An amino acid at position 512 in β-glucosidase from Clavibacter michiganensis determines the regioselectivity for hydrolyzing gypenoside XVII

Abstract

A recombinant β-glucosidase from Clavibacter michiganensis specifically hydrolyzed the outer and inner glucose linked to the C-3 position in protopanaxadiol (PPD)-type ginsenosides and the C-6 position in protopanaxatriol (PPT)-type ginsenosides except for the hydrolysis of gypenoside LXXV (GypLXXV). The enzyme converted gypenoside XVII (GypXVII) to GypLXXV by hydrolyzing the inner glucose linked to the C-3 position. The substrate-binding residues obtained from the GypXVII-docked homology models of β-glucosidase from C. michiganensis were replaced with alanine, and the amino acid residue at position 512 was selected because of the changed regioselectivity of W512A. Site-directed mutagenesis for the amino acid residue at position 512 was performed. W512A and W512K hydrolyzed the inner glucose linked to the C-3 position and the outer glucose linked to the C-20 position of GypXVII to produce GypLXXV and F2. W512R hydrolyzed only the outer glucose linked to the C-20 position of GypXVII to produce F2. However, W512E and W512D exhibited no activity for GypXVII. Thus, the amino acid at position 512 is a critical residue to determine the regioselectivity for the hydrolysis of GypXVII. These wild-type and variant enzymes produced diverse ginsenosides, including GypXVII, GypLXXV, F2, and compound K, from ginsenoside Rb1. To the best of our knowledge, this is the first report of the alteration of regioselectivity on ginsenoside hydrolysis by protein engineering.

Combinatorial Enzymatic Catalysis for Bioproduction of Ginsenoside Compound K

“…It is thought-provoking that the extended spatial conformation of the substrates prevents its entry into the active pocket. Shin et al performed targeted mutagenesis of amino acid residues at position 512 of β-glucosidase from Clavibacter michiganensis, which broadened the regioselectivity of the mutant and gave the mutant enzyme the ability to convert Rb 1 into CK (which enzyme could not do before) . The yield of CK was enhanced from 306 to 843 mg L –1 h –1 by the semirational design of β-glycosidase derived from S.…”

“…Shin et al performed targeted mutagenesis of amino acid residues at position 512 of β-glucosidase from Clavibacter michiganensis, which broadened the regioselectivity of the mutant and gave the mutant enzyme the ability to convert Rb 1 into CK (which enzyme could not do before). 47 The yield of CK was enhanced from 306 to 843 mg L −1 h −1 by the semirational design of β-glycosidase derived from S. solfataricus. 48 In the future, these substrate-binding residues can be engineered by protein engineering to optimize the catalytic properties and physicochemical functions of proteins and the resulting improvement in BG23 performance will make the proposed technology closer to industrial application.…”

Combinatorial enzymatic catalysis for bioproduction of ginsenoside Compound K

“…To some extent, the occurrence of catalysis was limited. Shin et al performed targeted mutagenesis of amino acid residues at position 512 of β-glucosidase from Clavibacter michiganensis, which broadened the regioselectivity of the mutant and gave the mutant enzyme the ability to convert Rb 1 into CK (which it could not do before) 51 . The yield of CK was enhanced from 306 mg•L − 1 •h − 1 to 843 mg•L − 1 •h − 1 by the semirational design of β-glycosidase derived from S. solfataricus 50 .…”

Improving the Substrate Affinity and Catalytic Efficiency of β-Glucosidase Bgl3A from Talaromyces leycettanus JCM12802 by Rational Design

“…The Q201E mutant of AaBGL1, generated by site-saturation mutagenesis, was found to have 2.7-times higher k cat / K m toward cellobiose than the WT enzyme [ 21 ]. Substitution of Trp512 in β-glucosidase from Clavibacter michiganensis has been shown to transform the regioselectivity for hydrolyzing gypenoside XVII [ 22 ]. Furthermore, the catalytic efficiency in quercetin-4’-glucoside hydrolysis of Thermotoga maritima β-glucosidase A was enhanced by site-directed mutagenesis [ 23 ].…”