Ho-Yung Lee

Purification, characterization, and primary structure of a chitinase from Pseudomonas sp. YHS-A2

Abstract A chitinase gene (chiA) from Pseudomonas sp. YHS-A2 was cloned into Escherichia coli using pUC19. The nucleotide sequence determination revealed a single open reading frame of chiA comprised of 1902 nucleotide base pairs and 633 deduced amino acids with a molecular weight of 67,452 Da. Amino acid sequence alignment showed that ChiA contains two putative chitin-binding domains and a single catalytic domain. Two proline-threonine repeat regions, which are linkers between catalytic and substrate-binding domains in some cellulases and xylanases, were also found. From E. coli, ChiA was purified 12.8-fold relative to the periplasmic fraction. The Michaelis constant and maximum initial velocity for p-nitrophenyl-N,N′-diacetylchitobiose were 1.06 mM and 44.4 μmol/h per mg protein, respectively. The purified ChiA binds not only to colloidal chitin but also to other substrates (avicel, chitosan, and xylan), but the binding affinity of avicel, chitosan, and xylan is around 10 times lower than that of colloidal chitin. The reaction of ChiA with colloidal chitin and chitooligosaccharides (trimer-hexamer) produced an end product of N,N′-diacetylchitobiose, indicating that ChiA is a chitobiosidase.

Partial characterization and cloning of leuconocin J, a bacteriocin produced by Leuconostoc sp. J2 isolated from the Korean fermented vegetable Kimchi

Leuconostoc sp. J2, isolated from naturally fermented Kimchi, produced a bacteriocin which was named leuconocin J. This bacteriocin exhibited an inhibitory activity against several lactic acid bacteria and some food‐borne pathogens. The antimicrobial substance was secreted into the medium during the late log phase. It appears to be proteinaceous since its activity was completely inactivated by a range of proteolytic enzymes, and it was also relatively heat‐stable. The bacteriocin was partially purified by ammonium sulphate precipitation, following dialysis. The apparent molecular mass of partially purified bacteriocin, as indicated by activity detection after Tricine‐SDS‐PAGE, was 2·5–3·5 kDa. Leuconostoc sp. J2 plasmid DNA digested by EcoRI was cloned into pUC118 and transformed into Escherichia coli DH5α. Phenotypic expression of the bacteriocin production was detected in transformants harbouring pULBJ5·5. Finally, Southern blotting with the 2·3 kb insert as a probe against plasmid digests of Leuconostoc sp. J2 revealed that the cloned foreign DNA originated from Leuconostoc sp. J2.

Cloning and sequencing of ββ-1,4-endoglucanase gene (celA) from Pseudomonas sp. YD-15

A β‐1,4‐endoglucanase gene (celA) from Pseudomonas sp. YD‐15 was cloned in Escherichia coli DH5α and its nucleotide sequence determined. The open reading frame of celA was 1830 base pairs and the enzyme was composed of 609 amino acids with a molecular weight of 63 617 Da. The deduced amino acid sequence and putative active site of CelA had high amino acid homology with family E cellulases. By dot blot analysis, the induction of celA according to carbon sources was determined. The transcripts hybridizing to the internal fragment of celA were detected in total RNA isolated from Pseudomonas sp. YD‐15 cells grown on avicel and glycerol, but not from cells grown on glucose and cellobiose.

Icodextrin Improves the Serum Potassium Profile with the Enhancement of Nutritional Status in Continuous Ambulatory Peritoneal Dialysis Patients

The impact of glucose-free icodextrin (ID) for overnight dwell as compared to conventional glucose-containing dialysate (GD) on potassium (K+) metabolism in continuous ambulatory peritoneal dialysis (CAPD) patients has not yet been investigated. Serum K+ in a total of 255 stable patients (116 on GD and 139 on ID) on CAPD for more than 6 months and in 139 patients on ID before and after ID use (Pre-ID and Post-ID) were observed along with nutritional markers in a 2-year study period (Jan. 2006 to Dec. 2007). The prevalence of hypokalemia was similar between patients on GD and ID (16.7% vs 17.3%), but was lower on Post-ID than Pre-ID (17.3% vs 20.5%) without statistic significance. The mean serum K+ level was higher on ID than on GD (P<0.05) as well as Post-ID than Pre-ID (P<0.001). In the multivariate analysis, serum K+ levels were positively correlated with serum albumin, and creatinine in all patients (P<0.05), and ID-use in younger patients (age≤56, P<0.001). Serum albumin, creatinine, total CO2, and body mass index were significantly higher on Post-ID than Pre-ID. Icodextrin dialysate for chronic overnight dwell could increase serum K+ levels and lower the prevalence of hypokalemia compared to conventional glucose-containing dialysate. The improved chronic K+ balance in CAPD patients on icodextrin could be related to enhanced nutritional status rather than its impact on acute intracellular K+ redistribution.