Cloning, expression, and deletion analysis of large nanH of Clostridium perfringens ATCC 10543

• Published in: Enzyme and microbial technology Vol. 31; no. 6; pp. 794 - 803
• Main Authors: Sheu, Sheh-Yi, Tseng, Huen-juin, Huang, Shu-ping, Chien, Chin-hsiang
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier Inc 01.11.2002; Elsevier Science
• Subjects: Bacteriology; Biological and medical sciences; Clostridium perfringens ATCC 10543; Fundamental and applied biological sciences. Psychology; Genetics; Large sialidase gene ( nanH); Microbiology; Overexpression of large nanH; DEAE, diethylaminoethyl; SDS–PAGE, sodium dodecylsulfate–polyacrylamide gel eletrophoresis; Overexpression of large nanH; Large sialidase gene ( nanH); IPTG, isopropylthiogalactoside; 4-MU, 4-methylumbelliferone; nanH, gene of sialidase or neuraminidase; NanH, sialidase protein; 4-MU-NeuAc, 2′-(4-methylumbelliferyl)-α- d- N-acetylneuraminic acid; Clostridium perfringens ATCC 10543; Nucleotide sequence; Enzyme; Clostridiales; Large sialidase gene (nanH); Gene expression; Clostridium perfringens; Glycosidases; Exo-α-sialidase; Clostridiaceae; Bacteria; Hydrolases; O-Glycosidases
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/S0141-0229(02)00177-1

The large sialidase gene ( nanH) of Clostridium perfringens ATCC 10543 was cloned in pCRII and subcloned in pQE70 expression vector. The large nanH consists of 2082 bp nucleotides and encodes 694 amino acids; large NanH is with four repeated “Asp-boxes” and one RIP (Arg–Ile–Pro) region formed by amino acids 266–268 in the sequence. It showed 26% of sequence homology with the small NanH from the same species (ATCC 10543). The large nanH, with six His residues adding to the C-terminus of NanH protein, was constructed in pQE70 (recombinant plasmid named pQE70-LSC) and expressed in Escherichia coli M15. The large NanH expressed in the periplasmic space of bacteria M15 showed 430-fold increase in large NanH enzymatic activity upon IPTG induction, as compared to the culture without IPTG added. The recombinant large NanH was purified with Ni–NTA and N-( p-aminophenyl) oxamic acid–agarose column chromatography. The purified enzyme showed specific activity of 136.6 U/mg when assayed with 4-MU-NeuAc as substrate. The truncated NanH (pQE70-Ld1), deletion of amino acids 1–215, exhibits similar specific activity, the same optimum temperature and optimum pH compared with those of wild-type large NanH.