wherein the host cell is Pichia pastoris (Pichia pastoris) ((Pichia pastoris))Pichia pastoris）。
9. Use of an amylase mutant according to any of claims 2-4 in feed production.
10. Use of an amylase mutant according to any of claims 2-4 in food processing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210839096.5A CN115975991A (en) | 2022-07-18 | 2022-07-18 | Medium temperature amylase mutant and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210839096.5A CN115975991A (en) | 2022-07-18 | 2022-07-18 | Medium temperature amylase mutant and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115975991A true CN115975991A (en) | 2023-04-18 |
Family
ID=85961168
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210839096.5A Pending CN115975991A (en) | 2022-07-18 | 2022-07-18 | Medium temperature amylase mutant and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115975991A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102803481A (en) * | 2009-10-23 | 2012-11-28 | 丹尼斯科美国公司 | Methods for reducing blue saccharide |
| CN109182304A (en) * | 2018-09-18 | 2019-01-11 | 云南中烟工业有限责任公司 | A kind of alpha-amylase gene and its application |
| CN109810961A (en) * | 2019-03-20 | 2019-05-28 | 中粮集团有限公司 | A- amylase mutant and its encoding gene and their application for high concentration starch liquefacation |
-
2022
- 2022-07-18 CN CN202210839096.5A patent/CN115975991A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102803481A (en) * | 2009-10-23 | 2012-11-28 | 丹尼斯科美国公司 | Methods for reducing blue saccharide |
| CN109182304A (en) * | 2018-09-18 | 2019-01-11 | 云南中烟工业有限责任公司 | A kind of alpha-amylase gene and its application |
| CN109810961A (en) * | 2019-03-20 | 2019-05-28 | 中粮集团有限公司 | A- amylase mutant and its encoding gene and their application for high concentration starch liquefacation |
Non-Patent Citations (1)
| Title |
|---|
| 孙清扬: "金属离子对重组深渊滕黄单胞菌低温α-淀粉酶LamA 的活性和稳定性影响", 微生物学通报, vol. 46, no. 11, 31 December 2019 (2019-12-31), pages 2848 - 2856 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108118041B (en) | Phospholipase D mutant, recombinant genetic engineering bacterium, and preparation methods and applications thereof | |
| CN115851473B (en) | Construction and application of pichia pastoris strain with high methanol tolerance | |
| CN110713996B (en) | Trehalase, and carrier and application thereof | |
| CN113684198B (en) | Method for improving cellulase catalytic efficiency and mutant 5I77-M2 | |
| CN113337495A (en) | Method for improving sialic acid yield and application | |
| CN111088241B (en) | Genetically engineered human lysozyme | |
| CN114761553A (en) | Nucleic acids, vectors, host cells and methods for producing beta-fructofuranosidase from aspergillus niger | |
| CN109735518B (en) | Beta-glucuronidase mutant with optimum reaction pH value increased and glycyrrhizic acid conversion process thereof | |
| CN109182301B (en) | Disaccharide degrading enzyme gene and application thereof | |
| CN115975991A (en) | Medium temperature amylase mutant and application thereof | |
| CN114181927B (en) | Heparinase I | |
| CN114395544A (en) | High specific activity phytase mutant | |
| CN116103267B (en) | Medium-temperature amylase mutant and application thereof | |
| CN115011622B (en) | Screening method and application of D-psicose 3-epimerase mutant | |
| CN110819609B (en) | Mutant lipase with improved thermal stability as well as preparation method and application thereof | |
| CN114703165A (en) | Beta-glucosidase mutant and application thereof | |
| CN115820606A (en) | High specific activity amylase mutant and application thereof | |
| CN114746548A (en) | Nucleic acids, vectors, host cells and methods for producing fructosyltransferase from aspergillus japonicus | |
| CN115975990A (en) | High-specific-activity moderate-temperature amylase mutant | |
| CN115161334B (en) | Neutral phytase production strain and application thereof | |
| TWI752463B (en) | Oligosaccharide oxidase, preparing method thereof and method of conversion into chito-oligochitosan acid | |
| CN114752588B (en) | Heparinase II | |
| CN113444754B (en) | Method for hydrolyzing raw starch and application thereof | |
| CN115125220B (en) | Laccase EcLac and application thereof, laccase gene EcLac and application thereof | |
| CN110951717B (en) | L-arabinose isomerase isomer 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 |