Heterologous expression and biochemical characterization of a GHF9 endoglucanase from the termite Reticulitermes speratus in Pichia pastoris

Pengfei Zhang,Yuguang Du,Xianghua Yuan,Jian-Jun Li

doi:10.1186/s12896-018-0432-3

Pengfei Zhang, Yuguang Du + Show 2 more

Open Access

https://doi.org/10.1186/s12896-018-0432-3

Copy DOI

Abstract

BackgroundCellulases are of great significance for full utilization of lignocellulosic biomass. Termites have an efficient ability to degrade cellulose. Heterologous production of the termite-origin cellulases is the first step to realize their industrial applications. The use of P. pastoris for the expression of recombinant proteins has become popular. The endoglucanase from Reticulitermes speratus (RsEG), belonging to glycoside hydrolase family 9 (GHF9), has not been produced in P. pastoris yet.ResultsA mutant RsEGm (G91A/Y97W/K429A) was successfully overexpressed in P. pastoris. RsEGm, with optimum pH 5.0, was active over the pH range of 4.0 to 9.0, and exhibited superior pH stability over between pH 4.0 and pH 11.0. It displayed the highest activity and good stability at 40 °C, but lost activity quickly at 50 °C. The apparent kinetic parameters of RsEGm against Carboxymethyl Cellulose (CMC) were determined, with Km and Vmax of 7.6 mg/ml and 5.4 μmol/min•mg respectively. Co2+, Mn2+ and Fe2+ enhanced the activity of RsEGm by 32.0, 19.5 and 11.2% respectively, while Pb2+ and Cu2+ decreased its activity by 19.6 and 12.7% separately.ConclusionsRsEGm could be overexpressed in P. pastoris. It was stable between pH 4.0 and pH 11.0, and exhibited higher stability at temperatures ≤ 40 °C. This endoglucanase may have potential to be used in the field of laundry, textile and lignocellulose-based biofuels and chemicals.

Highlights

Cellulases are of great significance for full utilization of lignocellulosic biomass
Studies have demonstrated that the majority of cellulase activity of higher termites takes place in the midgut, suggesting that they mainly depend on endogenous cellulases for cellulose degradation [6, 7, 15]
The agarose gel electrophoresis results of the PCR products of nine transformants, corresponding to the size (~1800 bp) of RsEG mutant (RsEGm) plus α-factor signal sequence, c-myc epitope and His6 tag, confirmed that the RsEG gene was successfully inserted into genome P. pastoris GS115 (Fig. 1)

Summary

Introduction

Cellulases are of great significance for full utilization of lignocellulosic biomass. Studies have demonstrated that the majority of cellulase activity of higher termites takes place in the midgut, suggesting that they mainly depend on endogenous cellulases for cellulose degradation [6, 7, 15]. Metagenomic analysis revealed a diverse set of genes related to glycoside hydrolases in the hindgut of a higher termite Nasutitermes sp., implying that hindgut microbes play an important role for cellulose degradation [17]. Proteome analysis of the bacterial community in a higher termite Nasutitermes corniger indicated that bacterial enzymes play more significant roles in metabolism than in activities related to cellulose degradation [18]

Methods

Results

Discussion

Conclusion