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.

Cold-Active Microbial Cellulase: Novel Approach to Understand Mechanism and Its Applications in Food and Beverages Industry

“…Cold active enzymes play a significant role in many food industries as the process require mild condition to maintain the taste of products and to avoid spoilage of food materials (Hamid and Mohiddin 2018;Feller 2013;Gerday et al 2000;Margesin and Schinner 1994;Russell 1998). In biofuel production, cold-active cellulase can produce ethanol from cellulose at low temperature resulting in saving production costs (Li et al 2019). Cold-active enzymes has more flexibility in comparison to mesophilic and thermophilic enzymes (Adapa et al 2014;Methe et al 2005;Somero 2004).…”

Cold‐active microbial enzymes and their biotechnological applications

“…These enzymes have high catalytic efficiency and protein flexibility at low temperature along with low thermal stability due to their unique protein characteristics (Rojas-Contreras et al, 2015). On the other hand, some of the cold-active enzymes showed high thermal stability that have an added advantage for various industrial applications (Zhou et al, 2019). Microorganisms such as bacteria, fungi, and algae were found to be excellent sources of cold-active enzymes and their sources have been reviewed (Bhatia et al, 2021;Hamid et al, 2022;Santiago et al, 2016).…”

“…Despite the challenges that need to be addressed, the potential advantages offered by cold‐active enzymes in biopulping make it a promising avenue for ongoing research and development. The mesophilic enzymes used in biopulping such as xylanase for xylan degradation (Walia et al., 2017 ), laccase for lignin degradation (Zerva et al., 2019 ), cellulase for cellulose degradation (Li et al., 2019 ), and pectinase for pectin degradation (Haile & Ayele, 2022 ) may be replaced by cold‐active xylanase, laccase, cellulase, and pectinase, respectively; that improve pulp and paper quality and also decreases energy demands. Research efforts are continuing to optimize cold enzymes and their application in biopulping, making them an attractive option for the future of the pulp and paper industry (Mesbah, 2022 ).…”