No abstract

A study was undertaken to examine the effects of N-linked glycosylation on the structure-function of porcine pepsin. The N-linked motif was incorporated into four sites (two on the N-terminal domain and two on the C-terminal domain), and the recombinant protein expressed using Pichia pastoris. All four N-linked recombinants exhibited similar secondary and tertiary structure to nonglycosylated pepsin, that is, wild type. Similar K(m) values were observed, but catalytic efficiencies were approximately one-third for all mutants compared with the wild type; however, substrate specificity was not altered. Activation of pepsinogen to pepsin occurred between pH 1.0 to 4.0 for wild-type pepsin, whereas the glycosylated recombinants activated over a wider range, pH 1.0 to 6.0. Glycosylation on the C-terminal domain exhibited similar pH activity profiles to nonglycosylated pepsin, and glycosylation on the N-domain resulted in a change in activity profile. Overall, glycosylation on the C-domain led to a more global stabilization of the structure, which translated into enzymatic stability, whereas on the N-domain, an increase in structural stability had little effect on enzymatic stability. Finally, glycosylation on the flexible loop region also appeared to increase the overall structural stability of the protein compared with wild type. It is postulated that the presence of the carbohydrate residues added rigidity to the protein structure by reducing conformational mobility of the protein, thereby increasing the structural stability of the protein.

No abstract

SPsychrophilic bacteria, which grow on lactose as a carbon source, were isolated from Antarctic polar sea water. Among the psychrophilic bacteria isolated, strain KNOUC808 was able to grow on lactose at below 5ºC, and showed 0.867 unit of o-nitrophenyl -D-galactopyranoside(ONPG) hydrolyzing activity at 4ºC.The isolate was gram-negative, rod, aerobic, catalase positive and oxidase positive. Optimum growth was done at 20ºC, pH 6.8-7.2. The composition of major fatty acids in cell of KNOUC801 was C 12:0 (5.48%), C 12:0 3OH (9.21%), C 16:0 (41.83%), C 17:0 8 (7.24%) and C 18:1 7 (7.04%). All these results together suggest that it is affiliated with Pseudoalteromonas genus. The 16S rDNA sequence corroborate the phenotypic tests and the novel strain was designated as Pseudoalteromonas sp. KNOUC808. The optimum temperature and pH for lactose hydrolyzing enzyme was 20ºC and 7.8, respectively. The enzyme was stable at 4ºC for 7 days, but its activity decreased to about 50% of initial activity at 37ºC in 7 days.