CN103710325B - Alpha-amylase AmyASS and application thereof in raw starch degradation - Google Patents
Alpha-amylase AmyASS and application thereof in raw starch degradation Download PDFInfo
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- CN103710325B CN103710325B CN201410005765.4A CN201410005765A CN103710325B CN 103710325 B CN103710325 B CN 103710325B CN 201410005765 A CN201410005765 A CN 201410005765A CN 103710325 B CN103710325 B CN 103710325B
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- 102000004139 alpha-Amylases Human genes 0.000 title claims abstract description 73
- 108090000637 alpha-Amylases Proteins 0.000 title claims abstract description 73
- 229940024171 alpha-amylase Drugs 0.000 title claims abstract description 73
- 229920002472 Starch Polymers 0.000 title abstract description 64
- 235000019698 starch Nutrition 0.000 title abstract description 38
- 239000008107 starch Substances 0.000 title abstract description 38
- 230000015556 catabolic process Effects 0.000 title description 7
- 238000006731 degradation reaction Methods 0.000 title description 7
- 210000003000 inclusion body Anatomy 0.000 claims abstract description 35
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 33
- 238000000034 method Methods 0.000 claims abstract description 22
- 238000004153 renaturation Methods 0.000 claims abstract description 12
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 32
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 30
- 239000000872 buffer Substances 0.000 claims description 27
- 230000000694 effects Effects 0.000 claims description 24
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 20
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 18
- 239000000385 dialysis solution Substances 0.000 claims description 18
- 239000011780 sodium chloride Substances 0.000 claims description 16
- 238000000502 dialysis Methods 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 11
- 239000000203 mixture Substances 0.000 claims description 11
- 239000006228 supernatant Substances 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 229910001868 water Inorganic materials 0.000 claims description 9
- 239000006166 lysate Substances 0.000 claims description 8
- 238000001556 precipitation Methods 0.000 claims description 8
- 235000018102 proteins Nutrition 0.000 claims description 8
- YPZRWBKMTBYPTK-BJDJZHNGSA-N glutathione disulfide Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@H](C(=O)NCC(O)=O)CSSC[C@@H](C(=O)NCC(O)=O)NC(=O)CC[C@H](N)C(O)=O YPZRWBKMTBYPTK-BJDJZHNGSA-N 0.000 claims description 7
- 108010053070 Glutathione Disulfide Proteins 0.000 claims description 6
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 claims description 5
- 229920004890 Triton X-100 Polymers 0.000 claims description 5
- 239000013504 Triton X-100 Substances 0.000 claims description 5
- 238000006555 catalytic reaction Methods 0.000 claims description 5
- 238000004140 cleaning Methods 0.000 claims description 5
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- 229930064664 L-arginine Natural products 0.000 claims description 4
- 235000014852 L-arginine Nutrition 0.000 claims description 4
- 230000009514 concussion Effects 0.000 claims description 3
- 230000007928 solubilization Effects 0.000 claims description 3
- 238000005063 solubilization Methods 0.000 claims description 3
- 239000004475 Arginine Substances 0.000 claims description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 2
- 235000009697 arginine Nutrition 0.000 claims description 2
- 238000002242 deionisation method Methods 0.000 claims 1
- 238000011084 recovery Methods 0.000 claims 1
- 235000007164 Oryza sativa Nutrition 0.000 abstract description 19
- 235000009566 rice Nutrition 0.000 abstract description 19
- 230000000593 degrading effect Effects 0.000 abstract description 12
- 241000588724 Escherichia coli Species 0.000 abstract description 8
- 240000007594 Oryza sativa Species 0.000 abstract 1
- 125000003275 alpha amino acid group Chemical group 0.000 abstract 1
- 235000019890 Amylum Nutrition 0.000 description 26
- 102000004190 Enzymes Human genes 0.000 description 24
- 108090000790 Enzymes Proteins 0.000 description 24
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- 241000209094 Oryza Species 0.000 description 18
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- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 13
- 150000001413 amino acids Chemical group 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 8
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 description 8
- 229960003180 glutathione Drugs 0.000 description 7
- 239000000758 substrate Substances 0.000 description 7
- 241000894006 Bacteria Species 0.000 description 5
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- 235000001014 amino acid Nutrition 0.000 description 4
- 239000008187 granular material Substances 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 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 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 230000009471 action Effects 0.000 description 3
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- 239000013604 expression vector Substances 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 229920001592 potato starch Polymers 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 241000607525 Aeromonas salmonicida Species 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 235000009508 confectionery Nutrition 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 235000003969 glutathione Nutrition 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 230000031700 light absorption Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- DBTMGCOVALSLOR-UHFFFAOYSA-N 32-alpha-galactosyl-3-alpha-galactosyl-galactose Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(OC2C(C(CO)OC(O)C2O)O)OC(CO)C1O DBTMGCOVALSLOR-UHFFFAOYSA-N 0.000 description 1
- 241000251468 Actinopterygii Species 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
- 241000972773 Aulopiformes Species 0.000 description 1
- 241001143891 Bacillus aquimaris Species 0.000 description 1
- 241000194110 Bacillus sp. (in: Bacteria) Species 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- RXVWSYJTUUKTEA-UHFFFAOYSA-N D-maltotriose Natural products OC1C(O)C(OC(C(O)CO)C(O)C(O)C=O)OC(CO)C1OC1C(O)C(O)C(O)C(CO)O1 RXVWSYJTUUKTEA-UHFFFAOYSA-N 0.000 description 1
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 description 1
- 241000305071 Enterobacterales Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 239000002253 acid Substances 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
- 235000019418 amylase Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- JDGPQNJYYADFKP-UHFFFAOYSA-N aniline;n-phenylaniline Chemical class NC1=CC=CC=C1.C=1C=CC=CC=1NC1=CC=CC=C1 JDGPQNJYYADFKP-UHFFFAOYSA-N 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000005660 chlorination reaction Methods 0.000 description 1
- 238000004587 chromatography analysis 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
- 238000001816 cooling Methods 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 235000019688 fish Nutrition 0.000 description 1
- 235000019256 formaldehyde Nutrition 0.000 description 1
- 239000012634 fragment 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
- 229960000789 guanidine hydrochloride Drugs 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion 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
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- FYGDTMLNYKFZSV-UHFFFAOYSA-N mannotriose Natural products OC1C(O)C(O)C(CO)OC1OC1C(CO)OC(OC2C(OC(O)C(O)C2O)CO)C(O)C1O FYGDTMLNYKFZSV-UHFFFAOYSA-N 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- -1 pharmacy Substances 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 210000000582 semen Anatomy 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- WUWHFEHKUQVYLF-UHFFFAOYSA-M sodium;2-aminoacetate Chemical compound [Na+].NCC([O-])=O WUWHFEHKUQVYLF-UHFFFAOYSA-M 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- LEAHFJQFYSDGGP-UHFFFAOYSA-K trisodium;dihydrogen phosphate;hydrogen phosphate Chemical compound [Na+].[Na+].[Na+].OP(O)([O-])=O.OP([O-])([O-])=O LEAHFJQFYSDGGP-UHFFFAOYSA-K 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- FYGDTMLNYKFZSV-BYLHFPJWSA-N β-1,4-galactotrioside Chemical compound 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](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-BYLHFPJWSA-N 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)
- C12N9/24—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)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
- C12N9/2414—Alpha-amylase (3.2.1.1.)
- C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
-
- 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)
- C12Y302/01001—Alpha-amylase (3.2.1.1)
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses an alpha-amylase, the gene sequence of which is shown as SEQ ID NO. 2; the amino acid sequence coded by the gene sequence is shown as SEQ ID NO. 1; the gene sequence SEQ ID NO. 2 can successfully express the alpha-amylase in the form of inclusion body in the escherichia coli. The alpha-amylase obtained by the inclusion body renaturation method has the protein purity of more than 85 percent and the renaturation rate of more than 50 percent; the alpha-amylase can be used for quickly degrading raw starch, and is particularly suitable for degrading rice raw starch.
Description
Technical field
The present invention relates to biological technical field, the bag of α-amylase AmyASS in a kind of marine bacteria source
Recombinant expressed, the renaturing inclusion bodies acquisition of containing bodily form formula have the α-amylase of catalysis activity and in rice uncooked amylum is degraded
Application.
Background technology
α-amylase is a kind of cut-out sugar chain, enzyme of degradable starch on α-Isosorbide-5-Nitrae glycosidic bond.α-amylase is in each industry
Field is such as brewageed, fiber, pharmacy, detergent and food industry have a wide range of applications.Natural uncooked amylum granule has complexity
Fine and close structure.Glucose molecule by glycosidic bond formed long-chain, long-chain be folded into crystallization and unformed two kinds of structures, then this
Two kinds of structures interweave and form natural uncooked amylum granule.Therefore, natural uncooked amylum granule can not be by common starch degrading enzyme
Directly degraded.Traditional starch degradation is required for including starch gelatinization and amylorrhexis two step.Only break first with high temperature gelatinizing
The structure of bad uncooked amylum granule, promotes farinose crystalline texture to open, and namely uncooked amylum becomes ripe starch, and starch long-chain is
Effectively can be cut off by starch degrading enzyme.Starch gelatinization is a process consumed energy very much, therefore, if using a kind of special energy
Starch is degraded by farinose amylase of directly degrading, and saves starch gelatinization link, can energy efficient, reduction greatly
Production cost, moreover it is possible to traditional handicraft united two into one, shortening processes the time.The α-amylase of about 10% has the life of this uniqueness
Starch degrading activity, they overwhelming majority are from terrestrial life.These only have three α-shallow lakes from marine bacteria so far
Powder enzyme is in the news and has uncooked amylum degrading activity, is respectively from Bacillus sp.ALSHL3 and B.aquimaris
MKSC6.2 and from ocean the α-shallow lake belonging to the most newly-established glycoside hydrolase subfamily GH13_37 of grand genomic library
Powder enzyme (AmyP).
Although it have been found that much have the α-amylase of uncooked amylum degradation capability, but these α-amylase are main
It is that raw wheaten starch and Treatment are had good degradation capability.Rice is that the main food of our country in Southeast Asia is made
Thing, rice starch is with a wide range of applications in industries such as food, chemical industry and medicine.But the Study on degradation of rice starch and
Actual application is far from wheaten starch and corn starch deeply.One important reason has uncooked rice starch degradation ability
α-amylase is more rare.At present in document report, only have altogether the rice life that 10 α-amylase can directly be degraded natural
Starch.But the degradation efficiency of these enzymes is low, and need the long-time reaction of more than 24 hours.
Summary of the invention
It is an object of the invention to provide one can advantage degraded the farinose α-amylase of rice, to make up prior art
The weak point existed.
The invention provides the gene order encoding described α-amylase, sequence is SEQ ID NO:2；This gene order is compiled
The aminoacid sequence of code is SEQ ID NO:1.This aminoacid sequence is only than the aminoacid of serial number YP_001143410 in NCBI
Sequence has lacked 26 aminoacid of N end, and similarity is 98.8%；But serial number YP_ in gene order SEQ ID NO:2 Yu NCBI
The gene order similarity that the aminoacid sequence of 001143410 is corresponding is only 64.5%.This gene order SEQ ID NO:2 can
The α-amylase of inclusion bodies is successfully given expression in escherichia coli.
The invention provides the recombiant plasmid expressing above-mentioned α-amylase inclusion body, be to be SEQ ID NO by gene order:
The nucleotide fragments of 2 is inserted in expression vector pColdIII.
The invention provides and carry the recombinant bacterium expressing above-mentioned α-amylase inclusion body.
The invention provides above-mentioned recombinant bacterium and give expression to the abduction delivering condition of α-amylase inclusion body.
The invention provides above-mentioned α-amylase renaturing inclusion bodies method.The α-amylase inclusion body making inactive turns
Become the α-amylase with catalysis activity, i.e. with soluble starch for detecting during substrate that enzyme is lived.Washing including inclusion body
Wash the concentration of α-amylase before the temperature in the composition of liquid, lysate and dialysis solution, and whole renaturation process and renaturation.
Refolding method:
1) being washed 1-2 time by α-amylase inclusion body cleaning mixture, deionized water wash 1 time, described cleaning mixture consists of
The final concentration 50mM Tris-HCl buffer of pH8.0, final concentration 5mM EDTA, and final concentration 1%(v/v) Triton X-100；
2) lysate of the α-amylase inclusion body after step 1) is washed is dissolved, make the albumen after solubilization of inclusion bodies dense
Degree is 280-320 μ g/ml, the final concentration 100mM Tris-HCl consisting of final concentration 6M GdmCl, pH6.0 of described lysate
Buffer, final concentration 100mM NaCl, final concentration 100mM dithiothreitol and final concentration 1mM EDTA；Then at 4 DEG C
Under the conditions of place 24 hours, therebetween every 3 hours concussion once, to promote the dissolving of inclusion body；After dissolving, 12000r/min from
The heart 20 minutes, removes the inclusion body precipitation still cannot dissolved on a small quantity, takes supernatant；
3) by step 2) supernatant that obtained loads bag filter, carries out dialysis renaturation；Dialysis uses 4 kinds of different dialysis
Liquid, changed a kind of dialysis solution every 12 hours, and whole dialysis procedure is carried out at 4 DEG C；The dialysis solution used for the first time consists of
The final concentration 50mM Tris-HCl buffer of pH8.0, final concentration 10% (v/v) glycerol, final concentration 50mM NaCl, final concentration
0.5mM EDTA and final concentration 4M GdmCl；The dialysis solution that second time uses consists of the final concentration 50mM Tris-HCl of pH8.0
Buffer, final concentration 10% (v/v) glycerol, final concentration 50mM NaCl, final concentration 0.5mM EDTA and final concentration 2M GdmCl；The
The dialysis solution that three times use consists of the final concentration 50mM Tris-HCl buffer of pH8.0, final concentration 10% (v/v) glycerol, end
Concentration 50mM NaCl, final concentration 0.5mM EDTA, final concentration 1M GdmCl, final concentration 2%L-arginine, final concentration 1mM
GSSG and final concentration 5mM GSH；The dialysis solution that 4th time uses consists of the final concentration 50mM Tris-HCl of pH8.0
Buffer, final concentration 10% (v/v) glycerol, final concentration 50mM NaCl, final concentration 0.5mM EDTA, final concentration 2%L-
Arginine, final concentration 1mM GSSG and final concentration 5mM GSH；After dialysis terminates, the protein liquid in bag filter is taken out,
12000r/min, 4 DEG C centrifugal 20 minutes, remove a small amount of the most still cannot the inclusion body precipitation of renaturation, take supernatant and be and recovered to urge
Change the α-amylase of activity.
The above-mentioned α-amylase of the present invention can be used for various uncooked amylum of degrading, such as uncooked rice starch, raw wheaten starch, raw Semen Maydis
Starch, raw potato starch, raw pea starch and raw green starch, when degrading uncooked rice starch, its activity is higher than other kinds
More than one times of uncooked amylum activity.
During α-amylase of the present invention degraded uncooked amylum: the pH value of degraded uncooked amylum reaction system is 4.5-9.5, and optimum pH is
6.0；Farinose concentration is 12-25%, and optimum concentration is 12%；Reaction temperature is 10-70 DEG C, and optimum temperature is 40 DEG C；Reaction
Time is 6 hours.As the CaCl containing final concentration 5mM in reaction system2Time, when substrate is rice uncooked amylum, its enzyme is lived notable
Rise to 230%.
Accompanying drawing explanation
Fig. 1 illustrates the pColdIII-AmyASS plasmid map of the α-amylase of the present invention
Fig. 2 illustrates the reaction pH curve of the α-amylase of the present invention
Fig. 3 illustrates the reaction temperature curve of the α-amylase of the present invention
Fig. 4 illustrates temperature stabilization linearity curve during 40 DEG C of the α-amylase of the present invention
Fig. 5 illustrates the α-amylase of the present invention to various farinose degrading activity comparison diagrams
Fig. 6 illustrates the α-amylase of the present invention degrading activity comparison diagram to the uncooked rice starch of variable concentrations
Fig. 7 illustrates the α-amylase of the present invention and degrades the time graph of 12% uncooked rice starch
Fig. 8 illustrates the α-amylase CaCl at variable concentrations of the present invention2Time degrade the specific activity of 12% uncooked rice starch
Relatively scheme
Detailed description of the invention
Chemical reagent used in following example is existing known reagent, and chemical reagent abbreviation table is as follows: EDTA
(disodium EDTA)；Triton X-100(Triton X-100) GdmCl(guanidine hydrochloride)；NaCl(chlorination
Sodium)；Dithiothreitol(DTT, dithiothreitol, DTT) L arginine(L-arginine)；GSSG(oxidized form of glutathione)；
GSH(reduced glutathion).
Embodiment 1
The gene chemical synthesis of α-amylase AmyASS
Pathogen aeromonas salmonicida according to the marine fishes announced in NCBI kills salmon subspecies (Aeromonas
Salmonicida ssp.salmonicida) full-length genome information, find one " assuming that glycoside hydrolase Families albumen "
(aminoacid sequence YP_001143410).α-amylase AmyP of the aminoacid of this AmyASS albumen and experimental identification
(Liu et al.2012) similarity is 50%, is presumed to a α-amylase.AmyASS albumen n end is predicted as signal
26 aminoacid of peptide are removed, and have redesigned gene order according to colibacillary codon preference.This gene order
Gene order (gene order NC_009348.1) similarity announced in NCBI with " assuming that glycoside hydrolase Families albumen "
It is only 64.5%.NdeI and XhoI restriction site is added at these gene two ends.The text sequence of this gene is delivered to biotech firm
(Sheng Gong bio tech ltd, Shanghai) synthesizes.The AmyASS gene order of synthesis is cloned on carrier pUC57, insertion point
Cloning for T-A, recipient bacterium is e.colistraindh5α.Wherein the nucleotides sequence of α-amylase AmyASS of the present invention is classified as
SEQ ID NO:2, aminoacid sequence is SEQ ID NO:1.
Embodiment 2
α-amylase AmyASS inclusion body recombinant expressed
The expression vector pCold III that the pUC57 carrier comprising α-amylase AmyASS gene and cold shock suffered a shock is carried out respectively
After the double digestion in NdeI and XhoI site, AmyASS gene is connected on pColdIII carrier, it is thus achieved that recombinant expression plasmid
PColdIII-AmyASS, as shown in Figure 1.Plasmid pColdIII-AmyASS is proceeded to e. coli bl21 cell, is containing
In the LB fluid medium of 50ug/ml ampicillin, 200r/min, 37 DEG C of cultivations.When the OD600 of E. coli broth reaches
During to about 0.5, it is cooled to 16 DEG C by hurried for culture fluid, and adds 1mM IPTG, then 16 DEG C, 120rpm incubated overnight.
α-amylase AmyASS of inclusion bodies will be produced in this incubation.8000r/min is centrifuged 5 minutes and collects 16 DEG C overnight
Escherichia coli are resuspended in 50mM Tris-HCl buffer(pH8.0 by the Bacillus coli cells cultivated), ultrasonication, until
Muddy Bacillus coli cells suspension becomes limpid liquid.3000r/min is centrifuged 5 minutes, takes supernatant and abandons precipitation.Supernatant fluid
Be centrifuged 20 minutes at 12000r/min, it is thus achieved that precipitation be α-amylase AmyASS inclusion body.
Attempt the method that various optimum combination is expressed, all cannot realize the gene order of this α-amylase AmyASS greatly
Enterobacteria is expressed as the soluble protein with catalysis activity.This gene order can only be expressed as forgiving of catalytically inactive
Body.It addition, the gene order (gene order NC_009348.1) that " assuming that glycoside hydrolase Families albumen " is announced in NCBI
It is cloned in expression vector pET20b and pColdIII, all cannot go out any albumen at expression in escherichia coli.
Embodiment 3
The renaturation of α-amylase AmyASS inclusion body
Renaturing inclusion bodies refers to the albumen of the inclusion bodies of inactive is being changed into bioactive albumen.
By α-amylase AmyASS inclusion body cleaning mixture (50mM Tris-HCl buffer, pH8.0,5mM EDTA, 1%Triton
X-100) washing 2 times, deionized water wash 1 time, dissolve inclusion body with lysate, it is desirable to the protein concentration after solubilization of inclusion bodies is about
It is 300 μ g/ml, according to this protein concentration requirement, adds a certain amount of lysate.The composition of lysate is 6M GdmCl, 100mM
Tris-HCl buffer, pH6.0, pH6.0,100mM NaCl, 100mM dithiothreitol, 1mM EDTA.Then 4
Place under the conditions of DEG C 24 hours, therebetween every concussion in 3 hours once, to promote the dissolving of inclusion body.After dissolving, 12000r/min
Centrifugal 20 minutes, remove the inclusion body precipitation still cannot dissolved on a small quantity, take supernatant, load bag filter, carry out dialysis renaturation.One
Have employed 4 kinds of different dialysis solution altogether, changed a kind of dialysis solution every 12 hours, whole dialysis procedure is carried out at 4 DEG C.For the first time
Use dialysis solution composition be 50mM Tris-HCl buffer(pH8.0), 10% (v/v) glycerol, 50mM NaCl, 0.5mM
EDTA and 4M GdmCl；Second time use dialysis solution composition be 50mM Tris-HCl buffer(pH8.0), 10% (v/v) sweet
Oil, 50mM NaCl, 0.5mM EDTA and 2M GdmCl；The dialysis solution composition that third time uses is 50mM Tris-HCl
Buffer(pH8.0), 10% (v/v) glycerol, 50mM NaCl, 0.5mM EDTA, 1M GdmCl, 2%L-arginine, 1mM
GSSG and 5mM GSH；4th time use dialysis solution composition be 50mM Tris-HCl buffer(pH8.0), 10% (v/v) sweet
Oil, 50mM NaCl, 0.5mM EDTA, 2%L-arginine, 1mM GSSG and 5mM GSH.After dialysis terminates, by bag filter
Protein liquid take out, 12000r/min, 4 DEG C centrifugal 20 minutes, remove a small amount of the most still cannot the inclusion body precipitation of renaturation, take
It is clearly α-amylase AmyASS having recovered catalysis activity.
Use said method obtain α-amylase AmyASS, purity of protein reaches more than at least 85%, renaturation yield reach to
Few more than 50%.
Embodiment 4
The determination of activity of α-amylase
The activity of α-amylase uses 3, and 5-dinitrosalicylic acid (DNS) method measures.After the method is detection Starch Hydrolysis
The reducing sugar amount discharged, standard curve is with reducing sugar glucose as standard test.One standard reaction system includes 40 μ l enzymes
Liquid and 1% soluble starch of 560 μ l or uncooked amylum (uncooked amylum concentration is arranged according to different situations).Starch is all arranged in 100mM
In citric acid-sodium citrate buffer (pH6.0).Enzyme reacts 10 minutes at 40 DEG C with starch, adds 300 μ l DNS immediately molten
Liquid, is placed in the 0.3M Na of pre-cooling2CO3In solution 5 minutes.When substrate is soluble starch, directly boil in boiling water 15 minutes,
Then light absorption value is measured at A540nm.When substrate is uncooked amylum, needs first 4 DEG C of 12000r/min to be centrifuged 5 minutes, take supernatant and exist
Boiling water boils 15 minutes, then measure light absorption value at A540nm.One standard enzyme unit (U) of living is defined as per minute discharging 1 μ
Enzyme amount needed for mol reducing sugar.During with 1% soluble starch for substrate, the ratio of enzyme is lived as 45U/mg；With 12% uncooked rice starch it is
During substrate, the ratio of enzyme is lived as 30U/mg.
Embodiment 5
Various farinose degrading activities are compared by α-amylase AmyASS
By uncooked rice starch, raw wheaten starch, Treatment, raw potato starch, raw pea starch and raw green starch
Being arranged in 100mM citric acid-sodium citrate buffer (pH6.0), concentration is 4%.Measure according to the method that embodiment 4 describes
Enzyme is lived, and result is as shown in Figure 6.α-amylase AmyASS can be degraded uncooked rice starch, raw wheaten starch and raw green starch, no
Can degrade Treatment, raw potato starch and raw pea starch.α-amylase AmyASS is the highest to the activity of uncooked rice starch,
At least 5 times and 2.5 times of raw green starch of raw wheaten starch.
Embodiment 6
The degraded to uncooked rice starch of α-amylase AmyASS
(1) action pH scope and the suitableeest action pH
Being configured with the buffer of different pH respectively by soluble starch, concentration is 1%.PH4.0-6.0 uses 100mM
Citric acid sodium citrate buffer solution；PH7.0-8.0 uses 100mM disodium hydrogen phosphate phosphate sodium dihydrogen buffer solution；
PH9.0-10.0 uses 100mM glycine sodium hydrate buffer solution.Measure 40 DEG C of methods described according to embodiment 4
Enzyme is lived, and result is as shown in Figure 2.α-amylase AmyASS works at pH4.5-9.5, and the suitableeest action pH is 6.0.
(2) operative temperature scope and the best use of temperature
The method described according to embodiment 4, measures the activity of α-amylase AmyASS, result such as Fig. 3 respectively at 10-70 DEG C
Shown in.α-amylase AmyASS functions both in the range of 10-70 DEG C, and optimum temperature is 40 DEG C.
(3) temperature stability
α-amylase AmyASS is added in 100mM citric acid-sodium citrate buffer (pH6.0), hatches 24 at 40 DEG C
Hour, during this period, take out 40 μ l enzyme liquid at regular intervals, the method described according to embodiment 4 measures enzyme and lives, result such as figure
Shown in 4.α-amylase AmyASS is highly stable at 40 DEG C, and after within 24 hours, hatching, it shows the residual enzyme work being not less than 70%.
(4) the farinose concentration of rice
Rice uncooked amylum is arranged in 100mM citric acid-sodium citrate buffer (pH6.0), concentration is respectively 4,8,
12,16 and 20%, the method described according to embodiment 4 measures enzyme and lives, and result is as shown in Figure 6.In relatively low rice uncooked amylum concentration
Time, the activity of α-amylase AmyASS increases along with the increase of rice uncooked amylum concentration, and optimal degraded concentration is 12%.Rice
After uncooked amylum concentration is more than 12%, declining occurs in the activity of α-amylase AmyASS, and shows the depression effect of high concentration of substrate.
The highest rice uncooked amylum concentration that α-amylase AmyASS can be degraded is 25%.
(5) response time
Being arranged in by rice uncooked amylum in 8ml 100mM citric acid-sodium citrate buffer (pH6.0), concentration is 12%,
Adding 0.2% toluene prevents microorganism from polluting, and adds 600 μ l enzyme liquid, reacts 8 hours at 40 DEG C, during this period, every a timing
Between take out 600 μ l reactant liquors, add 300 μ l DNS solution immediately, according to embodiment 4 describe method measure enzyme live, result is such as
Shown in Fig. 7.In the starting stage of reaction, the reducing sugar amount that degraded rice uncooked amylum produces sharply increases over time；30 minutes
After, gathering way of reducing sugar amount substantially slows down；After 6 hours, reducing sugar amount remains unchanged.This shows, α-amylase AmyASS
The hydrolysis to uncooked rice starch is completed in 6 hours.
(6) CaCl is added2
Being arranged in by rice uncooked amylum in 100mM citric acid-sodium citrate buffer (pH6.0), concentration is 12%, instead
Answer system adds 0 respectively, 3,5,8,10mM CaCl2, the method described according to embodiment 4 measures enzyme and lives, result such as Fig. 8 institute
Show.CaCl2Existence can significantly improve α-amylase AmyASS degraded the farinose activity of rice, CaCl2Optimal add dense
Degree is 5mM, 2.3 times when the activity of α-amylase AmyASS at least rises to without adding.
(7) effect
Configure the rice uncooked amylum solution of 12% with 100mM citric acid-sodium citrate buffer (pH6.0), add 0.2% first
Benzene prevents microorganism from polluting, and adds enzyme liquid, reacts 6 hours at 40 DEG C.8000r/min is centrifuged 5 minutes, takes supernatant and is rice life
The hydrolyzate of starch.The analysis of hydrolyzate uses thin layer chromatography to carry out.Exhibition coating systems is the isopropyl of volume ratio 3:1:1
Alcohol: ethyl acetate: H2O.After chromatography terminates, uniformly spray aniline-diphenylamines phosphoric acid, 85 DEG C of baking sheets 10 minutes.Result shows
The farinose product of α-amylase AmyASS hydrolysed rice is glucose, maltose and maltotriose.
Claims (1)
1. a refolding method for α-amylase inclusion body, is characterized in that, described method is carried out as follows:
1) gene order α-amylase inclusion body as shown in SEQ ID NO:2 cleaning mixture is washed 1-2 time, deionization washing
Washing 1 time, described cleaning mixture consists of the final concentration 50mM Tris-HCl buffer of pH 8.0, final concentration 5mM EDTA, eventually the denseest
Degree 1% (v/v) Triton X-100 and water；
2) will be through step 1) α-amylase inclusion body lysate after washing dissolves, and makes the protein concentration after solubilization of inclusion bodies be
280-320 μ g/ml, described lysate consist of final concentration 6M GdmCl, the final concentration 100mM Tris-HCl of pH 6.0
Buffer, final concentration 100mM NaCl, final concentration 100mM dithiothreitol, final concentration 1mM EDTA and water；Then 4
Place under the conditions of DEG C 24 hours, therebetween every concussion in 3 hours once, to promote the dissolving of inclusion body；After dissolving, 12000r/min
Centrifugal 20 minutes, remove the inclusion body precipitation still cannot dissolved on a small quantity, take supernatant；
3) by step 2) supernatant that obtained loads bag filter, carries out dialysis renaturation；Dialysis uses 4 kinds of different dialysis solution,
Changed a kind of dialysis solution every 12 hours, whole dialysis procedure is carried out at 4 DEG C；The dialysis solution used for the first time consists of pH 8.0
Final concentration 50mM Tris-HCl buffer, final concentration 10% (v/v) glycerol, final concentration 50mM NaCl, final concentration 0.5mM
EDTA, final concentration 4M GdmCl and water；The dialysis solution that second time uses consists of the final concentration 50mM Tris-HCl of pH 8.0
Buffer, final concentration 10% (v/v) glycerol, final concentration 50mM NaCl, final concentration 0.5mM EDTA, final concentration 2M GdmCl and
Water；The dialysis solution that third time uses consists of the final concentration 50mM Tris-HCl buffer of pH 8.0, final concentration 10% (v/v)
Glycerol, final concentration 50mM NaCl, final concentration 0.5mM EDTA, final concentration 1M GdmCl, final concentration 2%L-arginine, eventually the denseest
Degree 1mM GSSG, final concentration 5mM GSH and water；The dialysis solution that 4th time uses consists of the final concentration 50mM Tris-of pH 8.0
HCl buffer, final concentration 10% (v/v) glycerol, final concentration 50mM NaCl, final concentration 0.5mM EDTA, final concentration 2%L-
Arginine, final concentration 1mM GSSG, final concentration 5mM GSH and water；After dialysis terminates, the protein liquid in bag filter is taken
Go out, 12000r/min, 4 DEG C centrifugal 20 minutes, remove a small amount of the most still cannot the inclusion body precipitation of renaturation, take supernatant and be recovery
The α-amylase of catalysis activity.
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