CN109750071A

CN109750071A - Method for synthesizing rebaudioside M through biocatalysis

Info

Publication number: CN109750071A
Application number: CN201910095402.7A
Authority: CN
Inventors: 贾红华; 李艳; 陈量量; 潘华祎; 欧阳平凯
Original assignee: Nanjing Tech University
Current assignee: Nanjing Tech University
Priority date: 2019-01-31
Filing date: 2019-01-31
Publication date: 2019-05-14

Abstract

The invention discloses a method for synthesizing rebaudioside M by biocatalysis, which comprises the following steps of firstly, utilizing tomato source UDP-glycosyltransferase and potato source sucrose synthase to carry out glycosylation reaction by taking stevioside as a raw material to synthesize rebaudioside E; and then, using stevia rebaudiana-derived UDP-glycosyltransferase and potato-derived sucrose synthase to further carry out glycosylation reaction by taking rebaudioside E as a raw material to synthesize rebaudioside M. The method of the invention utilizes molecular cloning technology to obtain escherichia coli genetic engineering bacteria of heterologous expression UDP-glycosyltransferase and sucrose synthase, directly performs catalytic reaction by using crude cell extract after enzyme production through fermentation, uses UDP-glucose obtained by decomposing the UDP and the sucrose additionally added in the crude extract under the action of the sucrose synthase as raw materials of glycosylation reaction, establishes a double-enzyme circulation reaction system, and effectively catalyzes stevioside to produce rebaudioside-M. The raw material cost is lower, the process steps are simple, and the method has important application value.

Description

A kind of method of biocatalysis synthesis Rebaudiodside A M

Technical field

The invention belongs to technical field of bioengineering, and in particular to a kind of method of biocatalysis synthesis Rebaudiodside A M.

Background technique

STEVIA REBAUDIANA can extract stevia rebaudianum abundant as a kind of many years book on Chinese herbal medicine plant for originating in South America in cauline leaf Glucoside is used as natural sweetener by South America people always for centuries^[1].Sweet tea is introduced from China in 1976 from Japan Start after Ye Ju is planted successfully, after the eighties to national popularizing planting.The ground such as Fujian, An Wei, Jiangsu have large area kind at present It plants, the gross area is up to 1,000,000 mu, it has also become the maximum stevioside producing country in the whole world and exported country^{[2, 3]}.Stevioside is used as at present The sweetener for the most sweet tea known, sugariness is 250 ~ 450 times of sucrose, and heat only has the 1/300 of sucrose, is slightly had slight puckery Taste.Its solubility is not low, and can be stabilized in the solution of acid and salt, and the holding time is long, will not agglomerate.In addition to this, sweet tea Synanthrin, which is also equipped with, assists pharmacological action to hypertension, obesity, diabetes etc.^{[4, 5]}, therefore, have been to be concerned by more and more people.

Stevioside is the general name of stevioside, and separation identifies more than 35 kinds steviosides from STEVIA REBAUDIANA at present^[6]。 Stevioside structure is skeleton around diterpene steviol, has been separately connected Portugal not etc. in C13 hydroxyls and C19 carboxyls Grape glycosyl group, wherein the carbon-carbon double bond on skeleton between C16-C17 is to provide the pharmacological base of stevioside sweet taste and function Group^{[6, 7]}.Rebaudiodside A M is extracted identification in report in 2009 from the leaf of STEVIA REBAUDIANA new varieties for the first time, and sugariness is sugarcane 400 times of sugar, better than rebaudioside A on sale in the market is showed, mouthfeel is also more clean.Although researcher has cultivated high yield Lay The STEVIA REBAUDIANA new varieties of Bao Di glycosides M, but its content in cured leaf piece is still low (~ 0.4-0.5%), it is straight using physical means merely Meet the Rebaudiodside A M that preparation high-purity is extracted from blade, economic benefit is and its low.

The method that a kind of enzyme process of patent application CN 201310353500.9(prepares rebaudioside M) and patent application CN The method that a kind of enzyme process of 201410019981.4(prepares rebaudioside M) in using with rebaudioside A or Rebaudiodside A D For substrate raw material, closed through UDP-glucose based transferase (UGT-A and/or from the UGT-B of rice) from stevia rebaudianum and sucrose At the coupling catalysed realization of enzyme, the cost of substrate rebaudioside A and Rebaudiodside A D are significantly larger than Stevioside.

The synthetic method and its intermediate product and synthetic method of patent application CN 201410553617.6(Rebaudiodside A M) With a kind of process for preparing Rebaudiodside A M of patent application CN 201410314371.7() in all refer to part chemistry examination Agent or chemical reaction, and synthesis step is loaded down with trivial details.This method is built by biological enzyme means using Stevioside sterling as raw material New Rebaudiodside A M synthesis path prepares Rebaudiodside A M through two step glycosylations, and cost of material is more cheap, processing step Simply, there is important application value.

Bibliography

[1] Yang Yuanzhi, Li Facai, jade pendant are contended in beauty, and wait the application status of stevioside and development prospect [J] fermentation science and technology logical News, 2011,40 (1): 40-44.

[2] Ni Junming, Li Junping stevioside industrial development status and prospect [J] modern food science and technology, 2004,20 (3): 156-158.

[3] emergence of Tang Zhifa stevioside and the industry of development strategy [J] Chinese food, 1999,2:52-55.

[4]KOVYLYAEVA G I, BAKALEINIK G A, STROBYKINA I Y, et al. Glycosides from Stevia rebaudiana [J]. Chemistry of Natural Compounds, 2007, 43(1): 81-85.

[5]KINGHORN A D, KINGHORN A D. Stevia: the genus Stevia [J]. Crc Press, 2002.

[6]OLSSON K, CARLSEN S, SEMMLER A, et al. Microbial production of next- generation stevia sweeteners [J]. Microb Cell Fact, 2016, 15(1): 207-220.

[7]UPRETI M, DUBOIS G, PRAKASH I. Synthetic study on the relationship between structure and sweet taste properties of steviol glycosides [J]. Molecules, 2012, 17(4): 4186-4196.

Summary of the invention

The purpose of the present invention is provide a kind of biology and urge aiming at the problem that preparing Rebaudiodside A M economic inefficiencies at present It is combined to the variation route of Rebaudiodside A M, first with glycosyl transferase UGTSL2 glycosylation, Stevioside is catalyzed and synthesizes Lay Bao Enlightening glycosides E, followed by glycosyl transferase UGT76G1 catalysis Rebaudiodside A E synthesize Rebaudiodside A M, be Rebaudiodside A M preparation and Purification provides strong basis.

To achieve the goals above, the technical solution adopted by the present invention are as follows:

A kind of method of biocatalysis synthesis Rebaudiodside A M, utilizes molecule clone technology, acquisition heterogenous expression UDP- glycosyl transfer The Recombinant organism of enzyme and sucrose synthase is directly catalyzed after self-induction enzymatic production with cell crude extract Reaction, without adding UDP-glucose and UDP, the sucrose using UDP in crude extract and additionally added establishes circular response body System is effectively catalyzed Stevioside and produces Rebaudiodside A M.

Inducer is lactose in the self-induction fermentation, and lactose concentration in fermentation culture is 0.01 ~ 0.5%.

This method be specifically divided into two steps progress, as shown in Fig. 2, (1) firstly, raw material Stevioside through UDP- glycosyl transferase UGTSL2 and sucrose synthase StSUS1 double-enzyme catalysis generate intermediate product Rebaudiodside A E；(2) intermediate product Rebaudiodside A E is passed through UDP- glycosyl transferase UGT76G1 and sucrose synthase StSUS1 double-enzyme catalysis generate final product Rebaudiodside A M.

The UDP- glycosyl transferase UGT-A is tomato source glycosyl transferase UGTSL2, is NCBI Reference Sequence:NC_015448. 3, gene order as shown in SEQ.No.1, amino acid sequence (Uniprot number: K4D508) as shown in SEQ.No.4；The UDP- glycosyl transferase UGT-B is STEVIA REBAUDIANA source glycosyl transferase UGT76G1, is NCBI Reference Sequence:AY345974. 1, gene order is as shown in SEQ.No.2, amino acid sequence (Uniprot number:Q6VAB4) is as shown in SEQ.No.5；The sucrose synthase SUS is potato source Sucrose synthesis Enzyme StSUS1 is NCBI Reference Sequence:M18745, and gene order is as shown in SEQ.No.3, amino acid sequence (Uniprot number:P10691) is as shown in SEQ.No.6.

Genetic engineering bacterium in step 1 is to co-express containing UDP- glycosyl transferase UGTSL2 and sucrose synthase StSUS1 The recombination bacillus coli of carrier, UGTSL2 genetic fragment are inserted in plasmid vectorNdeI andXhoBetween I restriction enzyme site, StSUS1 genetic fragment is inserted in plasmid vectorNcoI andEcoBetween R I restriction enzyme site, ultimately provide can external source be total to table Up to the recombinant plasmid of glycosyl transferase UGTSL2 and sucrose synthase StSUS1, pRSFDuet-UGTSL2-StSUS1.

Genetic engineering bacterium in step 2 is to be total to table containing UDP- glycosyl transferase UGT76G1 and sucrose synthase StSUS1 Up to the recombination bacillus coli of carrier, UGT76G1 genetic fragment is inserted in plasmid vectorNdeI andXhoBetween I restriction enzyme site, StSUS1 genetic fragment is inserted in plasmid vectorNcoI andEcoBetween R I restriction enzyme site, ultimately provide can external source be total to table Up to the recombinant plasmid of glycosyl transferase UGT76G1 and sucrose synthase StSUS1, pRSFDuet-UGT76G1-StSUS1；

Recombinant plasmid thermal shock is converted into e. coli bl21 (DE3) competent cell, converted product is coated on containing 50 On the plate of the kalamycin resistance of mg/L, 37 DEG C are incubated overnight, and obtain the recombination bacillus coli genetic engineering bacterium of coexpression.

The recombination bacillus coli genetically engineered bacteria induced expression condition that step 1) and step 2 construct are as follows: recombination is big Enterobacteria is inoculated into LB culture medium, presses 2% inoculum concentration in 16 ~ 37 DEG C, 8 ~ 10 h of 200rpm shaken cultivation, then by culture bacterium solution Access TB culture medium in, wherein inducer lactose in TB culture solution concentration be 0.01 ~ 0.5%, in 16 ~ 37 DEG C of Fiber differentiations 16 ~ 36 h, thalline were collected by centrifugation.

It is obtained containing UDP- sugar that the cell crude extract is that the obtained thallus of fermentation is crushed through ultrasound or cryogenic high pressure The crude enzyme liquid of based transferase and sucrose synthase contains micro UDP in crude extract.

The system of the circular response catalysis Stevioside production Rebaudiodside A M are as follows: the starting reaction density of substrate Stevioside For 2 ~ 60 g/L, the mass ratio of sucrose and Stevioside is 1 ~ 10, prepared by the genetic engineering bacterium by co-expressing UGTSL2 and StSUS1 Crude enzyme liquid total protein concentration in the reaction system be 1 ~ 25 mg/mL, add water to be settled to 1 ~ 100 mL, pH value is 6 ~ 8, in 20 ~ 50 DEG C of 1 ~ 48 h of reaction；Then the total protein concentration of the genetic engineering bacterium preparation of addition coexpression UGT76G1 and StSUS1 is 1 ~ 25 mg/mL crude enzyme liquid, the reaction was continued 1 ~ 48 h.

The utility model has the advantages that

The present invention uses biological enzyme, is catalyzed Stevioside by glycosyl transferase UGTSL2 glycosylation and synthesizes intermediate production first Object Rebaudiodside A E recycles glycosyl transferase UGT76G1 transglycosylation catalysis Rebaudiodside A E to synthesize Rebaudiodside A M.Wherein, Rebaudiodside A E catalyzes and synthesizes yield up to 65% through Stevioside；Rebaudiodside A M catalyzes and synthesizes yield up to 65% through Rebaudiodside A E More than.

Detailed description of the invention

Fig. 1 Rebaudiodside A E(A) and Rebaudiodside A M(B) structural formula；

Fig. 2 UDP- glycosyl transferase and the coupling catalysed Stevioside of sucrose synthase synthesize Rebaudiodside A M；

Fig. 3 Rebaudiodside A E(A) and Rebaudiodside A M(B) LC-MS qualification result.

Specific embodiment

According to following embodiments, the present invention may be better understood.However, as it will be easily appreciated by one skilled in the art that real It applies content described in example and is merely to illustrate the present invention, without this hair described in detail in claim should will not be limited It is bright.

Embodiment 1: the building of recombination bacillus coli genetic engineering bacterium

1) acquisition of the recombination bacillus coli genetic engineering bacterium of UGTSL2 and StSUS1, is co-expressed

UGTSL2 and StSUS1 gene complete sequence is synthesized by Nanjing Jin Sirui company, and is building up on plasmid pRSFDuet-1, In, UGTSL2 genetic fragment is inserted in plasmid vector RSFDuet-1(Novagen company)NdeI andXhoI restriction enzyme site it Between, StSUS1 genetic fragment is inserted in plasmid vector pRSFDuet-1(Novagen company)NcoI andEcoR I restriction enzyme site Between, ultimately provide can external source coexpression glycosyl transferase UGTSL2 and sucrose synthase StSUS1 recombinant plasmid, pRSFDuet-UGTSL2-StSUS1。

Add 40 uL ddH₂O sufficiently dissolves into recombinant plasmid pRSFDuet-UGTSL2-StSUS1 dry powder, takes 5 ul Thermal shock is converted into e. coli bl21 (DE3) competent cell, and it is anti-that converted product is coated on the kanamycins containing 50 mg/L Property plate on, 37 DEG C are incubated overnight, obtain recombination bacillus coli genetic engineering bacterium.

2) acquisition of the recombination bacillus coli genetic engineering bacterium of UGT76G1 and StSUS1, is co-expressed

UGT76G1 and StSUS1 gene complete sequence is synthesized by Nanjing Jin Sirui company, and is building up on plasmid pRSFDuet-1, In, UGT76G1 genetic fragment is inserted in plasmid vector pRSFDuet-1(Novagen company)NdeI andXhoI restriction enzyme site Between, StSUS1 genetic fragment is inserted in plasmid vector pRSFDuet-1(Novagen company)NcoI andEcoR I digestion position Point between, ultimately provide can external source coexpression glycosyl transferase UGT76G1 and sucrose synthase StSUS1 recombinant plasmid, pRSFDuet-UGT76G1-StSUS1。

Add 40 uL ddH₂O sufficiently dissolves into recombinant plasmid pRSFDuet-UGT76G1-StSUS1 dry powder, takes 5 Ul thermal shock is converted into e. coli bl21 (DE3) competent cell, and converted product is coated on the kanamycins containing 50 mg/L On the plate of resistance, 37 DEG C are incubated overnight, and obtain recombination bacillus coli genetic engineering bacterium.

Embodiment 2: the coexpression of the double enzymes of genetic engineering bacterium

Plasmid pRSFDuet-UGTSL2-StSUS1(or pRSFDuet-UGT76G1-StSUS1 will be contained) recombination engineering Bacterium is inoculated into LB culture medium (0.5 g/L yeast powder, 1 g/L sodium chloride, 1 g/L pancreas egg containing 50 mg/L kalamycin resistances White peptone), in 37 DEG C, 200 rpm shaken overnight cultures, then culture bacterium is accessed by 2% inoculum concentration and contains 100 mL TB culture mediums The 500 of (2.5 g/L yeast powders, 1 g/L sodium chloride, 1.5 g/L tryptones, 0.2 g/L glucose, 0.05 g/L lactose) In mL shaking flask, in 200 rpm, 37 DEG C of 2 h of culture, it is subsequently transferred to 25 DEG C and continues to cultivate 24 h, thalline were collected by centrifugation.Ultrasound is broken Broken thallus, centrifuging and taking supernatant are cell crude extract, are placed in 4 DEG C and save for use.

Embodiment 3: enzyme law catalysis Stevioside synthesizes Rebaudiodside A E

1), catalystic converter system

Add 20 g/L Steviosides, 60 g/L sucrose, 3 mM Mg²⁺And the cell of appropriate coexpression UGTSL2 and StSUS1 slightly mentions For liquid (~ 6 mg/mL total protein) in 50 mM potassium phosphates buffering (pH 7.2), total volume quantifies 20 mL.30 DEG C, 200 rpm are anti- 24 h, 500 uL of timing sampling are answered, 95 DEG C of 15 min of heating water bath are placed in, 12000 rpm room temperatures are centrifuged 1 min, separate supernatant Into new 1.5 mL EP pipe, 4 DEG C of preservations detect liquid to HPCL.With regard to (table 1) from the point of view of result, glycosyl transferase UGTSL2 is coupled sugarcane Sugared synzyme StSUS1 catalysis Stevioside synthesis Rebaudiodside A E is feasible, and when reacting 24 h, the conversion ratio of Stevioside is Reach 94.12%, the yield of Rebaudiodside A E is 66.32%.

2), PHLC detection method

Chromatographic column: Agilent TC-C18(4.6*250mm, 5um, 120A)；Ultraviolet detection wavelength: 210 nm；Column temperature: 55 DEG C； Mobile phase A is the acetonitrile containing 0.1% formic acid, and Mobile phase B is the Wahaha water containing 0.1% formic acid, liquid phase testing conditions: 0.000 min (25% A, 75% B), 15.000 min (47% A, 53% B), 20.000 min (100% A, 0% B), 20.010-25.000 min (100% A, 0% B), 25.010 min (25% A, 75% B), 25.010-30.000 min (25% A, 75% B)；10 uL of sample feeding, 1 mL/min of flow velocity.

Table 1:UGTSL2-StSUS1 is catalyzed Stevioside and synthesizes Rebaudiodside A E

Embodiment 4: enzyme law catalysis Rebaudiodside A E synthesizes Rebaudiodside A M

1), catalystic converter system

The cell crude extract (~ 5 mg/mL total protein) of 18 g/L sucrose and appropriate coexpression UGTSL2 and StSUS1 is added in containing In solution after having the termination described in embodiment 3 of 3 g/L or 6 g/L Rebaudiodside A E reaction.30 DEG C, 200 rpm reaction 24 H, 500 uL of timing sampling, are placed in 95 DEG C of 15 min of heating water bath, and 12000 rpm room temperatures are centrifuged 1 min, and separation supernatant is to new In 1.5 mL EP pipes, 4 DEG C of preservations are detected to HPCL.With regard to (table 2) from the point of view of result, under different concentration of substrate, glycosyl transferase When UGT76G1 is coupled sucrose synthase StSUS1 catalysis Rebaudiodside A E synthesis Rebaudiodside A M 12 h of reaction, Rebaudiodside A E's turns Rate has reached 90% or more, for 24 hours when, Rebaudiodside A E has been totally converted completely.When being catalyzed 3 g/L Rebaudiodside A E for 24 hours, The yield of Rebaudiodside A M can reach 80%；When being catalyzed 6 g/L Rebaudiodside A E for 24 hours, the yield of Rebaudiodside A M is 36%.

2), PHLC detection method

Table 2:UGT76G1-StSUS1 is catalyzed Rebaudiodside A E and synthesizes Rebaudiodside A M

Embodiment 5: enzyme law catalysis Stevioside synthesizes Rebaudiodside A M

1), catalystic converter system

Add 10 g/L Steviosides, 30 g/L sucrose, 3 mM Mg²⁺And the cell of appropriate coexpression UGTSL2 and StSUS1 slightly mentions For liquid (~ 6 mg/mL total protein) in 50 mM potassium phosphates buffering (pH 7.2), total volume quantifies 20 mL.30 DEG C, 200 rpm are anti- 12 h, 500 uL of timing sampling are answered, 95 DEG C of 15 min of heating water bath are placed in, 12000 rpm room temperatures are centrifuged 1 min, separate supernatant Into new 1.5 mL EP pipe, 4 DEG C of preservations detect liquid to HPCL.

After above-mentioned reaction 12h, the cell of addition 20 mL coexpression UGT76G1 and StSUS1 is continued directly in reaction solution Crude extract (~ 6 mg/mL total protein), in 30 DEG C, 200 rpm the reaction was continued 12 h, 500 uL of timing sampling are placed in 95 DEG C of water-baths 15 min are heated, 12000 rpm room temperatures are centrifuged 1 min, separate supernatant into new 1.5 mL EP pipe, 4 DEG C of preservations, to HPCL Detection.

It the results are shown in Table 3, Stevioside is through glycosyl transferase and the lower synthesis Rebaudiodside A M of sucrose synthase co-catalysis effect Feasible, in the two-step method, first step Stevioside can reach 95% through converting under UGTSL2 and StSUS1 catalytic action, and is produced from centre The yield of object Rebaudiodside A E reaches 65%；Second step intermediate product Rebaudiodside A E is through lower turn of UGT76G1 and StSUS1 catalytic action For rate up to 90% or more, the yield of product Rebaudiodside A M is 67%.Finally, using 10 g/L Steviosides as starting material enzymatic clarification In Rebaudiodside A M experiment, the yield of Rebaudiodside A M is 43.5%.

2), PHLC detection method

3 liang of step enzyme catalysis method Steviosides of table synthesize Rebaudiodside A M

Enzyme	Time (h)	Stevioside concentration (g/L)	Rebaudiodside A E concentration (g/L)	Rebaudiodside A M concentration (g/L)
					(step 1) UGTSL2-StSUS1	12	0.5 g/L	6.5 g/L
(step 2) UGT76G1-StSUS1	24		0.2 g/L	2.2 g/L

* in step 2, because of the crude enzyme liquid of addition coexpression UGT76G1 and StSUS1, reaction solution volume is doubled, is being calculated When conversion ratio and yield, using quality as measurement standard.

Sequence table

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cctgactcat gtactcttga gaagttcttg gggagaattc ctatggtttt caatgtggtt 840

atcctttccc ctcatggata ttttgctcaa gaaaatgtct tgggttatcc tgacaccggt 900

ggccaggttg tctacatttt agatcaagtt cccgccttgg agcgtgaaat gcttaagcgc 960

ataaaggagc aaggacttga tatcatcccc cgtattctta ttgttactcg tctgctcccc 1020

gatgcagttg gaaccacttg tggtcagagg attgagaagg tgtatggagc agaacactca 1080

catattctta gggtcccttt taggactgag aagggcattg ttcgcaaatg gatctctcgc 1140

tttgaagtgt ggccatacat ggagacattc attgaggatg ttgcaaaaga aatttctgca 1200

gaactgcagg ccaagccaga tttgataatc ggaaactaca gtgagggcaa tcttgctgct 1260

tctttgctag ctcacaagtt aggcgtaacg cagtgcacca ttgcccacgc gttggagaaa 1320

acgaagtatc ctgattccga catttactgg aaaaagtttg atgaaaaata ccatttctcg 1380

tcccagttta ccgctgatct cattgcaatg aatcacactg atttcatcat caccagcacc 1440

ttccaggaga tagcaggaag caaggacact gtgggacaat atgagagcca tatggcattc 1500

acaatgcctg gattgtacag agttgttcac ggcattaatg tgttcgaccc caaattcaac 1560

attgtctcac ctggagctga tattaacctc tacttctcgt actccgaaac ggagaagaga 1620

cttacagcat ttcaccctga aattgatgag ctgctgtata gtgatgttga gaatgacgaa 1680

catctgtgcg tgctcaagga caggactaaa ccaattttat tcacaatggc gaggttggat 1740

cgtgtgaaga atttaactgg acttgttgag tggtacgcca aaaatccacg gctaagggga 1800

ttggttaacc tggttgtagt tggcggagat cgaaggaagg aatccaaaga tttggaagag 1860

caggcagaga tgaagaagat gtatgagcta attgagactc ataatttgaa tggccaattc 1920

agatggattt cttcccagat gaaccgagtg aggaatggtg agctctaccg atacattgct 1980

gacactaagg gagctttcgt tcagcctgca ttctacgagg cttttggtct gactgttgtc 2040

gaagcaatga cttgtggttt gcctacattt gcaactaatc acggtggtcc agctgagatc 2100

atcgttcatg gaaagtccgg cttccacatt gatccatatc acggtgagca agctgctgat 2160

ctgctagctg atttctttga gaaatgcaag aaagatcctt cacattggga aaccatttcg 2220

atgggtggcc tgaagcgcat cgaagagaag tacacttggc aaatctactc cgaaagccta 2280

ttgacactgg ctgctgttta tgggttctgg aaacatgttt ctaagcttga tcgtctagaa 2340

atccgtcgct atcttgaaat gttttatgct ctcaagtacc gtaagatggc tgaagctgtt 2400

ccattggctg ctgagtga 2418

<210> 4

<211> 442

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 4

Met Ala Thr Asn Leu Arg Val Leu Met Phe Pro Trp Leu Ala Tyr Gly

1 5 10 15

His Ile Ser Pro Phe Leu Asn Ile Ala Lys Gln Leu Ala Asp Arg Gly

20 25 30

Phe Leu Ile Tyr Leu Cys Ser Thr Arg Ile Asn Leu Glu Ser Ile Ile

35 40 45

Lys Lys Ile Pro Glu Lys Tyr Ala Asp Ser Ile His Leu Ile Glu Leu

50 55 60

Gln Leu Pro Glu Leu Pro Glu Leu Pro Pro His Tyr His Thr Thr Asn

65 70 75 80

Gly Leu Pro Pro His Leu Asn Pro Thr Leu His Lys Ala Leu Lys Met

85 90 95

Ser Lys Pro Asn Phe Ser Arg Ile Leu Gln Asn Leu Lys Pro Asp Leu

100 105 110

Leu Ile Tyr Asp Val Leu Gln Pro Trp Ala Glu His Val Ala Asn Glu

115 120 125

Gln Asn Ile Pro Ala Gly Lys Leu Leu Thr Ser Cys Ala Ala Val Phe

130 135 140

Ser Tyr Phe Phe Ser Phe Arg Lys Asn Pro Gly Val Glu Phe Pro Phe

145 150 155 160

Pro Ala Ile His Leu Pro Glu Val Glu Lys Val Lys Ile Arg Glu Ile

165 170 175

Leu Ala Lys Glu Pro Glu Glu Gly Gly Arg Leu Asp Glu Gly Asn Lys

180 185 190

Gln Met Met Leu Met Cys Thr Ser Arg Thr Ile Glu Ala Lys Tyr Ile

195 200 205

Asp Tyr Cys Thr Glu Leu Cys Asn Trp Lys Val Val Pro Val Gly Pro

210 215 220

Pro Phe Gln Asp Leu Ile Thr Asn Asp Ala Asp Asn Lys Glu Leu Ile

225 230 235 240

Asp Trp Leu Gly Thr Lys His Glu Asn Ser Thr Val Phe Val Ser Phe

245 250 255

Gly Ser Glu Tyr Phe Leu Ser Lys Glu Asp Met Glu Glu Val Ala Phe

260 265 270

Ala Leu Glu Leu Ser Asn Val Asn Phe Ile Trp Val Ala Arg Phe Pro

275 280 285

Lys Gly Glu Glu Arg Asn Leu Glu Asp Ala Leu Pro Lys Gly Phe Leu

290 295 300

Glu Arg Ile Gly Glu Arg Gly Arg Val Leu Asp Lys Phe Ala Pro Gln

305 310 315 320

Pro Arg Ile Leu Asn His Pro Ser Thr Gly Gly Phe Ile Ser His Cys

325 330 335

Gly Trp Asn Ser Ala Met Glu Ser Ile Asp Phe Gly Val Pro Ile Ile

340 345 350

Ala Met Pro Ile His Asn Asp Gln Pro Ile Asn Ala Lys Leu Met Val

355 360 365

Glu Leu Gly Val Ala Val Glu Ile Val Arg Asp Asp Asp Gly Lys Ile

370 375 380

His Arg Gly Glu Ile Ala Glu Thr Leu Lys Ser Val Val Thr Gly Glu

385 390 395 400

Thr Gly Glu Ile Leu Arg Ala Lys Val Arg Glu Ile Ser Lys Asn Leu

405 410 415

Lys Ser Ile Arg Asp Glu Glu Met Asp Ala Val Ala Glu Glu Leu Ile

420 425 430

Gln Leu Cys Arg Asn Ser Asn Lys Ser Lys

435 440

<210> 5

<211> 458

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 5

Met Glu Asn Lys Thr Glu Thr Thr Val Arg Arg Arg Arg Arg Ile Ile

1 5 10 15

Leu Phe Pro Val Pro Phe Gln Gly His Ile Asn Pro Ile Leu Gln Leu

20 25 30

Ala Asn Val Leu Tyr Ser Lys Gly Phe Ser Ile Thr Ile Phe His Thr

35 40 45

Asn Phe Asn Lys Pro Lys Thr Ser Asn Tyr Pro His Phe Thr Phe Arg

50 55 60

Phe Ile Leu Asp Asn Asp Pro Gln Asp Glu Arg Ile Ser Asn Leu Pro

65 70 75 80

Thr His Gly Pro Leu Ala Gly Met Arg Ile Pro Ile Ile Asn Glu His

85 90 95

Gly Ala Asp Glu Leu Arg Arg Glu Leu Glu Leu Leu Met Leu Ala Ser

100 105 110

Glu Glu Asp Glu Glu Val Ser Cys Leu Ile Thr Asp Ala Leu Trp Tyr

115 120 125

Phe Ala Gln Ser Val Ala Asp Ser Leu Asn Leu Arg Arg Leu Val Leu

130 135 140

Met Thr Ser Ser Leu Phe Asn Phe His Ala His Val Ser Leu Pro Gln

145 150 155 160

Phe Asp Glu Leu Gly Tyr Leu Asp Pro Asp Asp Lys Thr Arg Leu Glu

165 170 175

Glu Gln Ala Ser Gly Phe Pro Met Leu Lys Val Lys Asp Ile Lys Ser

180 185 190

Ala Tyr Ser Asn Trp Gln Ile Leu Lys Glu Ile Leu Gly Lys Met Ile

195 200 205

Lys Gln Thr Lys Ala Ser Ser Gly Val Ile Trp Asn Ser Phe Lys Glu

210 215 220

Leu Glu Glu Ser Glu Leu Glu Thr Val Ile Arg Glu Ile Pro Ala Pro

225 230 235 240

Ser Phe Leu Ile Pro Leu Pro Lys His Leu Thr Ala Ser Ser Ser Ser

245 250 255

Leu Leu Asp His Asp Arg Thr Val Phe Gln Trp Leu Asp Gln Gln Pro

260 265 270

Pro Ser Ser Val Leu Tyr Val Ser Phe Gly Ser Thr Ser Glu Val Asp

275 280 285

Glu Lys Asp Phe Leu Glu Ile Ala Arg Gly Leu Val Asp Ser Lys Gln

290 295 300

Ser Phe Leu Trp Val Val Arg Pro Gly Phe Val Lys Gly Ser Thr Trp

305 310 315 320

Val Glu Pro Leu Pro Asp Gly Phe Leu Gly Glu Arg Gly Arg Ile Val

325 330 335

Lys Trp Val Pro Gln Gln Glu Val Leu Ala His Gly Ala Ile Gly Ala

340 345 350

Phe Trp Thr His Ser Gly Trp Asn Ser Thr Leu Glu Ser Val Cys Glu

355 360 365

Gly Val Pro Met Ile Phe Ser Asp Phe Gly Leu Asp Gln Pro Leu Asn

370 375 380

Ala Arg Tyr Met Ser Asp Val Leu Lys Val Gly Val Tyr Leu Glu Asn

385 390 395 400

Gly Trp Glu Arg Gly Glu Ile Ala Asn Ala Ile Arg Arg Val Met Val

405 410 415

Asp Glu Glu Gly Glu Tyr Ile Arg Gln Asn Ala Arg Val Leu Lys Gln

420 425 430

Lys Ala Asp Val Ser Leu Met Lys Gly Gly Ser Ser Tyr Glu Ser Leu

435 440 445

Glu Ser Leu Val Ser Tyr Ile Ser Ser Leu

450 455

<210> 6

<211> 805

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 6

Met Ala Glu Arg Val Leu Thr Arg Val His Ser Leu Arg Glu Arg Val

1 5 10 15

Asp Ala Thr Leu Ala Ala His Arg Asn Glu Ile Leu Leu Phe Leu Ser

20 25 30

Arg Ile Glu Ser His Gly Lys Gly Ile Leu Lys Pro His Glu Leu Leu

35 40 45

Ala Glu Phe Asp Ala Ile Arg Gln Asp Asp Lys Asn Lys Leu Asn Glu

50 55 60

His Ala Phe Glu Glu Leu Leu Lys Ser Thr Gln Glu Ala Ile Val Leu

65 70 75 80

Pro Pro Trp Val Ala Leu Ala Ile Arg Leu Arg Pro Gly Val Trp Glu

85 90 95

Tyr Ile Arg Val Asn Val Asn Ala Leu Val Val Glu Glu Leu Ser Val

100 105 110

Pro Glu Tyr Leu Gln Phe Lys Glu Glu Leu Val Asp Gly Ala Ser Asn

115 120 125

Gly Asn Phe Val Leu Glu Leu Asp Phe Glu Pro Phe Thr Ala Ser Phe

130 135 140

Pro Lys Pro Thr Leu Thr Lys Ser Ile Gly Asn Gly Val Glu Phe Leu

145 150 155 160

Asn Arg His Leu Ser Ala Lys Met Phe His Asp Lys Glu Ser Met Thr

165 170 175

Pro Leu Leu Glu Phe Leu Arg Ala His His Tyr Lys Gly Lys Thr Met

180 185 190

Met Leu Asn Asp Arg Ile Gln Asn Ser Asn Thr Leu Gln Asn Val Leu

195 200 205

Arg Lys Ala Glu Glu Tyr Leu Ile Met Leu Pro Pro Glu Thr Pro Tyr

210 215 220

Phe Glu Phe Glu His Lys Phe Gln Glu Ile Gly Leu Glu Lys Gly Trp

225 230 235 240

Gly Asp Thr Ala Glu Arg Val Leu Glu Met Val Cys Met Leu Leu Asp

245 250 255

Leu Leu Glu Ala Pro Asp Ser Cys Thr Leu Glu Lys Phe Leu Gly Arg

260 265 270

Ile Pro Met Val Phe Asn Val Val Ile Leu Ser Pro His Gly Tyr Phe

275 280 285

Ala Gln Glu Asn Val Leu Gly Tyr Pro Asp Thr Gly Gly Gln Val Val

290 295 300

Tyr Ile Leu Asp Gln Val Pro Ala Leu Glu Arg Glu Met Leu Lys Arg

305 310 315 320

Ile Lys Glu Gln Gly Leu Asp Ile Ile Pro Arg Ile Leu Ile Val Thr

325 330 335

Arg Leu Leu Pro Asp Ala Val Gly Thr Thr Cys Gly Gln Arg Ile Glu

340 345 350

Lys Val Tyr Gly Ala Glu His Ser His Ile Leu Arg Val Pro Phe Arg

355 360 365

Thr Glu Lys Gly Ile Val Arg Lys Trp Ile Ser Arg Phe Glu Val Trp

370 375 380

Pro Tyr Met Glu Thr Phe Ile Glu Asp Val Ala Lys Glu Ile Ser Ala

385 390 395 400

Glu Leu Gln Ala Lys Pro Asp Leu Ile Ile Gly Asn Tyr Ser Glu Gly

405 410 415

Asn Leu Ala Ala Ser Leu Leu Ala His Lys Leu Gly Val Thr Gln Cys

420 425 430

Thr Ile Ala His Ala Leu Glu Lys Thr Lys Tyr Pro Asp Ser Asp Ile

435 440 445

Tyr Trp Lys Lys Phe Asp Glu Lys Tyr His Phe Ser Ser Gln Phe Thr

450 455 460

Ala Asp Leu Ile Ala Met Asn His Thr Asp Phe Ile Ile Thr Ser Thr

465 470 475 480

Phe Gln Glu Ile Ala Gly Ser Lys Asp Thr Val Gly Gln Tyr Glu Ser

485 490 495

His Met Ala Phe Thr Met Pro Gly Leu Tyr Arg Val Val His Gly Ile

500 505 510

Asn Val Phe Asp Pro Lys Phe Asn Ile Val Ser Pro Gly Ala Asp Ile

515 520 525

Asn Leu Tyr Phe Ser Tyr Ser Glu Thr Glu Lys Arg Leu Thr Ala Phe

530 535 540

His Pro Glu Ile Asp Glu Leu Leu Tyr Ser Asp Val Glu Asn Asp Glu

545 550 555 560

His Leu Cys Val Leu Lys Asp Arg Thr Lys Pro Ile Leu Phe Thr Met

565 570 575

Ala Arg Leu Asp Arg Val Lys Asn Leu Thr Gly Leu Val Glu Trp Tyr

580 585 590

Ala Lys Asn Pro Arg Leu Arg Gly Leu Val Asn Leu Val Val Val Gly

595 600 605

Gly Asp Arg Arg Lys Glu Ser Lys Asp Leu Glu Glu Gln Ala Glu Met

610 615 620

Lys Lys Met Tyr Glu Leu Ile Glu Thr His Asn Leu Asn Gly Gln Phe

625 630 635 640

Arg Trp Ile Ser Ser Gln Met Asn Arg Val Arg Asn Gly Glu Leu Tyr

645 650 655

Arg Tyr Ile Ala Asp Thr Lys Gly Ala Phe Val Gln Pro Ala Phe Tyr

660 665 670

Glu Ala Phe Gly Leu Thr Val Val Glu Ala Met Thr Cys Gly Leu Pro

675 680 685

Thr Phe Ala Thr Asn His Gly Gly Pro Ala Glu Ile Ile Val His Gly

690 695 700

Lys Ser Gly Phe His Ile Asp Pro Tyr His Gly Glu Gln Ala Ala Asp

705 710 715 720

Leu Leu Ala Asp Phe Phe Glu Lys Cys Lys Lys Asp Pro Ser His Trp

725 730 735

Glu Thr Ile Ser Met Gly Gly Leu Lys Arg Ile Glu Glu Lys Tyr Thr

740 745 750

Trp Gln Ile Tyr Ser Glu Ser Leu Leu Thr Leu Ala Ala Val Tyr Gly

755 760 765

Phe Trp Lys His Val Ser Lys Leu Asp Arg Leu Glu Ile Arg Arg Tyr

770 775 780

Leu Glu Met Phe Tyr Ala Leu Lys Tyr Arg Lys Met Ala Glu Ala Val

785 790 795 800

Pro Leu Ala Ala Glu

805

Claims

1. a kind of method of biocatalysis synthesis Rebaudiodside A M, which is characterized in that utilize molecule clone technology, obtain heterologous table Up to the genetic engineering bacterium of UDP- glycosyl transferase and sucrose synthase, after self-induction enzymatic production with cell crude extract directly into Row catalysis reaction establishes double enzyme circular response bodies using the micro UDP contained in itself in crude extract and the sucrose additionally added System is effectively catalyzed Stevioside and produces Rebaudiodside A M.

2. the method for biocatalysis synthesis Rebaudiodside A M according to claim 1 a kind of, which is characterized in that catalysis stevia rebaudianum It includes following two step that glucoside, which produces Rebaudiodside A M:

(1) UDP- glycosyl transferase UGT-A and sucrose synthase SUS coexpression double-enzyme catalysis Stevioside synthesizes intermediate product Lay Bao Enlightening glycosides E；

(2) UDP- glycosyl transferase UGT-B and sucrose synthase SUS coexpression double-enzyme catalysis Rebaudiodside A E synthesizes final product Rebaudiodside A M.

3. a kind of method of biocatalysis synthesis Rebaudiodside A M according to claim 2, which is characterized in that the UDP- sugar Based transferase UGT-A is tomato source glycosyl transferase UGTSL2, and gene order as shown in SEQ.No.1, be shown in by amino acid sequence Shown in SEQ.No.4；The UDP- glycosyl transferase UGT-B is STEVIA REBAUDIANA source glycosyl transferase UGT76G1, and gene order is shown in Shown in SEQ.No.2, amino acid sequence is as shown in SEQ.No.5；The sucrose synthase SUS is potato source sucrose synthase StSUS1, gene order is as shown in SEQ.No.3, and amino acid sequence is as shown in SEQ.No.6.

4. the method for biocatalysis synthesis Rebaudiodside A M according to claim 1 a kind of, which is characterized in that the cell Crude extract, which is the obtained thallus of fermentation, to be crushed through ultrasound or cryogenic high pressure and obtained closes containing UDP- glycosyl transferase and sucrose At the crude enzyme liquid of enzyme.

5. the method for biocatalysis synthesis Rebaudiodside A M according to claim 1 a kind of, which is characterized in that the circulation Reaction system are as follows: UDP-glucose and Stevioside are through UGTSL2 and StSUS1 catalysis generation Rebaudiodside A E；UDP-glucose with Rebaudiodside A E generates Rebaudiodside A M through UGT76G1 and StSUS1 catalysis.

6. the method for biocatalysis synthesis Rebaudiodside A M according to claim 1 a kind of, which is characterized in that the gene Engineering bacteria inducing expression condition are as follows: recombinant bacterium is inoculated into LB culture medium, in 16 ~ 37 DEG C, 8 ~ 10 h of 200rpm shaken cultivation, Culture bacterium solution is accessed in TB culture medium again, wherein inducer concentration in TB culture solution is 0.01 ~ 0.5%, is lured in 16 ~ 37 DEG C 16 ~ 36 h of culture is led, thalline were collected by centrifugation.

7. the method for biocatalysis synthesis Rebaudiodside A M according to claim 2 a kind of, which is characterized in that described two Walk catalysis method are as follows: in the reaction solution containing substrate stevioside, sucrose, add the thick enzyme of coexpression UGTSL2 and StSUS1 first Liquid catalyzes and synthesizes intermediate product Rebaudiodside A E；Coexpression UGT76G1 and StSUS1 is added thereto again after reacting a period of time Crude enzyme liquid, directly catalysis Rebaudiodside A E synthesize Rebaudiodside A M.

8. the method for biocatalysis synthesis Rebaudiodside A M according to claim 2 a kind of, which is characterized in that in step 1), The starting reaction density of substrate Stevioside is 2 ~ 60 g/L, and the mass ratio of sucrose and Stevioside is 1 ~ 10, and two kinds of crude enzyme liquids are anti- Answering the total protein concentration in system is 1 ~ 25 mg/mL.

9. the method for biocatalysis synthesis Rebaudiodside A M according to claim 2 a kind of, which is characterized in that the catalysis Reaction uses aqueous phase reactions system, and pH value of water solution is 6 ~ 8.

10. the method for biocatalysis synthesis Rebaudiodside A M according to claim 2 a kind of, which is characterized in that the catalysis The reaction temperature of reaction is 20 ~ 50 DEG C, and front and back reaction time two stages is 1 ~ 48 h.