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Enhancement of proline-4-hydroxylase activity by site-saturation mutagenesis
Author(s): 
Pages: 193-198
Year: Issue:  4
Journal: Bulletin of Fermentation Science and Technology

Keyword:  L-proline-4-hydroxylasetrans-4-hydroxy-L-prolinebiocatalysissite-saturation mutagenesis;
Abstract: To increase L-proline-4-hydroxylase(P4 H)activity for biosynthesis of trans-4-hydroxy-L-proline(4-Hyp),recombinant P4 H constructed previously in our laboratory was utilized for protein engineering.Through homology modeling and analysis of"hotspot"amino acids,7 residues near"hydrophobic pocket"including Y205,K207,I241,F137,T142,V53 and R45 were selected for site-saturation mutagenesis.Two strains harboring mutant P4 HF137 Rand Y205 K with improved activity were obtained.Single mutation of F137 Rand Y205 Kin P4 H resulted in titers of 55.09 mg/L and 52.29 mg/L 4-Hyp at the substrate concentration of 0.35 g/L,which was 58% and 50% higher than that of wild-type P4 H.The results indicated that single mutation of F137 Ror Y205 Kcould increase its catalytic activity due to the increased affinity between P4 H and substrate or co-substrate through slight configuration change.Molecular docking indicated that the active center of mutant P4 H was not altered.The results provided important theoretical foundation for further investigation of 4-Hyp biosynthesis with P4 H.
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