Substrate-Binding Mode and Intermediate-Product Distribution Coguided Protein Design of Alginate Lyase AlyF for Altered End-Product Distribution.
J Agric Food Chem
; 2021 Jun 16.
Article
en En
| MEDLINE
| ID: mdl-34133153
ABSTRACT
Recently, we reported alginate lyase AlyF that predominantly produced trisaccharides (the trisaccharide content is 87.0%), and the determination of its substrate-binding mode facilitated its protein engineering for new product distribution. To clarify the relationship between the substrate-binding pocket and end-product distribution, the open binding pocket change was initially designed. The resulting F128T_W172R mutant of AlyF exhibited different intermediate-product distributions but still similar end-product distributions. However, these observations suggested that cleavage pattern changes for intermediate products might contribute to an altered end-product distribution. Structural analysis indicated that the sugar-binding affinity at subsite -2 should be redesigned to achieve this goal. Thus, residue Arg266, which is involved in sugar binding at subsite -2, was selected for site-saturation mutagenesis in the F128T_W172R mutant. The dominant end products of the F128T_W172R_R226H mutant were altered to disaccharides and trisaccharides (the disaccharide content increased to 40.5%).
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Colección:
01-internacional
Base de datos:
MEDLINE
Idioma:
En
Revista:
J Agric Food Chem
Año:
2021
Tipo del documento:
Article
País de afiliación:
China