title: Heterologous expression in Pichia pastoris and characterization of an endogenous thermostable and high-glucose-tolerant β-glucosidase from the termite Nasutitermes takasagoensis.
abstract: Termites are well-known cellulose decomposers and can give researchers insights into how to utilize lignocellulosic biomass in the actual scenario of energy consumption. In this work, an endogenous β-glucosidase from the midgut of the higher termite Nasutitermes takasagoensis was purified to homogeneity by Ni(2+) affinity chromatography and its properties were characterized. This β-glucosidase (G1mgNtBG1), which belongs to glycoside hydrolase family 1, is a homotrimer in its native form, with a molecular mass of 169.5 kDa, as demonstrated by gel filtration chromatography. The enzyme displayed maximum activity at pH 5.5 and had broad substrate specificities toward several saccharides, including cellobiose. G1mgNtBG1 showed a relatively high temperature optimum of 65°C and one of the highest levels of glucose tolerance among several β-glucosidases already characterized, with a K(i) of 600 mM glucose. To examine the applicability of G1mgNtBG1 in biomass conversion, we compared the thermostability and glucose tolerance of G1mgNtBG1 with those of Novozym 188. We found that G1mgNtBG1 was more thermostable after 5 h of incubation at 60°C and more resistant to glucose inhibition than Novozym 188. Furthermore, our result suggests that G1mgNtBG1 acts synergistically with Celluclast 1.5 L in releasing reducing sugars from Avicel. Thus, G1mgNtBG1 seems to be a potential candidate for use as a supplement in the hydrolysis of biomass.
authors.affiliation: Department of Biotechnology, The University of Tokyo, Tokyo, Japan.
authors.forename: Cristiane Akemi
authors.initials: CA
authors.lastname: Uchima
authors.forename: Gaku
authors.initials: G
authors.lastname: Tokuda
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Watanabe
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Kitamoto
authors.forename: Manabu
authors.initials: M
authors.lastname: Arioka
mesh_terms: D000818:Animals; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004795:Enzyme Stability; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D020049:Isoptera / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics*; D055503:Protein Multimerization; D013379:Substrate Specificity; D013696:Temperature; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D001617:beta-Glucosidase