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Method for synthesizing 2-O-alpha-D-glyceroglucoside by utilizing microorganisms

A technology of glycerol and biocatalyst, applied in the field of genetic engineering, can solve the problems of large demand, easy inactivation, and long conversion time of 2-O-α-D-glycerol glucoside, and achieve large-scale industrial application and improve The effect of industrial application potential

Pending Publication Date: 2020-09-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

2-O-α-D-glycerol glucoside plays an important role in cosmetics, food, medicine, health products and other fields, and has broad application and market prospects; however, due to the lack of economical and efficient 2-O-α-D- The production process of glycerol glucoside has caused a large demand for 2-O-α-D-glycerol glucoside in the market, and the price is expensive
[0004] In the synthesis of 2-O-α-D-glycerol glucoside catalyzed by sucrose phosphorylase, although it has the advantages of low substrate raw materials and simple products, it also has the advantages of long conversion time, poor stability of the enzyme under in vitro conditions, and easy inactivation. and other problems limit the industrial application of sucrose phosphorylase to a certain extent

Method used

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  • Method for synthesizing 2-O-alpha-D-glyceroglucoside by utilizing microorganisms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1 Contains the construction of the recombinant vector of sucrose phosphorylase mutant

[0035] Specific steps are as follows:

[0036] (1) Obtaining of mutant enzyme gene gtfAK138C: using the nucleotide sequence shown in SEQ ID NO.4 as a template, f-primer1 (sequence shown in SEQ ID NO.5), r-primer 1 (sequence shown in SEQ ID NO.5) shown in NO.6) as primers, the gene shown in SEQ ID NO.4 is used as a template to carry out PCR to obtain the recombinant gene shown in SEQ ID NO.3.

[0037] (2) The recombinant gene and pET-28a obtained in step (1) were digested with BamH I and EcoR I respectively, and purified with T 4 DNA ligase was ligated overnight at 16°C, and the sequencing work was completed by Shanghai Sangon.

Embodiment 2

[0038] Embodiment 2 produces sucrose phosphorylase mutant recombinant Corynebacterium glutamicum engineering bacterium construction

[0039] The recombinant plasmid pET-28a-gtfAK138C obtained in Example 1 was chemically transformed into E.coli competent cells, and the specific method was as follows:

[0040] The solution required for the conversion experiment is as follows (g / L):

[0041] LB medium: yeast extract 5, peptone 10, NaCl 10.

[0042] 50% Glycerol, 0.1M CaCl 2 , 115 ℃ damp heat sterilization.

[0043] (1) Inoculate Escherichia coli BL21 (DE3) in 50 mL of fresh LB culture medium and culture overnight at 37°C and 220 r / min.

[0044] (2) Take 1 mL of the overnight culture and inoculate it into 100 mL of fresh LB medium at 37° C. and shake at 220 r / min.

[0045] (3) Start to detect the OD of the culture medium with a spectrophotometer after culturing for 1 h 600value, detected every 20 minutes or so until OD 600 When the value reaches 0.6 (about 2h).

[0046] (4)...

Embodiment 3

[0051] Example 3 Construction of recombinant bacteria expressing wild-type sucrose phosphorylase

[0052] The gene shown in SEQ ID NO.4 and pET-28a were double-digested with BamH I and EcoR I respectively, and purified with T 4 DNA ligase was ligated overnight at 16°C to obtain the recombinant plasmid pET-28a-gtfA, and the sequencing work was completed by Shanghai Sangong.

[0053] The recombinant plasmid pET-28a-gtfA was transformed into E.coli competent cells according to the same method as in Example 2, cultivated, and positive transformants were picked for verification, and recombinant bacteria E.coli BL21 / pET-28a-gtfA were obtained.

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Abstract

The invention discloses a method for synthesizing 2-O-alpha-D-glyceroglucoside by utilizing microorganisms, and belongs to the technical field of gene engineering. An amino acid sequence of a mutant is as shown in SEQ ID NO:1. According to the mutant disclosed by the invention, site-specific mutagenesis is carried out to improve the enzyme activity of sucrose phosphorylase on the basis of sucrosephosphorylase derived from leuconostoc mesenteroides; a mutant enzyme is expressed in escherichia coli and is used as a whole-cell catalyst to produce the 2-O-alpha-D-glyceroglucoside; and a large amount of 2-O-alpha-D-glyceroglucoside can be produced within a short time at the level of a 5L fermentation tank, so that the industrial application prospect of production of the 2-O-alpha-D-glyceroglucoside by the sucrose phosphorylase can be easily expanded, and the large-scale industrial application is achieved.

Description

technical field [0001] The invention relates to a method for synthesizing 2-O-α-D-glycerol glucoside by using microorganisms, belonging to the technical field of genetic engineering. Background technique [0002] According to the EC classification standard, sucrose phosphorylase (EC 2.4.1.7, Sucrose Phosphorylase, Spase) is classified as a glycosyltransferase, which consists of about 500 amino acid residues and is in the form of a functional monomer or dimer exist. The catalytic activity of sucrose phosphorylase does not depend on other cofactors. It can reversibly catalyze sucrose and phosphoric acid to generate D-fructose and 1-phosphate-glucose. In addition to hydrolyzing sucrose, it also has the ability to catalyze the transfer of glucose groups. [0003] With the catalytic properties, the sucrose phosphorylase can use glycerol as an acceptor to catalyze the synthesis of 2-O-α-D-glycerol glucoside from sucrose, which is of great industrial value. 2-O-α-D-glycerol gluco...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P19/44C12R1/19
CPCC12N9/1051C12N15/70C12P19/44C12Y204/01007C12N2800/22
Inventor 饶志明张显段培枫易敢峰付维来杨套伟徐美娟邵明龙
Owner JIANGNAN UNIV
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