Molecular modification and highly efficient expression of L-asparaginase from Rhizomucor miehei / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 3242-3252, 2021.
Artigo
em Chinês
| WPRIM
| ID: wpr-921421
ABSTRACT
L-asparaginase hydrolyzes L-asparagine to produce L-aspartic acid and ammonia. It is widely distributed in microorganisms, plants and serum of some rodents, and has important applications in the pharmaceutical and food industries. However, the poor thermal stability, low catalytic efficiency and low yield hampered the further application of L-asparaginase. In this paper, rational design and 5' untranslated region (5'UTR) design strategies were used to increase the specific enzyme activity and protein expression of L-asparaginase derived from Rhizomucor miehei (RmAsnase). The results showed that among the six mutants constructed through homology modeling combined with sequence alignment, the specific enzyme activity of the mutant A344E was 1.5 times higher than the wild type. Subsequently, a food-safe strain Bacillus subtilis 168/pMA5-A344E was constructed, and the UTR strategy was used for the construction of recombinant strain B. subtilis 168/pMA5 UTR-A344E. The enzyme activity of B. subtilis 168/pMA5 UTR-A344E was 7.2 times higher than that of B. subtilis 168/pMA5-A344E. The recombinant strain B. subtilis 168/pMA5 UTR-A344E was scaled up in 5 L fermenter, and the final yield of L-asparaginase was 489.1 U/mL, showing great potential for industrial application.
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Asparaginase
/
Bacillus subtilis
/
Microbiologia Industrial
/
Engenharia de Proteínas
/
Alinhamento de Sequência
/
Rhizomucor
Idioma:
Chinês
Revista:
Chinese Journal of Biotechnology
Ano de publicação:
2021
Tipo de documento:
Artigo
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