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

ABSTRACT: Improving the substrate affinity and catalytic efficiency of β-glucosidase is necessary for better performance in the enzymatic saccharification of cellulosic biomass because of its ability to prevent cellobiose inhibition on cellulases. Bgl3A from Talaromyces leycettanus JCM12802, identified in our previous work, was considered a suitable candidate enzyme for efficient cellulose saccharification with higher catalytic efficiency on the natural substrate cellobiose compared with other β-glucosidase but showed insufficient substrate affinity. In this work, hydrophobic stacking interaction and hydrogen-bonding networks in the active center of Bgl3A were analyzed and rationally designed to strengthen substrate binding. Three vital residues, Met36, Phe66, and Glu168, which were supposed to influence substrate binding by stabilizing adjacent binding site, were chosen for mutagenesis. The results indicated that strengthening the hydrophobic interaction between stacking aromatic residue and the substrate, and stabilizing the hydrogen-bonding networks in the binding pocket could contribute to the stabilized substrate combination. Four dominant mutants, M36E, M36N, F66Y, and E168Q with significantly lower K_m values and 1.4-2.3-fold catalytic efficiencies, were obtained. These findings may provide a valuable reference for the design of other β-glucosidases and even glycoside hydrolases.

SUBMITTER: Xia W

PROVIDER: S-EPMC8699594 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe development of sustainable technologies for plant cell wall degradation greatly depends on enzymes with hydrolytic activities against carbohydrates. The waste by-products of agricultural cereals are important biomass sources because they contain large amounts of saccharides. Achieving efficient debranching and depolymerization are two important objectives for increasing the utilization of such renewable bioresources. GH51 α-L-arabinofuranosidases are important in biomass pretreatment because they act synergistically with other enzymes during hemicellulose hydrolysis.ResultsA GH51 α-L-arabinofuranosidase from Talaromyces leycettanus JCM12802 was heterologously expressed in Pichia pastoris GS115 and characterized. The recombinant α-L-arabinofuranosidase, TlAbf51, showed an optimum temperature and pH of 55-60 °C and 3.5-4.0, respectively, and remained stable at 50 °C and pH 3.0-9.0. TlAbf51 showed a higher catalytic efficiency (5712 mM-1 s-1) than most fungal α-L-arabinofuranosidases towards the substrate 4-nitrophenyl-α-L-arabinofuranoside. Moreover, TlAbf51 preferentially removed 1,2- or 1,3-linked arabinofuranose residues from arabinoxylan and acted synergistically with the bifunctional xylanase/cellulase TcXyn10A at an activity ratio of 5:1. The highest yields of arabinose and xylooligosaccharides were obtained when TlAbf51 was added after TcXyn10A or when both enzymes were added simultaneously. High-performance anion-exchange chromatography analyses showed that (i) arabinose and xylooligosaccharides with low degrees of polymerization (DP1-DP5) and (ii) arabinose and xylooligosaccharides (DP1-DP3) were the major hydrolysates obtained during the hydrolysis of sodium hydroxide-pretreated cornstalk and corn bran, respectively.ConclusionsIn contrast to other fungal GH51 α-L-arabinofuranosidases, recombinant TlAbf51 showed excellent stability over a broad pH range and high catalytic efficiency. Moreover, TlAbf51 acted synergistically with another hemicellulase to digest arabino-polysaccharides. These favorable enzymatic properties make TlAbf51 attractive for biomass pretreatment and biofuel production.

Dataset Information

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

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