Extracellular enzymes of Phytophthora infestans: endo-cellulase, β-glucosidases, and 1,3-β-glucanases

Abstract

An endo-cellulase, two β-glucosidases, and two 1,3-β-glucanases from Phytophthora infestans were partially purified from the culture filtrate and their biochemical properties determined. The molecular weights were estimated by chromatography on Sephacryl S-200 and were 21 000 (endo-cellulase), 160 000 – 230 000 and 32 000 (β-glucosidases I and II), 160 000 – 230 000 and 21 000 (β-glucanases I and II). The optimal pH of the endocellulase was 6.0. The other enzymes showed the following optimal pH and temperature values: β-glucosidase 1,5.5 and 48 °C; β-glucosidase II, 5.25 and 30 °C; 1,3-β-glucanase 1, 7.0and40 °C; and 1,3-β-glucanase II, 4.5 and 45 °C. The β-glucosidase II was unstable above 30 °C, while the other enzymes remained stable to 43 °C. The β-glucosidase I did not show Michaelis–Menten kinetics for p-nitrophenyl-glucopyranoside (pNPG) and gentiobiose as substrates. The extrapolated Km value for pNPG was 1.1 mmol/L and the Km value for cellobiose was 280 mmol/L. The Km values of the β-glucosidase II were 34 mmol/L for pNPG, 340 mmol/L for cellobiose, and 42 mmol/L for gentiobiose. Finally, the Km value of the 1,3-β-glucanase II for laminarin was 0.29 g/L. The isoelectric point of the enzymes were 3.2 (endo-cellulase), 3.3 (β-glucosidase I), 4.7 (β-glucosidase II), and 3.4 (the two 1,3-β-glucanases). At 10 mmol/L, Cu2+ inhibited the β-glucosidase I by 90%, and the β-glucosidase II by about 50%. The 1,3-(3-glucanase II was inhibited 75% by Mn2+ and 35% by Cu2+.