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

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Abstract
An endo-cellulase, 2 .beta.-glucosidases and 2 1,3-.beta.-glucanases from P. infestans were partially purified from the culture filtrate and their biochemical properties determined. The MW were estimated by chromatography on Sephancryl S-200 and were 21,000 (endo-cellulase), 160,000-230,000 and 32,000 (.beta.-glucosidases I and II), 160,000-230,000 and 21,000 (.beta.-glucanases I and II). The optimal pH of the endocellulase was 6.0. The other enzymes showed the following optimal pH and temperature values: .beta.-glucosidase I, 5.5 and 48.degree. C; .beta.-glucosidase II, 5.25 and 30.degree. C; 1,3-.beta.-glucanase I. 7.0 and 40.degree. C; and 1,3-.beta.-glucanase II, 4.5 and 45.degree. C. The .beta.-glucosidase II was unstable above 30.degree. C; while the other enzymes remained stable to 43.degree. C. The .beta.-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 .beta.-glucosidase II were 34 mmol/l for pNPG, 340 mmol/l for cellobiose and 42 mmol/l for gentiobiose. The Km value of the 1,3-.beta.-glucanase II for laminarin was 0.29 g/l. The isoelectric point of the enzymes were 3.2 (endo-cellulase), 3.3 (.beta.-glucosidase I), 4.7 (.beta.-glucosidase II) and 3.4 (the 2 1,3-.beta.-glucanases). At 10 mmol/l Cu2+ inhibited the .beta.-glucosidase I by 90% and the .beta.-glucosidase II by .apprx. 50%. The 1,3-.beta.-glucanase II was inhibited 75% by Mn2+ and 35% by Cu2+.