Cloning and Characterization of Chitin Deacetylase from Euphausia superba.

Chitin deacetylase (CDA) can catalyze the deacetylation of chitin to produce chitosan. In this study, we identified and characterized a chitin deacetylase gene from Euphausia superba (EsCDA-9k), and a soluble recombinant protein chitin deacetylase from Euphausia superba of molecular weight 45 kDa was cloned, expressed, and purified. The full-length cDNA sequence of EsCDA-9k was 1068 bp long and encoded 355 amino acid residues that contained the typical domain structure of carbohydrate esterase family 4. The predicted three-dimensional structure of EsCDA-9k showed a 67.32% homology with Penaeus monodon. Recombinant chitin deacetylase had the highest activity at 40 °C and pH 8.0 in Tris-HCl buffer. The enzyme activity was enhanced by metal ions Co2+, Fe3+, Ca2+, and Na+, while it was inhibited by Zn2+, Ba2+, Mg2+, and EDTA. Molecular simulation of EsCDA-9k was conducted based on sequence alignment and homology modeling. The EsCDA-9k F18G mutant showed a 1.6-fold higher activity than the wild-type enzyme. In summary, this is the first report of the cloning and heterologous expression of the chitin deacetylase gene in Euphausia superba. The characterization and function study of EsCDA-9k will serve as an important reference point for future application.

[Intrinsic prokaryotic promoter activity of SUMO gene and its applications in the protein expression system of Escherichia coli].

Nowadays, SUMO fusion system is important for recombinant protein production in Escherichia coli, yet a few aspects remain to be improved, including the efficacy for vector construction and protein solubility. In this study, we found the SUMO gene Smt3 (Sm) of Saccharomyces cerevisiae conferred an unexpected activity of constitutive prokaryotic promoter during its PCR cloning, and the gene coding regions of SUMOs in most species had a sigma70-dependent prokaryotic promoter embedded, through the prediction via the BPROM program developed by Softberry. By combining the characters of Sm promoter activity and the Stu I site (added at the 3'-terminal of Sm), and introducing a His-tag and a hyper-acidic solubility-enhancing tag, we further constructed a set of versatile vectors for gene cloning and expression on the basis of Sm'-LacZa fusion gene. Experimentally started from these vectors, several target genes were subcloned and expressed through blue-white screening and SDS-PAGE analysis. The results manifest a few of expectable advantages such as rapid vector construction, highly soluble protein expression and feasible co-expression of correlated proteins. Conclusively, our optimized SUMO fusion technology herein could confer a large potential in E. coli protein expression system, and the simultaneously established co-expression vector systems could also be very useful in studying the protein-protein interactions in vivo.