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A kind of sucrose phosphorylase mutant with improved enzyme activity and its construction method and application

A technology of phosphorylase mutants and mutants, which is applied in the field of genetic engineering, can solve problems such as low enzyme activity and limited applications, and achieve the effect of improving industrial application potential and realizing large-scale industrial applications

Active Publication Date: 2021-06-18
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The enzyme activity of sucrose phosphorylase is relatively low, which limits the industrial application of sucrose phosphorylase to produce 2-O-α-D-glycerol glucoside

Method used

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  • A kind of sucrose phosphorylase mutant with improved enzyme activity and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Construction of recombinant vector containing sucrose phosphorylase mutant

[0038] Specific steps are as follows:

[0039] (1) Obtaining of the K138C mutant: using the nucleotide sequence shown in SEQ ID NO: 4 as a template, F-primer 1 (sequence shown in SEQ ID NO: 5), R-primer 1 (sequence shown in SEQ ID NO Shown in: 6) is primer, carry out PCR and obtain the recombinant gene shown in SEQID NO: 3.

[0040] (2) The recombinant gene and pXMJ-19 were digested with BamH I and Ecol I, respectively, and ligated with T4 DNA ligase overnight at 16°C after purification. The sequencing work was completed by Shanghai Sangong.

Embodiment 2

[0042] Construction of recombinant Corynebacterium glutamicum engineering bacteria producing sucrose phosphorylase mutant

[0043] The recombinant plasmid pXMJ-19-K138C obtained in Example 1 was chemically transformed into E.coli competent cells, the specific method is as follows:

[0044] The solution required for the conversion experiment is as follows (g / L):

[0045] LB medium: yeast extract 5, peptone 10, NaCl 10.

[0046] 50% glycerol, 0.1M CaCl, 115°C moist heat sterilization.

[0047] (1) Inoculate Escherichia coli JM109 or Escherichia coli BL21(DE3) in 50 mL of fresh LB culture medium and culture overnight at 37°C and 220r / min.

[0048] (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.

[0049] (3) Start to detect the OD of the culture medium with a spectrophotometer after culturing for 1 h 600 value, detected every 20 minutes or so until 0D 600 When the value reaches 0.6 (about 2h).

[0050] (4...

Embodiment 3

[0057] High expression and activity determination of sucrose phosphorylase from recombinant strain C.gATCC 13032 / pXMJ-19-K138C

[0058] The recombinant bacteria C.gATCC 13032 / pXMJ-19-K138C constructed in Example 2 and the original strain C.gATCC 13032 / pXMJ-19-SP expressing the unmutated enzyme were inoculated in 10 mL of BHI medium containing chloramphenicol , shake culture at 30°C for 16-20h, transfer to the medium to induce expression according to 1% inoculum amount the next day, culture at 30°C for 14h, take the culture solution and centrifuge at 4°C, 10000r / min for 10min, and the supernatant of cell disruption is Intracellular crude enzyme solution, and then purified by Ni column to obtain pure enzyme solution for the determination of enzyme activity. To explore the enzymatic properties of the pure enzyme, the optimum reaction temperature of the mutant strain of sucrose phosphorylase is 35°C, and the optimum reaction pH is 7.0.

[0059] The results showed that the specifi...

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Abstract

The invention relates to a sucrose phosphorylase mutant with improved enzyme activity and its construction method and application, belonging to the technical field of genetic engineering. The amino acid sequence of the mutant of the present invention is shown in SEQ ID NO:1. The mutant of the present invention is based on the sucrose phosphorylase derived from Leuconostoc enterococcus, and carries out site-directed mutation to realize the improvement of the sucrose phosphorylase enzyme activity, and expresses the mutant in Corynebacterium glutamicum, and uses As a whole-cell catalyst to produce 2-O-α-D-glycerol glucoside, at the level of a 5L fermenter, it can efficiently produce a large amount of 2-O-α-D-glycerol glucoside in a short period of time, which is conducive to expanding the phosphorylation of sucrose Enzyme production of 2-O-α-D-glycerol glucoside has industrial application prospect and realizes large-scale industrial application.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a sucrose phosphorylase mutant with improved enzyme activity and its construction method and application. Background technique [0002] Sucrose phosphorylase (EC2.4.1.7, Sucrose Phosphorylase, Spase) is a specific enzyme that catalyzes the transfer of glucosidic bonds. It mainly catalyzes two types of reactions: one is to transfer the glucose group in 1-phosphate glucose to the acceptor, such as D-fructose as the acceptor, sucrose can be generated under the catalysis of this enzyme; the other is to transfer the glucose group in sucrose Transfer to receptors, including inorganic phosphoric acid, water, substances containing phenolic hydroxyl groups, alcoholic hydroxyl groups and carboxyl groups, such as phosphoric acid as receptors, can generate 1-phosphate glucose and D-fructose. [0003] Using this catalytic property, sucrose phosphorylase can use fructose, xylose, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/77C12N1/21C12R1/15
CPCC12N9/1051C12Y204/01007C12N15/77C12P19/44C12N1/20C12N15/63
Inventor 饶志明段培枫张佳宁张显杨套伟徐美娟邵明龙刘雨王紫薇陈妍陈奇
Owner JIANGNAN UNIV
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