noncellulosomal family 48 cellobiohydrolase from Clostridium phytofermentans ISDg: heterologous expression, characterization, and processivity

• Published in: Applied microbiology and biotechnology 2010-03, Vol.86 (2), p.525-533
• Main Authors: Zhang, Xiao-Zhou, Zhang, Zuoming, Zhu, Zhiguang, Sathitsuksanoh, Noppadon, Yang, Yunfeng, Zhang, Y.-H. Percival
• Format: Article
• Language: English
• Subjects: Bacillus subtilis - genetics; Bacteria; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biodiesel fuels; Biofuels; Biological and medical sciences; Biomedical and Life Sciences; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Calcium - metabolism; Cellobiose - metabolism; Cellulase; Cellulose; Cellulose - analogs & derivatives; Cellulose - metabolism; Cellulose 1,4-beta-Cellobiosidase - chemistry; Cellulose 1,4-beta-Cellobiosidase - genetics; Cellulose 1,4-beta-Cellobiosidase - metabolism; Cloning; Clostridium - enzymology; E coli; Enzyme Stability; Enzymes; Escherichia coli - genetics; Ethanol; Fundamental and applied biological sciences. Psychology; Gene Expression; Genetic engineering; Genomes; Glucose; Hydrogen-Ion Concentration; Kinetics; Laboratories; Life Sciences; Microbial Genetics and Genomics; Microbiology; Protein expression; RNA polymerase; Studies; Substrate Specificity; Temperature; Tetroses - metabolism
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-009-2231-1

Family 48 glycoside hydrolases (cellobiohydrolases) are among the most important cellulase components for crystalline cellulose hydrolysis mediated by cellulolytic bacteria. Open reading frame (Cphy_3368) of Clostridium phytofermentans ISDg encodes a putative family 48 glycoside hydrolase (CpCel48) with a family 3 cellulose-binding module. CpCel48 was successfully expressed as two soluble intracellular forms with or without a C-terminal His-tag in Escherichia coli and as a secretory active form in Bacillus subtilis. It was found that calcium ion enhanced activity and thermostability of the enzyme. CpCel48 had high activities of 15.1 U μmol⁻¹ on Avicel and 35.9 U μmol⁻¹ on regenerated amorphous cellulose (RAC) with cellobiose as a main product and cellotriose and cellotetraose as by-products. By contrast, it had very weak activities on soluble cellulose derivatives (e.g., carboxymethyl cellulose (CMC)) and did not significantly decrease the viscosity of the CMC solution. Cellotetraose was the smallest oligosaccharide substrate for CpCel48. Since processivity is a key characteristic for cellobiohydrolases, the new initial false/right attack model was developed for estimation of processivity by considering the enzyme's substrate specificity, the crystalline structure of homologous Cel48 enzymes, and the configuration of cellulose chains. The processivities of CpCel48 on Avicel and RAC were estimated to be ∼3.5 and 6.0, respectively. Heterologous expression of secretory active cellobiohydrolase in B. subtilis is an important step for developing recombinant cellulolytic B. subtilis strains for low-cost production of advanced biofuels from cellulosic materials in a single step.