Recombinant cellobiohydrolase of Myceliophthora thermophila: characterization and applicability in cellulose saccharification

Anica Dadwal,Tulasi Satyanarayana,Shilpa Sharma

doi:10.1186/s13568-021-01311-8

Anica Dadwal, Tulasi Satyanarayana + Show 1 more

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https://doi.org/10.1186/s13568-021-01311-8

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Abstract

A codon optimized cellobiohydrolase (CBH) encoding synthetic gene of 1188 bp from a thermophilic mold Myceliophthora thermophila (MtCel6A) was cloned and heterologously expressed in Escherichia coli for the first time. In silico analysis suggested that MtCel6A is a GH6 CBH and belongs to CBHII family, which is structurally similar to Cel6A of Humicola insolens. The recombinant MtCel6A is expressed as active inclusion bodies, and the molecular mass of the purified enzyme is ~ 45 kDa. The rMtCel6A is active in a wide range of pH (4–12) and temperatures (40–100 °C) with optima at pH 10.0 and 60 °C. It exhibits T1/2 of 6.0 and 1.0 h at 60 and 90 °C, respectively. The rMtCel6A is an extremozyme with organic solvent, salt and alkali tolerance. The Km, Vmax, kcat and kcat/Km values of the enzyme are 3.2 mg mL−1, 222.2 μmol mg−1 min−1, 2492 s−1 and 778.7 s−1 mg−1 mL−1, respectively. The product analysis of rMtCel6A confirmed that it is an exoenzyme that acts from the non-reducing end of cellulose. The addition of rMtCel6A to the commercial cellulase mix (Cellic CTec2) led to 1.9-fold increase in saccharification of the pre-treated sugarcane bagasse. The rMtCel6A is a potential CBH that finds utility in industrial processes such as in bioethanol, paper pulp and textile industries.Graphical

Highlights

Lignocellulosic biomass is the abundant and renewable source of energy
CBHI acts on reducing ends, while CBHII acts on non-reducing ends of oligosaccharides of varied length to produce cellobiose
This study suggests that the recombinant MtCel6A is a good biocatalyst of commercial value in the various biotechnological processes where alkaline conditions prevail in the saccharification of alkali-pretreated lignocellulosic biomass

Summary

Introduction

Lignocellulosic biomass is the abundant and renewable source of energy. In view of global warming and increasing demands for energy, the conversion of lignocellulosics to bioethanol is an attractive alternative (Liu et al 2019). Lignocellulosics are predominantly composed of cellulose, hemicellulose and lignin. The enzymes that convert the complex and recalcitrant structure of cellulose are termed cellulases that includes three components cellobiohydrolase (CBH), endoglucanase (EGL) and β-glucosidase (BGL) (Himmel et al 1999). Cellobiohydrolases (CBHs) are further classified as CBHI and CBHII based on their mode of action. All three enzymes act synergistically to saccharify cellulose (Horn et al 2012). According to the CAZy (Carbohydrate Active Enzymes) database (http://www.cazy.org), the cellulases are classified into different GH families. The CBHs typically belong to GH5-GH7, GH9 and GH48 families. The cellulases are produced naturally by various fungal, bacterial and archaeal species (Obeng et al 2017) and the cellulases produced by thermophilic microbes are typically thermostable

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