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Asparaginase mutant with improved enzyme activity

An asparaginase and asparagine technology, applied in the field of enzyme engineering, can solve the problem of low yield and the like, and achieve the effects of increasing yield, reducing production cost and improving catalytic efficiency

Inactive Publication Date: 2017-01-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Current research shows that genetic engineering is the main method to increase the production of asparaginase. Asparaginase from different sources has been expressed in Escherichia coli, Bacillus subtilis, yeast and other hosts, but the production is still not high

Method used

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  • Asparaginase mutant with improved enzyme activity
  • Asparaginase mutant with improved enzyme activity
  • Asparaginase mutant with improved enzyme activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Simulation of crystal structure of asparaginase derived from Bacillus subtilis

[0031] Using the reported Erwinia chrysanthemi asparaginase (PDB code: 1hg0) as a template (Structural basis for the activity and substrate specificity of Erwinia chrysanthemi L-Asparaginase, published in 2001) (the amino acid similarity between the two is 58.1%), using online simulation The software SWISS-MODEL simulates the crystal structure of asparaginase derived from Bacillus subtilis.

Embodiment 2

[0032] Embodiment 2 site-directed mutation E29 asparaginase strain construction

[0033] The designed upstream and downstream primers P1 and P2 (as shown in Table 1) were used to carry out PCR using the pP43H-D30 plasmid as a template to construct an E29 saturation mutant strain. The PCR conditions are: 98°C for 3min, 98°C for 30S, 55°C for 90S, 72°C for 8min, 34 cycles. PCR amplification system: template 1 μL, upstream and downstream primers 1 μL, dNTP Mix 4 μL, 5×primeSTARBuffer 10 μL, sterilized double distilled water 32.5 μL, prime STAR DNA polymerase 0.5 μL. The gel recovery kit was used to purify and recover the PCR product, and the concentration of the recovered product was checked by electrophoresis. DpnI digested and processed PCR recovered product, transformed it into competent E.coil JM109, and picked positive colony with ampicillin LB plate. After culturing on a shaker overnight at 37°C, the plasmid was extracted and named pP43H-D30 / E29X, and then transformed int...

Embodiment 3

[0034] Example 3 Verification of High Secretion Ability Asparaginase Production Strain

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Abstract

The invention discloses an asparaginase mutant with improved enzyme activity, and belongs to the technical field of enzyme engineering. Asparaginase shown as SEQ ID NO.1 is subjected to site-saturation mutagenesis, amino acid residues near protein molecule activity sites are changed, asparaginase catalysis efficiency is improved, and asparaginase yield is further improved. Enzyme activity of the asparaginase can be improved by 2.37 times as compared with that of original strains by recombinant bacillus subtilis with enhanced asparaginase secretion capacity. The enzyme producing ability of modified genetically engineered bacteria is remarkably improved, the enzyme activity of the asparaginase fermented by a shake flask reaches 320U / mL, the yield is the currently reported highest yield of the shake flask, the asparaginase mutant is more suitable for industrial application, production cost can be reduced, and production efficiency is improved.

Description

technical field [0001] The invention relates to an asparaginase mutant with improved enzyme activity, which belongs to the technical field of enzyme engineering. Background technique [0002] L-asparaginase (EC 3.5.1.1) is a protease with anticancer activity, which can specifically catalyze the hydrolysis of L-asparagine into aspartic acid and NH 3 . The physiological effect of L-asparaginase is mainly manifested as the inhibitory effect on some tumors, especially effective on acute leukemia and malignant lymphoma. L-asparaginase has become a very effective drug for treating leukemia and has no inhibitory effect on bone marrow cells. [0003] L-asparaginase can reduce the formation of acrylamide in food. Acrylamide is mainly produced by the Maillard reaction of reducing sugar and asparagine in food raw materials during high-temperature heating. Adding asparaginase to food can hydrolyze asparagine and reduce the formation of acrylamide from the source. [0004] Some micro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/82C12N15/55C12N1/21C12R1/125
CPCC12N9/82C12Y305/01001
Inventor 刘松冯岳蕉蕴陈坚堵国成王云龙
Owner JIANGNAN UNIV
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