β-GLUCOSIDASE PRODUCING BY STRAIN OF BACTERIUM ESCHERICHIA COLI VKPM B-3223 Russian patent published in 1994 - IPC

FIELD: biotechnology. SUBSTANCE: strain E. coli VKPM V-3223 is grown up to achievement of maximal level of enzyme specific activity, cells are lyzed, obtained lyzate is heated, and enzyme is precipitated from supernatant with organic solvent taken at ratio from 1: 1 to 1: 10. Obtained enzyme b-glucosidase shows the following properties: substrate specificity - enzyme hydrolyzes para-nitrophenyl-b-D-glucoside (PNPG) and cellobiose, but not - arbutin. Specific activity of enzyme is 150 and 6 U/mg protein with PNPG and cellobiose as substrates, respectively; mol. mass of enzyme is 75000 ± 5000 Da, isoelectric point at 5.5 + 1.1, pH optimum at 5.4 + 0.3, range of pH stability is 6-7; residual activity at pH 5.5 is 75% , at pH 7.5 - 70% . Thermal stability: residual activity after incubation for 1 h in tris-HCl buffer, pH 6.9: at 50 C - 100% , at 60 C - 98% , at 65 C - 90% ; value K_m is (1,2± 0,1)·10^-4 M (pH 5.4, substrate is PNPG), (3,5± 0,1)·10^-5 М (pH 7.0, substrate is PNPG). Inhibitors: delta-gluconolactone (at concentration above 10^-3 M); temperature optimum - 60± 2^°C. Prepared enzyme β-glucosidase shows enhanced thermal stability, specific activity and substrate affinity as compared with known enzyme b-glucosidase from Clostridium thermocellum that provides to use lower substrate concentrations. EFFECT: enhanced specific activity and thermal stability of enzyme. 2 tbl