A novel glucose-tolerant β-glucosidase from the salivary gland of the termite Nasutitermes takasagoensis

Abstract

Lignocellulose, a major constituent of plant biomass, is a potential feedstock for bioconversion. However, one of the main bottlenecks to produce bioethanol is the high cost of enzymes used for hydrolysis of insoluble substrates to the fermentable sugar glucose. Hence, research on cellulases that enable efficient hydrolysis of plant biomass is a key point. Among cellulases, β-glucosidase (EC 3.2.1.21) acts in the final step of cellulose saccharification, liberating glucose. Moreover, it reduces the inhibition of endoglucanase (EC 3.2.1.4) and cellobiohydrolase (EC 3.2.1.91) through hydrolysis of cellobiose and cello-oligosaccharides (Bhatia et al., 2002; Lynd et al., 2002). β-Glucosidase itself is also subjected to the product inhibition which makes the identification of β-glucosidases insensitive to or stimulated by glucose of special interest in bioconversion (Perezpons et al., 1995; Uchima et al., 2011, 2012). Interestingly, in nature, termites are insects with a great capacity/facility to degrade wood; 74‒99% of the cellulose ingested is hydrolyzed (Prins and Kreulen, 1991). The high efficiency of termites in the degradation of plant biomass motivated the development of the present work. The aim of this study is to analyze some features of an endogenous β-glucosidase from the salivary gland of the higher termite Nasutitermes takasagoensis, hereafter called G1sgNtBG1 (glycoside hydrolase family 1), that might be useful in bioethanol production. N. takasagoensis cDNA was used as a donor of the target gene encoding G1sgNtBG1 (GenBank accession no. AB508954). Escherichia coli DH5α was used for DNA manipulations, and Pichia pastoris strain KM71 (his4 aox1::ARG4, Invitrogen) was used as a host for heterologous expression of G1sgNtBG1. The episomal plasmid pBGP3 (Uchima and Arioka, 2012) was employed for the expression of mycand hexahistidine-tagged G1sgNtBG1 in P. pastoris; electroporation of the expression plasmid into P. pastoris was done according to the standard method (Cereghino and Cregg, 2000). Purification of G1sgNtBG1 was performed using a Ni2+-NTA Purification System following the manufacturer’s instructions (Qiagen). β-Glucosidase activity was routinely assayed using 10 μM of p-nitrophenyl-β-D-glucopyranoside (pNPG; Sigma, St. Louis, USA) as a substrate according to the method previously described (Uchima et al., 2011) and the release of p-nitrophenol (pNP) was measured at A410. G1sgNtBG1 activity was also determined by the release of glucose from saccharides and cello-oligosaccharides using glucose oxidase-mutarotase reagent (Glucose CII Test Wako; Wako Pure Chemical Co., Tokyo, Japan) according to the method previously deJ. Gen. Appl. Microbiol., 59, 141‒145 (2013)