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 Hg 2ϩ 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 -cyclodextrin.
To investigate the function of amylases in the fruit-body formation of an ectomycorrhizal fungus, Lyophyllum shimeji, we purified the extracellular amylase in the medium of this fungus. The purified enzyme was obtained from 1.7 l stationary culture filtrate, with 4.2% recovery, and showed a single protein band on SDS-PAGE. The molecular mass was about 25 kDa. The enzyme was most active at around 40°C and pH 5.0 and stable over pH 4.5-6.5 for 30 min at 37°C. This amylase was remarkably activated by the presence of Ca 2ϩ ion (7.7 times that of the control), but Ba 2ϩ and Ag ϩ completely inhibited the activity. The amylase readily hydrolyzed the α-1,4 glucosidic linkage such as dextrin and amylose A (MW, 2900), converting into glucose, and hydrolyzed the α-1,6 glucosidic linkage of isomaltohexaose and amylopectin. However, the enzyme did not hydrolyze the cyclic polysaccharides. On the other hand, when a low molecular mass amylose A was hydrolyzed by this amylase, -anomer glucose was produced. From these results, we concluded that the amylase from L. shimeji seems to be a glucoamylase.
To evaluate the potential of the production of the ectomycorrhizal fungus Tricholoma matsutake to produce carbohydrases, (1) the distribution of carbohydrase activities among the different strains (18 strains) was investigated and (2) the abilities of T. matsutake and saprophytic fungi to produce β-glucosidase were compared. The results showed that the carbohydrase productions patterns of T. matsutake still resemble one another. Moreover, this fungus exhibited markedly higher β-glucosidase than did the saprophytic mushrooms. Tricholoma matsutake showed weak production of α-amylase and α-glucosidase in a static culture fi ltrate. On the other hand, glucoamylase activity was not observed. Surprisingly, we discovered that β-glucosidase demonstrated strong activity. This fi nding suggests that this fungus has saprotrophic abilities. The carbohydrase production systems in T. matsutake were characterized from our experimental results. Also, we point out some weak points in the carbohydrase production systems of T. matsutake.
We studied the characteristics of the utilization of carbohydrate substrates and the production of those hydrolyzing enzymes of the Tricholoma matsutake J-1 strain isolated from hardwood (Quercus sp.). In the culture medium, 5% glucose inhibited mycelial growth. The growth inhibition rate was remarkable in the Z-1 strain from softwood (Pinus densifl ora) compared with that of the J-1 strain from hardwood. α-Amylase production varied with starches from different origins in contrast to mycelial growth. The range of the effect of 0.5%-15% soluble starch on vegetative mycelial growth was also investigated. The optimal concentration for mycelial growth was 15% for the J-1 strain but 10% for the Z-1 strain. Mycelial growth of the J-1 strain was strongly inhibited in PMML medium containing Sunpeal-CP prepared from sulfi te pulp softwood waste, but that of the Z-1 strain was not inhibited by Sunpeal-CP. Moreover, mycelial growth of the J-1 strain from Quercus sp. dramatically decreased with the addition of CNF-HWSF (hot watersoluble fractions from corn fi ber) to the PMML and PDL medium. However, inhibition by CNF-HWSF was not shown in the Z-1 strain from P. densifl ora.
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