Site-directed Mutagenesis of a β-Glycoside Hydrolase from Lentinula edodes

Abstract

The β-glycoside hydrolases (LXYL-P1−1 and LXYL-P1−2) from Lentinula edodes (strain M95.33) can specifically hydrolyze 7-β-xylosyl-10-deacetyltaxol (XDT) to form 10-deacetyltaxol for the semi-synthesis of Taxol. Our previous study showed that both the I368T mutation in LXYL-P1−1 and the T368E mutation in LXYL-P1−2 could increase the enzyme activity, which prompted us to investigate the effect of the I368E mutation on LXYL-P1−1 activity. In this study, the β-xylosidase and β-glucosidase activities of LXYL-P1−1I368E were 1.5 and 2.2 times higher than those of LXYL-P1−1. Most importantly, combination of I368E and V91S exerted the cumulative effects on the improvement of the enzyme activities and catalytic efficiency. The β-xylosidase and β-glucosidase activities of the double mutant LXYL-P1−1V91S/I368E were 3.2 and 1.7-fold higher than those of LXYL-P1−1I368E. Similarly, the catalytic efficiency of LXYL-P1−1V91S/I368E on 7-β-xylosyl-10-deacetyltaxol was 1.8-fold higher than that of LXYL-P1−1I368E due to the dramatic increase in the substrate affinity. Molecular docking results suggest that the V91S and I368E mutation might positively promote the interaction between enzyme and substrate through altering the loop conformation near XDT and increasing the hydrogen bonds among Ser91, Trp301, and XDT. This study lays the foundation for exploring the relationship between the structure and function of the β-glycoside hydrolases.

Forty years of directed evolution and its continuously evolving technology toolbox: A review of the patent landscape

“…It is founded on the principle of inaccurate amplification of genes. To achieve the higher error rate of Taq polymerase, several epPCR protocols have been developed, such as (i) methods that cut the DNA polymerase fidelity through unbalancing dNTPs concentrations, increasing the MgCl 2 concentration, and/or addition of MnCl 2 ; (ii) methods that employ nucleoside triphosphate analogs; (iii) methods that utilize “mutagenic” polymerases; (iv) alcohol‐mediated epPCR; and (v) combined methods and kits from Clontech and Stratagene (Chen et al, 2019; Cline & Hogrefe, 2000; Copp et al, 2014; Frendorf et al, 2019; Lin‐Goerke et al, 1997; Xu et al, 1999; Yao et al, 2019; Zaccolo et al, 1996).…”

“…(iv) alcohol-mediated epPCR; and (v) combined methods and kits from Clontech and Stratagene (Chen et al, 2019;Cline & Hogrefe, 2000;Copp et al, 2014;Frendorf et al, 2019;Lin-Goerke et al, 1997;Xu et al, 1999;Yao et al, 2019;Zaccolo et al, 1996).…”

“…Amongst the conventional focused mutagenesis techniques, site‐directed mutagenesis (SDM) is the most useful method being capable of introducing specific and targeted changes (insertions, deletions, and substitutions) in a double‐stranded plasmid DNA (Chen et al, 2019). Use of synthetic oligonucleotides (primers) to introduce a single base‐pair substitution at a specified position in a gene is a common strategy in all SDM protocols (Cobb et al, 2013; Liu & Naismith, 2008).…”

Mutational Analysis of Ocriplasmin to Reduce Proteolytic and Autolytic Activity in Pichia pastoris

“…The random types of these approaches include random mutagenesis and evolutionary techniques such as DNA shuffling. The rational methods include site-directed mutagenesis [ 22 , 23 ] as an effective yet simple method that introduces special amino acids into target genes using 2 oligonucleotide primers with the desired mutations that complement the opposite strands of a double-stranded DNA template [ 24 – 26 ].…”

Mutational analysis of ocriplasmin to reduce proteolytic and autolytic activity in Pichia pastoris

“…The random types of these approaches include random mutagenesis and evolutionary techniques such as DNA shu ing. The rational methods include site-directed mutagenesis (23,24) as an effective yet simple method that introduces special amino acids into target genes using 2 oligonucleotide primers with the desired mutations that complement the opposite strands of a double-stranded DNA template (25)(26)(27).…”