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.     

Chlorine isotope fractionation during reductive dechlorination of chlorinated ethenes by anaerobic bacteria

Chlorine isotope fractionation during reductive dechlorination of trichloroethene (TCE) and tetrachloroethene (PCE) to cis-1,2-dichloroethene (cDCE) by anaerobic bacteria was investigated. The changes in the 37Cl/35Cl ratio observed during the one-step reaction (TCE to cDCE) can be explained by the regioselective elimination of chlorine accompanied by the Rayleigh fractionation. The fractionation factors (alpha) of the TCE dechlorination by three kinds of anaerobic cultures were approximately 0.994-0.995 at 30 degrees C. The enrichment of 37Cl in the organic chlorine during the two-step reaction (PCE to cDCE) can be explained by the random elimination of one chlorine atom in the PCE molecule followed by the regioselective elimination of one chlorine atom in the TCE molecule. The fractionation factors for the first step of the PCE dechlorination with three kinds of anaerobic cultures were estimated to be 0.987-0.991 at 30 degrees C using a mathematical model. Isotope fractionation during the first step would be the primary factor for the chlorine isotope fractionation during the PCE dechorination to cDCE. The developed models can be utilized to evaluate the fractionation factors of regioselective and multistep reactions.     

Mating-induced mating-type cassette conversion in Saccharomyces cerevisiae

We have previously reported that the HMRa-bearing restriction fragment of a rho degrees sir4-11 strain (HMLalpha-MATalpha-HMRa), which acts as an alpha-mater because of being rho degrees , changes its electrophoretic mobility when the strain mates with a certain group of a-mating strains (HMLalpha-MATa-HMRa). In this study, we found that the sir4-11 strain being rho degrees was not essential for this phenomenon and also that the altered form of the fragment contained HMRalpha in place of HMRa. Furthermore, we observed conversion of HMLa to HMLalpha in the cross in which a sir4-11 HMLa-MATalpha-HMRalpha strain was mated with a representative of the above-mentioned a-mating strain. In addition, when this a-mating strain was mated with a SIR(+) HMLalpha-MATa-HMRalpha strain, the resultant diploid was found to be HMLalpha/HMLalpha MATa/MATalphaHMRa/HMRalpha, indicating that conversion of MATa to MATalpha had taken place in the course of mating. From all these observations, we conclude that there is a group of S. cerevisiae strains that carries factor(s) that induces conversion of a mating-type cassette of the mating partner to alpha mating-type cassette and that this mating type cassette conversion takes place in all three mating type loci, HML, MAT and HMR, if the loci are in the non-silenced condition.     

Efficient synthesis of nonnatural mutants in Escherichia coli S30 in vitro protein synthesizing system

Factors that affect the efficiency of in vitro synthesis of mutant proteins that contain nonnatural amino acids were investigated. The process of the nonnatural mutagenesis consists of chemical aminoacylation of a tRNA that contains a 4-base anticodon, followed by in vitro synthesis in the presence of an mRNA that contains the corresponding 4-base codon. Detailed studies on the time courses of the synthesis revealed two major factors that suppress the yield of nonnatural mutants compared with the wild-type protein. First, a cyclic tRNA that exists as a by-product of the chemical aminoacylation inhibits the protein synthesis. Second, the very short lifetime of a tRNA aminoacylated with a nonnatural amino acid limits the protein yield. As a simple and practical way of surmounting these factors, aminoacyl tRNA was added into the in vitro system at 5 min after the start of the synthesis. The addition increased the protein yield up to the level of conventional proteins in the in vitro system.