Design and characterization of new ß-glucuronidase active site variants with altered substrate specificity.
Biotechnol Lett
; 40(1): 111-118, 2018 Jan.
Article
em En
| MEDLINE
| ID: mdl-28980152
ABSTRACT
OBJECTIVE:
To isolate and characterize the kinetics of variants of E. coli ß-glucuronidase (GUS) having altered substrate specificity.RESULTS:
Two small combinatorial libraries of E. coli GUS variants were constructed and screened for improved activities towards the substrate p-nitrophenyl-ß-D-galactoside (pNP-gal). Nine of the most active variants were purified and their kinetic parameters were determined. These variants show up to 134-fold improved kcat/KM value towards pNP-gal compared to wild-type GUS, up to 9 × 108-fold shift in specificity from p-nitrophenyl-ß-D-glucuronide (pNP-glu) to pNP-gal compared to wild-type, and 103-fold increase in specificity shift compared to a previously evolved GUS variant.CONCLUSIONS:
The kinetic data collected for nine new GUS variants is invaluable for training computational protein design models that better predict amino acid substitutions which improve activity of enzyme variants having altered substrate specificity.Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Especificidade por Substrato
/
Domínio Catalítico
/
Escherichia coli
/
Proteínas Mutantes
/
Glucuronidase
Tipo de estudo:
Prognostic_studies
Idioma:
En
Ano de publicação:
2018
Tipo de documento:
Article