产胡萝卜素酵母的筛选

从被油污染的土样和植物的落花中分离筛选出几株产胡萝卜素的酵母菌,其中从椰子油污染的土样中分离获得的菌株ＣＯＳ－５的胡萝卜素产量最高。ＣＯＳ－５菌株经鉴定为红酵母（Ｒｈｏｄｏｔｏｒｕｌａ ｓｐ．）ＣＯＳ－５。该菌株以蔗糖为碳源,添加桔子皮和盐酸硫胺素的培养基２８℃振荡培养９６ｈ,细胞生物量为２７．５ｍｇ干重／ｍｌ发酵液,胡萝卜素产量为１３．５５μｇ／ｍｌ发酵液。菌体胡萝卜素含量达４９２．７μｇ／ｇ干     

Stereoselective synthesis of (<Emphasis Type="Italic">R</Emphasis>)-3-quinuclidinol through asymmetric reduction of 3-quinuclidinone with 3-quinuclidinone reductase of <Emphasis Type="Italic">Rhodotorula rubra</Emphasis>

A novel nicotinamide adenine dinucleotide phosphate-dependent carbonyl reductase, 3-quinuclidinone reductase, was isolated from Rhodotorula rubra JCM3782. The enzyme catalyzes the asymmetric reduction of 3-quinuclidinone to (R)-3-quinuclidinol. The gene encoding the enzyme was also cloned and sequenced. A 819-bp nucleotide fragment was confirmed to be the gene encoding the 3-quinuclidinone reductase by agreement of the internal amino acid sequences of the purified enzyme. The gene encodes a total of 272 amino acid residues, and the deduced amino acid sequence shows similarity to those of several short-chain dehydrogenase/reductase family proteins. An expression vector, pWKLQ, which contains the full length 3-quinuclidinone reductase gene was constructed. Using Escherichia coli cells coexpressing the 3-quinuclidinone reductase and glucose dehydrogenase (cofactor regeneration enzyme) genes, 618 mM 3-quinuclidinone was almost stiochiometrically converted to (R)-3-quinuclidinol with an >99.9% enantiomeric excess within 21 h of reaction.     

海洋中海藻糖产生菌的筛选及发酵条件优化

从180余份海水、海泥样品中筛选得到60株产海藻糖较高的菌株,编号为2-14的菌株海藻糖产量最高,为127.9mg/g cell。对2-14菌株进行形态特征、培养特征及生理生化试验,鉴定该菌株为红酵母属(Rhodotorula sp.)。研究摇瓶发酵条件对红酵母海藻糖产量的影响,结果为:初始pH5.5,发酵温度28℃,装液量75mL(250mL三角瓶中)。采用优化后发酵条件红酵母海藻糖产量为193.3mg/g cell,优化前对照值为132.1mg/g cell,优化后的结果是优化前的1.46倍。在5L发酵罐中培养得到最佳发酵时间为54h,发酵罐培养发酵液中海藻糖含量最高达2.5g/L,为摇瓶培养的1.6倍。     

粘红酵母产L-苯丙氨酸解氨酶发酵培养基的优化

通过单因子和正交试验 ,对粘红酵母产 L -苯丙氨酸解氨酶 ( PAL )培养基进行优化 ,L-苯丙氨酸的积累浓度可以从 2 .0 g/1 0 0 ml提高到 3 .3 g/1 0 0 ml,最终得到了 L-苯丙氨酸解氨酶发酵的最适条件     

The Production of γ-Decalactone by Fermentation of Castor Oil

A microbial process for the production of optically-active γ-decalactone from the ricinoleic acid present as triglycerides in castor oil has been developed, γ-decalactone (γDL) is a component of some fruit flavours, being an important organoleptic component of peach flavours. Screening showed two red yeast microorganisms, Rhodotorula glutinis and Sporobolomyces odortts to be especially suitable for this biotransformation. The process involves lipase-mediated hydrolysis of the castor oil to give free ricinoleic acid, uptake of the acid by the cells and aerobic fermentation to achieve abbreviated β-oxidation of the ricinoleic acid (12-hydroxyoleic acid) into 4-hydroxydecanoic acid (4HDA), lactonisation of the acid into γ-DL, followed by solvent extraction and distillation. γ-DL broth concentrations of 0.5-1.2g · 1^-t were obtained after 3-5 days from fermentation media containing 10 g · 1^-1 castor oil, representing an 8.3-20.0% theoretical yield. Intermediates detected were consistent with the operation of the β-oxidation pathway. Appreciable amounts of novel metabolites identified as cis and trans isomers of a tetrahydrofuran (C₁₀) were also produced. Their formation from 4HDA appeared to be non-enzymic and was favoured by anaerobic conditions. Yields of γ-DL were inversely proportional to the concentration of castor oil present in the medium, indicating that substrate inhibition takes place. The highest yields of γ-DL were obtained when castor oil was present from the beginning of the fermentation, rather than when added once the fermentation had become established, demonstrating that the β-oxidation pathway and/or transport system require continual induction. Significant amounts of γ-DL were not produced from other fatty acids, including ricinelaidic acid, the trans isomer of ricinoleic acid. γ-DL formation was dramatically inhibited by antibiotic inhibitors of oxidative phosphorylation, indicating the importance of intact β-oxidation pathways, whereas inhibitors of protein synthesis and cell-wall synthesis had much less marked effects. Selective extraction of 4HDA from the fermentation broths, and of γDL from broth lactonised by heating at low pH, could be achieved by adsorption to Amberlite XAD-1 and XAD-7 resins respectively. Some product could be recovered from the exit gases of the fermenter by passing through propylene glycol traps. This pathway is unusual in that it is a rare example of the truncated β-oxidation of a fatty acid by microorganisms. This effect probably occurs because of partial inhibition of one or more enzymes of the β-oxidation pathway by the C₁₀ hydroxylated fatty acid intermediate(s) allowing intracellular accumulation of the 4HDA, followed by leakage out of the cell; although further metabolism of this C₁₀ intermediate does take place slowly.     

Characterization of optimal growth conditions and carotenoid production of strain Rhodotorula mucilaginosa HP isolated from larvae of Pieris rapae

Yeasts have been studied because of their production of a pigment known as carotenoid with potential application in food and feed supplements. A carotenoid‐producing yeast was isolated from the larvae of Pieris rapae, named HP. The strain HP was identified as Rhodotorula mucilaginosa classified by its carbohydrate fermentation pattern and physiological tests. Rhodotorula mucilaginosa HP produces several exogenous enzymes: alkaline phosphatase, esterase, leucine arylamidase, valine arylamidase, acid phosphatase and β‐glucosidase. Using response surface methodology, selected medium components (yeast extract, malt extract, peptone, glucose) were tested to find the optimum conditions for carotenoid production and the growth of R. mucilaginosa HP. Central composite design was used to control the concentrations of medium components. Peptone and glucose had the largest effects on carotenoid production and cell growth of R. mucilaginosa HP, respectively. The estimated optimal growth conditions of R. mucilaginosa HP were: yeast extract 3.23%, malt extract 2.84%, peptone 6.99% and glucose 12.86%. The estimated optimal conditions for carotenoid production were: yeast extract 2.17%, malt extract 2.11%, peptone 5.79% and glucose 12.46%. These results will assist in the formulation of an appropriate culture medium for optimal carotenoid production of R. mucilaginosa HP for commercial use.     

三种拮抗酵母菌对苹果采后青霉病的抑制效果

从苹果果实上分离获得的50余种酵母菌中筛选出了能够有效地抑制苹果青霉病(Penicilium expansum Link)的丝孢酵母(Trichosporon pullulans (Lindner.) Diddens and Lodder)、罗伦隐球酵母(Cryptococcus laurentii (Kuffer.) Skinner)和粘红酵母(Rhodotorula glutinis (Fresen.) F. C. Harrison).其中,抑病效果最好的T. pullulans 是一种用于采后果实生物防治的新型拮抗菌.研究了这三种拮抗菌不同浓度处理和外加营养物质以及与钙配合使用对苹果青霉病的抑病效果.实验结果表明:酵母菌浓度越高,抑病作用越强;外源营养物质的加入削弱了酵母菌的拮抗效果;在C. laurentii的细胞悬浮液中加入0.18 mol/L 的CaCl2能显著提高其抑病能力,但增加CaCl2 对T. pullulans 和R. glutinis 的抑病效果却没有明显作用.     

粘红酵母乙酰辅酶A羧化酶（ACC）基因的克隆及CT功能域基因的原核表达

利用简并PCR结合染色体步移法首次克隆获得粘红酵母乙酰辅酶A羧化酶（ACC）基因的全长序列信息。序列分析表明,该基因包含2个内含子,分别位于42～147 bp和315～677 bp处,编码区域总长为6 801bp,推导的氨基酸序列进行二级结构分析具备乙酰辅酶A羧化酶典型的3个功能域：生物素羧化酶（BC）、生物素羧基载体蛋白（BCCP）和羧基转移酶（CT）。克隆该基因的CT功能域基因,连接到原核表达载体pET-28a上,在Escherichia coli BL21（DE3）中成功表达,利用Ni-NTA树脂柱纯化获得CT的可溶性重组蛋白,浓度为1.8mg/mL,为研究ACC的功能和针对CT作用的除草剂机理研究提供了有价值的材料。     

Rhodotorula svalbardensis sp. nov., a novel yeast species isolated from cryoconite holes of Ny-Ålesund,Arctic

A psychrophilic yeast species was isolated from glacier cryoconite holes of Svalbard. Nucleotide sequences of the strains were studied using D1/D2 domain, ITS region and partial sequences of mitochondrial cytochrome b gene. The strains belonged to a clade of psychrophilic yeasts, but showed marked differences from related species in the D1/D2 domain and biochemical characters. Effects of temperature, salt and media on growth of the cultures were also studied. Screening of the cultures for amylase, cellulase, protease, lipase, urease and catalase activities was carried out. The strains expressed high amylase and lipase activities. Freeze tolerance ability of the isolates indicated the formation of unique hexagonal ice crystal structures due to presence of ‘antifreeze proteins’ (AFPs). FAME analysis of cultures showed a unique trend of increase in unsaturated fatty acids with decrease in temperature. The major fatty acids recorded were oleic acid, linoleic acid, linolenic acid, palmitic acid, stearic acid, myristic acid and pentadecanoic acid. Based on sequence data and, physiological and morphological properties of the strains, we propose a novel species, Rhodotorula svalbardensis and designate strains MLB-I (CCP-II) and CRY-YB-1 (CBS 12863, JCM 19699, JCM 19700, MTCC 10952) as its type strains (Etymology: sval.bar.den′sis. N.L. fem. adj. svalbardensis pertaining to Svalbard).     

Highly enantiomeric reduction of acetophenone and its derivatives by locally isolated Rhodotorula glutinis

Ninety isolates of microorganisms belonging to different taxonomical groups (30 bacteria, 20 yeast, and 40 fungi) were previously isolated from various samples. These isolates were screened as reducing agents for acetophenone 1a to phenylethanol 2a . It was found that the isolate EBK‐10 was the most effective biocatalyst for the enantioselective bioreduction of acetophenone. This isolate was identified as Rhodotorula glutinis by the VITEK 2 Compact system. The various parameters (pH 6.5, temperature 32°C, and agitation 200 rpm) of the bioreduction reaction was optimized, which resulted in conversions up to 100% with >99% enantiomeric excesses (ee) of the S‐configuration. The preparative scale bioreduction of acetophenone 1a by R. glutinis EBK‐10 gave (S)‐1‐phenylethanol 2a in 79% yield, complete conversion, and >99% ee. In addition, R.glutinis EBK‐10 successfully reduced various substituted acetophenones. Chirality, 2010. © 2010 Wiley‐Liss, Inc.