CN117431229B - Glycosyltransferase UGT91C1 mutant and application thereof - Google Patents
Glycosyltransferase UGT91C1 mutant and application thereof Download PDFInfo
- Publication number
- CN117431229B CN117431229B CN202311452527.3A CN202311452527A CN117431229B CN 117431229 B CN117431229 B CN 117431229B CN 202311452527 A CN202311452527 A CN 202311452527A CN 117431229 B CN117431229 B CN 117431229B
- Authority
- CN
- China
- Prior art keywords
- ugt91c1
- glycosyltransferase
- mutant
- rebaudioside
- sus
- 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.)
- Active
Links
- 108700023372 Glycosyltransferases Proteins 0.000 title claims abstract description 46
- 102000051366 Glycosyltransferases Human genes 0.000 title claims abstract description 46
- 101100101354 Arabidopsis thaliana UGT91C1 gene Proteins 0.000 title claims abstract description 44
- 102000004190 Enzymes Human genes 0.000 claims abstract description 37
- 108090000790 Enzymes Proteins 0.000 claims abstract description 37
- RPYRMTHVSUWHSV-CUZJHZIBSA-N rebaudioside D Chemical compound O([C@H]1[C@H](O)[C@@H](CO)O[C@H]([C@@H]1O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)O[C@]12C(=C)C[C@@]3(C1)CC[C@@H]1[C@@](C)(CCC[C@]1([C@@H]3CC2)C)C(=O)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O RPYRMTHVSUWHSV-CUZJHZIBSA-N 0.000 claims abstract description 24
- 108010043934 Sucrose synthase Proteins 0.000 claims abstract description 22
- 241000235058 Komagataella pastoris Species 0.000 claims abstract description 19
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 18
- HELXLJCILKEWJH-UHFFFAOYSA-N entered according to Sigma 01432 Natural products C1CC2C3(C)CCCC(C)(C(=O)OC4C(C(O)C(O)C(CO)O4)O)C3CCC2(C2)CC(=C)C21OC(C1OC2C(C(O)C(O)C(CO)O2)O)OC(CO)C(O)C1OC1OC(CO)C(O)C(O)C1O HELXLJCILKEWJH-UHFFFAOYSA-N 0.000 claims abstract description 14
- 235000019203 rebaudioside A Nutrition 0.000 claims abstract description 14
- HELXLJCILKEWJH-NCGAPWICSA-N rebaudioside A Chemical compound O([C@H]1[C@H](O)[C@@H](CO)O[C@H]([C@@H]1O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)O[C@]12C(=C)C[C@@]3(C1)CC[C@@H]1[C@@](C)(CCC[C@]1([C@@H]3CC2)C)C(=O)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O HELXLJCILKEWJH-NCGAPWICSA-N 0.000 claims abstract description 14
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 13
- 239000001512 FEMA 4601 Substances 0.000 claims abstract description 9
- HELXLJCILKEWJH-SEAGSNCFSA-N Rebaudioside A Natural products O=C(O[C@H]1[C@@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1)[C@@]1(C)[C@@H]2[C@](C)([C@H]3[C@@]4(CC(=C)[C@@](O[C@H]5[C@H](O[C@H]6[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O6)[C@@H](O[C@H]6[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O6)[C@H](O)[C@@H](CO)O5)(C4)CC3)CC2)CCC1 HELXLJCILKEWJH-SEAGSNCFSA-N 0.000 claims abstract description 9
- 238000002741 site-directed mutagenesis Methods 0.000 claims abstract description 7
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 6
- 238000006243 chemical reaction Methods 0.000 claims description 43
- 239000013612 plasmid Substances 0.000 claims description 19
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 16
- 238000006555 catalytic reaction Methods 0.000 claims description 14
- 229930006000 Sucrose Natural products 0.000 claims description 13
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 13
- 239000005720 sucrose Substances 0.000 claims description 13
- 230000015572 biosynthetic process Effects 0.000 claims description 9
- 239000008057 potassium phosphate buffer Substances 0.000 claims description 9
- 238000003786 synthesis reaction Methods 0.000 claims description 9
- 239000011780 sodium chloride Substances 0.000 claims description 8
- 239000013604 expression vector Substances 0.000 claims description 6
- 238000003259 recombinant expression Methods 0.000 claims description 6
- 101150090011 UGT91C1 gene Proteins 0.000 claims description 5
- 108020004705 Codon Proteins 0.000 claims description 4
- 108700010070 Codon Usage Proteins 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 241000894006 Bacteria Species 0.000 claims description 2
- 238000005520 cutting process Methods 0.000 claims description 2
- 239000000758 substrate Substances 0.000 claims description 2
- 230000002194 synthesizing effect Effects 0.000 claims description 2
- 230000001131 transforming effect Effects 0.000 claims description 2
- 239000013598 vector Substances 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 14
- HSCJRCZFDFQWRP-JZMIEXBBSA-N UDP-alpha-D-glucose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OP(O)(=O)OP(O)(=O)OC[C@@H]1[C@@H](O)[C@@H](O)[C@H](N2C(NC(=O)C=C2)=O)O1 HSCJRCZFDFQWRP-JZMIEXBBSA-N 0.000 abstract description 9
- HSCJRCZFDFQWRP-UHFFFAOYSA-N Uridindiphosphoglukose Natural products OC1C(O)C(O)C(CO)OC1OP(O)(=O)OP(O)(=O)OCC1C(O)C(O)C(N2C(NC(=O)C=C2)=O)O1 HSCJRCZFDFQWRP-UHFFFAOYSA-N 0.000 abstract description 9
- 239000007788 liquid Substances 0.000 abstract description 4
- 238000000746 purification Methods 0.000 abstract description 4
- 230000002255 enzymatic effect Effects 0.000 abstract description 3
- 239000000348 glycosyl donor Substances 0.000 abstract description 3
- 230000008929 regeneration Effects 0.000 abstract description 3
- 238000011069 regeneration method Methods 0.000 abstract description 3
- 125000004122 cyclic group Chemical group 0.000 abstract description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 18
- 239000000243 solution Substances 0.000 description 17
- 238000000855 fermentation Methods 0.000 description 14
- 230000004151 fermentation Effects 0.000 description 14
- 239000006228 supernatant Substances 0.000 description 12
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 12
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 10
- 235000019202 steviosides Nutrition 0.000 description 9
- 241001052560 Thallis Species 0.000 description 8
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 6
- 238000001816 cooling Methods 0.000 description 6
- 238000010438 heat treatment Methods 0.000 description 6
- 229910000160 potassium phosphate Inorganic materials 0.000 description 6
- 235000011009 potassium phosphates Nutrition 0.000 description 6
- 238000005070 sampling Methods 0.000 description 6
- 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 description 5
- UEDUENGHJMELGK-HYDKPPNVSA-N Stevioside Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@]12C(=C)C[C@@]3(C1)CC[C@@H]1[C@@](C)(CCC[C@]1([C@@H]3CC2)C)C(=O)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O UEDUENGHJMELGK-HYDKPPNVSA-N 0.000 description 5
- 238000001914 filtration Methods 0.000 description 5
- 238000004811 liquid chromatography Methods 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 229940013618 stevioside Drugs 0.000 description 5
- OHHNJQXIOPOJSC-UHFFFAOYSA-N stevioside Natural products CC1(CCCC2(C)C3(C)CCC4(CC3(CCC12C)CC4=C)OC5OC(CO)C(O)C(O)C5OC6OC(CO)C(O)C(O)C6O)C(=O)OC7OC(CO)C(O)C(O)C7O OHHNJQXIOPOJSC-UHFFFAOYSA-N 0.000 description 5
- 239000001888 Peptone Substances 0.000 description 4
- 108010080698 Peptones Proteins 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 239000004383 Steviol glycoside Substances 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 235000011187 glycerol Nutrition 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 235000019319 peptone Nutrition 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 235000019411 steviol glycoside Nutrition 0.000 description 4
- 229930182488 steviol glycoside Natural products 0.000 description 4
- 150000008144 steviol glycosides Chemical class 0.000 description 4
- 244000228451 Stevia rebaudiana Species 0.000 description 3
- 238000007792 addition Methods 0.000 description 3
- 235000003599 food sweetener Nutrition 0.000 description 3
- 239000003765 sweetening agent Substances 0.000 description 3
- 108010006654 Bleomycin Proteins 0.000 description 2
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 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
- 235000006092 Stevia rebaudiana Nutrition 0.000 description 2
- 238000005273 aeration Methods 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 229960001561 bleomycin Drugs 0.000 description 2
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 description 2
- PASHVRUKOFIRIK-UHFFFAOYSA-L calcium sulfate dihydrate Chemical compound O.O.[Ca+2].[O-]S([O-])(=O)=O PASHVRUKOFIRIK-UHFFFAOYSA-L 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000000265 homogenisation Methods 0.000 description 2
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 2
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 235000013615 non-nutritive sweetener Nutrition 0.000 description 2
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 description 2
- 229910052939 potassium sulfate Inorganic materials 0.000 description 2
- 235000011151 potassium sulphates Nutrition 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000000600 sorbitol Substances 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- 239000007221 ypg medium Substances 0.000 description 2
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 241000235648 Pichia Species 0.000 description 1
- GIPHUOWOTCAJSR-UHFFFAOYSA-N Rebaudioside A. Natural products C1CC2C3(C)CCCC(C)(C(=O)OC4C(C(O)C(O)C(CO)O4)O)C3CCC2(C2)CC(=C)C21OC1OC(CO)C(O)C(O)C1OC(C1O)OC(CO)C(O)C1OC1OC(CO)C(O)C(O)C1O GIPHUOWOTCAJSR-UHFFFAOYSA-N 0.000 description 1
- 240000004922 Vigna radiata Species 0.000 description 1
- 235000010721 Vigna radiata var radiata Nutrition 0.000 description 1
- 235000011469 Vigna radiata var sublobata Nutrition 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 208000002925 dental caries Diseases 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000021472 generally recognized as safe Nutrition 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000027939 micturition Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 235000021096 natural sweeteners Nutrition 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
Abstract
The invention discloses a glycosyltransferase UGT91C1 mutant, which is (a) protein with an amino acid sequence shown as SEQ ID NO. 3; or (b) a protein derived from (a) which has been substituted, deleted or added with one or more amino acid sequences in the amino acid sequence shown as SEQ ID NO.3 and has an enzymatic activity of catalyzing rebaudioside A to rebaudioside D. The invention also discloses a gene sequence and the like for encoding the glycosyltransferase UGT91C1 mutant as claimed in claim 1. According to the invention, the enzyme activity of the glycosyltransferase UGT91C1 is obviously improved through site-directed mutagenesis, and compared with the wild glycosyltransferase UGT91C1, the optimal mutant enzyme activity is improved by 4.42 times, so that the use amount of the enzyme can be obviously reduced. According to the invention, the glycosyltransferase UGT91C1 mutant and sucrose synthase SUS are respectively subjected to heterologous expression in Pichia pastoris, purification is not needed, a cyclic regeneration system of UDP-UDPG is constructed by using crude enzyme liquid, and the addition of UDPG as a glycosyl donor is avoided, so that the cost can be obviously reduced.
Description
Technical Field
The invention belongs to the technical field of biological enzyme engineering, and particularly relates to a glycosyltransferase UGT91C1 mutant and application thereof.
Background
In recent years, worldwide there has been an increasing risk of caries, obesity, diabetes, hypertension, cardiovascular disease and, therefore, consumer demand for low-calorie or non-caloric sweeteners has been increasing. Steviol glycosides extracted from stevia rebaudiana are currently considered the most attractive sweetener because of their high sweetness (50-450 times that of sucrose), non-caloric, and safe. In addition, steviol glycosides have been found to have important pharmacological activities such as lowering blood sugar, lowering blood pressure, promoting urination, anti-inflammatory, anti-tumor and immunomodulating effects. Sixty kinds of steviol glycosides have been found from stevia rebaudiana, of which stevioside (5-10% by dry weight of leaves) and rebaudioside a (Reb a, 2-4% by dry weight of leaves) are the two most abundant components, and are the main components of steviol glycoside additives currently commercially available on the market. However, stevioside and rebaudioside A have obvious after-bitter taste, and compared with the stevioside D (Reb D) has better taste characteristics, so that the stevioside sweetener is a novel development direction, and toxicity researches of structural substances related to stevioside also prove that the Reb D is safe to eat and can be used in the fields of foods, medicines and the like. In 2017, the U.S. food and drug administration FDA also certified Reb D as a "GRAS (GENERALLY RECOGNIZED AS SAFE, generally regarded as safe)" grade. Reb D is thus a better quality potential natural sweetener than Reb a.
With the increase of the taste requirements of sweeteners, the requirements for high-end products such as Reb D and the like are increasing. However, in stevia plants, the content of Reb D is extremely low, and is only about 0.5% of the dry leaf weight, so that the separation and extraction cost is high, and the market demand cannot be met. Glycosyltransferase UGT91C1 can catalyze Reb A to generate Reb D in the presence of uridine diphosphate glucose (UDPG), but the enzyme activity of wild type enzyme is lower, and meanwhile, UGT91C1 also needs to use UDPG as a glycosyl donor, so that the UDPG has high market price, and the industrialized production is limited.
Disclosure of Invention
In order to solve the problems in the prior art, the invention successfully obtains the mutant with high enzyme activity by carrying out structural transformation on glycosyltransferase UGT91C1, combines the optimal mutant with sucrose synthase SUS, establishes a UDPG circulation regeneration system, and avoids the additional addition of expensive UDPG, thereby providing a new method for the production of Reb D.
Accordingly, in a first aspect of the present invention, there is provided a glycosyltransferase UGT91C1 mutant, said mutant being:
(a) A protein with an amino acid sequence shown as SEQ ID NO. 3; or alternatively
(B) A protein derived from (a) which has been substituted, deleted or added with one or more amino acid sequences in the amino acid sequence shown in SEQ ID NO.3 and has the enzymatic activity of catalyzing rebaudioside A to rebaudioside D.
In a second aspect of the invention, a gene sequence encoding said glycosyltransferase UGT91C1 mutant is provided.
In a third aspect of the present invention, there is provided a method for preparing the glycosyltransferase UGT91C1 mutant, comprising the steps of:
s1: according to the accession number: XP_015629141.1 obtains the amino acid sequence of glycosyltransferase UGT91C1 from Genbank, optimizes the sequence according to the codon preference of pichia pastoris, and obtains the codon optimized UGT91C1 gene sequence;
S2: carrying out total gene synthesis and connecting the total gene synthesis with a polyclonal enzyme cutting site of the vector pPICZA to obtain a recombinant plasmid pPICZA-UGT91C1;
s3: carrying out full plasmid PCR by taking a recombinant plasmid pPICZA-UGT91C1 as a template, and sequentially carrying out four rounds of site-directed mutagenesis to construct a corresponding recombinant plasmid carrying mutants;
S4: the recombinant plasmid identified as correct is transformed into host bacteria to obtain a transformant;
s5: fermenting the obtained transformant to obtain a glycosyltransferase UGT91C1 mutant;
the primer pair sequences for four rounds of site-directed mutagenesis in step S3 are as follows:
L150H-F：ATGTTGCATGGTTCTGCTCATATGATTGCTTCTATT；(SEQ ID NO.4)
L150H-R：AGCAGAACCATGCAACATCATAGCACATGGAACTTTATG；(SEQ ID NO.5)
A205D-F：TCTTTGGATGAAAGATTTTCTTTGACTTTGTCTAGATCTTCT；(SEQ ID NO.6)
A205D-R：AAATCTTTCATCCAAAGACATACCAGAAGAACCTTTAG；(SEQ ID NO.7)
V284D-F：CTGAAGGATCATTGGGTGTTGAAAAAGTTCATGAA；(SEQ ID NO.8)
V284D-R：ACCCAATGGATCTTCAGAACCCAAAGCAACATAAACAAC；(SEQ ID NO.9)
G380A-F：CCAATTTTTGCTGATCAAGGTCCAAATGCTAGATTG；(SEQ ID NO.10)
G380A-R：TTGATCAGCAAAAATTGGCAACATAATCAATGGATG。(SEQ ID NO.11)
According to a preferred embodiment of the present invention, the sequence of the codon optimized UGT91C1 gene described in step S1 is shown in SEQ ID No. 1.
In a fourth aspect of the invention, a recombinant expression vector is provided, which is cloned with a gene sequence encoding the glycosyltransferase UGT91C1 mutant.
In a fifth aspect of the invention, there is provided a recombinant strain of Pichia pastoris, said recombinant strain being cloned with said recombinant expression vector.
According to a preferred embodiment of the present invention, the recombinant strain is obtained by transforming the recombinant expression vector into pichia pastoris X33.
In a sixth aspect of the present invention, a method for synthesizing rebaudioside D is provided, wherein the method uses the glycosyltransferase UGT91C1 mutant and sucrose synthase SUS in combination, and uses rebaudioside a as a substrate to perform a catalytic reaction to obtain rebaudioside D.
According to a preferred embodiment of the present invention, the sequence of the gene encoding the sucrose synthase SUS is shown as SEQ ID NO. 2.
According to a preferred embodiment of the present invention, the reaction temperature of the catalytic reaction is 40 ℃, and the reaction system in 10mL comprises:
100mM potassium phosphate buffer (pH 8.0, containing 100mM NaCl), 300mM sucrose, 0.8mM UDP, 10g/LReb A, 10mg sucrose synthase SUS, 50mg glycosyltransferase UGT91C1.
In a seventh aspect of the present invention, there is provided the use of the glycosyltransferase UGT91C1 mutant for catalyzing the synthesis of rebaudioside D from rebaudioside a.
The invention has the following beneficial effects:
1. According to the invention, the enzyme activity of the glycosyltransferase UGT91C1 is obviously improved through site-directed mutagenesis, and compared with the wild glycosyltransferase UGT91C1, the optimal mutant enzyme activity is improved by 4.42 times, so that the use amount of the enzyme can be obviously reduced.
2. According to the invention, the glycosyltransferase UGT91C1 mutant and sucrose synthase SUS are respectively subjected to heterologous expression in Pichia pastoris, purification is not needed, a cyclic regeneration system of UDP-UDPG is constructed by using crude enzyme liquid, and the addition of UDPG as a glycosyl donor is avoided, so that the cost can be obviously reduced.
3. The invention further improves the conversion rate of the rebaudioside A (Reb A) and the yield of the rebaudioside D (Reb D) through optimizing the catalytic reaction conditions, and has good application prospect.
Detailed Description
The following specific examples are provided to further illustrate the invention, but are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Unless otherwise indicated, the reagents and materials used in the following examples are commercially available or may be prepared by conventional methods known in the art.
The Pichia host X33 used in the examples below is a commercial strain and is available by conventional commercial means.
The plasmid vector pPICZA used in the following examples is a commercial plasmid, and is available by conventional commercial routes.
The methods used in the following examples are conventional in the art, unless otherwise specified, or are performed in accordance with the product specifications.
Example 1 acquisition of glycosyltransferase UGT91C1 Gene and construction of mutant
The amino acid sequences of rice-derived glycosyltransferase UGT91C1 (accession number: XP_ 015629141.1) and mung bean-derived sucrose synthase SUS (accession number: BAA 01108.1) were downloaded from Genbank, respectively, and the sequences were optimized according to the codon preference of Pichia pastoris to obtain codon-optimized UGT91C1 and SUS genes, the sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.
The recombinant plasmids pPICZA-UGT91C1 and pPICZA-SUS were obtained by total gene synthesis from the Souzhou Jin Weizhi Biotechnology Co., ltd.
Taking a recombinant plasmid pPICZA-UGT91C1 as a template, respectively carrying out full plasmid PCR by using the following primer pairs, and sequentially carrying out one or more rounds of site-directed mutagenesis to construct a corresponding recombinant plasmid carrying mutants:
L150H-F：ATGTTGCATGGTTCTGCTCATATGATTGCTTCTATT；
L150H-R：AGCAGAACCATGCAACATCATAGCACATGGAACTTTATG；
L150F-F：ATGTTGTTTGGTTCTGCTCATATGATTGCTTCTATT；
L150F-R：AGCAGAACCAAACAACATCATAGCACATGGAACTTTATG；
I159D-F：GCTTCTGATGCTGATAGAAGATTGGAAAGAGCTGAA；
I159D-R：TCTATCAGCATCAGAAGCAATCATATGAGCAGAACCCAACAA；
I159H-F：GCTTCTCATGCTGATAGAAGATTGGAAAGAGCTGAA；
I159H-R：TCTATCAGCATGAGAAGCAATCATATGAGCAGAACCCAACAA；
A205D-F：TCTTTGGATGAAAGATTTTCTTTGACTTTGTCTAGATCTTCT；
A205D-R：AAATCTTTCATCCAAAGACATACCAGAAGAACCTTTAG；
I242A-F：GGTAAACCAGCTACTTTTTTGGGTTTGATGCCACCA；
I242A-R：AAAAGTAGCTGGTTTACCTCTCAAAGTAGACAACAATG；
V284D-F：CTGAAGGATCATTGGGTGTTGAAAAAGTTCATGAA；
V284D-R：ACCCAATGGATCTTCAGAACCCAAAGCAACATAAACAAC；
L320A-F：GCTGATGCTTTGCCTGCTGGTTTTGAAGAAAG；
L320A-R：AGCAGGCAAAGCATCAGCATCAGAAACACCAGTTGG；
G380A-F：CCAATTTTTGCTGATCAAGGTCCAAATGCTAGATTG；
G380A-R：TTGATCAGCAAAAATTGGCAACATAATCAATGGATG；
G380D-F：CCAATTTTTGATGATCAAGGTCCAAATGCTAGATTG；
G380D-R：TTGATCATCAAAAATTGGCAACATAATCAATGGATG。
The obtained mutant plasmid was sequenced and identified by Jin Weizhi Biotechnology Inc. of Suzhou, the plasmid pPICZA-UGT91C1-L150H、pPICZA-UGT91C1-L150F、pPICZA-UGT91C1-I159D、pPICZA-UGT91C1-I159H、pPICZA-UGT91C1-A205D、pPICZA-UGT91C1-I242A、pPICZA-UGT91C1-V284D、pPICZA-UGT91C1-L320A、pPICZA-UGT91C1-G380A、pPICZA-UGT91C1-G380D、pPICZA-UGT91C1-L150H/G380A、pPICZA-UGT91C1-I159D/G380A、pPICZA-UGT91C1-A205D/G380A、pPICZA-UGT91C1-V284D/G380A、pPICZA-UGT91C1-L150H/V284D/G380A、pPICZA-UGT91C1-I159D/V284D/G380A、pPICZA-UGT91C1-A205D/V284D/G380A、pPICZA-UGT91C1-L150H/A205D/V284D/G380A、pPICZA-UGT91C1-I159D/A205D/V284D/G380A、 with correct sequencing and the wild type plasmid pPICZA-UGT91C1 were respectively subjected to enzyme tangential digestion and purification, then were electrically transformed into Pichia pastoris host X33 competent cells, and were spread onto YPDS solid plates (20 g/L peptone, 10g/L yeast powder, 20g/L glucose, 182.17g/L sorbitol) containing 100. Mu.g/mL bleomycin, and were cultured upside down at 30℃to screen positive transformants, thereby obtaining the corresponding 20 recombinant Pichia pastoris strains designated ：X33-L150H、X33-L150F、X33-I159D、X33-I159H、X33-A205D、X33-I242A、X33-V284D、X33-L320A、X33-G380A、X33-G380D、X33-L150H/G380A、X33-I159D/G380A、X33-A205D/G380A、X33-V284D/G380A、X33-L150H/V284D/G380A、X33-I159D/V284D/G380A、X33-A205D/V284D/G380A、X33-L150H/A205D/V284D/G380A、X33-I159D/A205D/V284D/G380A、 and X33-UGT91C1, respectively.
Example 2 recombinant Pichia pastoris Strain induced expression and crude enzyme solution preparation
The 20 recombinant Pichia pastoris strains obtained in example 1 were inoculated into YPG (20 g/L peptone, 10g/L yeast powder, 10g/L glycerol) medium, and were shake-cultured at 220rpm at 30℃for 20-22 hours to obtain a first seed solution.
The primary seed solution was inoculated into 200mL of YPG medium at a ratio of 10% (v/v), and cultured overnight at 220rpm and 30℃to give a secondary seed solution.
Inoculating the secondary seed solution into a fermentation tank filled with a fermentation medium according to the proportion of 10% (v/v) for culture, wherein the formula of the fermentation medium is as follows: 26.07mL/L of phosphoric acid, 1.18g/L of calcium sulfate dihydrate, 18.2g/L of potassium sulfate, 14.9g/L of magnesium sulfate heptahydrate, 4.13g/L of potassium hydroxide, 40g/L, PTM g/8.7 mL/L of glycerin.
Controlling the rotation speed of the fermentation tank to be less than 700rpm, the aeration ratio to be 1V/V/min, controlling the pH to be 5.0 at the temperature of 30 ℃, culturing for 96 hours, and obtaining the fermentation liquor after the fermentation is finished. The fermentation broth is centrifuged at 8000rpm and 4 ℃ for 30min to collect thalli, then the thalli is washed with ultrapure water for three times, finally the thalli is resuspended with 100mM potassium phosphate buffer (pH 8.0), and after high pressure homogenization and crushing, the thalli is centrifuged at 8000rpm and 4 ℃ for 40min, and the supernatant is taken as crude enzyme solution.
And respectively obtaining 20 glycosyltransferase UGT91C1 crude enzyme solutions of pichia pastoris strains according to the method, wherein the glycosyltransferase UGT91C1 crude enzyme solutions are respectively marked as L150H、L150F、I159D、I159H、A205D、I242A、V284D、L320A、G380A、G380D、L150H/G380A、I159D/G380A、A205D/G380A、V284D/G380A、L150H/V284D/G380A、I159D/V284D/G380A、A205D/V284D/G380A、L150H/A205D/V284D/G380A、I159D/A205D/V284D/G380A、 wild types.
Example 3 enzyme Activity assay of mutants
The 20 glycosyltransferase UGT91C1 crude enzyme solutions obtained in example 2 were taken respectively to prepare a reaction system for measuring the enzyme activity, wherein the wild type crude enzyme solution was used as a control, and 1mL reaction system comprises the following components:
50mM Tris-HCl buffer (pH 8.0), 2mM MgCl 2, 2mM UDPG, 2mM Reb A, 0.5mg glycosyltransferase UGT91C1.
Reacting at 40 ℃ and 200rpm for 30min; after the reaction, sampling, heating at 95 ℃ for 5min to terminate the reaction, cooling, adding 4 times of methanol, centrifuging at 12000rpm for 5min, filtering the supernatant with a 0.22 mu M filter membrane, and detecting by liquid chromatography to calculate the enzyme activity. The measurement results are shown in Table 1.
Table 1: UGT91C1 mutant enzyme activity
As shown in the results of Table 1, in the 19 mutant crude enzyme solutions, the catalytic activity of 5 mutant enzymes is obviously reduced compared with that of the wild type enzyme, and the activity of the mutant enzymes in the rest 14 mutant enzymes is obviously increased, wherein the highest mutant enzyme carries four mutations L150H/A205D/V284D/G380A, the enzyme activity is improved by 4.42 times compared with that of the wild type enzyme, and the improvement effect is very obvious.
And (3) selecting L150H/A205D/V284D/G380A (the amino acid sequence is shown as SEQ ID NO. 3) with the highest enzyme activity, and respectively examining the influence of the reaction temperature, pH, sucrose concentration and UDP concentration on the catalytic reaction so as to screen the optimal reaction condition.
Those skilled in the art will readily appreciate that proteins which still have the enzymatic activity of catalyzing the synthesis of rebaudioside D from rebaudioside A are readily available by conventional means such as substitution, deletion or addition of one or more amino acid sequences, etc., based on the amino acid sequence shown in SEQ ID NO. 3, and thus these mutants are also within the scope of the present invention.
EXAMPLE 4 preparation of sucrose synthase SUS crude enzyme solution
The plasmid pPICZA-SUS obtained in example 1 was subjected to enzyme-tangential digestion and purification, then, was electrotransformed into Pichia pastoris host X33 competent cells, and was plated onto YPDS solid plates (20 g/L peptone, 10g/L yeast powder, 20g/L glucose, 182.17g/L sorbitol) containing 100. Mu.g/mL bleomycin, and cultured upside down at 30℃to obtain positive transformants, thereby obtaining the corresponding recombinant Pichia pastoris strain X33-SUS.
The recombinant Pichia pastoris strain is inoculated into YPG (20 g/L peptone, 10g/L yeast powder and 10g/L glycerol) culture medium, and is subjected to shaking culture at 220rpm and 30 ℃ for 20-22 hours to obtain first-stage seed liquid.
The primary seed solution was inoculated into 200mL of YPG medium at a ratio of 10% (v/v), and cultured overnight at 220rpm and 30℃to give a secondary seed solution.
Inoculating the secondary seed solution into a fermentation tank filled with a fermentation medium for culture according to the proportion of 10% (v/v), wherein the fermentation culture formula comprises the following steps: 26.07mL/L of phosphoric acid, 1.18g/L of calcium sulfate dihydrate, 18.2g/L of potassium sulfate, 14.9g/L of magnesium sulfate heptahydrate, 4.13g/L of potassium hydroxide, 40g/L, PTM g/8.7 mL/L of glycerin.
Controlling the rotation speed of the fermentation tank to be less than 700rpm, the aeration ratio to be 1V/V/min, controlling the pH to be 5.0 at the temperature of 30 ℃, culturing for 96 hours, and obtaining the fermentation liquor after the fermentation is finished. The fermentation broth is centrifuged at 8000rpm and 4 ℃ for 30min to collect thalli, then the thalli is washed with ultrapure water for three times, finally the thalli is resuspended with 100mM potassium phosphate buffer (pH 8.0), and after high pressure homogenization and crushing, the thalli is centrifuged at 8000rpm and 4 ℃ for 40min, and the supernatant is taken as crude enzyme solution.
A sucrose synthase SUS crude enzyme solution was obtained as described above.
Example 5 influence of the reaction temperature on the catalytic reaction
A reaction system was prepared from the glycosyltransferase L150H/A205D/V284D/G380A obtained in example 2 and the sucrose synthase SUS obtained in example 4, and 10mL of the reaction system contained the following components:
100mM potassium phosphate buffer (pH 8.0, containing 100mM NaCl), 200mM sucrose, 2mM UDP, 10g/L Reb A, 10mg sucrose synthase SUS, 50mg glycosyltransferase UGT91C1.
The reactions were carried out at 30, 35, 40, 45, 50℃and 200rpm, respectively, for 24h. After the reaction, sampling, heating at 95 ℃ for 5min to terminate the reaction, cooling, adding 4 times of methanol, centrifuging at 12000rpm for 5min, filtering the supernatant with a 0.22 mu M filter membrane, and then using the filtered supernatant for liquid chromatography detection to calculate the content of Reb D. The measurement results are shown in Table 2.
Table 2: reb D content at different reaction temperatures
| Temperature (. Degree. C.) | Reb D(g/L) |
| 30 | 4.56 |
| 35 | 7.66 |
| 40 | 8.38 |
| 45 | 6.98 |
| 50 | 3.24 |
The results in Table 2 show that the optimum reaction temperature for this catalytic reaction is 40℃and the corresponding yield of Reb D reaches 8.38g/L.
Example 6 influence of the reaction pH on the catalytic reaction
A reaction system was prepared from the glycosyltransferase L150H/A205D/V284D/G380A obtained in example 2 and the sucrose synthase SUS obtained in example 4, and 10mL of the reaction system contained the following components:
buffer, 200mM sucrose, 2mM UDP, 10g/L Reb A, 10mg sucrose synthase SUS, 50mg glycosyltransferase UGT91C1.
The buffer solution is selected as follows: 100mM potassium phosphate buffer pH 6.0-8.0 (containing 100mM NaCl), 100mM Tris-HCl buffer pH 8.0-9.0 (containing 100mM NaCl).
The reaction was carried out at 40℃and 200rpm for 24 hours. After the reaction, sampling, heating at 95 ℃ for 5min to terminate the reaction, cooling, adding 4 times of methanol, centrifuging at 12000rpm for 5min, filtering the supernatant with a 0.22 mu M filter membrane, and then using the filtered supernatant for liquid chromatography detection to calculate the content of Reb D. The measurement results are shown in Table 3.
Table 3: reb D content at different reaction pH
| Buffer pH | Reb D(g/L) |
| Potassium phosphate pH 6.0 | 2.35 |
| Potassium phosphate pH 6.5 | 3.56 |
| Potassium phosphate pH 7.0 | 5.35 |
| Potassium phosphate pH 7.5 | 7.46 |
| Potassium phosphate pH 8.0 | 8.40 |
| Tris-HCl pH 8.0 | 6.45 |
| Tris-HCl pH 8.5 | 5.46 |
| Tris-HCl pH 9.0 | 4.26 |
The results in Table 3 show that the optimum reaction buffer for this catalytic reaction is potassium phosphate (pH 8.0), with a corresponding yield of Reb D of 8.40g/L.
EXAMPLE 7 Effect of sucrose concentration on catalytic reactions
A reaction system was prepared from the glycosyltransferase L150H/A205D/V284D/G380A obtained in example 2 and the sucrose synthase SUS obtained in example 4, and 10mL of the reaction system contained the following components:
100mM potassium phosphate buffer (pH 8.0, containing 100mM NaCl), sucrose (100, 200, 300, 400, 500 mM), 2mM UDP, 10g/L Reb A, 10mg sucrose synthase SUS, 50mg glycosyltransferase UGT91C1.
The reaction was carried out at 40℃and 200rpm for 24 hours. After the reaction, sampling, heating at 95 ℃ for 5min to terminate the reaction, cooling, adding 4 times of methanol, centrifuging at 12000rpm for 5min, filtering the supernatant with a 0.22 mu M filter membrane, and then using the filtered supernatant for liquid chromatography detection to calculate the content of Reb D. The measurement results are shown in Table 4.
Table 4: reb D content at different sucrose concentrations
| Sucrose concentration (mM) | Reb D(g/L) |
| 100 | 7.01 |
| 200 | 8.40 |
| 300 | 9.01 |
| 400 | 8.61 |
| 500 | 8.45 |
The results in Table 4 show that the optimum sucrose concentration for this catalytic reaction is 300mM, with a corresponding yield of Reb D of 9.01g/L.
Example 8 influence of UDP concentration on catalytic reaction
A reaction system was prepared from the glycosyltransferase L150H/A205D/V284D/G380A obtained in example 2 and the sucrose synthase SUS obtained in example 4, and 10mL of the reaction system contained the following components:
100mM potassium phosphate buffer (pH 8.0, containing 100mM NaCl), 300mM sucrose, UDP (0.4, 0.8, 1.2, 1.6, 2.0 mM), 10g/L Reb A, 10mg sucrose synthase SUS, 50mg glycosyltransferase UGT91C1.
The reaction was carried out at 40℃and 200rpm for 24 hours. After the reaction, sampling, heating at 95 ℃ for 5min to terminate the reaction, cooling, adding 4 times of methanol, centrifuging at 12000rpm for 5min, filtering the supernatant with a 0.22M filter membrane, and then using the filtered supernatant for liquid chromatography detection to calculate the content of Reb D. The measurement results are shown in Table 5.
Table 5: reb D content at various UDP concentrations
| UDP concentration | Reb D(g/L) |
| 0.4 | 9.06 |
| 0.8 | 9.11 |
| 1.2 | 9.08 |
| 1.6 | 9.06 |
| 2.0 | 9.01 |
The results in Table 5 show that the optimum UDP concentration for this catalytic reaction is 0.8mM, and the yield of the corresponding Reb D reaches 9.11g/L.
As can be seen from examples 5-8 above, the optimal reaction conditions are:
The crude enzyme liquid L150H/A205D/V284D/G380A obtained after the induced expression of the recombinant Pichia pastoris strain X33-L150H/A205D/V284D/G380A is adopted for reaction. The 10mL reaction system comprises:
100mM potassium phosphate buffer (pH 8.0, containing 100mM NaCl), 300mM sucrose, 0.8mM UDP, 10g/LReb A, 10mg sucrose synthase SUS, 50mg glycosyltransferase UGT91C1.
The reaction was carried out at 40℃and 200 rpm. After 24h of reaction, sampling, heating at 95 ℃ for 5min to terminate the reaction, cooling, adding 4 times of methanol, centrifuging at 12000rpm for 5min to obtain supernatant, wherein the yield of Reb D is the highest and is 9.11g/L, and the conversion rate reaches 95%.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (11)
1. A glycosyltransferase UGT91C1 mutant is characterized in that the mutant is a protein with an amino acid sequence shown as SEQ ID NO. 3.
2. A gene sequence encoding the glycosyltransferase UGT91C1 mutant of claim 1.
3. Method for the preparation of a glycosyltransferase UGT91C1 mutant according to claim 1, characterized in that it comprises the steps of:
s1: according to the accession number: XP_015629141.1 obtains the amino acid sequence of glycosyltransferase UGT91C1 from Genbank, optimizes the sequence according to the codon preference of pichia pastoris, and obtains the codon optimized UGT91C1 gene sequence;
S2: carrying out total gene synthesis and connecting the total gene synthesis with a polyclonal enzyme cutting site of the vector pPICZA to obtain a recombinant plasmid pPICZA-UGT91C1;
s3: carrying out full plasmid PCR by taking a recombinant plasmid pPICZA-UGT91C1 as a template, and sequentially carrying out four rounds of site-directed mutagenesis to construct a corresponding recombinant plasmid carrying mutants;
S4: the recombinant plasmid identified as correct is transformed into host bacteria to obtain a transformant;
s5: fermenting the obtained transformant to obtain a glycosyltransferase UGT91C1 mutant;
the primer pair sequences for four rounds of site-directed mutagenesis in step S3 are as follows:
L150H-F：ATGTTGCATGGTTCTGCTCATATGATTGCTTCTATT；
L150H-R：AGCAGAACCATGCAACATCATAGCACATGGAACTTTATG；
A205D-F：TCTTTGGATGAAAGATTTTCTTTGACTTTGTCTAGATCTTCT；
A205D-R：AAATCTTTCATCCAAAGACATACCAGAAGAACCTTTAG；
V284D-F：CTGAAGGATCATTGGGTGTTGAAAAAGTTCATGAA；
V284D-R：ACCCAATGGATCTTCAGAACCCAAAGCAACATAAACAAC；
G380A-F：CCAATTTTTGCTGATCAAGGTCCAAATGCTAGATTG；
G380A-R：TTGATCAGCAAAAATTGGCAACATAATCAATGGATG。
4. The method of claim 3, wherein the sequence of the codon optimized UGT91C1 gene in step S1 is set forth in SEQ ID No. 1.
5. A recombinant expression vector cloned with a gene sequence encoding the glycosyltransferase UGT91C1 mutant of claim 1.
6. A recombinant strain of Pichia pastoris, wherein the recombinant strain is cloned with the recombinant expression vector of claim 5.
7. The recombinant strain of pichia pastoris according to claim 6, wherein the recombinant strain is obtained by transforming the recombinant expression vector of claim 5 into pichia pastoris X33.
8. A method for synthesizing rebaudioside D, characterized in that the method utilizes the glycosyltransferase UGT91C1 mutant and sucrose synthase SUS as a combination, and uses rebaudioside A as a substrate to carry out catalytic reaction to obtain the rebaudioside D.
9. The method of synthesis according to claim 8, wherein the sequence of the gene encoding sucrose synthase SUS is shown in SEQ ID NO. 2.
10. The method according to claim 8, wherein the reaction temperature of the catalytic reaction is 40 ℃, and the reaction system in 10mL comprises:
100 mM potassium phosphate buffer (pH 8.0, containing 100 mM NaCl), 300 mM sucrose, 0.8mM UDP, 10 g/L Reb A, 10 mg sucrose synthase SUS, 50 mg glycosyltransferase UGT91C1.
11. Use of the glycosyltransferase UGT91C1 mutant according to claim 1 for catalyzing the synthesis of rebaudioside D from rebaudioside a in combination with sucrose synthase SUS.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311452527.3A CN117431229B (en) | 2023-11-03 | Glycosyltransferase UGT91C1 mutant and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311452527.3A CN117431229B (en) | 2023-11-03 | Glycosyltransferase UGT91C1 mutant and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117431229A CN117431229A (en) | 2024-01-23 |
| CN117431229B true CN117431229B (en) | 2024-07-09 |
Family
ID=
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111344399A (en) * | 2018-11-16 | 2020-06-26 | 邦泰生物工程(深圳)有限公司 | UDP-glucosyltransferase mutant, application thereof and method for preparing rebaudioside D by using same |
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111344399A (en) * | 2018-11-16 | 2020-06-26 | 邦泰生物工程(深圳)有限公司 | UDP-glucosyltransferase mutant, application thereof and method for preparing rebaudioside D by using same |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109750072B (en) | Method for preparing rebaudioside E by enzyme method | |
| CN111518782B (en) | Glycosyltransferase UGTZJ1 mutant and application thereof | |
| CN104726523B (en) | A kind of method that enzyme process prepares Rebaudiodside A M | |
| CN109402158B (en) | Recombinant expression plasmid vector for producing fucosyllactose, metabolic engineering bacteria and production method | |
| CN112852796A (en) | Cellobiose epimerase mutant and application thereof in preparation of lactulose | |
| WO2020097922A1 (en) | Udp-glucosyltransferase mutant, use thereof and method for preparing rebaudioside d | |
| CN113249287B (en) | Bacillus subtilis engineering strain for expressing D-psicose 3-epimerase and application thereof | |
| CN117660385A (en) | Method for efficiently biosynthesizing rebaudioside D by utilizing glycosyltransferase | |
| CN109517864A (en) | A kind of preparation method of Momordia grosvenori aglycone | |
| CN114574460B (en) | Method for efficiently biosynthesizing rebaudioside M by utilizing glycosyltransferase UGT76G1 mutant | |
| CN117431229B (en) | Glycosyltransferase UGT91C1 mutant and application thereof | |
| CN117431229A (en) | Glycosyltransferase UGT91C1 mutant and application thereof | |
| CN115725484A (en) | Enzyme mutation expression engineering bacterium for synthesizing D-psicose and application thereof | |
| CN115418358B (en) | Glycosyltransferase and application thereof | |
| CN115449514A (en) | Beta-1, 2-glycosyltransferase and application thereof | |
| CN113151132B (en) | Gluconobacter oxydans genetically engineered bacterium for producing 5-ketofructose, construction method and application thereof | |
| CN110872586A (en) | Immobilized glucosyltransferase, preparation method thereof and method for catalytically producing rebaudioside D | |
| CN114561417B (en) | Corynebacterium glutamicum engineering strain for preparing psicose and application thereof | |
| CN116042562B (en) | Recombinant saccharomycetes and application thereof | |
| CN115478060B (en) | Glycosyltransferase and application thereof | |
| CN116286770B (en) | D-psicose-3-epimerase from clostridium and application thereof | |
| CN116515925A (en) | Method for efficiently synthesizing uridine diphosphate and application thereof | |
| CN116004417A (en) | Bacillus subtilis and application thereof | |
| JP2024500432A (en) | Glycosyltransferase and method for producing steviol glycoside using the same | |
| CN118147103A (en) | Alpha 1, 3/4-fucosyltransferase mutant and method for biosynthesis of difucosyl lactose by using same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant |