Small neuraminidase gene of Clostridium perfringens ATCC 10543: cloning, nucleotide sequence, and production.

The small nanH gene encoding a neuraminidase from Clostridium perfringens ATCC 10543 was cloned in JM109 using pUC19 as a vector. Sequence analysis revealed an ORF, nt 310-1455, encoding 382 amino acids that was proceeded by a typical Shine-Dalgarno sequence, GGACGAGA. The nt sequence in the 15-402 region had in vivo promoter activity in an Escherichia coli promoter probe plasmid pKK232-8, which suggested that the small nanH promoter is functional in E. coli. Four regions of amino acids demonstrated great similarity to the "Asp boxes" (-Ser-X-Asp-X-Gly-X-Thr-Trp-) of other bacterial nanH proteins. The small nanH expressing clone, pCPN-1, which was cultured under aerobic conditions resulted in NanH activity which was 203-fold in culture and 211-fold in intracellular fraction compared to that of C. perfringens which has to be cultured under anaerobic conditions. Production of small NanH was also induced by adding sialyllactose to the culture medium of JM109 [pCPN-1]. The enzyme activity could be detected in the periplasmic fraction and the culture medium of JM109 [pCPN-1] after culturing to the stationary phase. The molecular weight, K(m), and optimum pH and pI of the cloned enzyme are identical to those of the parent strain. The cloned, small nanH could be used to study the structure-functional relationship of nanH, while the pCPN-1 clone could be used in the aerobic production of neuraminidase.