Enzymatic identification and functional sites study of a novel cold-active cellulase (MkCel5) from Microbacterium kitamiensea

Abstract

A strain of Microbacterium kitamiense capable of decomposing cellulose was obtained from the soil of Shigatse region in Tibet. Low temperature and cellulase gene MkCel5 was cloned and sequenced. The size of the open-reading frame was 1449 bp; it was predicted to encode a polypeptide with 482 residues with a molecular weight of 51.38 kDa. Sequence analysis indicated that it belongs to the glycoside hydrolase family 5 (GH5), which has high homology with reported endoglucanases. The predicted protein structure had a typical carbohydrate-binding domain and a catalytic domain of cellulase. Site mutagenesis experiments showed that the two glutamates at positions 312 and 404 were crucial in the catalytic process. MkCel5 was identified as a cold-adaptive and neutral enzyme with a maximum activity at 25 °C and pH 7.0. These properties suggested that MkCel5 has potential for application in the cellulose processing industry requiring low temperature.

Keywords

• cellulose
• cold-active enzyme
• microbacterium kitamiense
• site-directed mutagenesis
• protein modelling