In order to confirm the presence of putative glucoamylase gene in Tricholoma matsutake genome, the genomic DNA was prepared from T. matsutake NBRC30773 strain and was used as template to clone the glucoamylases gene (TmGlu1). We obtained the nucleotide sequence of TmGlu1 and its franking region. The coding region (from ATG to stop codon) is 2,186 bp. The locations of exons and introns were determined from the nucleotide sequences of 3’- and 5’-RACE PCR and RT- PCR products. On the other hand, to investigate the relationship between composition of medium and glucoamylase expression, we checked the expression level of glucoamylase gene by realtime reverse transcription PCR and measurement of glucoamylase enzyme activity. It was found that enzyme activity of glucoamylase was very low in different medium. Expression of glucoamylases gene appeared to not be affected by different carbon source.

[Objective] The color is one of the important factor for commercial value of edible mushroom. Pleurotus cornucopiae is edible mushroom, naturally distributed in the northern part of Japan. Due to its beautiful yellowish color, P. cornucopiae is sometimes called as “Golden-Shimeji”. The unique flavor of this mushroom also help to enhacement its commercial value and the consumption of this mushroom has gradually increased. The yellow color of this mushroom is resistant against high temperature because the color is not diminished after deep-frying. These facts suggest that the structure and biosynthesis of this yellow color is very interesting. However, the report concerning about these profiles are none at all. In the present study, we tried to isolate yellow pigment from P. cornucopiae fruit body and some properties of the pigments were also investigated. [Methods and Results] The fruit body of P. cornucopiae was kindly given from Tanaka Machinery Corporation and kept in the freezer (-20°C) until use. The fruit body was homogenized with twice amount of ice cold distilled water with a mixer and subsequently filtered to be crude pigment solution. According to the result of ultra filtration by CentriPrep YM-10, molecular weight of the pigment was under 10,000. The resultant filtrate of YM-10 was concentrated by lyophilization and subsequently subjected to size exclusion chromatography (SEC) by Sephadex LH-20. Two different pigment fractions, brown and yellow, were obtained from SEC separation. According to the retention time of HPLC analysis with TSKgel G2500PWXL, the molecular weights of these two pigments are larger than size exclusion limit (Ca. 5,000). Moreover, detectable spot with ninhydrin reagent on thin layer chromatography (TLC) analysis suggest that these pigments are guessed as peptide compounds. In addition, no color changes were observed after heating for 20 min at 100℃.

[Object] Gamma-Amino Butyric Acid (GABA), a four-carbon non protein amino acid, is widely distributed in nature and acts as the major suppressive neurotransmitter in the mammalian central nervous system. Recently, GABA has been reported to have several physiological functions such as antihypertensive, diuretic, relaxing and antidiabetic effects. Due to these unique biological functions, GABA has been used as functional ingredients for functional foods. Glutamic Acid Decarboxylase (GAD), which catalyzes decarboxylation of L-glutamic acid to GABA, has been purified from mammals, higher plants, and some microorganisms and their properties have already been reported. On the other hand, mushrooms are commonly appreciated as healthy food due to their nutritional properties, such as low calorie, rich in fiber, vitamin and mineral. L-Glutamic acid and GABA are also commonly distributed in edible mushrooms. The fact that mushrooms accumulate GABA suggests the existence of GAD. However, the enzyme property of mushroom GAD has not been reported. In the present study, we tried to evaluate the property of Flammulina veltipes GAD, and the purification and characterization of enzyme is also investigated. [Methods and Results] Mushroom (fruit-body of Flammulina veltipes) used in this study was purchased in local market. GAD activity was determined by formation of GABA from L-glutamic acid in the presence of pyridoxal-5’- phosphate (PLP). The activity of enzyme was determined semi-quantitatively by color intensity of GABA on TLC analysis. Fruit-body was crushed and then centrifuged to separate supernatant and precipitate. To investigate the location of GAD, both supernatant and precipitate were subjected to enzyme reaction after dialysis. The enzyme activity of precipitate is stronger than that of supernatant. The formation of GABA was observed between pH 4 and 6 and the maximum color intensity of GABA was observed at pH 6. However, GAD activity was lost after dialysis for overnight against buffer of pH 6-11. These results suggest that GAD from Flammulina veltipes is stable at pH 4-5 in spite of its optimum pH for GABA production is around 6.

Gamma-Amino Butyric Acid (GABA), a four-carbon non protein amino acid, is widely distributed in nature and acts as the major suppressive neurotransmitter in the mammalian central nervous system. Recently, GABA has been reported to have several physiological functions such as antihypertensive, diuretic, relaxing and antidiabetic effects. Due to these unique biological functions, GABA has been used as functional ingredients for functional foods. Glutamic Acid Decarboxylase (GAD), which catalyzes decarboxylation of L-glutamic acid to GABA, has been purified from mammals, higher plants, and some microorganisms and their properties have already been reported. On the other hand, mushrooms are commonly appreciated as healthy food due to their nutritional properties, such as low calorie, rich in fiber, vitamin and mineral. L-Glutamic acid and GABA are also commonly distributed in edible mushrooms. The fact that mushrooms accumulate GABA suggests the existence of GAD. However, the enzyme property of mushroom GAD has not been reported. In the present study, we tried to purify and characterize the property of Flammulina veltipes. [Methods and Results] Mushroom (fruit-body of Flammulina veltipes) used in this study was purchased in local market. GAD activity was determined by formation of GABA from L-glutamic acid in the presence of pyridoxal-5’š- phosphate (PLP). The GAD activity was determined by the formation of GABA from L-glutamic acid. Fruit- body was crushed and then centrifuged to separate supernatant and cell debris. To investigate the localization of GAD, both supernatant and precipitate were subjected to enzyme reaction after dialysis. The cell debris showed stronger GAD activity than that of supernatant. The formation of GABA was observed between pH 4 and 6 and the maximum GAD activity was observed at pH 6. However, GAD activity was lost after dialysis for overnight against buffer of pH 6-11. These results suggest that GAD from Flammulina veltipes is stable at pH 4-5 in spite of its optimum pH for GABA production is around 6.

Effect of trehalose for the mycelial growth of Tricholoma matsutake were examined. When T. matsutake Z-1 strain was cultured in the partially modified matsutake liquid (PMML) medium and the Hamada matsutake liquid (HML) medium supplemented with trehalose at 24 ℃ for 80days, the vegetative mycelial dry weights showed higher value compared with those of PMML medium and HML medium supplemented with glucose (control). The range of the effect of 1.0-8.0% carbohydrate substrate on vegetative mycelial growth was investigated. The optimal concentration for mycelial growth was 2.0% for the glucose medium but 8.0% for the trehalose medium. To evaluate the potential of the production of trehalase from T. matsutake, the extracellular trehalase activity during the vegetative mycelial growth was measured. The activity of the extracellular trehalase increased during vegetative mycelial growth of T. matsutake and was maximal 70 days after inoculation. This extracellular enzyme was investigated for the purification and the characterization. The partially purified trehalase was obtained from about 1.53l static culture filtrate, with 19.1% recovery, and about 2,940 fold purification. The molecular mass was about 62.6kDa (SDS-PAGE) and 70kDa (Gel-filtration). The enzyme was most active around 40℃ and pH 5.0 and stable over a pH of 4.5~ 6.5 for 30min at 37℃. The enzyme readily hydrolyzed trehalose having α -1,1 glucosidic bond. However, it did not hydrolyze disaccharides such as maltose, iso-maltose, cellobiose, saccharose and lactose.