耐乙酸粘红酵母合成油脂

乙酸是木质纤维素在水解过程中的主要副产物,高浓度的乙酸严重影响产油微生物的生长和油脂合成。本文研究了粘红酵母对乙酸的耐受性及其利用乙酸合成微生物油脂的能力。结果表明,在初始葡萄糖、木糖浓度分别为6 g/L和44 g/L的混合糖培养基中,乙酸浓度低于10 g/L时,不会对菌体生长产生抑制作用,油脂合成还得到了促进。当乙酸添加量为10 g/L时,生物量、油脂产量、油脂含量较对照组分别提高了21.5%、171.2%和121.6%。进一步研究表明,粘红酵母具备利用乙酸合成油脂的能力,当以乙酸为唯一碳源,浓度为25 g/L时,油脂产量达到3.20 g/L,油脂质量得率为13%。微生物油脂成分分析表明,粘红酵母以乙酸为底物制得的油脂可以作为制备生物柴油的油脂原料,其主要成分为棕榈酸、硬脂酸、油酸、亚油酸和亚麻酸,其中饱和脂肪酸和不饱和脂肪酸含量分别为40.9%和59.1%。由于粘红酵母具有利用乙酸合成微生物油脂的能力,在以木质纤维素水解液为原料生产微生物油脂的脱毒过程中,一定浓度的乙酸可以不必脱除。     

粘红酵母在味精废水中发酵生产油脂

对粘红酵母菌株进行驯化得到一株优良菌株Rh8,利用其在味精废水中发酵以去除废水中的化学需氧量(COD)并生产油脂;考察废水pH以及添加葡萄糖母液、营养因子等对菌株Rh8在味精废水中生长、产油和COD去除效果的影响,发现将废水稀释4倍、调节pH至5.5时,菌株可以较好地生长;而添加废葡萄糖母液、酵母粉、KH2PO4、MgSO4、MnSO4均能够促进菌体的生长、产油和废水中的COD去除,在250 mL摇瓶中,生物量最高可达15.6 g/L,干菌体中油脂质量分数达到29.61%,COD去除率达到45.1%。     

Affinity purification and characterization of a yeast epoxide hydrolase

Purification of the membrane-associated epoxide hydrolase from the yeast Rhodosporidium toruloides CBS 0349 to electrophoretic homogeneity was achieved in a single chromatographic step employing the affinity ligand adsorbent Mimetic Green. More than 68% of the total epoxide hydrolase activity present in the whole cells was recovered from the membrane fraction. The enzyme was purified 26-fold with respect to the solubilized membrane proteins and was obtained in a 90% yield. The purified epoxide hydrolase has an apparent monomeric molecular weight of 54 kDa, and a pI of 7.3. The enzyme was optimally active at 30–40 °C, and pH 7.3–8.5. The enzyme is highly glycosylated with a carbohydrate content >42%. The specific activity of the purified enzyme for (±)-1,2-epoxyoctane is 172 mol min^–1 mg protein^–1. The amino acid composition of the protein was determined. This is the first report of a yeast epoxide hydrolase purified to homogeneity in milligram amounts.     

Effect of an exponential feeding regime on the production of Rhodotorula araucariae epoxide hydrolase in Yarrowia lipolytica

Aims: To evaluate the effect of and exponential feeding regime on the production of epoxide hydrolase (EH) enzyme in recombinant Yarrowia lipolytica in comparison to a constant feed strategy. Methods and Results: An exponential feed model was developed and fermentations were fed at six different exponential rates. A twofold increase in EH productivity and a 15% increase in volumetric EH activity was obtained by applying exponential glucose feed rates in fed‐batch cultivation. These responses were modelled to obtain a theoretical optimum feed rate that was validated in duplicate fermentations. The model optimum of 0·06 h^?1 resulted in a volumetric EH activity of c. 5500 U l^?1 h^?1 and a maximum activity of 206 000 U l^?1. This correlated well with model predictions, with a variance of <10%. Conclusions: The use of an exponential feed strategy at a rate of 0·06 h ^{? 1} yielded best results for all key responses which show a clear improvement over a constant feed strategy. Significance and Impact of the Study: The study was the first evaluation of an exponential feed strategy on recombinant Y. lipolytica for the production of EH enzyme. The results suggest a strategy for the commercial production of a valuable pharmaceutical enzyme.     

影响黏红酵母黏附牙鲆肠黏液的因素研究

目的研究pH、温度、二价阳离子对黏红酵母黏附作用的影响以及不同生长阶段的黏红酵母对牙鲆肠黏液的黏附特点。方法以牙鲆肠黏液作为黏附模型,采用MTT比色法测定黏红酵母在上述影响因素作用下的黏附能力。结果在10~30℃黏红酵母对牙鲆肠黏液的黏附随温度升高而增强,中性偏酸的环境有利于黏红酵母的黏附;Ca2 能显著增加黏红酵母的黏附,但Mg2 促黏附作用不明显;处于对数生长期的黏红酵母对牙鲆肠黏液的黏附能力最强,其次是延滞期的酵母,而稳定期和衰亡期的黏红酵母黏附能力显著减弱。结论黏红酵母对牙鲆肠黏液的黏附作用不仅受多种环境因子的影响,也受其自身生理状态的影响,其黏附作用具有可控性,这对黏红酵母在牙鲆肠道内黏附、定植的调控有一定的参考价值。     

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     

Stability of beta-carotene in spray dried preparation of Rhodotorula glutinis mutant 32

AIMS: To obtain beta-carotene-rich dry cell preparation from mutant 32 of Rhodotorula glutinis and determination of its pigment stability. METHODS AND RESULTS: The mutant 32 of R. glutinis was grown in a 14 l stirred tank fermenter. Cell mass was concentrated 10-fold by cross-flow microfiltration and then spray dried. Butylated hydroxy toluene (BHT) and d-tocopherol were used as protecting agents. A two-level, three-variable, factorial optimization was performed to achieve moisture-free, non-viable and beta-carotene-rich feed additive. CONCLUSIONS: The beta-carotene and cell mass in stirred tank fermenter were found to be 54 +/- 5 mg l-1 and 12.8 +/- 2 g l-1, respectively. In the presence of BHT, 97 +/- 3% (w/w) beta-carotene was recovered for all the inlet temperatures studied. The best beta-carotene and yeast powder recoveries were obtained at 160 degrees C, 11.6% (w/v) cell mass concentration and 1 g l-1 BHT. The pigments inside dried yeast powder were stable in dark and cold condition for at least 10 weeks. The purified beta-carotene got almost totally denatured, under similar conditions of storage, within 76 h. SIGNIFICANCE AND IMPACT OF THE STUDY: Spray dried and stable preparation of beta-carotene-rich yeast, R. glutinis can provide alternative source of beta-carotene for use in animal nutrition.     

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.     

粘红酵母和酿酒酵母联合处理味精废水

为了解决味精废水中高NH4+浓度抑制油脂微生物的生长和油脂积累问题,采用粘红酵母和酿酒酵母联合处理味精废水的方法:首先利用酿酒酵母降解味精废水(MSG)中NH4+,然后将处理后的废水进一步发酵培养合成油脂。研究结果表明:用经酿酒酵母预处理过的味精废水作为粘红酵母的培养基发酵时,粘红酵母的生物量为33.3 g/L,油脂产率为18.16%,COD降解率为50.6%,NH4+的降解率为93.9%。比粘红酵母单独处理味精废水,NH4+的降解率提高了6.14倍,生物量、油脂产率和COD降解率分别提高了8.1%、30.06%和9.58%。     

Novel aliphatic epoxide hydrolase activities from dematiaceous fungi

Abstract Epoxide hydrolases were found to be constitutively expressed in dematiaceous fungi coincident with secondary metabolite pigment production in stationary or idiophase. Washed-cell preparations of two fungi, Ulodadium atrum CMC 3280 and Zopfiella karachiensis CMC 3284, exhibited affinity for 2,2-dialkylated oxiranes, for which contrasting enantioselectivities were observed, but not for aromatic styrene oxide or alicyclic cyclohexene oxide type substrates. Lyophilised preparations of soluble epoxide hydrolase activities proved to be effective catalysts for the mild hydrolysis of aliphatic epoxides.     

粘红酵母和钝顶螺旋藻混合培养生产微生物油脂培养基优化

通过单因素实验和正交实验对粘红酵母和钝顶螺旋藻混合培养的培养基进行了优化,得到的优化培养基于5L发酵罐中实验,葡萄糖质量浓度低于10．0g／L时,开始流加600g／L葡萄糖,控制葡萄糖质量浓度在（20±5）g/L,最终得到的油脂产量达到16．0g／L。     

Polyphasic taxonomy of the basidiomycetous yeast genus Rhodotorula: Rh. glutinis sensu stricto and Rh. dairenensis comb. nov

The phenotypic and genetic heterogeneity of the basidiomycetous yeast species Rhodotorula glutinis was investigated in a group of 109 isolates. A polyphasic taxonomic approach was followed which included PCR fingerprinting, determination of sexual compatibility, 26S and ITS rDNA sequence analysis, DNA-DNA reassociation experiments and reassessment of micromorphological and physiological attributes. The relationships with species of the teleomorphic genus Rhodosporidium were studied and isolates previously identified as Rh. glutinis were found to belong to Rhodosporidium babjevae, Rhodosporidium diobovatum and Rhodosporidium sphaerocarpum. Other isolates included in the study were found to belong to Rh. glutinis var. dairenensis, which is elevated to the species level, or to undescribed species. The concept of Rh. glutinis sensu stricto is proposed due to the close phenetic and phylogenetic proximity detected for Rh. glutinis, Rhodotorula graminis and R. babjevae.     

糠醛对粘红酵母生长与油脂积累的影响

为了探究纤维素水解液中常见的发酵抑制物糠醛对粘红酵母Rhodotorula glutinis生长与油脂积累的影响,对比了不同的糠醛浓度(0.1、0.4、0.6、1.5 g/L)下粘红酵母的生物量和油脂积累情况,并探究了1.0 g/L的糠醛对粘红酵母不同碳源(葡萄糖和木糖)利用的影响。研究表明,当糠醛浓度达1.5 g/L时,粘红酵母的延迟期延长至96 h,残糖高达17.7 g/L,生物量最高6.6 g/L,仅为正常积累量的47%,油脂含量也减少了约50%;以木糖为碳源时,糠醛对粘红酵母的抑制程度小于葡萄糖为碳源时的情况;在糠醛存在的逆境中,粘红酵母倾向于生成更多的18碳脂肪酸或18碳不饱和脂肪酸。     

Attenuation of cisplatin nephrotoxicity by inhibition of soluble epoxide hydrolase

Cisplatin is a highly effective chemotherapeutic agent against many tumors; however, it is also a potent nephrotoxicant. Given that there have been no significant advances in our ability to clinically manage acute renal failure since the advent of dialysis, the development of novel strategies to ablate nephrotoxicity would represent a significant development. In this study, we investigated the ability of an inhibitor of soluble epoxide hydrolase (sEH), n-butyl ester of 12-(3-adamantan-1-yl-ureiido)-dodecanoic acid (nbAUDA), to attenuate cisplatin-induced nephrotoxicity. nbAUDA is quickly converted to AUDA and results in maintenance of high AUDA levels in vivo. Subcutaneous administration of 40 mg/kg of nbAUDA to C3H mice every 24 h resulted in elevated blood levels of AUDA; this protocol was also associated with attenuation of nephrotoxicity induced by cisplatin (intraperitoneal injection) as assessed by BUN levels and histological evaluation of kidneys. This is the first report of the use of sEH inhibitors to protect against acute nephrotoxicity and suggests a therapeutic potential of these compounds.     

Covalent attachment of epoxide hydrolase to dextran

Epoxide hydrolase (EC 3.3.2.3) purified from rat liver microsomes has been immobilized by covalent linking to dextran activated by imidazolyl carbamate groups, under mild conditions. K^app_m values of free and dextran bound epoxide hydrolase toward benzo(a)pyrene-4,5-oxide were 0.5 and 0.35 μM respectively, while V^app_max was lowered from 300 to 120 nmol min^?1mg^?1protein. The activity lost upon coupling could not be restored by digestion of the support by dextranase (1,6-α-d-glucan 6-glucanohydrolase, EC 3.2.1.11) treatment. This fact, along with the similarity of the activation energy values for both native and bound epoxide hydrolase, indicated that steric hindrance effects due to the polymer support played only a minor role in this loss of activity. Evidences of changes in the conformation of epoxide hydrolase were obtained by a comparative study of u.v. circular dichroism and tryptophan fluorescence emission spectra of the native and dextran bound enzymes. On the other hand, the enzyme conjugate showed greater resistance than the free enzyme to thermal inactivation.     

Correlation between the physicochemical properties of organic solvents and their biocompatibility toward epoxide hydrolase activity in whole-cells of a yeast, Rhodotorula sp.

Epoxides are often highly hydrophobic substrates and the presence of an organic co-solvent within an aqueous bioreactor is in such cases indicated. The effect of 40 water-miscible and -immiscible organic solvents on epoxide hydrolase activity in whole-cells of the yeast Rhodotorula sp. UOFS Y-0448 was investigated. No formal correlation between solvent biocompatibility and physicochemical properties was deductible, although the introduction of hydroxyl groups increased biocompatibility. 1-Pentanol, 2-methylcyclohexanol and 1-octanol were the most biocompatible resulting in relatively low activity losses when used at up to 20% (v/v).     

Physico-chemical properties of the epoxide hydrolase from Rhodosporidium toruloides

Residue-specific chemical modification of amino acid residues of the microsomal epoxide hydrolase (mEH) from Rhodosporidium toruloides UOFS Y-0471 revealed that the enzyme is inactivated through modification of Asp/Glu and His residues, as well as through modification of Ser. Since Asp acts as the nucleophile, and Asp/Glu and His serve as charge relay partners in the catalytic triad of microsomal and soluble epoxide hydrolases during epoxide hydrolysis, inactivation of the enzyme by modification of the Asp/Glu and His residues agrees with the established reaction mechanism of these enzymes. However, the inactivation of the enzyme through modification of Ser residues is unexpected, suggesting that a Ser in the catalytic site is indispensable for substrate binding by analogy of the role of Ser residues in the related L-2-haloacid dehalogenases, as well as the ATPase and phosphatase enzymes. Co²⁺, Hg²⁺, Ag⁺, Mg²⁺ and Ca²⁺ inhibited enzyme activity and EDTA increased enzyme activity. The activation energy for inactivation of the enzyme was 167 kJ mol^–1. Kinetic constants for the enzyme could not be determined since unusual behaviour was displayed during hydrolysis of 1,2-epoxyoctane by the purified enzyme. Enantioselectivity w as strongly dependent on substrate concentration. When the substrate was added in concentrations ensuring two-phase conditions, the enantioselectivity was greatly enhanced. On the basis of these results, it is proposed that this enzyme acts at an interface, analogous to lipases.     

Fluorometric assay for determining epoxide hydrolase activity

We have developed a rapid screening procedure that enables the screening of hundreds of enzyme samples or variants for epoxide hydrolase activity towards any substrate. The procedure detects the products of the enzymatic reaction via periodate cleavage and remaining fluorescence of carboxyfluorescein.     

红酵母RY-4中虾青素的分离纯化研究

本文主要研究了红酵母RY-4菌株中天然虾青素的提取和纯化工艺。通过各种破壁提取方法的比较分析,得出了酸热法破壁丙酮提取为较经济理想的粗色素提取方法,提取的粗色素油中虾青素含量达51.7%。通过对各种柱层析条件的摸索研究,得出了以硅胶为吸附剂,Ⅴ(石油醚)∶Ⅴ(乙酸乙酯)混和液为洗脱剂梯度洗脱从粗色素油中分离纯化虾青素的方法,所得虾青素纯度达到97%。     

Structure of an atypical epoxide hydrolase from Mycobacterium tuberculosis gives insights into its function

Epoxide hydrolases are vital to many organisms by virtue of their roles in detoxification, metabolism and processing of signaling molecules. The Mycobacterium tuberculosis genome encodes an unusually large number of epoxide hydrolases, suggesting that they might be of particular importance to these bacteria. We report here the first structure of an epoxide hydrolase from M.tuberculosis, solved to a resolution of 2.5 A using single-wavelength anomalous dispersion (SAD) from a selenomethionine-substituted protein. The enzyme features a deep active-site pocket created by the packing of three helices onto a curved six-stranded beta-sheet. This structure is similar to a previously described limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis and unlike the alpha/beta-hydrolase fold typical of mammalian epoxide hydrolases (EH). A number of changes in the mycobacterial enzyme create a wider and deeper substrate-binding pocket than is found in its Rhodococcus homologue. Interestingly, each structure contains a different type of endogenous ligand of unknown origin bound in its active site. As a consequence of its wider substrate-binding pocket, the mycobacterial EH is capable of hydrolyzing long or bulky lipophilic epoxides such as 10,11-epoxystearic acid and cholesterol 5,6-oxide at appreciable rates, suggesting that similar compound(s) will serve as its physiological substrate(s).