Takao Terashita

Purification and Characterization of Thermostable Pectate Lyase with Protopectinase Activity from Thermophilic Bacillus sp. TS 47

A strain of thermophilic bacterium, Bacillus sp., with pectolytic activity has been isolated. It produced an extracellular endo-polygalacturonate trans-eliminase (PL, EC 4.2.2.1) when grown at 60°C on a medium containing polygalacturonate (PGA). The PL was purified by hydrophobic, cation exchange, and size exclusion column chromatographies. The molecular mass of the enzyme was 50 kDa by SDS-PAGE. The isoelectric point of the enzyme was pH 5.3. The enzyme had a half-life of 13 and 1 h at 65 and 70°C, respectively, and showed optimal activity around at 70°C and pH 8.0. It had protopectinase activity, besides PL activity, on lemon protopectin and cotton fibers. The first 20 amino acids sequence of the enzyme had significant similarity with that of PL from methophilic Bacillus subtilis, with 50% identity.

Molecular Cloning, DNA Sequence, and Expression of the Gene Encoding for Thermostable Pectate Lyase of Thermophilic Bacillus sp. TS 47

The gene that encodes a thermostable pectate lyase (called PL 47), from Bacillus sp. TS 47, was cloned, sequenced, and expressed in mesophilic B. subtilis. The gene contained an open reading frame consisting of 1326 bp, which encoded 441 amino acids. The deduced amino acid sequence of the mature enzyme (416 amino acids with a calcuated molecular mass of 47, 262 Da), showed 52% similarity with PL (BsPel) from mesophilic B. subtilis SO113. The structure-based alignment of the deduced amino acid sequence of PL 47 with that of BsPel suggested that PL 47 might have a parallel β-helix structure with three long loops. The amino acids making up PL 47 are richer in hydrophobic amino acids and glutamic acid than BsPel. The hydropathy profile of PL 47 indicated that the amino acid sequences around putative calcium binding sites are more hydrophobic than the same region of BsPel. The gene product expressed in B. subtilis as the host was stable up to 70°C and the reaction was optimal around 70°C, as well as native PL 47.

Productivity of hydrolytic enzymes by mycorrhizal mushrooms

To survey the potential for production of extracellular hydrolytic enzymes by mycorrhizal mushrooms, productivities of these exo-enzymes from mycelia on potato-dextrose liquid medium were determined.Tricholoma matsutake produced relatively high levels of CM-cellulase and avicelase activities in all test strains. It also produced higher activity of acid proteinase than neutral proteinase. Its xylanase activities seemed to be higher than those of the other carbohydrases. The productivities ofLyophyllum shimeji strains were at similar levels to those ofT. matsutake strains. CM-cellulase and avicelase activities ofL. shimeji were higher than those ofT. matsutake. Neutral proteinase inL. shimeji strains showed higher activity levels than acid proteinase. The relative productivities of hydrolytic enzymes between the groups of mycorrhizal mushrooms and wood-rotting mushrooms were also examined.T. matsutake andL. shimeji both produce many kinds of hydrolytic enzymes in their culture broth, and the potential for production of hydrolytic enzymes by mycorrhizal mushrooms was judged to be relatively high.

Detection of β-glucosidase as saprotrophic ability from an ectomycorrhizal mushroom, Tricholoma matsutake

We investigated extracellular carbohydrase production in the medium of an ectomycorrhizal fungus, Tricholoma matsutake, to reveal its ability to utilize carbohydrates such as starch as a growth substrate and to survey the saprotrophic aspects. We found β-glucosidase activity in the static culture filtrate of this fungus. The β-glucosidase was purified and characterized. The purified enzyme was obtained from about 2.1 l static culture filtrate, with 9.0% recovery, and showed a single protein band on SDS-PAGE. Molecular mass was about 160 kDa. The enzyme was most active around 60°C and pH 5.0, and stable over a pH of 4.0–8.0 for 30 min at 37°C. The purified enzyme was activated by the presence of Ca2+ and Mn2+ ions (about 2–3 times that of the control). The enzyme readily hydrolyzed oligosaccharides having a β-1,4-glucosidic linkage such as cellobiose and cellotriose. However, it did not hydrolyze polysaccharides such as avicel and CM-cellulose or oligosaccharides having an α-glucosidic linkage. Moreover, cellotriose was hydrolyzed by the enzyme for various durations, and the resultant products were analyzed by TLC. We concluded that the enzyme from T. matsutake seems to be a β-glucosidase because cellotriose with a β-1,4-glucosidic linkage decomposed to glucose during the enzyme reaction.

Antimutagenic activity of extracts from Japanese eggplant

Using the Salmonella/microsome assay, the antimutagenic effects of specific components of the extracts from eggplant fruits were investigated. The eggplant fruit juice exhibited an antimutagenic activity against 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) induced mutagenicity. In some of the fractions extracted with several organic solvents (acetone, petroleum ether, ethyl acetate; and methanol), the activity was recognized. No mutagenicity or toxicity for Salmonella typhimurium TA98 in the presence of S9 mixture was observed with any of the extracts. It is suggested that there are multiple components of the activities that exist in the eggplant fruit. We isolated lutein from the 84% methanol (methanol/water, v/v) layer, pheophorbide or chlorophyllide from the 70% methanol layer and tannins containing sugar-moieties from the water layer. Pheophytin a and b, Mg-free derivatives of chlorophyll a and b, were isolated from the petroleum ether layer as possible antimutagens. The pheophytin a with S9 mix inhibited by 30-40% the mutagenicity of Trp-P-2.

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

AbstractΑ-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.

Use of genetically engineered Salmonella typhimurium OY1002/1A2 strain coexpressing human cytochrome P450 1A2 and NADPH-cytochrome P450 reductase and bacterial O-acetyltransferase in SOS/umu assay.

The major pathway of bioactivation of procarcinogenic heterocyclic aromatic amines (HCAs) is cytochrome P450 1A2 (CYP1A2)–catalyzed N‐hydroxylation and subsequent esterification by O‐acetyltransferase (O‐AT). We have previously reported that an umu tester strain, Salmonella typhimurium OY1001/1A2, endogenously coexpressing human CYP1A2 and NADPH‐P450 reductase (reductase), is able to detect the genotoxicity of some aromatic amines [Aryal et al., 1999, Mutat Res 442:113–120]. To further enhance the sensitivity of the strain toward HCAs, we developed S. typhimurium OY1002/1A2 by introducing pCW″/1A2:hNPR (a bicistronic construct coexpressing human P450 1A2 and the reductase) and pOA102 (constructed by subcloning the Salmonella O‐AT gene in the pOA101‐expressing umuC″lacZ gene) in S. typhimurium TA1535. In addition, as an O‐AT–deficient strain, we developed the OY1003/1A2 strain by introducing pCW″/1A2:hNPR and pOA101 into O‐AT–deficient S. typhimurium TA1535/1,8‐DNP. Strains OY1001/1A2, OY1002/1A2, and OY1003/1A2 expressed, respectively, about 150, 120, and 140 nmol CYP1A2/l culture (in whole cells), and respective cytosolic preparations acetylated 15, 125, and ≧0 nmol isoniazid/min/mg protein as the O‐AT activities of cytosolic preparations, respectively. We compared the induction of umuC gene expression as a measure of genotoxicity and observed that the OY1002/1A2 strain was more sensitive than OY1001/1A2 strain toward the genotoxicity of 2‐amino‐1,4‐dimethylimidazo[4,5‐f]quinoline (MeIQ), 2‐amino‐3‐methylimidazo[4,5‐f]quinoline (IQ), 2‐amino‐3,8‐dimethylimidazo[4,5‐f]quinoxaline (MeIQx), 2‐aminoanthracene, 2‐amino‐6‐methyldipyrido[1,2‐a::3,2′‐d]imidazole, 3‐amino‐1,4‐dimethyl‐5H‐pyrido[4,3‐b]indole, and 3‐amino‐1‐methyl‐5H‐pyrido[4,3‐a]indole. However, the genotoxicity of MeIQ, IQ, and MeIQx was not detected with the OY1003/1A2 strain. These results indicate that the newly developed strain OY1002/1A2 can be employed in detecting potential genotoxic aromatic amines requiring bioactivation by CYP1A2 and O‐acetyltransferase. Environ. Mol. Mutagen. 36:121–126, 2000.

Characterization of extracellular glucoamylase from the ectomycorrhizal mushroom Lyophyllum shimeji

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 Ca2+ ion (7.7 times that of the control), but Ba2+ and Ag+ 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, β-anomer glucose was produced. From these results, we concluded that the amylase from L. shimeji seems to be a glucoamylase.

Amanita ibotengutake sp. nov., a poisonous fungus from Japan

On the basis of molecular phylogeny, it is clear that the fungi treated as Amanita pantherina in Japan are confused, i.e., A. pantherina and a misidentified species. As for morphology, the misidentified species differs from A. pantherina by having clamp connections on hyphae and basidia, a larger-sized fruitbody, ascending volval rings of stipe base and deciduous annulus. It is clear that the misidentified species is a poisonous fungus containing ibotenic acid and muscimol and causes the Pantherina syndrome. On these characters, it is concluded that the misidentified species is the unexplored Amanita species treated under the Japanese name ‘ibotengutake’ after which ibotenic acid was named. Here, we described it as a new species Amanita ibotengutake.

Molecular Cloning of the Gene Encoding Thermostable Endo-1,5-α-L-arabinase of Bacillus thermodenitrificans TS-3 and Its Expression in Bacillus subtilis

The gene that encodes a thermostable endo-arabinase (called ABN-TS) from Bacillus thermodenitrificans TS-3 was cloned, sequenced, and expressed in the mesophilic B. subtilis. The gene contained an open reading frame consists of 939 bp, which encodes 313 amino acids. The deduced amino acid sequence of the enzyme showed 50, 46, and 36% similarity with endo-arabinase from B. subtilis IFO 3134 (PPase-C), Pseudomonas fluorescens (ArbA), and Aspergillus niger (ABNA), respectively. The hydrophobic and acidic amino acids making up ABN-TS outnumbered those in PPase-C. The gene product expressed in B. subtilis, as the host, had substantially the same characteristics, and was stable up to 70°C, and the reaction was optimal around 70°C, as well as native ABN-TS.