Characterization and mutagenesis of a novel Mycobacterium smegmatis-derived glutamate decarboxylase active at neutral pH.
World J Microbiol Biotechnol
; 38(5): 75, 2022 Mar 17.
Article
en En
| MEDLINE
| ID: mdl-35298707
γ-aminobutyric acid (GABA) has various physiological functions and is widely used in medicine, food, and other fields. Glutamate decarboxylase (GAD) is a key enzyme that catalyzes the decarboxylation of L-glutamate to synthesize GABA. However, the industrial application of microorganism-derived GAD is limited by its rapid loss of enzymatic activity with pH approaching neutrality. In this study, a novel glutamate decarboxylase, GADMSM, from Mycobacterium smegmatis was overexpressed and purified. On the basis of homologous modeling and substrate molecular docking, several GADMSM mutants were constructed, and their enzymatic properties were analyzed. The results showed that the optimal pH of wild-type GADMSM is 5.4; at pH 6.2, 22.8% enzymatic activity was retained. The T211I replacement in GAD and C-terminal deletion mutant GADMSMΔC showed relatively high catalytic activity in a pH range of 5.0-7.0. The Vmax and Km values of GADMSMΔC were 14.69 and 5.70, respectively, at pH 5.5, and 9.87 and 6.17, respectively, at pH 7.0. Compared with the wild-type GAD, GADMSMΔC maintained higher affinity and enzymatic activity of the substrate, maintaining 78.5% of the highest enzymatic activity even at pH 7.0, which is the highest reported activity retention for GAD under neutral pH condition. Therefore, GADMSMΔC can be used for the transformation of high-yielding strains and industrial production of GABA.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Mycobacterium smegmatis
/
Glutamato Descarboxilasa
Idioma:
En
Revista:
World J Microbiol Biotechnol
Año:
2022
Tipo del documento:
Article
País de afiliación:
China
Pais de publicación:
Alemania