CN110592043A - UDP-glucosyltransferase mutant and application thereof - Google Patents
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Abstract
The invention discloses a UDP-glucosyltransferase mutant and application thereof, wherein a site-directed mutagenesis technology is utilized to carry out directed evolution on wild UDP-glucosyl, the UDP-glucosyltransferase mutant has 9 site-directed mutagenesis compared with an original gene, and can carry out single-point or multi-point combined mutagenesis to artificially transform and evolve the wild enzyme. After mutation, the enzyme catalyzes the rebaudioside A with higher efficiency and purity of more than 97 percent, reduces the production cost, belongs to a green and environment-friendly biological enzyme method, and is suitable for industrial production.
Description
Technical Field
The invention belongs to the technical field of enzymology, and particularly relates to a UDP-glucosyltransferase mutant and application thereof.
Background
The stevioside is extracted from stevia rebaudiana Bertoni, is a natural sweetener with low calorie and high sweetness, is used as a substitute of natural cane sugar which is advocated for health, is widely applied to food industries such as beverages, candies, cakes and the like, and has the cost which is more than 50 percent lower than that of the cane sugar. It is 300 times to 450 times as sweet as sucrose and only 1/300 times as hot as sucrose. However, the directly extracted stevioside has high sweetness but has obvious aftertaste, directly influences the taste and limits the application. The structural formula of stevioside is shown in figure 1, wherein the higher the Rebaudioside A (RA) content is, the better the taste and sweetness is, as shown in Table 1.
In the prior art, the technological means for purification and refining rebaudioside A mainly comprise: high performance liquid chromatography, liquid drop countercurrent distribution chromatography, thin layer chromatography, capillary electrophoresis, supercritical extraction, membrane separation, recrystallization, etc. The process has the disadvantages of multiple processes, high energy consumption, high pollution, and low yield and product purity, and is not favorable for industrial production.
Disclosure of Invention
The invention aims to provide a UDP-glucosyltransferase mutant and a method for synthesizing rebaudioside A by using the mutant.
The technical scheme for realizing the purpose of the invention is as follows:
a UDP-glucosyltransferase mutant, wherein the DNA sequence of the UDP-glucosyltransferase mutant is shown as GenBank: KX657833.1, the amino acid sequence is shown as Seq No.2, or the amino acid sequence shown as Seq No.2 is obtained by at least one mutation of the following:
respectively mutating methionine at position 56 and glutamine at position 58 into leucine and histidine, M56L + Q58H for short;
or/and mutation of asparagine at position 106 into glutamine, N106Q for short;
or/and mutation of valine at position 237 into threonine, namely V237T;
or/and mutating glutamine 250 and glutamine 251 to glutamic acid and histidine respectively, which is Q250E + Q251H for short;
or/and mutation of the 287 th arginine into histidine, R287H for short;
or/and the 300 th glycine is mutated into serine, G300S for short;
or/and mutation of 353 rd glutamine into histidine, Q353H for short;
or/and mutation of 361 th aspartic acid into lysine, D361K for short;
or/and mutation of asparagine 415 to aspartic acid, N415D for short.
Another objective of the invention is to provide application of the UDP-glucosyltransferase mutant in synthesis of rebaudioside A.
A method for synthesizing rebaudioside A by applying the UDP-glucosyltransferase mutant comprises the following steps:
(1) enzyme expression
UGT22 gene Seq ID No.1 is connected to pET30a, and the recombinant plasmid is named as pET30a-UGT 22;
then the recombinant plasmid is transformed into an escherichia coli host Rosseta (DE 3), the Escherichia coli host Rosseta is subjected to shaking culture at the temperature of 30 ℃ and the rpm of 200 until the OD is 0.4 ~ 0.8.8, and IPTG induction is carried out to obtain thalli containing enzyme;
finally, adding water with the volume 4 times of the weight of the thalli into the thalli containing the enzyme, breaking the wall by using a homogenizer to obtain a crude enzyme solution, and detecting the expression by using polyacrylamide gel electrophoresis;
(2) enzyme catalysis
10G of stevioside, 0.02G of UDP uridine diphosphate or glucose uridine diphosphate UDP-G, 40mL of crude enzyme solution, 0.1G of sucrose synthase (derived from Arabidopsis NP-197583), 8G of sucrose were added to 0.1M phosphate buffer, water was added to the total system to 100mL, the pH was adjusted to 7.0, the temperature was controlled at 40 ℃, the reaction was stirred for 8 hours, the conversion rate was detected to be 88.3%, the enzyme was denatured by heat treatment after the reaction was completed, the reaction was heated to 100 ℃ for 30min, the protein was removed by filtration, the filtrate was collected and purified to obtain 8.7G of Rebaudioside A (RA), the purity was > 97%.
Sucrose synthase is used to transfer the glucose group on sucrose to UDP to synthesize UDPG.
The efficiency of the wild UDP-glucosyl enzyme catalysis substrate can reach 88.3 percent, high-purity RA can be obtained, and the method has certain application prospect in industrial application. In order to further reduce the production cost, the invention utilizes the site-directed mutagenesis technology to carry out directed evolution on the wild UDP-glucosyl, the UDP-glucosyl transferase mutant has 9 site-directed mutagenesis compared with the original gene, and can carry out single-point or multi-point combined mutagenesis to artificially transform and evolve the wild enzyme. After mutation, the enzyme catalyzes the rebaudioside A with higher efficiency and purity of more than 97 percent, and the production cost is reduced.
Drawings
FIG. 1 is a general structural formula of rebaudioside A;
FIG. 2 is a scheme showing the synthesis of rebaudioside A using UDP-glucosyltransferase mutants;
FIG. 3 shows the recombinant plasmid pET30a-UGT 22.
Detailed Description
The present invention will be further described with reference to the following examples, but the present invention is not limited thereto.
Example 1:
acquisition of UDP-glucosyltransferase mutant
The DNA sequence of the UDP-glucosyltransferase mutant is shown as GenBank: KX657833.1, the amino acid sequence is shown as Seq No.2, or the amino acid sequence shown as Seq No.2 is obtained by at least one mutation condition as follows:
respectively mutating methionine at position 56 and glutamine at position 58 into leucine and histidine, M56L + Q58H for short;
or/and mutation of asparagine at position 106 into glutamine, N106Q for short;
or/and mutation of valine at position 237 into threonine, namely V237T;
or/and mutating glutamine 250 and glutamine 251 to glutamic acid and histidine respectively, which is Q250E + Q251H for short;
or/and mutation of the 287 th arginine into histidine, R287H for short;
or/and the 300 th glycine is mutated into serine, G300S for short;
or/and mutation of 353 rd glutamine into histidine, Q353H for short;
or/and mutation of 361 th aspartic acid into lysine, D361K for short;
or/and mutation of asparagine 415 to aspartic acid, N415D for short.
Example 2:
referring to fig. 2, the method for synthesizing rebaudioside a using the UDP-glucosyltransferase mutant comprises the steps of:
(1) enzyme expression
UGT22 gene Seq ID No.1 is connected to pET30a, and the recombinant plasmid is named as pET30a-UGT22, as shown in FIG. 3;
then the recombinant plasmid is transformed into an escherichia coli host Rosseta (DE 3), the Escherichia coli host Rosseta is subjected to shaking culture at the temperature of 30 ℃ and the rpm of 200 until the OD is 0.4 ~ 0.8.8, and IPTG induction is carried out to obtain thalli containing enzyme;
finally, adding water with the volume 4 times of the weight of the thalli into the thalli containing the enzyme, breaking the wall by using a homogenizer to obtain a crude enzyme solution, and detecting the expression by using polyacrylamide gel electrophoresis;
(2) enzyme catalysis
10G of stevioside, 0.02G of UDP uridine diphosphate or glucose uridine diphosphate UDP-G, 40mL of crude enzyme solution, 0.1G of sucrose synthase (derived from Arabidopsis NP-197583), 8G of sucrose were added to 0.1M phosphate buffer, water was added to the total system to 100mL, the pH was adjusted to 7.0, the temperature was controlled at 40 ℃, the reaction was stirred for 8 hours, the conversion rate was detected to be 88.3%, the enzyme was denatured by heat treatment after the reaction was completed, the reaction was heated to 100 ℃ for 30min, the protein was removed by filtration, the filtrate was collected and purified to obtain 8.7G of Rebaudioside A (RA), the purity was > 97%.
Sucrose synthase is used to transfer the glucose group on sucrose to UDP to synthesize UDPG.
Example 3:
the efficiency of each mutant enzyme for catalyzing the synthesis of rebaudioside a was examined as shown in the following table:
| enzyme type | Substrate conversion/% |
| Wild type | 88.3% |
| M56L+Q58H | 95.2% |
| N106Q | 98.6% |
| V237T | 97.4% |
| Q250E+Q251H | 99.2% |
| R287H | 99.5% |
| G300S | 97.8% |
| Q353H | 99.0% |
| D361K | 96.4% |
| N415D | 97.5% |
As can be seen from the table, the conversion rate of the wild UDP-glucosyl enzyme to the substrate is 88.3%, and the conversion rate of the enzyme mutant to the substrate is more than 95% and as high as 99.2%, which proves that the UDP-glucosyl transferase mutant can greatly improve the catalytic efficiency.
Sequence listing
<110> university of Guangxi Master
<120> UDP-glucosyltransferase mutant and application thereof
<141> 2019-11-01
<160> 2
<170> SIPOSequenceListing 1.0
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atggagctct gcaagcacat ggcctccagg gacttcaaaa ccaccctcct catttcctcc 60
aacctctctt cttctgtccc ctcctctttc cgccaggtct ctctactcca tgtcttggaa 120
atcccatcgc ctccaccgcc tcctccccag cctggatccg accccatgca ccaacaccgt 180
agacaccacg gcgacctggc tctaggcctt gaaaacctcc tctcggcccg agccaaaagc 240
ccaaacccag agctgccagt ttgtgccatc ctcgacgtca tgatgagctg gagcgctgaa 300
gtttttcaaa aattcaatgt cccaacggtc gctttcttca catccggcgc ctgctctgcg 360
gccatagagc atgcgatgtg gaaggcccag ccgctggata ttcaacctgg agaggtccgg 420
ttactccccg gattacctga agacatggct ctcacccaat cggacctcaa gcaacggcct 480
catgggccac cgggagggcc accggggccg ccaccgccaa ccggtggtgc cccaccaggc 540
cggggtgccg atttccatcc acccccgggt cccggaccga agatgatggg cccacccaaa 600
cccggtcagc atccaccgtg ggtggaggag gtgaagaaca cgatcgcgct gctgatcaac 660
acgtgcgacg atcttgagcg tccatttatc agttacctca cacatcaagt cgggaaaccg 720
gtctggggcg tcggcccgct tctgccacag cagtactggc agtcgtcccg ttcacttctc 780
cacgaccggg aaatccgaac caatcgacgg tcgaacgtca cggaggacga agtgatcgaa 840
tggctggagt cgaagccacg cgcgtccgtc ctctacgttt cgttcgggag tgaggtgggt 900
ccaaccatgg aagagtaccc acgtctagcc gacgcactag aagcctcaaa ccggccattc 960
atatgggtaa tccaacccgg tgcaggcaga tcgggtccac ctcgtcaatt actcgggaac 1020
aaacccggtt cagaagaaga agagggttac ttcccacagg gcttggatga acgagttggc 1080
gacaggggtt tgataatacg cggatgggca ccgcagctgc tgatactgag tcacccatca 1140
accggaggat tcttgtctca ctgcggttgg aactctacgg tggaggcgat agggcgtggg 1200
gtcccatttt tggtgtggcc catcaggggt gaccaatact ataatgggaa gttggtggtg 1260
gtccatctca aagttgggta catggtttct gatgatcttt cggaacccat aaacaaggaa 1320
gatataggaa aaggaataga gaagctcatg ggggatgaag atgtgaagaa gcgagctttg 1380
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Met Glu Leu Cys Lys His Met Ala Ser Arg Asp Phe Lys Thr Thr Leu
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Leu Ile Ser Ser Asn Leu Ser Ser Ser Val Pro Ser Ser Phe Arg Gln
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Val Ser Leu Leu His Val Leu Glu Ile Pro Ser Pro Pro Pro Pro Pro
35 40 45
Pro Gln Pro Gly Ser Asp Pro Met His Gln His Arg Arg His His Gly
50 55 60
Asp Leu Ala Leu Gly Leu Glu Asn Leu Leu Ser Ala Arg Ala Lys Ser
65 70 75 80
Pro Asn Pro Glu Leu Pro Val Cys Ala Ile Leu Asp Val Met Met Ser
85 90 95
Trp Ser Ala Glu Val Phe Gln Lys Phe Asn Val Pro Thr Val Ala Phe
100 105 110
Phe Thr Ser Gly Ala Cys Ser Ala Ala Ile Glu His Ala Met Trp Lys
115 120 125
Ala Gln Pro Leu Asp Ile Gln Pro Gly Glu Val Arg Leu Leu Pro Gly
130 135 140
Leu Pro Glu Asp Met Ala Leu Thr Gln Ser Asp Leu Lys Gln Arg Pro
145 150 155 160
His Gly Pro Pro Gly Gly Pro Pro Gly Pro Pro Pro Pro Thr Gly Gly
165 170 175
Ala Pro Pro Gly Arg Gly Ala Asp Phe His Pro Pro Pro Gly Pro Gly
180 185 190
Pro Lys Met Met Gly Pro Pro Lys Pro Gly Gln His Pro Pro Trp Val
195 200 205
Glu Glu Val Lys Asn Thr Ile Ala Leu Leu Ile Asn Thr Cys Asp Asp
210 215 220
Leu Glu Arg Pro Phe Ile Ser Tyr Leu Thr His Gln Val Gly Lys Pro
225 230 235 240
Val Trp Gly Val Gly Pro Leu Leu Pro Gln Gln Tyr Trp Gln Ser Ser
245 250 255
Arg Ser Leu Leu His Asp Arg Glu Ile Arg Thr Asn Arg Arg Ser Asn
260 265 270
Val Thr Glu Asp Glu Val Ile Glu Trp Leu Glu Ser Lys Pro Arg Ala
275 280 285
Ser Val Leu Tyr Val Ser Phe Gly Ser Glu Val Gly Pro Thr Met Glu
290 295 300
Glu Tyr Pro Arg Leu Ala Asp Ala Leu Glu Ala Ser Asn Arg Pro Phe
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Ile Trp Val Ile Gln Pro Gly Ala Gly Arg Ser Gly Pro Pro Arg Gln
325 330 335
Leu Leu Gly Asn Lys Pro Gly Ser Glu Glu Glu Glu Gly Tyr Phe Pro
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Gln Gly Leu Asp Glu Arg Val Gly Asp Arg Gly Leu Ile Ile Arg Gly
355 360 365
Trp Ala Pro Gln Leu Leu Ile Leu Ser His Pro Ser Thr Gly Gly Phe
370 375 380
Leu Ser His Cys Gly Trp Asn Ser Thr Val Glu Ala Ile Gly Arg Gly
385 390 395 400
Val Pro Phe Leu Val Trp Pro Ile Arg Gly Asp Gln Tyr Tyr Asn Gly
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Lys Leu Val Val Val His Leu Lys Val Gly Tyr Met Val Ser Asp Asp
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Leu Ser Glu Pro Ile Asn Lys Glu Asp Ile Gly Lys Gly Ile Glu Lys
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Leu Met Gly Asp Glu Asp Val Lys Lys Arg Ala Leu Leu Leu Ser Thr
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Glu Phe Gln Arg Gly Phe Pro Ala Ser Ser Val Ala Ala Leu Asp Ala
465 470 475 480
Phe Arg Asp Phe Ile Asn Gln Ile Val Ala
485 490
Claims (2)
1. A UDP-glucosyltransferase mutant, comprising: the DNA sequence of the UDP-glucosyltransferase mutant is shown as GenBank: KX657833.1, the amino acid sequence is shown as Seq No.2, or the amino acid sequence shown as Seq No.2 is obtained by at least one mutation condition as follows:
respectively mutating methionine at position 56 and glutamine at position 58 into leucine and histidine, M56L + Q58H for short;
or/and mutation of asparagine at position 106 into glutamine, N106Q for short;
or/and mutation of valine at position 237 into threonine, namely V237T;
or/and mutating glutamine 250 and glutamine 251 to glutamic acid and histidine respectively, which is Q250E + Q251H for short;
or/and mutation of the 287 th arginine into histidine, R287H for short;
or/and the 300 th glycine is mutated into serine, G300S for short;
or/and mutation of 353 rd glutamine into histidine, Q353H for short;
or/and mutation of 361 th aspartic acid into lysine, D361K for short;
or/and mutation of asparagine 415 to aspartic acid, N415D for short.
2. Use of the beta-glucosidase mutant of claim 1 for the synthesis of rebaudioside a.
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Cited By (2)
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| WO2020249138A1 (en) * | 2019-06-14 | 2020-12-17 | 中国科学院分子植物科学卓越创新中心 | Glycosyltransferase mutant and use therefor |
| CN112375750A (en) * | 2020-12-02 | 2021-02-19 | 南京工业大学 | Glycosyltransferase mutant and method for catalytically synthesizing rebaudioside A by using same |
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| CN112375750A (en) * | 2020-12-02 | 2021-02-19 | 南京工业大学 | Glycosyltransferase mutant and method for catalytically synthesizing rebaudioside A by using same |
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| CN110592043B (en) | 2022-10-18 |
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