title: Cloning and expression of the cold-adapted endo-1,4-β-glucanase gene from Eisenia fetida.
abstract: Biofuel production from plant-derived lignocellulosic material using fungal cellulases is facing cost-effective challenges related to high temperature requirements. The present study identified a cold-adapted cellulase named endo-1,4-β-glucanase (EF-EG2) from the earthworm Eisenia fetida. The gene was cloned in the cold-shock expression vector (pCold I) and functionally expressed in Escherichia coli ArcticExpress RT (DE3). The gene consists of 1,368 bp encoding 456 amino acid residues. The amino acid sequence shares sequence homology with the endo-1,4-β-glucanases of Eisenia andrei (98%), Pheretima hilgendorfi (79%), Perineresis brevicirris (63%), and Strongylocentrotus nudus (58%), which all belong to glycoside hydrolase family 9. Purified recombinant EF-EG2 hydrolyzed soluble cellulose (carboxymethyl cellulose), but not insoluble (powdered cellulose) or crystalline (Avicel) cellulose substrates. Thin-layer chromatography analysis of the reaction products from 1,4-β-linked oligosaccharides of various lengths revealed a cleavage mechanism consistent with endoglucanases (not exoglucanases). The enzyme exhibited significant activity at 10°C (38% of the activity at optimal 40°C) and was stable at pH 5.0-9.0, with an optimum pH of 5.5. This new cold-adapted cellulase could potentially improve the cost effectiveness of biofuel production.
authors.affiliation: Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan. Electronic address: mueda@biochem.osakafu-u.ac.jp.
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Ueda
authors.forename: Akihiro
authors.initials: A
authors.lastname: Ito
authors.forename: Masami
authors.initials: M
authors.lastname: Nakazawa
authors.forename: Kazutaka
authors.initials: K
authors.lastname: Miyatake
authors.forename: Minoru
authors.initials: M
authors.lastname: Sakaguchi
authors.forename: Kuniyo
authors.initials: K
authors.lastname: Inouye
mesh_terms: D000222:Adaptation, Physiological; D000595:Amino Acid Sequence; D000818:Animals; D055162:Biocatalysis; D002480:Cellulase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D003080:Cold Temperature; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006868:Hydrolysis; D007221:Industry; D008969:Molecular Sequence Data; D009835:Oligochaeta / Q000201:enzymology* / Q000235:genetics* / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017421:Sequence Analysis
chemical_list: D011994:Recombinant Proteins; D002480:Cellulase
keywords: CBB; CMC; Cloning; Cold-adapted enzyme; Coomassie Brilliant Blue; EF-EG2; Eisenia fetida; Endo-1,4-β-glucanase; Expression; GH; SDS-PAGE; carboxymethyl cellulose; dNTP; deoxynucleotide triphosphate; endo-1,4-β-glucanase from Eisenia fetida; glycoside hydrolase; sodium dodecyl sulfate-polyacrylamide gel electrophoresis