Title:
THERMOPHILIC L-ASPARAGINASE MUTANT AND SCREENING AND FERMENTATION METHOD THEREFOR
Document Type and Number:
WIPO Patent Application WO/2019/113965
Kind Code:
A1
Abstract:
Disclosed in the present invention are a thermophilic L-asparaginase mutant and a screening and fermentation method therefor, relating to the field of genetic engineering, enzyme engineering and fermentation engineering. In Bacillus subtilis 168, an L-asparaginase-encoding gene derived from thermophilus Pyrococcus yayanosii CH1 is used as a template to construct a mutant library by means of error-prone PCR technology, and mutants having increased enzyme activities are screened by means of a high-throughput screening method of "synchronized cell disruption and enzyme activity assay"; mutant residues contained in the obtained mutants are analyzed to construct a composite mutant S17G/A90S/R156S/K272A having increased enzyme activities; the expression level of the composite mutant in Bacillus subtilis 168 is increased by means of strong promoter P43 and RBS optimization; and finally, the fermentation yield of the L-asparaginase mutant is increased by means of a coupled strategy of optimization of medium and pH feed.
Inventors:
RAO ZHIMING (CN)
LI XU (CN)
ZHANG XIAN (CN)
XU SHUQIN (CN)
HU JINGYI (CN)
XU MEIJUAN (CN)
YANG TAOWEI (CN)
LI XU (CN)
ZHANG XIAN (CN)
XU SHUQIN (CN)
HU JINGYI (CN)
XU MEIJUAN (CN)
YANG TAOWEI (CN)
Application Number:
PCT/CN2017/116568
Publication Date:
June 20, 2019
Filing Date:
December 15, 2017
Export Citation:
Assignee:
UNIV JIANGNAN (CN)
International Classes:
C12N9/82; C12N15/55; C12N15/70; C12N15/75; C12N15/81
Domestic Patent References:
| WO2014170811A2 | 2014-10-23 |
Foreign References:
| CN104146270A | 2014-11-19 | |||
| CN104371993A | 2015-02-25 | |||
| CN105062997A | 2015-11-18 |
Attorney, Agent or Firm:
HARBIN SHINEIP INTELLECTUAL PROPERTY AGENCY CO., LTD. (CN)
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