A themostable intracellular β-galactosidase from a thermophilic Bacillus stearothermophilus was purified by a combination of (NH4)2SO4 fractionation, ion-exchange (DEAE-22)and gel filtration (Sephades G-75). The optimum temperature and pH of the enzyme acivity were 60Cand pH6.4 respectively. The β-galatosidase activity exhibited thermosttability at 50 C. The enzyme was significaantly activated by alkali and alkali-earth metal ions. The activity was inhibited by Zn2+ 、 Fe3+ 、 Cu2+Reducing agents enhanced β- galactosidase activity. Thiol-binding agents drastically decreased the enzyme activity. The enzyme was specific for β-D glycosidic linkages,and the identity of the aglycone moiety also influenced enzyme activity. At 55Cthe Km for O-nitrophenyl-β-D-galactosidase (ONPG)and lactose were 2. 63mmol/L and 4.39mmol/L, respectively,and Vmax for both substrates were 1.93 × 10-5mmol. min-1 mg-1protein6.54 ×105 mmol. min-1. mg-1protein,respectively. The enzyme was inhibited by glucose (the products of lactose hydrolysis,ki 2.47mmol/L),but not by galactose. In addition,the enzyme possessed transgalactosylation activity. Galacto-oligosaccharides,both tri- and tetrasaccharide,were involved in the products during lactose hydrolysi

A themostable intracellular ? galactosidase from a thermophilic Bacillus stearothermophilus was purified by a combination of (NH 4) 2SO 4 fractionation,ion exchange (DEAE 22)and gel filtration (Sephades G 75).The optimum temperature and pH of the enzyme acivity were 60℃and pH6.4 respectively.The ? galatosidase activity exhibited thermosttability at 50 ℃.The enzyme was significaantly activated by alkali and alkali earth metal ions.The activity was inhibited by Zn 2+ 、 Fe 3+ 、 Cu 2+ Reducing agents enhanced ? galactosidase activity.Thiol binding agents drastically decreased the enzyme activity.The enzyme was specific for ? D glycosidic linkages,and the identity of the aglycone moiety also influenced enzyme activity.At 55℃the Km for O nitrophenyl ? D galactosidase(ONPG)and lactose were 2.63mmol/L and 4.39mmol/L, respectively,and Vmax for both substrates were 1.93?10 5 mmol.min 1 .mg 1 protein6.54?10 5 mmol.min 1 .mg 1 protein,respectively.The enzyme was inhibited by glucose (the products of lactose hydrolysis,ki 2.47mmol/L),but not by galactose.In addition,the enzyme possessed transgalactosylation activity.Galacto oligosaccharides,both tri and tetrasaccharide,were involved in the products during lactose hydrolysis.

Heparinase III is an enzyme that specifically cleaves certain sequences of heparan sulfate. Previous reports showed that this enzyme expressed in Escherichia coli was highly prone to aggregation in inclusion bodies and lacks detectable biological activity. In this paper, we fused a glutathione-S-transferase (GST) tag to the N-terminus of heparinase III gene and expressed the fusion protein in Escherichia coli to develop an expression system of soluble heparinase III. As a result, approximately 80% of the fusion protein was soluble. The protein was then purified to near homogeneity via one-step affinity chromatography. A 199.4-fold purification was achieved and the purified enzyme had a specific activity of 101.7 IU/mg protein. This represented 32.3% recovery of the total activity of recombinant GST-heparinase III. The maximum enzyme production was achieved when bacteria were induced with 0.5 mmol/L isopropyl-beta-D-thiogalactoside at 15 degrees C for 12 h. The enzyme showed maximum activity at 30 degrees C and pH 7.5. And the enzyme activity was stimulated by 1 mmol/L Ca2+ and 150 mmol/L NaCl.
Escherichia coli ; genetics ; metabolism ; Flavobacterium ; enzymology ; genetics ; growth & development ; Glutathione Transferase ; biosynthesis ; genetics ; Heparin Lyase ; biosynthesis ; genetics ; isolation & purification ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

Objective To assess and compare the efficacy and safety of ecabet sodium-based quadruple therapy versus bismuth-based quadruple therapy for Helicobacter pylori (Hp) eradication.Methods A multicenter,randomized,positive controlled clinical trial was carried out.The object of the study were chronic gastritis patients at 8 hospitals in Xi'an,Beijing,Shanghai and Guangzhou from June 2009 to June 2011.All patients were divided into treatment group and control group.In treatment group,patients received ecabet sodium-based quadruple therapy (two times per day,omeprazole magnesium 20 mg,amoxicillin 1000 mg,clarithromycin 500 mg and ecabet sodium 1.0 g each time for 10 days.In control group,patients were assigned to receive bismuth-based quadruple therapy (two times per day; omeprazole magnesium 20 mg,amoxicillin 1000 mg,clarithromycin 500 mg and bismuth potassium citrate 220 mg each time) for 10 days.The Hp eradication was determined by 13C or 14C urea breath test at the 38th day after the treatment and the eradication rate was calculated.Side effects were recorded and analyzed.The data were analyzed by chi square test and Fisher's exact test.Results A total of 311 patients were recruited,and 155 patients were allatted in treatment group and 156 in control group.The per-protocol (PP) analysis indicated that the eradication rates of treatment group arid control group were 75.71％(106/140) and 77.37％(106/137) respectively,and there was no significant difference x2 =0.106,P=0.745).The intention-to-treat (ITT) analysis indicated that the eradication rates of treatment group and control group were 68.39％ (106/155) and 67.95％ (106/156) respectively,and there was no significant difference x2 =0.007,P=0.934).The side effects rates of treatment group and control group were 20.00％ (31/155) and 25.64％(40/156) respectively,and the difference was not statistically significant (Fisher's exact test,P=0.280).No serious side effect was observed in two groups.Conclusion The efficacy and safety of ecabet sodium-based quadruple therapy for Hp eradication in chronic gastritis patients may be the same as bismuth-based quadruple therapy.