CN102827815A - A group of cyclodextrin glucosyltransferase, and coding gene and application thereof - Google Patents
A group of cyclodextrin glucosyltransferase, and coding gene and application thereof Download PDFInfo
- Publication number
- CN102827815A CN102827815A CN2012103031661A CN201210303166A CN102827815A CN 102827815 A CN102827815 A CN 102827815A CN 2012103031661 A CN2012103031661 A CN 2012103031661A CN 201210303166 A CN201210303166 A CN 201210303166A CN 102827815 A CN102827815 A CN 102827815A
- Authority
- CN
- China
- Prior art keywords
- sequence
- terminal
- sequence table
- amino acids
- sports
- 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
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 61
- 229920000858 Cyclodextrin Polymers 0.000 title claims abstract description 13
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 title claims abstract description 12
- 102000000340 Glucosyltransferases Human genes 0.000 title abstract 3
- 108010055629 Glucosyltransferases Proteins 0.000 title abstract 3
- HFHDHCJBZVLPGP-RWMJIURBSA-N alpha-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO HFHDHCJBZVLPGP-RWMJIURBSA-N 0.000 claims abstract description 56
- 235000018102 proteins Nutrition 0.000 claims abstract description 45
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 45
- 230000035772 mutation Effects 0.000 claims abstract description 22
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims abstract description 21
- 235000004279 alanine Nutrition 0.000 claims abstract description 21
- 229940024606 amino acid Drugs 0.000 claims description 76
- 235000001014 amino acid Nutrition 0.000 claims description 76
- 150000001413 amino acids Chemical group 0.000 claims description 76
- 239000002773 nucleotide Substances 0.000 claims description 75
- 125000003729 nucleotide group Chemical group 0.000 claims description 75
- 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 52
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 52
- 239000013612 plasmid Substances 0.000 claims description 30
- 230000008859 change Effects 0.000 claims description 25
- 241000894006 Bacteria Species 0.000 claims description 19
- 230000008521 reorganization Effects 0.000 claims description 16
- 235000014304 histidine Nutrition 0.000 claims description 12
- 229920002472 Starch Polymers 0.000 claims description 10
- 239000008107 starch Substances 0.000 claims description 10
- 235000019698 starch Nutrition 0.000 claims description 10
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 8
- 239000013604 expression vector Substances 0.000 claims description 8
- 150000002411 histidines Chemical class 0.000 claims description 8
- 238000003259 recombinant expression Methods 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 5
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 4
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims description 4
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 claims description 4
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 claims description 4
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 claims description 4
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims description 4
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 claims description 4
- XUYPXLNMDZIRQH-LURJTMIESA-N N-acetyl-L-methionine Chemical compound CSCC[C@@H](C(O)=O)NC(C)=O XUYPXLNMDZIRQH-LURJTMIESA-N 0.000 claims description 4
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims description 4
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 4
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims description 4
- 239000004473 Threonine Substances 0.000 claims description 4
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 4
- 239000006035 Tryptophane Substances 0.000 claims description 4
- 229960002989 glutamic acid Drugs 0.000 claims description 4
- 235000013905 glycine and its sodium salt Nutrition 0.000 claims description 4
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 4
- 229930182817 methionine Natural products 0.000 claims description 4
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 229960004799 tryptophan Drugs 0.000 claims description 4
- 229930092411 Swietenocoumarin D Natural products 0.000 claims description 3
- 238000003780 insertion Methods 0.000 claims description 3
- 230000037431 insertion Effects 0.000 claims description 3
- 230000000968 intestinal effect Effects 0.000 claims description 3
- 239000013598 vector Substances 0.000 claims description 3
- 230000009261 transgenic effect Effects 0.000 claims description 2
- 230000037430 deletion Effects 0.000 claims 1
- 238000012217 deletion Methods 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 241000178960 Paenibacillus macerans Species 0.000 abstract description 3
- 125000000539 amino acid group Chemical group 0.000 abstract 2
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 abstract 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 abstract 1
- 239000004474 valine Substances 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 83
- 102000053602 DNA Human genes 0.000 description 64
- 239000000203 mixture Substances 0.000 description 61
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 description 52
- 239000006228 supernatant Substances 0.000 description 47
- 108091008146 restriction endonucleases Proteins 0.000 description 25
- 238000012163 sequencing technique Methods 0.000 description 20
- 239000000047 product Substances 0.000 description 14
- 108090000790 Enzymes Proteins 0.000 description 10
- 102000004190 Enzymes Human genes 0.000 description 10
- 239000000126 substance Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 239000007864 aqueous solution Substances 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- KZMAWJRXKGLWGS-UHFFFAOYSA-N 2-chloro-n-[4-(4-methoxyphenyl)-1,3-thiazol-2-yl]-n-(3-methoxypropyl)acetamide Chemical compound S1C(N(C(=O)CCl)CCCOC)=NC(C=2C=CC(OC)=CC=2)=C1 KZMAWJRXKGLWGS-UHFFFAOYSA-N 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 230000008034 disappearance Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- GDSRMADSINPKSL-HSEONFRVSA-N gamma-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO GDSRMADSINPKSL-HSEONFRVSA-N 0.000 description 3
- 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 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 229920001353 Dextrin Polymers 0.000 description 2
- 239000004375 Dextrin Substances 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 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 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 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 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- -1 cyclic oligosaccharides Chemical class 0.000 description 2
- 235000019425 dextrin Nutrition 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 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
- 230000031700 light absorption Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 229920001542 oligosaccharide Polymers 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- WHGYBXFWUBPSRW-UHFFFAOYSA-N Cycloheptaamylose Natural products O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO WHGYBXFWUBPSRW-UHFFFAOYSA-N 0.000 description 1
- 108010025880 Cyclomaltodextrin glucanotransferase Proteins 0.000 description 1
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 1
- 239000001116 FEMA 4028 Substances 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
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 241001609931 bacterium 20 Species 0.000 description 1
- 235000011175 beta-cyclodextrine Nutrition 0.000 description 1
- 229960004853 betadex Drugs 0.000 description 1
- 235000019658 bitter taste Nutrition 0.000 description 1
- RYYVLZVUVIJVGH-UHFFFAOYSA-N caffeine Chemical compound CN1C(=O)N(C)C(=O)C2=C1N=CN2C RYYVLZVUVIJVGH-UHFFFAOYSA-N 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000012847 fine chemical Substances 0.000 description 1
- 238000005187 foaming Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 229930182478 glucoside Natural products 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 125000003147 glycosyl group Chemical group 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- BULVZWIRKLYCBC-UHFFFAOYSA-N phorate Chemical compound CCOP(=S)(OCC)SCSCC BULVZWIRKLYCBC-UHFFFAOYSA-N 0.000 description 1
- 238000005375 photometry Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 108010046845 tryptones Proteins 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
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
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a group of cyclodextrin glucosyltransferase, and a coding gene and an application thereof. The cyclodextrin glucosyltransferase provided by the invention comes from Bacillus macerans, and is a protein obtained by mutating a protein as shown in a sequence 1 of a sequence table through the following two mutations: (a) mutating an amino acid residue at 536th site of N-end of the sequence 1 in the sequence table from alanine to valine, and (b) mutating an amino acid residue at 167th site of N-end of the sequence 1 in the sequence table. The protein provided by the invention is relatively high in specificity of producing alpha-CD, relatively high in production efficiency, and has great value to production.
Description
Technical field
The present invention relates to one group of cyclodextrine Transglucosylase and encoding sox and application.
Background technology
The English of cyclodextrine is cyclodextrin, is called for short CD.Schardinger dextrins is the product that Schardinger dextrins transglucosylase (CGTase) acts on starch; Be that six above glucose are with α-1; 4-glycosidic link banded member cyclic oligosaccharides; Wherein the most common, research is maximum to be that (((γ-CD) is made up of six, seven and eight glucose molecules respectively alpha-cylodextrin, is the molecule of big relatively and relative flexibility for β-CD), γ-Huan Hujing for α-CD), beta-cyclodextrin.
Cyclohexaamylose is one type of high-valued product of starch deep processing, passes through the cyclic oligosaccharide that α-1,4 glucoside bond is formed by connecting by six glucones.Structurally; The three-dimensional arrangement of α-CD cylindrical (end is big, and an end is little), have " outer hydrophilic; in hydrophobic " singularity and nontoxic premium properties; Can adopt proper method to carry out inclusion with multiple guest compound, thereby change, some very useful functions are provided by physico-chemical properties such as inclusion solubility of substances, volatility and chemical reaction performances.
α-CD is a kind of potential foodstuff additive, particularly can be used as the cycloheptaamylose the produced at present (substitute products of β-CD).β-CD has scale operation, but finds that in use it has the sedimentary risk of internal organs, and bag connects the problem of inefficiency.
Summary is got up, and α-CD has following application prospect in foodstuffs industry: the high encapsulation compound of water solubility is processed with water-insoluble or the low compound of solubleness in (1); (2) make the compound of sealing have satisfactory stability property (as protect look, protect fragrant, heat-resisting, acidproof, hydrolysis, anti-oxidant, volatilization prevention etc.); (3) shielding effect (covering unhappy smell and bitter taste in the food); (4) remove unwanted composition (like theine, SUV etc.) in the food; (5) have emulsification, foaming effect, can make emulsifying agent and whipping agent; (6) liquid, oily, volatile material are solidified.
At present, the biggest obstacle of α-CD development and application is to lack to have the zymin that high alpha-cylodextrin transforms vigor, makes the cost and price of this product exceed three times of β-CD, has limited its large-scale application.
The generation of α-CD mainly is to be raw material with starch, through the glycosyl transformation of cyclodextrine Transglucosylase (cyclodextrin glucano-transferase is called for short CGTases).The specificity of CGTases is lower, in most of the cases produces α-CD, β-CD and three kinds of products of γ-CD simultaneously.Three kinds of proportion of products differences that adopt different CGTases to produce, the main converted product of existing C GTases is β-CD, also produces a certain amount of α-and γ-CD simultaneously.Existing research direction mainly is two aspects: the first is sought the better or higher enzyme of specificity of catalytic effect from natural resources; Thereby it two is existing zymoprotein to be carried out directional transformation obtain the better or higher enzyme of specificity of catalytic effect.
Summary of the invention
The purpose of this invention is to provide one group of cyclodextrine Transglucosylase and encoding sox and application.
Cyclodextrine Transglucosylase provided by the invention from soft rotten genus bacillus (Bacillus macerans), is that protein shown in the sequence 1 of sequence table is carried out as follows (a) and (b) two protein that sudden change obtains:
(a) sequence 1 with sequence table is Xie Ansuan from N-terminal the 536th amino acids residue by alanine mutation;
(b) sequence 1 of sequence table is suddenlyd change from N-terminal the 167th amino acids residue.
Sport as follows in (1) to (17) any one described in said (b):
(1) sequence 1 with sequence table sports three successive Histidines from N-terminal the 167th amino acids residue by tyrosine;
(2) sequence 1 with sequence table sports two successive Histidines from N-terminal the 167th amino acids residue by tyrosine;
(3) sequence 1 with sequence table sports Histidine from N-terminal the 167th amino acids residue by tyrosine;
(4) sequence 1 with sequence table lacks from N-terminal the 167th amino acids residue (tyrosine);
(5) sequence 1 with sequence table sports proline(Pro) from N-terminal the 167th amino acids residue by tyrosine;
(6) sequence 1 with sequence table sports glycocoll from N-terminal the 167th amino acids residue by tyrosine;
(7) sequence 1 with sequence table sports Threonine from N-terminal the 167th amino acids residue by tyrosine;
(8) sequence 1 with sequence table sports L-glutamic acid from N-terminal the 167th amino acids residue by tyrosine;
(9) sequence 1 with sequence table sports l-asparagine from N-terminal the 167th amino acids residue by tyrosine;
(10) sequence 1 with sequence table sports Serine from N-terminal the 167th amino acids residue by tyrosine;
(11) sequence 1 with sequence table sports phenylalanine(Phe) from N-terminal the 167th amino acids residue by tyrosine;
(12) sequence 1 with sequence table sports Xie Ansuan from N-terminal the 167th amino acids residue by tyrosine;
(13) sequence 1 with sequence table sports halfcystine from N-terminal the 167th amino acids residue by tyrosine;
(14) sequence 1 with sequence table sports l-arginine from N-terminal the 167th amino acids residue by tyrosine;
(15) sequence 1 with sequence table sports leucine from N-terminal the 167th amino acids residue by tyrosine;
(16) sequence 1 with sequence table sports Methionin from N-terminal the 167th amino acids residue by tyrosine;
(17) sequence 1 with sequence table sports tryptophane from N-terminal the 167th amino acids residue by tyrosine.
Said mutation is the single amino acids residue like no specified otherwise.
The gene of code for said proteins also belongs to protection scope of the present invention.
Said gene specifically can be protein shown in the sequence 2 of sequence table is carried out as follows (c) and (d) two dna moleculars that sudden change obtains:
(c) sequence 2 with sequence table sports GTG from 5 ' terminal 1606-1608 position Nucleotide by GCG;
(d) sequence 2 of sequence table is suddenlyd change from 5 ' terminal 499-501 position Nucleotide.
Said in said (d) sports any one in following (1) to (17) as follows:
(1) sequence 2 with sequence table sports CACCACCAC from 5 ' terminal 499-501 position Nucleotide by TAC;
(2) sequence 2 with sequence table sports CACCAC from 5 ' terminal 499-501 position Nucleotide by TAC;
(3) sequence 2 with sequence table sports CAC from 5 ' terminal 499-501 position Nucleotide by TAC;
(4) sequence 2 with sequence table lacks from 5 ' terminal 499-501 position Nucleotide (TAC);
(5) sequence 2 with sequence table sports CCA from 5 ' terminal 499-501 position Nucleotide by TAC;
(6) sequence 2 with sequence table sports GGT from 5 ' terminal 499-501 position Nucleotide by TAC;
(7) sequence 2 with sequence table sports ACA from 5 ' terminal 499-501 position Nucleotide by TAC;
(8) sequence 2 with sequence table sports GAG from 5 ' terminal 499-501 position Nucleotide by TAC;
(9) sequence 2 with sequence table sports AAT from 5 ' terminal 499-501 position Nucleotide by TAC;
(10) sequence 2 with sequence table sports TCA from 5 ' terminal 499-501 position Nucleotide by TAC;
(11) sequence 2 with sequence table sports TTT from 5 ' terminal 499-501 position Nucleotide by TAC;
(12) sequence 2 with sequence table sports GTA from 5 ' terminal 499-501 position Nucleotide by TAC;
(13) sequence 2 with sequence table sports TGC from 5 ' terminal 499-501 position Nucleotide by TAC;
(14) sequence 2 with sequence table sports CGA from 5 ' terminal 499-501 position Nucleotide by TAC;
(15) sequence 2 with sequence table sports CTA from 5 ' terminal 499-501 position Nucleotide by TAC;
(16) sequence 2 with sequence table sports AAG from 5 ' terminal 499-501 position Nucleotide by TAC;
(17) sequence 2 with sequence table sports TGG from 5 ' terminal 499-501 position Nucleotide by TAC.
The recombinant expression vector, expression cassette, transgenic cell line or the reorganization bacterium that contain said gene all belong to protection scope of the present invention.
Said recombinant expression vector specifically can be the recombinant plasmid that the MCS with said gene insertion vector pET-22b (+) obtains.Said recombinant expression vector specifically can be the recombinant plasmid that obtains between the BamHI of said gene insertion vector pET-22b (+) and the XhoI restriction enzyme site.
Said reorganization bacterium specifically can be said recombinant expression vector is imported the reorganization bacterium that intestinal bacteria obtain.Said intestinal bacteria specifically can be e. coli bl21 (DE3).
The present invention also protects the said method of protein of a kind of preparation, is to cultivate said reorganization bacterium, obtains said protein.The process of said cultivation is specific as follows: said reorganization bacterium is seeded to the TB substratum, and 37 ℃, 220rpm shaking culture are to OD600
Nm=0.6 (0.6-0.8 all can); Add IPTG, making its concentration in culture system is 0.01mM, 16 ℃ then, 220rpm shaking culture 96h; With 4 ℃ of culture systems, centrifugal 10 minutes of 8000rpm, collect supernatant.
The present invention also protects said proteinic application, is (I) or (II) or (III) as follows:
(I) preparation cyclodextrine Transglucosylase;
(II) degraded starch (like Zulkovsky starch);
(III) produced cyclohexaamylose.
Protein provided by the invention, the specificity of producing α-CD is higher, and production efficiency is higher, and production is had great value.
Description of drawings
Fig. 1 is the structural representation of recombinant plasmid first.
Fig. 2 is α-CD typical curve.
Fig. 3 is β-CD typical curve.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.Zulkovsky starch: modern east, Beijing fine chemicals ltd, lot number: 20070216.Carrier pET-22b (+): Novagen, catalog number is 69744-3.E. coli bl21 (DE3): the Beijing Quanshijin Biotechnology Co., Ltd, catalog number is CD601-01.
TB substratum (g/L): Tryptones 12, yeast extract cream 24, glycerine 4mL, zero(ppm) water constant volume.
The discovery of embodiment 1, mutain and encoding sox thereof
Screen the soft rotten genus bacillus of a strain (Bacillus macerans) from nature, can produce Maltose 4-glucosyltransferase, the Maltose 4-glucosyltransferase gene in this bacterial strain shown in the sequence 2 of sequence table, the protein shown in the sequence 1 of code sequence tabulation.
With the α of protein called after shown in the sequence 1-CGTase-1 albumen, with its encoding sox called after α-CGTase-1 gene (shown in sequence 2).With the α of protein called after shown in the sequence 3-CGTase-WT albumen (disclosed sequence among the NCBI), with its encoding sox called after α-CGTase-WT gene (shown in sequence 4).
With double-stranded DNA shown in the sequence 1 of sequence table is template, carries out fallibility PCR, obtains mutant library.Carry out enzyme evaluation alive through albumen, found that the product specificity of a series of enzymes is different from proteic mutain of α-CGTase-WT shown in the sequence 3 and encoding sox thereof each genetic expression in the mutant library.
Embodiment 2, enzyme are lived and are identified
One, the following double chain DNA molecule of difference synthetic:
(1) double chain DNA molecule 1: with the difference of dna molecular shown in the sequence 2 of sequence table be with from 5 ' terminal 499-501 position Nucleotide by the TAC sudden change for TGG, and will sport GTG by GCG from 5 ' terminal 1606-1608 position Nucleotide; Accordingly the 167th amino acids residue in the sequence 1 is sported tryptophane by tyrosine, and be Xie Ansuan by alanine mutation the 536th amino acids residue.Double chain DNA molecule 1 encoded protein called after albumen 1.
(2) double chain DNA molecule 2: with the difference of dna molecular shown in the sequence 2 of sequence table be with from 5 ' terminal 499-501 position Nucleotide by the TAC sudden change for TGC, and will sport GTG by GCG from 5 ' terminal 1606-1608 position Nucleotide; Accordingly the 167th amino acids residue in the sequence 1 is sported halfcystine by tyrosine, and be Xie Ansuan by alanine mutation the 536th amino acids residue.Double chain DNA molecule 2 encoded protein called after albumen 2.
(3) double chain DNA molecule 3: with the difference of dna molecular shown in the sequence 2 of sequence table be with from 5 ' terminal 499-501 position Nucleotide by the TAC sudden change for CTA, and will sport GTG by GCG from 5 ' terminal 1606-1608 position Nucleotide; Accordingly the 167th amino acids residue in the sequence 1 is sported leucine by tyrosine, and be Xie Ansuan by alanine mutation the 536th amino acids residue.Double chain DNA molecule 3 encoded protein called after albumen 3.
(4) double chain DNA molecule 4: with the difference of dna molecular shown in the sequence 2 of sequence table be with from 5 ' terminal 499-501 position Nucleotide by the TAC sudden change for GTA, and will sport GTG by GCG from 5 ' terminal 1606-1608 position Nucleotide; Accordingly the 167th amino acids residue in the sequence 1 is sported Xie Ansuan by tyrosine, and be Xie Ansuan by alanine mutation the 536th amino acids residue.Double chain DNA molecule 4 encoded protein called after albumen 4.
(5) double chain DNA molecule 5: with the difference of dna molecular shown in the sequence 2 of sequence table be with from 5 ' terminal 499-501 position Nucleotide by the TAC sudden change for GGT, and will sport GTG by GCG from 5 ' terminal 1606-1608 position Nucleotide; Accordingly the 167th amino acids residue in the sequence 1 is sported glycocoll by tyrosine, and be Xie Ansuan by alanine mutation the 536th amino acids residue.Double chain DNA molecule 5 encoded protein called after albumen 5.
(6) double chain DNA molecule 6: with the difference of dna molecular shown in the sequence 2 of sequence table be with from 5 ' terminal 499-501 position Nucleotide by the TAC sudden change for CGA, and will sport GTG by GCG from 5 ' terminal 1606-1608 position Nucleotide; Accordingly the 167th amino acids residue in the sequence 1 is sported l-arginine by tyrosine, and be Xie Ansuan by alanine mutation the 536th amino acids residue.Double chain DNA molecule 6 encoded protein called after albumen 6.
(7) double chain DNA molecule 7: with the difference of dna molecular shown in the sequence 2 of sequence table be with from 5 ' terminal 499-501 position Nucleotide by the TAC sudden change for GAG, and will sport GTG by GCG from 5 ' terminal 1606-1608 position Nucleotide; Accordingly the 167th amino acids residue in the sequence 1 is sported L-glutamic acid by tyrosine, and be Xie Ansuan by alanine mutation the 536th amino acids residue.Double chain DNA molecule 7 encoded protein called after albumen 7.
(8) double chain DNA molecule 8: with the difference of dna molecular shown in the sequence 2 of sequence table be with from 5 ' terminal 499-501 position Nucleotide by the TAC sudden change for AAT, and will sport GTG by GCG from 5 ' terminal 1606-1608 position Nucleotide; Accordingly the 167th amino acids residue in the sequence 1 is sported l-asparagine by tyrosine, and be Xie Ansuan by alanine mutation the 536th amino acids residue.Double chain DNA molecule 8 encoded protein called after albumen 8.
(9) double chain DNA molecule 9: with the difference of dna molecular shown in the sequence 2 of sequence table be with from 5 ' terminal 499-501 position Nucleotide by the TAC sudden change for CAC, and will sport GTG by GCG from 5 ' terminal 1606-1608 position Nucleotide; Accordingly the 167th amino acids residue in the sequence 1 is sported Histidine by tyrosine, and be Xie Ansuan by alanine mutation the 536th amino acids residue.Double chain DNA molecule 9 encoded protein called after albumen 9.
(10) double chain DNA molecule 10: with the difference of dna molecular shown in the sequence 2 of sequence table be with from 5 ' terminal 499-501 position Nucleotide by the TAC sudden change for CCA, and will sport GTG by GCG from 5 ' terminal 1606-1608 position Nucleotide; Accordingly the 167th amino acids residue in the sequence 1 is sported proline(Pro) by tyrosine, and be Xie Ansuan by alanine mutation the 536th amino acids residue.Double chain DNA molecule 10 encoded protein called after protein 10s.
(11) double chain DNA molecule 11: with the difference of dna molecular shown in the sequence 2 of sequence table be with from 5 ' terminal 499-501 position Nucleotide by the TAC sudden change for ACA, and will sport GTG by GCG from 5 ' terminal 1606-1608 position Nucleotide; Accordingly the 167th amino acids residue in the sequence 1 is sported Threonine by tyrosine, and be Xie Ansuan by alanine mutation the 536th amino acids residue.Double chain DNA molecule 11 encoded protein called after protein 11s.
(12) double chain DNA molecule 12: with the difference of dna molecular shown in the sequence 2 of sequence table be with from 5 ' terminal 499-501 position Nucleotide by the TAC sudden change for TCA, and will sport GTG by GCG from 5 ' terminal 1606-1608 position Nucleotide; Accordingly the 167th amino acids residue in the sequence 1 is sported Serine by tyrosine, and be Xie Ansuan by alanine mutation the 536th amino acids residue.Double chain DNA molecule 12 encoded protein called after protein 12s.
(13) double chain DNA molecule 13: with the difference of dna molecular shown in the sequence 2 of sequence table be with from 5 ' terminal 499-501 position Nucleotide by the TAC sudden change for TTT, and will sport GTG by GCG from 5 ' terminal 1606-1608 position Nucleotide; Accordingly the 167th amino acids residue in the sequence 1 is sported phenylalanine(Phe) by tyrosine, and be Xie Ansuan by alanine mutation the 536th amino acids residue.Double chain DNA molecule 13 encoded protein called after albumen 13.
(14) double chain DNA molecule 14: with the difference of dna molecular shown in the sequence 2 of sequence table be with from 5 ' terminal 499-501 position Nucleotide by the TAC sudden change for AAG, and will sport GTG by GCG from 5 ' terminal 1606-1608 position Nucleotide; Accordingly the 167th amino acids residue in the sequence 1 is sported Methionin by tyrosine, and be Xie Ansuan by alanine mutation the 536th amino acids residue.Double chain DNA molecule 14 encoded protein called after protein 14s.
(15) double chain DNA molecule 15: be from 5 ' terminal 499-501 position Nucleotide (TAC) disappearance with the difference of dna molecular shown in the sequence 2 of sequence table, and will sport GTG by GCG from 5 ' terminal 1606-1608 position Nucleotide; Is Xie Ansuan accordingly with the disappearance of the 167th amino acids residue (tyrosine) in the sequence 1, and with the 536th amino acids residue by alanine mutation.Double chain DNA molecule 15 encoded protein called after protein 15s.
(16) double chain DNA molecule 16: with the difference of dna molecular shown in the sequence 2 of sequence table be with from 5 ' terminal 499-501 position Nucleotide by the TAC sudden change for CACCAC, and will sport GTG by GCG from 5 ' terminal 1606-1608 position Nucleotide; Accordingly the 167th amino acids residue in the sequence 1 is sported two successive Histidines by tyrosine, and be Xie Ansuan by alanine mutation the 536th amino acids residue.Double chain DNA molecule 16 encoded protein called after protein 16s.
(17) double chain DNA molecule 17: with the difference of dna molecular shown in the sequence 2 of sequence table be with from 5 ' terminal 499-501 position Nucleotide by the TAC sudden change for CACCACCAC, and will sport GTG by GCG from 5 ' terminal 1606-1608 position Nucleotide; Accordingly the 167th amino acids residue in the sequence 1 is sported three successive Histidines by tyrosine, and be Xie Ansuan by alanine mutation the 536th amino acids residue.Double chain DNA molecule 17 encoded protein called after protein 17s.
(18) double chain DNA molecule 18: be sporting GTG from 5 ' terminal 1606-1608 position Nucleotide by GCG with the difference of dna molecular shown in the sequence 2 of sequence table; Corresponding is Xie Ansuan with the 536th amino acids residue in the sequence 1 by alanine mutation.Double chain DNA molecule 18 encoded protein called after protein 18s.
(19) double chain DNA molecule 19: i.e. double chain DNA molecule shown in the sequence 2 of sequence table.
(20) double chain DNA molecule 20: i.e. double chain DNA molecule shown in the sequence 4 of sequence table.
Two, construction of recombinant plasmid (structural representation is seen Fig. 1)
1, designs a pair of primer, form by S2 and A3.
S2：5’-CGC
GGATCCG
-3’；
A3：5'-CGG
CTCGAG 
-3’。
Among the S2, underscore mark BamHI restriction endonuclease recognition sequence, the zone that the square frame mark is corresponding with target sequence.Among the A3, underscore mark XhoI restriction endonuclease recognition sequence, the zone that the square frame mark is corresponding with target sequence.
2, be template with each double chain DNA molecule of step 2 synthetic respectively, the primer of forming with S2 and A3 obtains pcr amplification product to carrying out pcr amplification.
3, with the pcr amplification product of restriction enzyme BamHI and XhoI double digestion step (1), obtain enzyme and cut product.
4,, reclaim carrier framework (about 5.4kb) with restriction enzyme BamHI and XhoI double digestion carrier pET-22b (+).
5, the carrier framework of the enzyme of step (3) being cut product and step (4) is connected, and obtains recombinant plasmid.
According to the numbering of double chain DNA molecule, called after recombinant plasmid 1 to recombinant plasmid 20 successively.
It is following according to sequencing result recombinant plasmid 1 to be carried out structrual description: between the BamHI of carrier pET-22b (+) and XhoI restriction enzyme site, inserted double chain DNA molecule 1.
It is following according to sequencing result recombinant plasmid 2 to be carried out structrual description: between the BamHI of carrier pET-22b (+) and XhoI restriction enzyme site, inserted double chain DNA molecule 2.
It is following according to sequencing result recombinant plasmid 3 to be carried out structrual description: between the BamHI of carrier pET-22b (+) and XhoI restriction enzyme site, inserted double chain DNA molecule 3.
It is following according to sequencing result recombinant plasmid 4 to be carried out structrual description: between the BamHI of carrier pET-22b (+) and XhoI restriction enzyme site, inserted double chain DNA molecule 4.
It is following according to sequencing result recombinant plasmid 5 to be carried out structrual description: between the BamHI of carrier pET-22b (+) and XhoI restriction enzyme site, inserted double chain DNA molecule 5.
It is following according to sequencing result recombinant plasmid 6 to be carried out structrual description: between the BamHI of carrier pET-22b (+) and XhoI restriction enzyme site, inserted double chain DNA molecule 6.
It is following according to sequencing result recombinant plasmid 7 to be carried out structrual description: between the BamHI of carrier pET-22b (+) and XhoI restriction enzyme site, inserted double chain DNA molecule 7.
It is following according to sequencing result recombinant plasmid 8 to be carried out structrual description: between the BamHI of carrier pET-22b (+) and XhoI restriction enzyme site, inserted double chain DNA molecule 8.
It is following according to sequencing result recombinant plasmid 9 to be carried out structrual description: between the BamHI of carrier pET-22b (+) and XhoI restriction enzyme site, inserted double chain DNA molecule 9.
It is following according to sequencing result recombinant plasmid 10 to be carried out structrual description: between the BamHI of carrier pET-22b (+) and XhoI restriction enzyme site, inserted double chain DNA molecule 10.
It is following according to sequencing result recombinant plasmid 11 to be carried out structrual description: between the BamHI of carrier pET-22b (+) and XhoI restriction enzyme site, inserted double chain DNA molecule 11.
It is following according to sequencing result recombinant plasmid 12 to be carried out structrual description: between the BamHI of carrier pET-22b (+) and XhoI restriction enzyme site, inserted double chain DNA molecule 12.
It is following according to sequencing result recombinant plasmid 13 to be carried out structrual description: between the BamHI of carrier pET-22b (+) and XhoI restriction enzyme site, inserted double chain DNA molecule 13.
It is following according to sequencing result recombinant plasmid 14 to be carried out structrual description: between the BamHI of carrier pET-22b (+) and XhoI restriction enzyme site, inserted double chain DNA molecule 14.
It is following according to sequencing result recombinant plasmid 15 to be carried out structrual description: between the BamHI of carrier pET-22b (+) and XhoI restriction enzyme site, inserted double chain DNA molecule 15.
It is following according to sequencing result recombinant plasmid 16 to be carried out structrual description: between the BamHI of carrier pET-22b (+) and XhoI restriction enzyme site, inserted double chain DNA molecule 16.
It is following according to sequencing result recombinant plasmid 17 to be carried out structrual description: between the BamHI of carrier pET-22b (+) and XhoI restriction enzyme site, inserted double chain DNA molecule 17.
It is following according to sequencing result recombinant plasmid 18 to be carried out structrual description: between the BamHI of carrier pET-22b (+) and XhoI restriction enzyme site, inserted double chain DNA molecule 18.
It is following according to sequencing result recombinant plasmid 19 to be carried out structrual description: between the BamHI of carrier pET-22b (+) and XhoI restriction enzyme site, inserted the double chain DNA molecule shown in the sequence 2 of sequence table.
It is following according to sequencing result recombinant plasmid 20 to be carried out structrual description: between the BamHI of carrier pET-22b (+) and XhoI restriction enzyme site, inserted the double chain DNA molecule shown in the sequence 4 of sequence table.
Three, the structure of reorganization bacterium
Each recombinant plasmid that step 2 is made up imports e. coli bl21 (DE3) respectively, obtains each reorganization bacterium.According to the numbering of recombinant plasmid, called after reorganization bacterium 1 is to the bacterium 20 of recombinating successively.
Four, the fermentation of reorganization bacterium
Each reorganization bacterium that step 3 is made up is seeded to respectively in the TB substratum, and 37 ℃, 220rpm shaking culture are to OD600
Nm=0.6 (0.6-0.8 all can); Add IPTG, making its concentration in culture system is 0.01mM, 16 ℃ then, 220rpm shaking culture 96h; With 4 ℃ of culture systems, centrifugal 10 minutes of 8000rpm, collect supernatant.
According to the numbering of reorganization bacterium, called after supernatant 1 to supernatant 20 successively.
Five, enzyme is lived and is identified and product analysis
1, enzyme activity determination
(1) the Zulkovsky starch aqueous solution of preparation 0.25g/100mL.
(2) experimental group: add the 0.4mL Zulkovsky starch aqueous solution in the test tube, be incubated 15min in 40 ℃ of water-baths, add 0.1mL solution to be measured then; Control group group: before adding solution to be measured, add the 1.5mL 0.1mol/L HCl aqueous solution earlier, other same experimental group; Experimental group and control group mixing are placed in 40 ℃ of waters bath with thermostatic control and are incubated 10min, and experimental group is added the 1.5mL 0.1mol/L HCl aqueous solution then; Add 3mL 0.1mol/L I
2Liquid (solvent is a water) and adding 5mL zero(ppm) water, mixing, rapidly in 700nm place photometry absorption value, and the record experimental data.
Enzyme (U/mL)=(a-b)/a alive * 100 * extension rate; A is the light absorption value of control group, and b is the light absorption value of experimental group.
2, product analysis
Each supernatant that respectively step 4 is obtained (solution to be measured) is tested as follows: the Zulkovsky starch aqueous solution of 2g/100mL is boiled 10min; Get 2mL after the cooling and add in the new EP pipe, add solution to be measured by the 400U/g substrate then, be settled to 4mL, in 40 ℃ of water-baths, left standstill 24 hours then with zero(ppm) water; Boil 10min then, the centrifugal 10min of 12000rpm gets supernatant; Supernatant is got 20uL after with the 0.45um ultrafiltration membrance filter to carry out HPLC and analyzes.
The parameter that HPLC analyzes: Waters600HPLC chromatographic instrument, Waters manual injector, chromatographic column Lichrosorb NH
2(4.6mm * 150mm), Waters2414 differential detector, moving phase is made up of 73 parts by volume acetonitriles and 27 parts by volume water, flow velocity 1mL/min, 40 ℃ of column temperatures.
Use α-CD standard substance, β-CD standard substance production standard curve respectively.The appearance time of α-CD standard substance is 8.5min, and typical curve is seen Fig. 2, and the typical curve equation is y=908404x, R
2=0.9954, y represents the concentration (g/100ml) of α-CD, and y represents peak area.The appearance time of β-CD standard substance is 9.8min, and typical curve is seen Fig. 3, and the typical curve equation is y=772166x-65169, R
2=0.9978, y represents the concentration (g/100ml) of β-CD, and y represents peak area.The concentration ÷ of concentration=α of the α-CD-CD (concentration of concentration+β of α-CD-CD * 100%.The concentration ÷ of concentration=β of β-CD-CD (concentration of content+β of α-CD-CD) * 100%.
Adopt supernatant 1 to carry out step 5 and obtain in the supernatant, the percentage composition of α-CD is 73.8 ± 1.8%, and the percentage composition of β-CD is 26.2 ± 1.8%, and the percentage composition ratio of α-CD and β-CD is 2.8 ± 0.3.(tryptophane)
Adopt supernatant 2 to carry out step 5 and obtain in the supernatant, the percentage composition of α-CD is 81.5 ± 0.5%, and the percentage composition of β-CD is 18.5 ± 0.5%, and the percentage composition ratio of α-CD and β-CD is 4.4 ± 0.2.(halfcystine)
Adopt supernatant 3 to carry out step 5 and obtain in the supernatant, the percentage composition of α-CD is 75.8 ± 2.1%, and the percentage composition of β-CD is 24.2 ± 2.1%, and the percentage composition ratio of α-CD and β-CD is 3.2 ± 0.3.(leucine)
Adopt supernatant 4 to carry out step 5 and obtain in the supernatant, the percentage composition of α-CD is 81.9 ± 0.8%, and the percentage composition of β-CD is 18.1 ± 0.8%, and the percentage composition ratio of α-CD and β-CD is 4.5 ± 0.3.(Xie Ansuan)
Adopt supernatant 5 to carry out step 5 and obtain in the supernatant, the percentage composition of α-CD is 85.7 ± 0.7%, and the percentage composition of β-CD is 14.3 ± 0.7%, and the percentage composition ratio of α-CD and β-CD is 6.0 ± 0.3.(glycocoll)
Adopt supernatant 6 to carry out step 5 and obtain in the supernatant, the percentage composition of α-CD is 82.5 ± 1.7%, and the percentage composition of β-CD is 17.5 ± 1.7%, and the percentage composition ratio of α-CD and β-CD is 4.4 ± 0.2.(l-arginine)
Adopt supernatant 7 to carry out step 5 and obtain in the supernatant, the percentage composition of α-CD is 83.5 ± 0.2%, and the percentage composition of β-CD is 16.5 ± 0.2%, and the percentage composition ratio of α-CD and β-CD is 5.0 ± 0.1.(L-glutamic acid)
Adopt supernatant 8 to carry out step 5 and obtain in the supernatant, the percentage composition of α-CD is 83.3 ± 0.9%, and the percentage composition of β-CD is 16.7 ± 0.9%, and the percentage composition ratio of α-CD and β-CD is 5.0 ± 0.7.(l-asparagine)
Adopt supernatant 9 to carry out step 5 and obtain in the supernatant, the percentage composition of α-CD is 88.2 ± 0.6%, and the percentage composition of β-CD is 11.8 ± 0.6%, and the percentage composition ratio of α-CD and β-CD is 7.5 ± 0.1.(Histidine)
Adopt supernatant 10 to carry out step 5 and obtain in the supernatant, the percentage composition of α-CD is 85.8 ± 1.1%, and the percentage composition of β-CD is 14.2 ± 1.0%, and the percentage composition ratio of α-CD and β-CD is 6.1 ± 0.6.(proline(Pro))
Adopt supernatant 11 to carry out step 5 and obtain in the supernatant, the percentage composition of α-CD is 83.9 ± 1.0%, and the percentage composition of β-CD is 16.1 ± 1.0%, and the percentage composition ratio of α-CD and β-CD is 5.3 ± 0.4.(Threonine)
Adopt supernatant 12 to carry out step 5 and obtain in the supernatant, the percentage composition of α-CD is 83 ± 1.0%, and the percentage composition of β-CD is 17 ± 1.0%, and the percentage composition ratio of α-CD and β-CD is 4.9 ± 0.3.(Serine)
Adopt supernatant 13 to carry out step 5 and obtain in the supernatant, the percentage composition of α-CD is 83.8 ± 0.6%, and the percentage composition of β-CD is 16.2 ± 0.6%, and the percentage composition ratio of α-CD and β-CD is 4.9 ± 0.6.(phenylalanine(Phe))
Adopt supernatant 14 to carry out step 5 and obtain in the supernatant, the percentage composition of α-CD is 75.5 ± 2.5%, and the percentage composition of β-CD is 24.5 ± 2.5%, and the percentage composition ratio of α-CD and β-CD is 3.1 ± 0.4.(Methionin)
Adopt supernatant 15 to carry out step 5 and obtain in the supernatant, the percentage composition of α-CD is 87.6 ± 0.3%, and the percentage composition of β-CD is 12.4 ± 0.3%, and the percentage composition ratio of α-CD and β-CD is 7.1 ± 0.2.(disappearance tyrosine)
Adopt supernatant 16 to carry out step 5 and obtain in the supernatant, the percentage composition of α-CD is 90 ± 0.5%, and the percentage composition of β-CD is 10 ± 0.5%, and the percentage composition ratio of α-CD and β-CD is 9.0 ± 0.5.(two Histidines)
Adopt supernatant 17 to carry out step 5 and obtain in the supernatant, the percentage composition of α-CD is 93 ± 0.4%, and the percentage composition of β-CD is 7 ± 0.4, and the percentage composition ratio of α-CD and β-CD is 13.3 ± 0.9.(three Histidines)
Adopt supernatant 18 to carry out step 5 and obtain in the supernatant, the percentage composition of α-CD is 77.8 ± 0.4%, and the percentage composition of β-CD is 22.2 ± 0.4%, and the percentage composition ratio of α-CD and β-CD is 3.5 ± 0.1.(tyrosine)
Adopt supernatant 19 to carry out step 5 and obtain in the supernatant, the percentage composition of α-CD is 77.3 ± 0.4%, and the percentage composition of β-CD is 22.7 ± 0.4%, and the percentage composition ratio of α-CD and β-CD is 3.4 ± 0.1.(wild pair of mutant)
Adopt supernatant 20 to carry out step 5 and obtain in the supernatant, the percentage composition of α-CD is 77.8 ± 0.4%, and the percentage composition of β-CD is 22.2 ± 0.4%, and the percentage composition ratio of α-CD and β-CD is 3.5 ± 0.1.(wild no mutant)
Claims (10)
1. protein shown in the sequence 1 of sequence table is carried out as follows (a) and (b) two protein that sudden change obtains:
(a) sequence 1 with sequence table is Xie Ansuan from N-terminal the 536th amino acids residue by alanine mutation;
(b) sequence 1 of sequence table is suddenlyd change from N-terminal the 167th amino acids residue.
2. protein as claimed in claim 1 is characterized in that: said in said (b) sports as follows any one in (1) to (17):
(1) sequence 1 with sequence table sports three successive Histidines from N-terminal the 167th amino acids residue by tyrosine;
(2) sequence 1 with sequence table sports two successive Histidines from N-terminal the 167th amino acids residue by tyrosine;
(3) sequence 1 with sequence table sports Histidine from N-terminal the 167th amino acids residue by tyrosine;
(4) sequence 1 with sequence table lacks from N-terminal the 167th amino acids residue;
(5) sequence 1 with sequence table sports proline(Pro) from N-terminal the 167th amino acids residue by tyrosine;
(6) sequence 1 with sequence table sports glycocoll from N-terminal the 167th amino acids residue by tyrosine;
(7) sequence 1 with sequence table sports Threonine from N-terminal the 167th amino acids residue by tyrosine;
(8) sequence 1 with sequence table sports L-glutamic acid from N-terminal the 167th amino acids residue by tyrosine;
(9) sequence 1 with sequence table sports l-asparagine from N-terminal the 167th amino acids residue by tyrosine;
(10) sequence 1 with sequence table sports Serine from N-terminal the 167th amino acids residue by tyrosine;
(11) sequence 1 with sequence table sports phenylalanine(Phe) from N-terminal the 167th amino acids residue by tyrosine;
(12) sequence 1 with sequence table sports Xie Ansuan from N-terminal the 167th amino acids residue by tyrosine;
(13) sequence 1 with sequence table sports halfcystine from N-terminal the 167th amino acids residue by tyrosine;
(14) sequence 1 with sequence table sports l-arginine from N-terminal the 167th amino acids residue by tyrosine;
(15) sequence 1 with sequence table sports leucine from N-terminal the 167th amino acids residue by tyrosine;
(16) sequence 1 with sequence table sports Methionin from N-terminal the 167th amino acids residue by tyrosine;
(17) sequence 1 with sequence table sports tryptophane from N-terminal the 167th amino acids residue by tyrosine.
3. coding claim 1 or 2 said proteinic genes.
4. gene as claimed in claim 3 is characterized in that: said gene is for carrying out protein shown in the sequence 2 of sequence table as follows (c) and (d) two dna moleculars that sudden change obtains:
(c) sequence 2 with sequence table sports GTG from 5 ' terminal 1606-1608 position Nucleotide by GCG;
(d) sequence 2 of sequence table is suddenlyd change from 5 ' terminal 499-501 position Nucleotide.
5. gene as claimed in claim 4 is characterized in that: said in said (d) sports any one in following (1) to (17) as follows:
(1) sequence 2 with sequence table sports CACCACCAC from 5 ' terminal 499-501 position Nucleotide by TAC;
(2) sequence 2 with sequence table sports CACCAC from 5 ' terminal 499-501 position Nucleotide by TAC;
(3) sequence 2 with sequence table sports CAC from 5 ' terminal 499-501 position Nucleotide by TAC;
(4) with the sequence 2 of sequence table from 5 ' terminal 499-501 position nucleotide deletion;
(5) sequence 2 with sequence table sports CCA from 5 ' terminal 499-501 position Nucleotide by TAC;
(6) sequence 2 with sequence table sports GGT from 5 ' terminal 499-501 position Nucleotide by TAC;
(7) sequence 2 with sequence table sports ACA from 5 ' terminal 499-501 position Nucleotide by TAC;
(8) sequence 2 with sequence table sports GAG from 5 ' terminal 499-501 position Nucleotide by TAC;
(9) sequence 2 with sequence table sports AAT from 5 ' terminal 499-501 position Nucleotide by TAC;
(10) sequence 2 with sequence table sports TCA from 5 ' terminal 499-501 position Nucleotide by TAC;
(11) sequence 2 with sequence table sports TTT from 5 ' terminal 499-501 position Nucleotide by TAC;
(12) sequence 2 with sequence table sports GTA from 5 ' terminal 499-501 position Nucleotide by TAC;
(13) sequence 2 with sequence table sports TGC from 5 ' terminal 499-501 position Nucleotide by TAC;
(14) sequence 2 with sequence table sports CGA from 5 ' terminal 499-501 position Nucleotide by TAC;
(15) sequence 2 with sequence table sports CTA from 5 ' terminal 499-501 position Nucleotide by TAC;
(16) sequence 2 with sequence table sports AAG from 5 ' terminal 499-501 position Nucleotide by TAC;
(17) sequence 2 with sequence table sports TGG from 5 ' terminal 499-501 position Nucleotide by TAC.
6. the recombinant expression vector, expression cassette, transgenic cell line or the reorganization bacterium that contain claim 3 or 4 or 5 said genes.
7. recombinant expression vector as claimed in claim 6 is characterized in that: the recombinant plasmid that said recombinant expression vector obtains for the MCS with said gene insertion vector pET-22b (+).
8. reorganization bacterium as claimed in claim 6 is characterized in that: said reorganization bacterium is that the said recombinant expression vector of claim 7 is imported the reorganization bacterium that intestinal bacteria obtain.
9. one kind prepares claim 1 or 2 said method of protein, is to cultivate claim 6 or 8 said reorganization bacterium, obtains claim 1 or 2 said protein.
10. claim 1 or 2 said proteinic application are (I) or (II) or (III) as follows:
(I) preparation cyclodextrine Transglucosylase;
(II) degraded starch;
(III) produced cyclohexaamylose.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210303166.1A CN102827815B (en) | 2012-08-23 | 2012-08-23 | A group of cyclodextrin glucosyltransferase, and coding gene and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210303166.1A CN102827815B (en) | 2012-08-23 | 2012-08-23 | A group of cyclodextrin glucosyltransferase, and coding gene and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102827815A true CN102827815A (en) | 2012-12-19 |
| CN102827815B CN102827815B (en) | 2014-01-01 |
Family
ID=47331128
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210303166.1A Expired - Fee Related CN102827815B (en) | 2012-08-23 | 2012-08-23 | A group of cyclodextrin glucosyltransferase, and coding gene and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102827815B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103232981A (en) * | 2013-05-06 | 2013-08-07 | 中国科学院微生物研究所 | A group of cyclodextrine glucosyltransferases and encoding gene and application thereof |
| CN103589697A (en) * | 2013-10-17 | 2014-02-19 | 北京航空航天大学 | One group of cyclodextrine glucosyltransferase as well as coding gene and application thereof |
| CN113801860A (en) * | 2020-06-16 | 2021-12-17 | 中国科学院微生物研究所 | Application of protein CGTase as cyclodextrin glycosyltransferase |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101294149A (en) * | 2008-05-14 | 2008-10-29 | 江南大学 | Alpha-cyclodextrin glucosyl transferase gene clone and expression |
-
2012
- 2012-08-23 CN CN201210303166.1A patent/CN102827815B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101294149A (en) * | 2008-05-14 | 2008-10-29 | 江南大学 | Alpha-cyclodextrin glucosyl transferase gene clone and expression |
Non-Patent Citations (5)
| Title |
|---|
| 《重组alpha-环糊精葡糖基转移酶表达条件的优化及其产物专一性的分析》 20120131 宋炳红 等 重组alpha-环糊精葡萄糖基转移酶表达条件的优化及其产物专一性的分析 第10卷, 第1期 * |
| 《食品科学》 20110215 王宁 等 利用来源于Paenibacillus macerans的alpha-CGTase突变体Y89D制备alpha-环糊精 第32卷, 第3期 * |
| COSTA H ET AL: "Structure-function relationship in cyclodextrin glycosyltransferase from bacillus circulans DF 9R", 《CARBOHYDRATE RESEARCH》, vol. 344, no. 1, 27 September 2008 (2008-09-27) * |
| 宋炳红 等: "重组α-环糊精葡萄糖基转移酶表达条件的优化及其产物专一性的分析", 《重组Α-环糊精葡糖基转移酶表达条件的优化及其产物专一性的分析》, vol. 10, no. 1, 31 January 2012 (2012-01-31) * |
| 王宁 等: "利用来源于Paenibacillus macerans的α-CGTase突变体Y89D制备α-环糊精", 《食品科学》, vol. 32, no. 3, 15 February 2011 (2011-02-15) * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103232981A (en) * | 2013-05-06 | 2013-08-07 | 中国科学院微生物研究所 | A group of cyclodextrine glucosyltransferases and encoding gene and application thereof |
| CN103232981B (en) * | 2013-05-06 | 2014-10-08 | 中国科学院微生物研究所 | A group of cyclodextrine glucosyltransferases and encoding gene and application thereof |
| CN103589697A (en) * | 2013-10-17 | 2014-02-19 | 北京航空航天大学 | One group of cyclodextrine glucosyltransferase as well as coding gene and application thereof |
| CN103589697B (en) * | 2013-10-17 | 2016-10-19 | 北京航空航天大学 | One group of cyclodextrin glucosyltransferase and encoding gene thereof and application |
| CN113801860A (en) * | 2020-06-16 | 2021-12-17 | 中国科学院微生物研究所 | Application of protein CGTase as cyclodextrin glycosyltransferase |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102827815B (en) | 2014-01-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Raza et al. | Optimization and characterization of a polysaccharide produced by Pseudomonas fluorescens WR-1 and its antioxidant activity | |
| CN105821020B (en) | A kind of 'beta '-mannase mRmMan5A and its encoding gene and application | |
| KR101239624B1 (en) | Preparation method of Hydrolyzed Vegetable Protein | |
| US8748123B2 (en) | Fucose-containing bacterial biopolymer | |
| Selbmann et al. | Exopolysaccharide production by filamentous fungi: the example of Botryosphaeria rhodina | |
| CN101294149A (en) | Alpha-cyclodextrin glucosyl transferase gene clone and expression | |
| da Silva et al. | Xanthan: biotechnological production and applications | |
| CN102971432B (en) | Bacterium living beings condensate containing fucose | |
| CN102827815B (en) | A group of cyclodextrin glucosyltransferase, and coding gene and application thereof | |
| Cahyaningtyas et al. | Statistical optimization of halophilic chitosanase and protease production by Bacillus cereus HMRSC30 isolated from Terasi simultaneous with chitin extraction from shrimp shell waste | |
| CN102250931B (en) | Gene for coding beta-cyclodextrin glucosyltransferase and application thereof | |
| Dev et al. | Mutagenesis enhances gellan gum production by a novel Sphingomonas spp.: upstream optimization, kinetic modeling, and structural and physico-functional evaluation | |
| CN103232981B (en) | A group of cyclodextrine glucosyltransferases and encoding gene and application thereof | |
| EP4423187A1 (en) | Heat stable and/or heat activated biopolymers | |
| JPWO2003027304A1 (en) | Method for producing inulin | |
| CN103589697A (en) | One group of cyclodextrine glucosyltransferase as well as coding gene and application thereof | |
| JP2008531003A (en) | Methods and means for the production of hyaluronan | |
| CN114752613B (en) | Pectinase gene, pectinase, recombinant vector and application of pectinase gene and pectinase in blueberry processing | |
| WO2017221905A1 (en) | β-GLUCAN-PRODUCING FUNGUS WITHOUT MELANIN PIGMENT FORMATION ABILITY OR WITH LOW MELANIN PIGMENT FORMATION ABILITY, METHOD FOR ARTIFICIALLY PRODUCING SAID FUNGUS, β-GLUCAN PRODUCED UTILIZING SAID FUNGUS, AND METHOD FOR PRODUCING SAID Β-GLUCAN | |
| WO2004078959A1 (en) | Pullulan breakdown enzyme, method for production thereof and use thereof | |
| Agnes et al. | α-and β-cyclodextrin production by cyclodextrin glucosyl transferase from Bacillus species using different carbon substrate | |
| Cancella et al. | Xanthan gum produced from milk permeate and deproteinized cheese whey: A comparative analysis with commercial xanthan gums | |
| EP2758528A1 (en) | 3s-rhamnose-glucuronyl hydrolase, method of production and uses | |
| JP2010148409A (en) | Method for treating waste water by using blanching enzyme | |
| Abd-Aziz et al. | Studies on the enzymatic hydrolysis of rice protein using commercial enzymes in batch reactor |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140101 |