title: Cloning and expression of low temperature active endoglucanase EG5C from Paenibacillus sp. IHB B 3084.
abstract: The endoglucanase gene designated as EG5C encoding cold active endoglucanase produced by Paenibacillus sp. IHB B 3084 was cloned and expressed in Escherichia coli BL21(DE3). The gene consisting of 1719bp open reading frame encoded a protein of 573 amino acids with a predicted molecular weight of 63.5kDa. The presence of N-terminal catalytic domain of the glycosyl hydrolase family 5 (GH5) and C-terminal carbohydrate binding X2 domain suggested the modular nature of the enzyme. The native signal peptide of EG5C was capable of efficiently secreting the enzyme with near equal activities in the cytoplasmic and extracellular fractions. The recombinant enzyme purified 9.46 fold to homogeneity with 22.33% yield gave 7.758IU/mg specific activity. The enzyme was stable over the broad pH range of 4-12 with more than 50% residual activity. The optimal activity was at 40°C with 70% relative activity at 5°C. The low temperature activity despite the shorter linker region suggested a novel cold adaptation mechanism by the enzyme. The enzyme displayed higher activity on carboxymethylcellulose than avicel which is useful in maintaining the tensile strength of fiber. The efficient secretion and low temperature activity offer prospect for large-scale production and industrial application of the endoglucanase.
authors.affiliation: Academy of Scientific and Innovative Research, New Delhi 110 025, India; CSIR-Institute of Himalayan Bioresource Technology, Palampur, HP 176 061, India.
authors.forename: Hena
authors.initials: H
authors.lastname: Dhar
authors.affiliation: CSIR-Institute of Himalayan Bioresource Technology, Palampur, HP 176 061, India.
authors.forename: Ramesh C
authors.initials: RC
authors.lastname: Kasana
authors.affiliation: CSIR-Institute of Himalayan Bioresource Technology, Palampur, HP 176 061, India.
authors.forename: Som
authors.initials: S
authors.lastname: Dutt
authors.affiliation: Academy of Scientific and Innovative Research, New Delhi 110 025, India; CSIR-Institute of Himalayan Bioresource Technology, Palampur, HP 176 061, India. Electronic address: arvindgulati@ihbt.res.in.
authors.forename: Arvind
authors.initials: A
authors.lastname: Gulati
mesh_terms: D020134:Catalytic Domain; D002480:Cellulase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D003080:Cold Temperature; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008958:Models, Molecular; D056507:Paenibacillus / Q000145:classification / Q000201:enzymology* / Q000235:genetics*; D010802:Phylogeny; D011487:Protein Conformation; D011994:Recombinant Proteins; D017422:Sequence Analysis, DNA
chemical_list: D011994:Recombinant Proteins; C097231:endoglucanase V; D002480:Cellulase
keywords: Endoglucanase; Low temperature active; Paenibacillus sp.