Preliminary characterisation of an Escherichia coli K5 lyase-deficient strain producing the K5 polysaccharide.
Escherichia coli K5 polysaccharide has structural analogies with N-acetylheparosan, a non-sulphated precursor of heparin and, for this reason, can be considered an attractive precursor for the production of semi-synthesis heparin analogues. This polysaccharide has two components: a high molecular weight (HMW) one and a low molecular weight (LMW) one, whose ratio varies depending on the action of a lyase enzyme synthesized by the same K5 producer strain. The present paper reports the production of the K5 polysaccharide by a spontaneous E. coli mutant strain lacking the lyase activity. Similar K5 polysaccharide yields, 180 mg l(-1) after 16 h fermentation, were obtained by both the wild and mutant strains, though K5 lyase activity was only observed in the culture filtrates from the wild strain. The time course of the specific filtrate volume (1 m(-2)) and of the specific filtrate flux rate (1 m(-2) h(-1)) during ultrafiltration (UF) of culture filtrates where the lyase enzyme acted on the K5 chain, showed a decrease of UF performance, probably because of membrane fouling by the LMW K5 fraction. In particular, the specific filtrate volume and specific filtrate flux rate of wild strain samples reached respectively 13 l m(-2) and 4 l m(-2) h(-1), compared to 25 l m(-2) and 15 l m(-2) h(-1) obtained from the mutant strain samples. PCR molecular analysis of the DNA region encoding for the lyase enzyme showed that, in the mutant strain, molecular rearrangements occurred in both regulatory and structural regions.
Manzoni
Matilde
M
Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Sezione di Microbiologia Industriale, Uni- ersità degli Studi, Via G. Celoria 2, 20133 Milano, Italy.
Rollini
Manuela
M
Piran
Elena
E
Parini
Carlo
C
D016667:Bacterial Capsules; D001709:Biotechnology / Q000379:methods*; D003470:Culture Media / Q000494:pharmacology; D004247:DNA / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D005285:Fermentation; D008190:Lyases / Q000235:genetics / Q000502:physiology*; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D011133:Polysaccharide-Lyases / Q000737:chemistry* / Q000235:genetics; D011134:Polysaccharides / Q000737:chemistry*; D011135:Polysaccharides, Bacterial / Q000737:chemistry; D013997:Time Factors; D014462:Ultrafiltration
D003470:Culture Media; D029968:Escherichia coli Proteins; D011134:Polysaccharides; D011135:Polysaccharides, Bacterial; C050673:capsular polysaccharide K5; D004247:DNA; D008190:Lyases; C411971:KflA protein, E coli; D011133:Polysaccharide-Lyases