Thermophilic L-asparaginase mutant and screening and fermentation methods thereof
US11001825B2 · kind B2 · utility
Assignee
Inventors
Key dates
| Filing date | Jan 24, 2019 |
| Grant date | May 11, 2021 |
| Priority date | — |
| Expiry date | May 10, 2039 |
Classification
- Technology area (CPC C)Chemistry; Metallurgy
- CPC primaryC12Y305/01001
- WIPO fieldBiotechnology
- WIPO sectorChemistry
Abstract
The present disclosure discloses a thermophilic L-asparaginase mutant and screening and fermentation methods thereof, and belongs to the field of gene engineering, enzyme engineering and fermentation engineering. In Bacillus subtilis 168, a Pyrococcus yayanosii CH1-derived L-asparaginase encoding gene is used as a template, and a mutation library is constructed by an error-prone PCR (epPCR) technology. A mutant strain with improved specific enzyme activity is screened through a high-flux screening method of synchronous cell disruption and enzyme activity measurement. Mutated residues included in a positive mutant are analyzed to construct a composite mutant strain S17G/A90S/R156S/K272A with improved specific enzyme activity and specific enzyme activity of 3108 U/mg. An expression quantity of the composite mutant strain in the Bacillus subtilis 168 is increased through measures of a strong promoter P43 and RBS optimization. Finally, the Bacillus subtilis 168 with a gene of the L-asparaginase composite mutant strain is subjected to enzyme production fermentation in a 5 L fermentation tank through culture medium optimization and pH and feeding coupling strategies. The enzyme activity …
Source: USPTO / EPO open patent data. Objective bibliographic and citation counts.