Improvement of thermostability of beta-1,3-1,4-glucanase from Bacillus amyloliquefaciens BS5582 through in vitro evolution / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 1293-1301, 2010.
Artigo
em Chinês
| WPRIM
| ID: wpr-351593
ABSTRACT
In vitro evolution methods are often used to modify protein with improved characteristics. We developed a directed evolution protocol to enhance the thermostability of the beta-1,3-1,4-glucanase. The thermostability of the enzyme was significantly improved after two rounds of directed evolution. Three variants with higher thermostability were obtained. The mutant enzymes were further analyzed by their melting temperature, halftime and kinetic parameters. Comparing to intact enzyme, the T50 of mutant enzymes 2-JF-01, 2-JF-02 and 2-JF-03 were increased by 2.2 degrees C, 5.5 degrees C and 3.5 degrees C, respectively, the halftime (t1/2, 60 degrees C) of mutant enzymes 2-JF-01, 2-JF-02 and 2-JF-03 were shortened by 4,13 and 17 min, respectively, the V(max) of mutant enzymes were decreased by 8.3%, 2.6% and 10.6%, respectively, while K(m) of mutant enzymes were nearly unchanged. Sequence analysis revealed seven single amino acid mutant happened among three mutant enzymes, such as 2-JF-01 (N36S, G213R), 2-JF-02 (C86R, S115I, N150G) and 2-JF-03 (E156V, K105R). Homology-modeling showed that five of seven substituted amino acids were located on the surface of or in hole of protein. 42.8% of substituted amino acids were arginine, which indicated that arginine may play a role in the improvement of the thermostability of the beta-1,3-1,4-glucanase.This study provide some intresting results of the structural basis of the thermostability of beta-1,3-1,4-glucanase,and provide some new point of view in modifying enzyme for future industrial use.
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Bacillus
/
Estabilidade Enzimática
/
Proteínas Recombinantes de Fusão
/
Dados de Sequência Molecular
/
Química
/
Sequência de Aminoácidos
/
Evolução Molecular Direcionada
/
Endo-1,3(4)-beta-Glucanase
/
Genética
/
Temperatura Alta
Tipo de estudo:
Estudo prognóstico
Idioma:
Chinês
Revista:
Chinese Journal of Biotechnology
Ano de publicação:
2010
Tipo de documento:
Artigo
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