The gene encoding a thermostable β-D-xylosidase (GbtXyl43B) from Geobacillus thermoleovorans IT-08 was cloned in pET30a and expressed in Escherichia coli; additionally, characterization and kinetic analysis of GbtXyl43B were carried out. The gene product was purified to apparent homogeneity showing M r of 72 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme exhibited an optimum temperature and pH of 60 °C and 6.0, respectively. In terms of stability, GbtXyl43B was stable at 60 °C at pH 6.0 for 1 h as well as at pH 6-8 at 4 °C for 24 h. The enzyme had a catalytic efficiency (k cat/K M) of 0.0048 ± 0.0010 s(-1) mM(-1) on p-nitrophenyl-β-D-xylopyranoside substrate. Thin layer chromatography product analysis indicated that GbtXyl43B was exoglycosidase cleaving single xylose units from the nonreducing end of xylan. The activity of GbtXyl43B on insoluble xylan was eightfold higher than on soluble xylan. Bioinformatics analysis showed that GbtXyl43B belonging to glycoside hydrolase family 43 contained carbohydrate-binding module (CBM; residues 15 to 149 forming eight antiparallel β-strands) and catalytic module (residues 157 to 604 forming five-bladed β-propeller fold with predicted catalytic residues to be Asp287 and Glu476). CBM of GbtXyl43B dominated by the Phe residues which grip the carbohydrate is proposed as a novel CBM36 subfamily.

No abstract

roteins hydrolyzed from melinjo seeds (Gnetum gnemon) at green (GM), yellow (YM) and red (RM) stages of maturity were studied for their effectiveness in antioxidant and antidiabetic activities. The seed protein extract was hydrolyzed using alcalase 2.4L, and the resulting hydrolysates with the highest degree of hydrolysis, protein profile, and the most potent contributors to antioxidant and invitro antidiabetic activities were identified. The degree of hydrolysis value of hydrolysates ranged from 52-84%, and the SDS-PAGE protein profile showed two distinct bands in which the band with molecular weight of 30 kDa degraded more intensively. Antioxidant capacity was measured using different standard methods, including radical cation 2,2-azinobis-(3-ethylbenzothizoline-6-sulphonate) (ABTS•+) assay, hydroxyl radical (OH•), and superoxide anion (O2•-) scavenging. The green hydrolysate (GMH) had significantly higher (p<0.05) free radical scavenging (ABTS•+, OH•, and O2•-) activities than that of the yellow hydrolysate (YMH) and red hydrolysate (RMH). However, invitro antidiabetic testing was performed based on the inhibitory activity of α-amylase and α-glucosidase. GMH was found to be more effective than YMH and RMH. These results showed that the antioxidant and antidiabetic activity in hydrolyzed GM protein has high potential to be utilized as natural nutraceuticals.

No abstract

No abstract