A 31P NMR study of the internal pH of yeast peroxisomes

The internal pH of peroxisomes in the yeasts Hansenula polymorpha, Candida utilis and Trichosporon cutaneum X4 was estimated by ³¹P nuclear magnetic resonance (NMR) spectroscopy. ³¹P NMR spectra of suspensions of intact cells of these yeasts, grown under conditions of extensive peroxisomal proliferation, displayed two prominent P_i-peaks at different chemical shift positions. In control cells grown on glucose, which contain very few peroxisomes, only a single peak was observed. This latter peak, which was detected under all growth conditions, was assigned to cytosolic P_i at pH 7.1. The additional peak present in spectra of peroxisome-containing cells, reflected P_i at a considerably lower pH of approximately 5.8–6.0. Experiments with the protonophore carbonyl cyanide m-chlorophenylhydrazon (CCCP) and the ionophores valinomycin and nigericin revealed that separation of the two P_i-peaks was caused by a pH-gradient across a membrane separating the two pools. Experiments with chloroquine confirmed the acidic nature of one of these pools. In a number of transfer experiments with the yeast H. polymorpha it was shown that the relative intensity of the P_i-signal at the low pH-position was correlated to the peroxisomal volume fraction. These results strongly suggest that this peak has to be assigned to P_i in peroxisomes, which therefore are acidic in nature. The presence of peroxisome-associated P_i was confirmed cytochemically.Abbreviations CCCP Carbonyl cyanide m-chlorophenylhydrazon - DCCD N,N-dicyclohexylcarbodiimide     

Oxidation of amines by yeasts grown on 1-aminoalkanes or putrescine as the sole source of carbon,nitrogen and energy

The maximum growth rate of Trichosporon cutaneum CBS 8111 in chemostat cultures was 0.185 h^-1 on ethylamine and 0.21 h^-1 on butylamine, that of Candida famata CBS 8109 was 0.32 h^-1 on putrescine.The amine oxidation pattern of the ascomycetous strains studied, viz. Candida famata CBS 8109, Stephanoascus ciferrii CBS 4856 and Trichosporon adeninovorans CBS 8244 was independent of the amine that had been used as the growth substrate. It resembled that of benzylamine/putrescine oxidase found in other ascomycetous yeasts. However, differences in pH optimum and substrate specificity were observed between the amine-oxidizing systems of these three species.The amine oxidation pattern of cell-free extracts of Trichosporon cutaneum CBS 8111 varied with the amine that was used as growth substrate. The enzyme system produced by Cryptococcus laurentii CBS 7140 failed to oxidize isobutylamine and benzylamine, and showed a high pH optimum.The synthesis of amine oxidase in the four yeast strains studied was not repressed by ammonium chloride and was weakly repressed by glucose but was strongly repressed if both compounds were present in the growth medium.     

丝孢酵母高甲硫氨酸突变株的选育及营养调控

以丝孢酵母(Trichosporon Behr)ST851为原始菌株,经紫外线诱变,在含乙硫氨酸的双层平板上筛选到多株抗乙硫氨酸突变株。其中ST851-10株抗乙硫氨酸浓度达到350μg/ml,其菌体蛋白质含量由40.5％提高到44.3％,菌体甲硫氨酸含量由20.45mg/g-DCW增加到29.32mg/g-DCW。在以苹果渣为碳源、尿素为氮源、硫酸镁作硫源的最适培养条件下,固态发酵24h后,蛋白质和甲硫氨酸含量较原始菌株分别提高了15.8％和44.9％。培养基中C/N值低有利于甲硫氨酸的合成,C/N值高则适合于菌体生长。在苹果渣固态发酵过程中,适当补加氮源既有利于菌体生长和甲硫氨酸的合成,又可起到调节培养基pH值的作用。     

Biotransformation of diphenyl ether by the yeastTrichosporon beigelii SBUG 752

Trichosporon beigelii SBUG 752 was able to transform diphenyl ether. By TLC, HPLC, GC, GC-MS, NMR- and UV-spectroscopy, several oxidation products were identified. The primary attack was initiated by a monooxygenation step, resulting in the formation of 4-hydroxydiphenyl ether, 2-hydroxydiphenyl ether and 3-hydroxydiphenyl ether (48:47:5). Further oxidation led to 3,4-dihydroxydiphenyl ether. As a characteristic product resulting from the cleavage of an aromatic ring, the lactone of 2-hydroxy-4-phenoxymuconic acid was identified. The possible mechanism of ring cleavage to yield this metabolite is discussed.     

国内首例皮瘤丝孢酵母真菌血症分离株的表型及rDNA序列鉴定

目的通过对1例“脂膜炎”患者外周血分离株的表型及分子生物学鉴定,报道国内首例皮瘤丝孢酵母菌血症。方法采集患者的血及鼻咽结节组织标本通过真菌培养获得分离株,经沙氏培养基、马铃薯培养基、科玛嘉念珠菌显色培养后形态学观察及API 20C AUX系统鉴定,最后经PCR扩增rDNA基因测序证实。采用CLSI制定的M27-A2微量稀释法对病原菌株进行7种药物的体外药敏试验。结果分离株通过表型和分子生物学鉴定为“皮瘤丝孢酵母”。药敏结果显示菌株对两性霉素B耐药,氟康唑、伏立康唑、伊曲康唑、5-氟胞嘧啶敏感。卡泊芬净的抑菌浓度较低,而米卡芬净的抑菌浓度相对较高。患者因多种并发症死亡。结论皮瘤丝孢酵母引起的真菌血症为我国首例报道。API 20C AUX酵母鉴定系统可作为鉴定皮瘤丝孢酵母快速而简便的方法。通过分子生物学DNA序列检测分析能鉴定皮瘤丝孢酵母。     

Trichosporon mycotoxinivorans sp. nov., a new yeast species useful in biological detoxification of various mycotoxins

A yeast strain isolated from the hindgut of the lower termite Mastotermes darwiniensis (Mastotermitidae) was found to represent a new member of the genus Trichosporon. Trichosporon mycotoxinivorans is closely related to T. loubieri on the basis of the phylogenetic trees based on the D1/D2 region of 26S rDNA, an approx. 600 bp fragment of the 18S rDNA and both ITS regions. However, the two species differ at nine positions in the D1/D2 region of 26S rDNA. The IGS1 region of T. mycotoxinivorans is 401 bp long. T. mycotoxinivorans is distinguished from T. loubieri by its ability to assimilate inulin and galactitol, and its inability to grow at 40 °C. The name of this newly isolated strain refers to an important characteristics of T. mycotoxinivorans to detoxify mycotoxins such as ochratoxin A and zearalenone. Therefore this strain can be used for the deactivation of the respective mycotoxins in animal feeds.     

响应面法优化产酰胺酶发酵培养基

采用响应面分析方法,对阿萨希丝孢酵母（Trichosporon asahii）ZZB-1产酰胺酶的发酵培养基进行了优化。运用单N子试验筛选出麦芽糖和酵母浸膏为最适碳源、氮源,金属离子Ca^2＋、Mn^2＋可提高发酵酰胺酶产量;通过最陡爬坡实验逼近以上4个因子的最大响应区域后,采用Box—Behnken响应面分析法,确定产酰胺酶最佳发酵培养基为麦芽糖18．84g／L、酵母浸膏9．55g／L、NaC15g／L、KH2PO41g／L、MgSO4·7H2O0．2g／L、FeS040．001g／L、CaC0370．84μmol／L、MnS0465．39肚mo[／L（1％丙烯酸诱导）,NH4·H2O调节pH至7．0。培养基优化后酰胺酶产量由初始2554U／L提高到4156U／L,为原始发酵培养基配方酶活产量的1．63倍。     

粘性丝孢酵母的诱变及以玉米秸秆水解液为原料产油条件优化

对粘性丝胞酵母进行紫外诱变,获得一株产油率较高的菌株,较原菌株提高了1.53倍。将该菌株接种于用1%硫酸和酶水解处理并浓缩至还原糖浓度为5%的玉米秸秆水解液中培养,生长较好。通过四因素三水平正交实验,确定培养条件为初始pH值7.0、接种量1%、发酵温度30℃、发酵时间5 d时产油率最高。对最佳产油条件进行验证,测得油脂含量为21.3%。从而为利用农业废弃物大规模生产微生物油脂提供了试验数据。气相色谱-质谱联用分析仪显示油脂脂肪酸组成为棕榈酸28.36%,油酸55.86%,10-十八烯酸9.23%,硬脂酸6.70%,可以作为原料生产生物柴油。     

Trichosporon siamense sp. nov. isolated from insect frass in Thailand

A strain of yeast isolated from insect frass collected in Thailand was found to represent a hitherto undescribed species of a basidiomycetous anamorphic genus Trichosporon. It is described as Trichosporon siamense. In the phylogenetic tree based on the D1/D2 region sequences of 26S rDNA, this yeast constitutes a cluster with several Q-9 having species of Trichosporon including T. otae and T. brassicae but is clearly differentiated from these species by 1.8% or more base substitutions. In the internal transcribed spacer region (ITS1 and ITS2), this species differs from T. scarabaeorum, the nearest species, by 6.5% base substitution.     

Efficient lipid production with Trichosporon fermentans and its use for biodiesel preparation

Effects of medium components and culture conditions on biomass and lipid production of Trichosporon fermentans were studied. The optimal nitrogen source, carbon source and C/N molar ratio were peptone, glucose and 163, respectively. The favorable initial pH of the medium and temperature were 6.5 and 25 degrees C. Under the optimized conditions, a biomass of 28.1 g/l and a lipid content of 62.4% could be achieved after culture for 7 days, which were much higher than the original values (19.4 g/l and 50.8%) and the results reported by other groups. T. fermentans could grow well in pretreated waste molasses and a lipid yield of 12.8 g/l could be achieved with waste molasses of 15% total sugar concentration (w/v) at pH 6.0, representing the best result with oleaginous microorganisms on agro-industrial residues. Addition of various sugars to the pretreated molasses could efficiently enhance the accumulation of lipid and the lipid content reached as high as above 50%. Similar to vegetable oils, the lipid mainly contains palmitic acid, stearic acid, oleic acid and linoleic acid and the unsaturated fatty acids amount to about 64% of the total fatty acids. The microbial oil with an acid value of 5.6 mg KOH/g was transesterified to biodiesel by base catalysis after removal of free fatty acids and a high methyl ester yield of 92% was obtained.