Yuji Kawase

Molecular cloning and characterization of a novel bacteriophage-associated sialidase

Bacteriophage 63D, previously isolated from sewage, is associated with alpha-2,8-linked polysialic acid degrading activity. We cloned a DNA fragment containing the sialidase gene from a 63D phage genomic library and the enzyme was functionally expressed in Escherichia coli. Determination of the nucleotide sequence of the fragment revealed that it contained one open reading frame (ORF) coding for a 108-kDa polypeptide consisting of 984 amino acid residues. The fragment had promoter sequences similar to the E. coli consensus promoters for sigma70. The deduced amino acid sequence of the central region of the ORF showed homology to those of phages K1F (51.6% identity) and PK1E (51.7% identity) endosialidases. Two Asp-box motifs that are widely found in sialidases were conserved. Purification of the soluble enzyme from lysed culture broth of infected E. coli yielded a 90-kDa protein upon SDS polyacrylamide gel electrophoresis, suggesting that the primary translational product is processed to the mature 90-kDa protein. The molecular mass of the enzyme was determined as 360 kDa by gel filtration, indicating that the native enzyme was probably a tetramer of identical 90-kDa subunits.

Colominic acid production from Escherichia coli in a fed-batch culture under the control of ammonium ions using an FIA system

Abstract A fed-batch culture was applied for the production of colominic acid from Escherichia coli . It was found that the copper ion, which is a trace element in medium and an essential element in N -acetylneuraminic acid synthase, affected the colominic acid production and the highest production was obtained using 1.6 mg/ l CuCl 2 . Ammonia formed from nitrogen source utilization greatly affected colominic acid production and only slightly affected the cell growth. A flow injection analysis (FIA) system was fabricated and applied to monitor and control the ammonia concentration in the culture broth. It was found that a concentration of 0.3 g/ l of ammonia was optimal for colominic acid production where 1.9 g/ l was obtained.