Phenol and cresol mixture degradation by the yeast <Emphasis Type="Italic">Trichosporon cutaneum</Emphasis>

Most industrial wastes contain different organic mixtures, making important the investigation on the microbial destruction of composite substrates. The capability of microbes to remove harmful chemicals from polluted environments strongly depends on the presence of other carbon and energy substrates. The effect of mixtures of phenol- and methyl-substituted phenols (o-, m-, p-cresol) on the growth behaviour and degradation capacity of Trichosporon cutaneum strain was investigated. The cell-free supernatants were analysed by HPLC. It was established that the presence of o-, m- and p- cresol has not prevented complete phenol assimilation but had significant delaying effect on the phenol degradation dynamics. The mutual influence of phenol and p-cresol was investigated. We developed the kinetic model on the basis of Haldane kinetics, which used model parameters from single-substrate experiments to predict the outcome of the two-substrate mixture experiment. The interaction coefficients indicating the degree to which phenol affects the biodegradation of p-cresol and vice versa were estimated. Quantitative estimation of interaction parameters is essential to facilitate the application of single or mixed cultures to the bio-treatment of hazardous compounds.     

利用转座标签mTn-lacZ/leu2插入突变发酵性丝孢酵母2.1368-Leu?筛选高效产油突变株

为提高微生物油脂产率,降低其生产成本,以转座标签mTn-lacZ/leu2插入突变发酵性丝孢酵母2.1368-Leu?筛选高效产油突变株。利用LacZ显色反应、脂肪酸合成酶抑制剂Cerulenin和磷酸香草醛反应,最终在玉米秸秆糖化液中筛选出一株高效产油突变株2.1368-Leu?-7。结果表明其油脂含量为38.30％,比对照的29.33％高了8.97％,而其产油率为8.35％,比对照的6.92％提高了20.63％;在玉米秸秆糖化液中的糖利用率为77％,每100 g玉米秸秆可转化油脂8.32 g。可为未来生物柴油产业提供了廉价原料。     

阿萨希毛孢子菌生物膜构建及水杨酸干预策略研究

目的在医院内常用生物材料聚氯乙烯（PVC）表面构建阿萨希毛孢子菌的生物膜,评估该生物膜对几种临床抗真菌药物的耐药性,并观察水杨酸是否对阿萨希毛孢子菌生物膜的形成有干预作用。方法菌株鉴定采用API20CAUX并经PCR鉴定复核;使用PVC于RPM11640-MOPS中培养进行阿萨希毛孢子菌生物膜构建;MIC测定采用法国生物梅里埃公司ATB Fungus-3真菌药敏试剂条以及微量液体稀释法,并观察水杨酸对生物膜构建的影响。结果阿萨希毛孢子菌可以通过几个不连续阶段在聚氯乙烯表面形成生物膜,且已使用PVC块上附着的生物膜细胞比未使用PVC块上黏附的生物膜细胞明显密集;固着相即生物膜细胞的MIC比浮游相成倍提高;24h两性霉素B的MIC〉512μ/ml,且经两性霉素B的药物刺激后,阿萨希毛孢子明显可见芽管延长,菌丝交织;水杨酸作用后阿萨希毛孢子菌的菌丝明显变短,孢子短小。结论介入性器械可以作为阿萨希毛孢子菌生物膜构建的黏附基质,使微生物群体黏附于细胞外多聚材料表面而造成持续播散感染,因此生物膜干预对阿萨希毛孢子菌深部感染的治疗有很重要的意义。     

对产胞内脂肪酶的发酵性丝孢酵母超声波透性化处理的条件优化

发酵性丝孢酵母产胞内脂肪酶,为了把细胞整体作为脂肪酶催化剂,需对细胞进行透性化处理.利用超声波进行细胞透性化的适宜条件为:超声波输出功率180 W,每次辐射时间2s(间歇时间5 s),工作总时间1.2 min,菌体浓度40 g/L,此条件下细胞通透性可明显改善,透性化细胞脂肪酶表现出较高活力.     

Physiological role of microbodies in the yeast Trichosporon cutaneum during growth on ethylamine as the source of energy,carbon and nitrogen

Compartmentation of the metabolism of ethylamine in Trichosporon cutaneum X4 was studied in cells, grown on this compound as the sole source of energy, carbon, and nitrogen. Transfer experiments indicated that an amine oxidase is involved in the early metabolism of ethylamine. The synthesis of this enzyme was induced by primary amines and was subject to partial carbon catabolite repression. Repression by ammonium ions was not observed. Adaptation of glucose-grown cells to growth on ethylamine was associated with the development of many microbodies, which developed from already existing organelles present in the inoculum cells and multiplied by division. Cytochemical experiments indicated that the organelles contained amine oxidase and catalase. Therefore, they were considered to play a key role in the metabolism of ethylamine. The physiological significance of the microbodies was investigated by fractionation studies of homogenized protoplasts from ethylamine-grown cells by differential- and sucrose-gradient centrifugation of subcellular organelles. Intact microbodies were only obtained when the isolation procedure was performed at pH 5.8 in the absence of Mg²⁺-ions. Analysis of the different fractions indicated that the key enzymes of the glyoxylate cycle, namely isocitrate lyase and malate synthase, cosedimented together with catalase and amine oxidase. In addition, activities of malate dehydrogenase, glutamate:oxaloacetate aminotransferase (GOT) and (NAD-dependent) glutamate dehydrogenase were detected in these fractions. Electron microscopy revealed that they mainly contained microbodies. Cytochemical experiments indicated that the above enzymes were all present in the same organelle. These findings suggest that microbodies of ethylamine-grown T. cutaneum X4 produce aspartate, so allowing NADH generated in the oxidation of malate by malate dehydrogenase to be quantitatively reoxidized inside the organelles in a series of reactions involving GOT and glutamate dehydrogenase. Aspartase and fumarase were not detected in the microbodies; activities of these two enzymes were present in the cytoplasm.Abbreviations ABTS 2,2-Azino-di(3-ethylbenzthiazoline sulfonate [6]) - DTT dithiothreitol - GOT glutamate:oxaloacetate aminotransferase - DTNB 5,5-dithiobis-2-nitrobenzoate - DAB diaminobenzidine - BSPT 2-(2-benzothiazolyl)-3-(4-phthalhydrazidyl)-t-styryl-sH-tetrazolium chloride - PF convex fracture face - EF concave fracture face     

酮基布洛芬拆分用酯酶产生菌的筛选及其催化特性

从土壤中筛选获得一株可以高对映选择性水解酮基布洛芬乙酯的酵母KET4,经鉴定为芸苔丝孢酵母（Trichosporon brassicae）。研究了该菌的生长和产酶过程,考察了其静息细胞对酮基布洛芬乙酯水解的催化特性。用该菌催化酯水解时,转化率为41%时,产物的对映体过量值为91%,对映选择率达到45。     

Biocatalytic resolution of nitro-substituted phenoxypropylene oxides with Trichosporon loubierii epoxide hydrolase and prediction of their enantiopurity variation with reaction time

Biocatalytic resolution of 3-(2′-nitrophenoxy)propylene oxide (1a), 3-(3′-nitrophenoxy)propylene oxide (1b) and 3-(4′-nitrophenoxy)propylene oxide (1c) were exploited by using lyophilized cells of yeast Trichosporon loubierii ECU1040 with epoxide hydrolase (EH) activity, which preferentially hydrolyzes (S)-enantiomers of the epoxides (1a–c), yielding (S)-diols and (R)-epoxides. The activity increased as the nitro group in the phenyl ring was shifted from 4′-position (1c) to 2′-position (1a). When the substrate concentration of 1a was increased from 10 to 80 mM, the E-value increased at first, until reaching a peak at 40 mM, and then decreased at higher concentrations (>40 mM). The optically active epoxide (R)-1a was prepared at gram-scale (97% ee, 41% yield). Furthermore, a simple method was developed to predict the enantiomeric excess of substrate (ee_s) at any time of the whole reaction course based on the ee_s value determined at a certain reaction time at a relatively lower substrate concentration. This will be helpful for terminating the reaction at a proper time to get both higher optical purity and higher yield of the remaining epoxides.     

Purification and Characterization of a (R)-1-Phenyl-1,3-propanediol-producing Enzyme from Trichosporon fermentans AJ-5152 and Enzymatic (R)-1-Phenyl-1,3-propanediol Production

An (R)-1-phenyl-1,3-propanediol-producing enzyme was purified from Trichosporon fermentans AJ-5152. It was NADPH-dependent and converted 3-hydroxy-1-phenylpropane-1-one (HPPO) to (R)-1-phenyl-1,3-propanediol [(R)-PPD] with anti-Prelog’s specificity. It showed maximum activity at pH 7.0 and 40 °C. Its K _m and V _max values toward HPPO were 20.1 mM and 3.4 μmol min^?1 mg protein^?1 respectively. The relative molecular weight of the enzyme was estimated to be 68,000 on gel filtration and 32,000 on SDS-polyacrylamide gel electrophoresis. An (R)-PPD-producing reaction using the (R)-PPD-producing enzyme and an NADPH recycling system was carried out by successive feeding of HPPO. A total (R)-PPD yield of 8.9 g/l was produced in 16 h. The molar yield was 76%, and the optical purity of the (R)-PPD produced was over 99% e.e.     

Structural studies of the antigen III cell wall polysaccharide of Trichosporon domesticum

Cell wall and soluble polysaccharides that reacted with Trichosporon domesticum factor III serum were isolated from the type strain of T. domesticum. The fractions contained O-acetyl groups, which contributed to the serological reactivity. The antigenic structure was characterized by chromatographic and spectroscopic methods. The polysaccharide has an alpha-(1-->3)-D-mannan backbone with hetero-oligosaccharide side chains consisting of a 2-O-substituted beta-D-glucuronic acid residue bound to O-2 of the mannose residue, beta-D-xylopyranosyl residues located in the middle of the side chain, and a nonreducing terminal alpha-L-arabinopyranosyl residue bound to 0-4 of xylose. The mannan backbone is O-acetylated at O-6 of the mannose residues.