GH-16 Type β-1,3-Glucanase from Lysobacter sp. MK9-1 Enhances Antifungal Activity of GH-19 Type Chitinase,and Its Glucan-binding Domain Binds to Fungal Cell-wall

The GH-16 type β-1,3-glucanase (BgluC16MK) gene of Lysobacter sp. MK9-1 was cloned to study its antifungal activities. BgluC16MK displays amino acid sequence similarity with GluC from L. enzymogenes strain N4-7. BgluC16MK includes a signal sequence, a catalytic domain and carbohydrate-binding module family 6-type β-glucan binding domain (B-GBD). The expression of the BgluC16MK gene in Escherichia coli without the signal sequence resulted in antifungal activity at a dose of 0.6-0.8 nmol/disk. However, BgluC16MK displayed antifungal activity at a dose of 0.025 nmol/disk in combination with Chi19MK. Substrate-specific assay revealed that purified BgluC16MK hydrolyzed insoluble curdlan more readily than the soluble substrate. Furthermore, to explore the binding selectivity of B-GBD of BgluC16MK, we constructed a fusion protein (B-GBD-GFP) using the B-GBD and green fluorescent protein. The activity of the fusion protein against various substrates indicates that B-GBD was selective for glucans with β-1,3-linkages. An additional study demonstrated the binding ability of B-GBD-GFP to the cell-wall of living fungi, such as T. reesei and Aspergillus oryzae. These findings suggest that BgluC16MK can be utilized to generate antifungal enzyme preparations and that the fusion protein B-GBD-GFP can be used to identify the fungal cell surface structure using β-glucans.     

大豆脂肪氧化酶酶活性变化研究

采用分光光度法对大豆中三种脂肪氧化酶（同工酶）进行检测并测定脂肪氧化酶活性,研究表明：大豆粉碎后随着贮存时间的延长,脂肪氧化酶活性逐渐降低,大豆发芽后脂肪氧化酶活性降低了43%.     

A Comparative Biochemical Characterization of Microbial Transglutaminases: Commercial vs. a Newly Isolated Enzyme from <Emphasis Type="Italic">Streptomyces</Emphasis> Sp.

A new microbial transglutaminase (MTGase or MTG, EC 2.3.2.13) from a Streptomyces sp. strain isolated from Brazilian soil samples was characterized in crude and purified forms. The aim of this work is to provide relevant information about a new transglutaminase and to compare its characteristics with the well-known commercial transglutaminase from Ajinomoto Co. Inc. (Activa® TG-BP). The enzyme from Streptomyces sp., in both crude and pure forms, exhibited optimal activity in the 6.0–6.5 pH range and at 35–40°C. The results for the commercial enzyme were the same. A second maximum of activity was observed at pH 10.0 with both the crude Streptomyces sp. enzyme and the commercial enzyme. This interesting fact has not been reported in the literature previously. The fact that this second maximum of activity does not appear on the purified form of the enzyme may suggest the presence of an isoenzyme on the crude extract. All of the enzymes tested were stable over the pH range from 4.5 to 8.0 and up to 45°C. The decline in activity of the commercial transglutaminase above 45°C and pH 8.0 was more gradual. The activities of all the MTG samples were independent of Ca⁺² concentration, but they were elevated in the presence of K⁺, Ba²⁺, and Co²⁺ and inhibited by Cu²⁺ and Hg²⁺, which suggests the presence of a thiol group in the MTG’s active site. The purified enzyme presented a K _m of 6.37 mM and a V _max of 1.7 U/mL, while the crude enzyme demonstrated a K _m of 6.52 mM and a V _max of 1.35 U/mL.     

Crystallization kinetics of polymorphic forms of a molecular compound constructed by SOS (1,3-distearoyl-2-oleoyl-sn-glycerol) and SSO (1,2-distearoyl-3-oleoyl-rac-glycerol)

Crystallization kinetics of binary mixture phases of SOS (1,3-distearoyl-2-oleoyl-sn-glycerol) and SSO (1,2-distearoyl-3-oleoyl-rac-glycerol) were examined with time-resolved X-ray diffraction (XRD) and DSC methods. Formation of three polymorphic forms, named _c, β′_c and β_c, of a molecular compound was observed at a SOS/SSO=50/50 concentration ratio. The XRD data showed that all three polymorphs were arranged in a double chain-length structure, although a triple chain-length structure is formed in and β′ forms of SSO, and β′ and β forms of SOS. The time-resolved XRD studies showed that _c and β_c forms of the molecular compound crystallized more rapidly than the corresponding polymorphic forms of SOS and SSO. The results were discussed in terms of molecular chain–chain interactions of SOS and SSO, by assuming that kinetic processes of nucleation may prefer the formation of the _c and β_c forms arranged in the double chain-length structure.     

Isolation and Characterization of Two Types of Xyloglucanases from a Phytopathogenic Fungus,Verticillium dahliae

Xyloglucan is a major hemicellulosic component in plant cell walls. Phytopathogenic fungi secrete cell wall-degrading enzymes on their infection to hosts, while the nature of the cell wall-lytic enzymes of such fungi are yet to be fully understood. Verticillium dahliae is a soil-borne fungus that causes vascular wilt diseases in a variety of commercially important crops worldwide. We purified two types of xyloglucanases, XEG12A and XEG74B, from the culture of naturally isolated Verticillium dahliae strain 2148. XEG12A showed a molecular size of 23 kDa with its maximal activity at pH 7.5. XEG12A specifically hydrolyzed xyloglucan with no activity on other β-glucans. XEG74B had a molecular size of 110 kDa with its optimum pH at 6.0. XEG74B primarily hydrolyzed xyloglucan, with a slight activity on β-1,3-1,4-glucan. Analysis of hydrolytic products of xyloglucanooligasaccharide (XXXGXXXG) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) revealed that the both enzymes cleaved β-1,4-glucosidic linkage at the position of unbranched chain, while XEG74B showed a little fluctuation with the cleavage site. Both enzymes did not hydrolyzed xyloglucanoheptasaccharide (XXXG) at all. N-Terminal and internal amino acid sequencing of the enzymes revealed that XEG12A and XEG74B belonged to Glycoside Hydrolase (GH) Families 12 and 74, respectively. Based on these results we concluded that V. dahliae XEG12A and XEG74B were xyloglucan-specific endo-β-1,4-glucanases (EC 3.2.1.151).     

High‐fat Diet‐induced Intestinal Hyperpermeability is Associated with Increased Bile Acids in the Large Intestine of Mice

Metabolic syndrome is characterized by low‐grade chronic systemic inflammation, which is associated with intestinal hyperpermeability. This study examined the effects of 3 high‐fat diets (HFDs) composed of different fat sources (soybean oil and lard) on the intestinal permeability, tight junction (TJ) protein expression, and cecal bile acid (BA) concentrations in mice, and then analyzed their interrelations. C57/BL6 mice were fed the control diet, HFD (soybean oil), HFD (lard), and HFD (mix; containing equal concentrations of soybean oil and lard) for 8 wk. Glucose tolerance, intestinal permeability, TJ protein expression, and cecal BA concentration were evaluated. Feeding with the 3 HDFs similarly increased body weight, liver weight, and fat pad weight, and induced glucose intolerance and intestinal hyperpermeability. The expression of TJ proteins, zonula occludens‐2 and junctional adhesion molecule‐A, were lower in the colons of the 3 HFD groups than in the control group (P < 0.05), and these changes appeared to be related to intestinal hyperpermeability. Feeding with HFDs increased total secondary BA (SBA) and total BA concentrations along with increases in some individual BAs in the cecum. Significant positive correlations between intestinal permeability and the concentrations of most SBAs, such as deoxycholic acid and ω‐muricholic acids, were detected (P < 0.05). These results suggest that the HFD‐induced intestinal hyperpermeability is associated with increased BA secretion. The abundance of SBAs in the large intestine may be responsible for the hyperpermeability.     

Evaluation of the Anti-Proliferative Activity of Rare Aldohexoses against MOLT-4F and DU-145 Human Cancer Cell Line and Structure-Activity Relationship of D-Idose

D-Allose (D-All), a C-3 epimer of D-glucose (D-Glc), is a naturally rare monosaccharide, which shows anti-proliferative activity against several human cancer cell lines. Unlike conventional anticancer drugs, D-All targets glucose metabolism and is non-toxic to normal cells. Therefore, it has attracted attention as a unique “seed” compound for anticancer agents. However, the anti-proliferative activities of the other rare aldohexoses have not been examined yet. In this study, we evaluated the anti-proliferative activity of rare aldohexoses against human leukemia MOLT-4F and human prostate cancer DU-145 cell lines. We found that D-All and D-idose (D-Ido) at 5 mM inhibited cell proliferation of MOLT-4F cells by 46 % and 60 %, respectively. On the other hand, the rare aldohexoses at 5 mM did not show specific anti-proliferative activity against DU-145 cells. To explore the structure–activity relationship of D-Ido, we evaluated the anti-proliferative activity of D-sorbose (D-Sor), 6-deoxy-D-Ido, and L-xylose (L-Xyl) against MOLT-4F cells and found that D-Sor, 6-deoxy-D-Ido, and L-Xyl showed no inhibitory activity at 5 mM, suggesting that the aldose structure and the C-6 hydroxy group of D-Ido are important for its activity. Cellular glucose uptake assay and western blotting analysis of thioredoxin-interacting protein (TXNIP) expression suggested that the anti-proliferative activity of D-Ido is induced by inhibition of glucose uptake via TXNIP-independent pathway.     

Effects of Phosphoryl Oligosaccharides of Calcium (POs-Ca) on Mycelial Growth and Fruiting Body Development of the Edible Mushroom,Pleurotus ostreatus

Phosphoryl oligosaccharides of calcium (POs-Ca) is a calcium salt of phosphoryl maltooligosaccharides made from potato starch. POs-Ca is highly water-soluble and can supply both the calcium ion and acidic oligosaccharides in an aqueous solution. In this study, we investigated the effects of POs-Ca on the mycelial growth and fruiting body yield of Pleurotus ostreatus , which is one of the most widely cultivated edible mushrooms in the world. We cultivated the mushroom using both potato dextrose agar (PDA) medium and sawdust-based medium, with added calcium salts. The addition of POs-Ca into the PDA medium with a calcium concentration of 10 mg increased mycelial growth significantly ( p < 0.05, vs . control). POs-Ca addition to the sawdust-based medium at concentrations of 1.0 to 3.0 g/100 g medium increased the amount of calcium in the fruiting bodies but did not affect the length of the cultivation period or the weight of the fruiting body. The calcium content in the fruiting body increased 12-fold when compared to the control. On the other hand, neither the CaHPO ₄ ･2H ₂ O group nor the CaHPO ₄ ･2H ₂ O with oligosaccharides group showed changes in the calcium content of the fruiting bodies. Our results indicate that the use of POs-Ca in mushroom cultivation allows for the possibility of developing new functional foods like calcium-enriched edible mushrooms. This is the first report describing the effects of POs-Ca on mushroom cultivation.