Cel5M was identified as a cold-active cellulase with an optimal temperature of 30 °C and it was active within a narrow pH range with an optimum at pH 4.5. Phylogenetic analysis showed that Cel5M represented a new subfamily of the glycosyl hydrolase family 5, representing an opportunity for research into and applications of novel cold-active cellulases. Glycoside hydrolases (GHs) have been classified into more than 100 families according to similarities in their amino acid sequence (Henrissat & Davies, 1997) and into clans according to their three-dimensional structures.

GH5, which belongs to glycoside hydrolase clan A, is a superfamily with a conserved overall structure and mechanism (Leggio & Larsen, 2002). Cold-active cellulases have gained considerable attention for both industrial applications and fundamental research because of their unique structural and catalytic characteristics (Zeng et al., 2006). Only Raf targets a few cold-active cellulases have been reported so far, CelG from Pseudoalteromonas haloplanktis (Violot et al., 2003)

and CelX from Pseudoalteromonas sp. DY3 (Zeng et al., 2006). Both CelG and CelX belong to GH5 and consist of a catalytic module (CM) and a carbohydrate-binding module (CBM), separated by a linker region GPCR & G Protein inhibitor (LR) that plays a key role in cold adaptation of cold-active cellulases (Sonan et al., 2007). In the present study, a gene encoding a novel cold-active endo-β-1,4-glucanase (named Cel5M) from psychrophilic deep-sea bacteria Pseudomonas sp. MM15 was isolated. The deduced protein sequence lacked the typical cellulase domain structures of CBM and LR, providing an opportunity for investigating its novel cold-adaptation mechanism. Phylogenetic analysis showed that Cel5M represents a new subfamily in GH5. Carboxymethyl cellulase (CMCase) producing Pseudomonas sp. MM15, deposited in

the China Center of Industrial Culture Collection under strain collection number CICC 10441, was isolated from the deep-sea sediment of the Southern Okinawa Trough using the method described by Ibrahim & El-Diwany (2007). The in situ environment of the deep-sea sediments with a water depth of 1245 m was characterized by a strong terrestrial input of organic matters, thus favoring the activity of various Urease extracellular enzyme-producing bacteria (Dang et al., 2009). A genomic library of Pseudomonas sp. MM15 was constructed using plasmid pUC19 (TaKaRa, Japan) and Escherichia coli DH 5α following the procedure described by Chen et al. (2011). After 14 h incubation at 37 °C, the colonies were transferred onto carboxymethyl cellulose (CMC; Sigma) plates (1 g L−1 KH2PO4; 5 g L−1 NaCl; 10 g L−1 yeast extract; 10 g L−1 peptone; 10 g L−1 CMC and 15 g L−1 agar). After another 14 h growth at 37 °C, the plates were stained with Congo red (1 g L−1) for 15 min and then washed with 1 M NaCl solution for 5 min.