Purification and some properties of Α-amylase from an ectomycorrhizal fungus, Tricholoma matsutake

Abstract

Α-Amylase from a still culture filtrate of Tricholoma matsutake, an ectomycorrhizal fungus, was isolated and characterized. The enzyme was purified to a homogeneous preparation with Toyopearl-DEAE, gel filtration, and Mono Q column chromatography. The Α-amylase was highly purified (3580 fold) with a recovery of 10.5% and showed a single protein band by SDS-PAGE. The enzyme was most active at pH 5.0–6.0 toward soluble starch and stable within the broad pH range 4.0–10.0. This Α-amylase was a relatively thermostable enzyme (optimum temperature, 60°C; thermal stability, 50°C). The molecular mass was 34 kDa by size-exclusion chromatography and 46 kDa by SDS-PAGE. This enzyme was not inhibited by the Hg²⁺ ion. Measurement of viscosity and TLC and HPLC analysis of the hydrolysates obtained from amylose showed that the amylase from T. matsutake is an endo-type (Α-amylase). Substrate specificity was tested using amylose with different polysaccharides. This Α-amylase readily hydrolyzed the Α-1,4 glucoside bond in soluble starch and amylose A (MW, 2900), but did not hydrolyze the Α-1,6 bond and cyclic polysaccharides such as Α- and Β-cyclodextrin.