Streptococcus agalactiae amylomaltase offers insight into the transglycosylation mechanism and the molecular basis of thermostability among amylomaltases.
Sci Rep
; 11(1): 6740, 2021 03 24.
Article
en En
| MEDLINE
| ID: mdl-33762620
ABSTRACT
Amylomaltase (AM) catalyzes transglycosylation of starch to form linear or cyclic oligosaccharides with potential applications in biotechnology and industry. In the present work, a novel AM from the mesophilic bacterium Streptococcus agalactiae (SaAM), with 18-49% sequence identity to previously reported AMs, was characterized. Cyclization and disproportionation activities were observed with the optimum temperature of 30 °C and 40 °C, respectively. Structural determination of SaAM, the first crystal structure of small AMs from the mesophiles, revealed a glycosyl-enzyme intermediate derived from acarbose and a second acarbose molecule attacking the intermediate. This pre-transglycosylation conformation has never been before observed in AMs. Structural analysis suggests that thermostability in AMs might be mainly caused by an increase in salt bridges since SaAM has a lower number of salt bridges compared with AMs from the thermophiles. Increase in thermostability by mutation was performed. C446 was substituted with A/S/P. C446A showed higher activities and higher kcat/Km values for starch in comparison to the WT enzyme. C446S exhibited a 5 °C increase in optimum temperature and the threefold increase in half-life time at 45 °C, most likely resulting from H-bonding interactions. For all enzymes, the main large-ring cyclodextrin (LR-CD) products were CD24-CD26 with CD22 as the smallest. C446S produced more CD35-CD42, especially at a longer incubation time.
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Streptococcus agalactiae
/
Sistema de la Enzima Desramificadora del Glucógeno
Idioma:
En
Revista:
Sci Rep
Año:
2021
Tipo del documento:
Article
País de afiliación:
Tailandia