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.
Artículo
en Chino
| 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:
Disponible
Índice:
WPRIM (Pacífico Occidental)
Asunto principal:
Bacillus
/
Estabilidad de Enzimas
/
Proteínas Recombinantes de Fusión
/
Datos de Secuencia Molecular
/
Química
/
Secuencia de Aminoácidos
/
Evolución Molecular Dirigida
/
Endo-1,3(4)-beta-Glucanasa
/
Genética
/
Calor
Tipo de estudio:
Estudio pronóstico
Idioma:
Chino
Revista:
Chinese Journal of Biotechnology
Año:
2010
Tipo del documento:
Artículo
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