Expression and characterization of family 40 Carbohydrate Binding Module (CBM) from Vibrio cholerae Non-O1 sialidase

Abstract

Carbohydrate Binding Module (CBM) is a non-catalytic protein domain found in carbohydrate-active enzyme (glycoside hydrolase) and its role is to bring carbohydrates in close proximity to the enzyme catalytic site for complete hydrolysis. The removal of this CBM from most protein domains often leads to reduced enzyme activity and efficiency. In this study, a gene encoding for family 40 CBM from Vibrio cholerae Non-O1 sialidase was cloned and successfully expressed in E. coli BL21 (DE3) strain. The CBM40 encoded 195 amino acids with 585 bp of nucleotide sequence. The protein was successfully expressed at 18°C when induced with 1 mM IPTG. Maximum expression was achieved at 20 hours after post-induction time. For purification of the protein, an anionic denaturing detergent method was used containing 1% SDS and 0.1% sarkosyl with gradient affinity elution at 50 mM imidazole concentrations. SDS-PAGE analysis of the purified CBM40 protein displayed a protein band with a molecular mass of 21 kDa. Protein characterization showed optimum stability in 100 mM citrate buffer pH 5.5, with the highest Tm value of 40 °C. The protein was stable between pH 5.5–6.2 and able to retain its activity at 27–56°C. The addition of Mn2+ and Mg2+ increased the protein melting temperature to 56°C. Meanwhile, the addition of reagents, such as 1% SDS and 1 M urea increased the protein melting temperature (Tm) to approximately 55°C. Protein stability can be influenced by many factors, including different buffers, pHs, temperatures, ionic strengths, and chemical reagents used in a study. The optimum characterization conditions established would further lead to the discovery of CBM40 protein true potential in enhancing substrate binding affinity and protein-carbohydrate recognition, which underpins its broad applications in biotechnology and protein engineering fields.