Sucrose hosphorylase mutant with improved enzyme activity and construction method and application of sucrose phosphorylase mutant

A technology for phosphorylase mutants and construction methods, which is applied in the field of sucrose phosphorylase mutants and their construction, can solve the problems of limited application and low enzyme activity, achieve the effect of improving industrial application potential and realizing large-scale industrial application

Active Publication Date: 2020-01-31
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 th

Method used

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  • Sucrose hosphorylase mutant with improved enzyme activity and construction method and application of sucrose phosphorylase mutant

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 a construction method and application of the sucrose phosphorylase mutant, and belongs to the technical fieldof genetic engineering. The amino acid sequence of the mutant is shown in SEQ ID NO: 1. Based on sucrose phosphorylase which is derived from Leuconostoc mesenteroides, site-directed mutagenesis is performed on the mutant so as to improve the enzyme activity of sucrose phosphorylase, the mutant is expressed in Corynebacterium glutamicum, and is used as a whole-cell catalyst for production of 2-O-alpha-D-glycosylglycerol, and a large amount of 2-O-alpha-D-glycosylglycerol can be produced efficiently in a short period of time at the level of a fermenter of 5 L, so that industrial application prospects for 2-O-alpha-D-glycosylglycerol production from sucrose phosphorylase is expanded, and large-scale industrial application is achieved.

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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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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