Cloning and expression of low temperature active endoglucanaseEG5C from Paenibacillus sp. IHB B 3084

Abstract

tThe endoglucanase gene designated as EG5C encoding cold active endoglucanase produced by Paenibacil-lus sp. IHB B 3084 was cloned and expressed in Escherichia coli BL21(DE3). The gene consisting of 1719 bpopen reading frame encoded a protein of 573 amino acids with a predicted molecular weight of 63.5 kDa.The presence of N-terminal catalytic domain of the glycosyl hydrolase family 5 (GH5) and C-terminalcarbohydrate binding X2 domain suggested the modular nature of the enzyme. The native signal peptideof EG5C was capable of efficiently secreting the enzyme with near equal activities in the cytoplasmic andextracellular fractions. The recombinant enzyme purified 9.46 fold to homogeneity with 22.33% yieldgave 7.758 IU/mg specific activity. The enzyme was stable over the broad pH range of 4–12 with morethan 50% residual activity. The optimal activity was at 40◦C with 70% relative activity at 5◦C. The lowtemperature activity despite the shorter linker region suggested a novel cold adaptation mechanism bythe enzyme. The enzyme displayed higher activity on carboxymethylcellulose than avicel which is usefulin maintaining the tensile strength of fiber. The efficient secretion and low temperature activity offerprospect for large-scale production and industrial application of the endoglucanase.