In vitro synthesis of β-(1-3)-glucan with a membrane fraction of Botrytis cinerea

Botrytis cinerea is known as a β-(1-3)(1-6)-d-glucan-overproducer with an exceptionally high degree of β-(1-6)-branches in the glucan. To localize and characterize the glucan synthase, the incorporation of ["%C]glucose, activated by uridyl diphosphate and incubated with mycelial extracts, into ethanol-precipitable material was studied. Whereas crude extracts were found to produce a- and β-glucans, a pure β-(1-3)-d-glucan was formed by the membrane fraction. This was monitored by the complete cleavage into glucose with a purified β-(1-3)-glucanase and by the detection of laminaribiose, a β-(1-3)-linked dimer of glucose, in incomplete digestions. Degradation products with β-(1-6)-linkages, e.g. gentiobiose, which is found after degradation of the native polymer, were not detectable. β-(1-3)-glucan synthase activity was optimal at 22 °C and pH 7·2 with a K m of 0·8mm and a V max of 0·24 mU mg − protein. GTP ( K a fl 4·2 lm), cellobiose, BSA and EGTA enhanced the reaction whereas UDP ( K i fl 0·45 mm) and Ca 2+ inhibited it.