Stabilization of d-amino acid oxidase from Rhodosporidium toruloides by immobilization onto magnetic nanoparticles

d-Amino acid oxidase from Rhodosporidium toruloides was immobilized onto glutaraldehyde-activated magnetic nanoparticles. Approximately four enzyme molecules were attached to one magnetic nanoparticle when the weight ratio of the enzyme to the support was 0.12. After immobilization, the T _m was increased from 45°C of the free form to 55°C. In the presence of 20 mM H₂O₂, the immobilized form retained 93% of its activity after 5 h while the free form was completely inactivated after 3.5 h.     

Alkyl-substituted methoxysilanes enhance the activity and stability of d-amino acid oxidase encapsulated in biomimetic silica

Several alkyl-substituted methoxysilanes were evaluated as potential activity and stability enhancing agents for biomimetic silicification of Rhodosporidium toruloides D-amino acid oxidase (RtDAO). When methyl-substituted silanes along with tetramethoxysilane were used as silicic acid precursors for polyallylamine (PAA)--or R5 peptide-catalyzed silicic encapsulation, the RtDAO activity increased with the degree of substitution and the molar ratio up to 15 % of methyl-substituted silanes added. In the presence of 15 mol% trimethylmethoxysilane, the specific activities of encapsulated RtDAO catalyzed by PAA and R5 increased by 1.4- and 4.8-fold, respectively. For PAA-catalyzed encapsulation, a 2.4-fold increase occurred with 30 mol% n-propyltrimethoxysilane; this modification increased the T (m) value by 10 °C and gave a threefold longer half-life in the presence of 10 mM H(2)O(2) as compared to the encapsulation using tetramethoxysilane only.     

Activity enhancement and stabilization of lipase from Pseudomonas cepacia in polyallylamine-mediated biomimetic silica

Triacylglycerol lipase from Pseudomonas cepacia and Fe(3)O(4) magnetic nanoparticles were encapsulated simultaneously within biomimetic silica through the catalysis of polyallylamine. The encapsulation efficiency reached 96% with an activity recovery of 51%. After 5 h at 37°C, the activities of the free and encapsulated lipases decreased by 77 and 16%, respectively. Addition of 10 and 15 mol% trimethylmethoxysilane to tetramethoxysilane during encapsulation doubled the lipase activity while inclusion of 50 and 60 mol% γ-(methacryloxypropyl)-trimethoxysilane tripled the activity. Thus, such encapsulation not only stabilized P. cepacia lipase but also could enhance the activity by varying silane additives.     

Affinity-binding immobilization of d-amino acid oxidase on mesoporous silica by a silica-specific peptide

Journal of Industrial Microbiology & Biotechnology - Enzyme immobilization is widely used for large-scale industrial applications. However, the weak absorption through physical methods limits...     

Transformation of Rhodosporidium toruloides

Rhodosporidium toruloides protoplasts could be transformed, in the presence of polyethylene glycol (PEG), at frequencies of approx. 1 X 10(3) transformants/micrograms of DNA. The plasmid used, pHG2, which contains the phenylalanine ammonia-lyase (PAL)-coding gene (PAL) of R. toruloides, could replicate as an unstable plasmid in the yeast, or could integrate at the PAL locus to give stable transformants. Plasmids that function in R. toruloides were constructed using either the PAL gene or LEU2 gene of Saccharomyces cerevisiae as dominant selectable markers. R. toruloides transformed with pHG8, which contains both genes, coinherited the two markers. It is also shown that the 2mu replicon of S. cerevisiae does not function in R. toruloides; neither is the PAL gene expressed in S. cerevisiae.     

Biosynthesis of lipids by Rhodosporidium toruloides ATCC 10788

The limiting amount of nitrogen required to trigger lipid accumulation in the oleaginous yeast Rhodosporidium toruloides ATCC 10788 was studied, batchwise, by subjecting washed mid-exponentially grown cells to nitrogen at levels of 10⁻² M down to 10⁻⁴ M per g l⁻¹ of lean cells (2–5% fat content) in a mineral medium where glucose was present at 35 g l⁻¹. The results showed that lipid accumulation always started sometime after nitrogen reached a level of 3 × 10⁻⁵ M and the specific initial lipid productivity was constant. Furthermore, the cells were subjected to nine combinations of temperature and pH, from (25° C, pH 4.5) to (35° C, pH 7.5) in the mineral medium supplemented with 0.5 g l⁻¹ of yeast extract and 1 g l⁻¹ (NH₄)₂SO₄. As was expected, lipid content in the cells was higher at 25° C, but pH around 6.0–7.5 slightly enhanced the effect of lower temperature. The effect of pH was also noticed to affect the size of changes in the temporal profiles of the oil's fatty acid distribution prior to nitrogen depletion, whereas no significant difference in the fatty acid composition of the oil was shown after exhaustion of nitrogen from the medium for all combinations of temperature and pH.     

Fluorimetric determination of d-amino acid oxidase

1. A sensitive fluorimetric procedure for the assay of d-amino acid oxidase has been developed. 2. The method depends on the formation of a fluorescent derivative, 2-hydroxy-3-methylquinoxaline, on condensation of pyruvate with o-phenylenediamine in acid medium. 3. 2-Hydroxy-3-methylquinoxaline fluoresces strongly in 50% (v/v) sulphuric acid after excitation at 375mmu. A single emission peak is observed at 480mmu. 4. Formation of the quinoxaline is dependent on time, temperature, acidity and relative concentration of reactants. 5. A particulate preparation from mouse kidney required FAD for optimum activity at pH8.5; K(m) was 3.8x10(-3)m; K(FAD) was 1.4x10(-7)m and the reaction was strongly inhibited by p-chloromercuribenzoate and phenylmercuric acetate. 6. Subcellular fractionation on a sucrose density gradient confirmed that the d-amino acid oxidase was localized on small granules.     

圆红冬孢酵母的密码子用法分析

运用CodonW等软件,分析了圆红冬孢酵母Rhodosporidium toruloides基因组中191个蛋白质编码基因的密码子使用模式,包括密码子3个位置上的GC含量、有效密码子数和密码子使用频率。圆红冬孢酵母有效密码子数ENc值为38.9,密码子GC含量为63%,密码子第三位GC含量为78.3%,且偏好使用G或C结尾的密码子,确定了圆红冬孢酵母R. toruloides的21个高表达优越密码子。研究发现,圆红冬孢酵母与毕赤酵母、酿酒酵母、大肠杆菌和拟南芥在密码子使用频率上有较大差异,而与解脂耶氏酵母和果蝇差异相对较小。研究结果对提高外源基因在圆红冬孢酵母中表达效率及相关代谢工程和合成生物学研究有一定意义。     

Metabolism of dihydrouracil in Rhodosporidium toruloides.

Previous studies, including those done with a similar species, have indicated that dihydrouracil is formed by the breakdown of uracil and is degraded into N-carbamyl-beta-alanine. (Fink et al., J. Biol. Chem. 201:349-355, 1953; S. R. Vilks and M. Y. Vitols, Mikrobiologiya 42:567-583, 1973; O. A. Milstein and M. L. Bekker, J. Bacteriol. 127:1-6, 1976). In the present work the conversion of dihydrouracil to uracil is studied in Rhodosporidium toruloides, and the growth characteristics of mutants that have lost the ability to use dihydrouracil as a source of nitrogen are examined. It is concluded that dihydrouracil must be converted to uracil before catabolism of the pyrimidine ring can take place.     

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.     

圆红冬孢酵母新颖脂肪酸合酶的重组表达、纯化与活性检测

脂肪酸合酶(Fatty acid synthase,FAS)催化乙酰辅酶A和丙二酸单酰辅酶A反应生成脂肪酸,是油脂合成代谢途径中最重要的酶之一。在高产油脂的圆红冬孢酵母Rhodosporidium toruloides中发现了一种新颖的FAS,它含两个亚基,与其他物种的FAS相比,具有独特的结构域组成,尤其是含两个酰基载体蛋白(ACP)结构域。由于ACP在脂肪酸合成反应中起辅因子作用,推测多个ACP有利于提高FAS的催化活性,为研究该FAS的生物化学和结构特征,构建了表达FAS两个亚基的载体,并转化大肠杆菌Escherichia coli BL21(DE3),含pET22b-FAS1和pET24-FAS2质粒的重组菌株ZWE06可同时高表达两个亚基,经硫酸铵沉淀、蔗糖密度梯度离心和阴离子交换层析纯化,得到的重组FAS比活力达到548 mU/mg。纯化的FAS复合物可用于后续酶动力学和蛋白结构研究,且表达与纯化方法的建立对研究其他ACP的功能具有参考价值。     

Sequential liquid-liquid extraction of d-amino acid oxidase from Trigonopsis variabilis

A method for isolation of d-amino acid oxidase (DAAO) from disrupted Trigonopsis variabilis cells has been developed. In an aqueous two-phase system consisting of PEG6000 (220 g l^–1), potassium phosphate (110 g l^–1, K₂HPO₄ + KH₂PO₄ = 10.1:1, mol mol^–1) and dl-methionine (11 g l^–1), the major portion of cellular proteins (87%) was partitioned into the salt phase. By sequential extraction, 48% of DAAO was recovered in PEG phase, giving a yield of 211 U mg protein^–1.     

Interaction of steroids with d-amino acid oxidase

• 1.1. Progesterone hibited d-amino acid oxidase (d-amino acid : O₂ oxidoreductase (deaminating), EC 1.4.3.3.) in competition with its substrate, d-alanine, Binding of progesterone brought about the increase in both fluorescence intensity and fluorescence polarization of FAD, which indicates that the environment surrounding FAD chromophore is modified due to a conformational change in the apoenzyme.
• 2.2. Ethinyl estradiol testosterone, testosterone propionate, corticosterone and aldosterone also inhibited the enzyme slightly in the same manner. Their binding also produced a slight increase in FAD fluorescence without decreasing the fluorescence polarization.
• 3.3. Cholesterol did not inhibit the enzyme, though it increased the fluorescence polarization of FAD. This indicates the binding of cholesterol with the enzyme at a site other than the substrate binding site.

     

Microbial lipid production by Rhodosporidium toruloides under sulfate-limited conditions

Novel biochemical approaches remain to be developed to improve microbial lipid technology. This study demonstrated that sulfate limitation was effective to promote accumulating substantial amounts of intracellular lipid by the oleaginous yeast Rhodosporidium toruloides Y4. When it was cultivated using a medium with an initial carbon-to-sulfur (C/S) molar ratio of 46,750, cellular lipid content reached up to 58.3%. The time courses of cell growth, lipid accumulation and nutrient depletion were analyzed and discussed in terms of lipid biosynthesis. Moreover, lipid accumulation under sulfate-limited conditions was effective regardless of the presence of a high concentration of nitrogen sources. Thus, lipid contents almost held constant at near 57% in the media with an initial C/S molar ratio of 11,380 although the carbon-to-nitrogen molar ratio ranged from 28.3 to 5.7. Taken together, our results established the sulfate-limitation approach to control lipid biosynthesis, which should be valuable to explore nitrogen-rich raw materials as the feedstock for lipid production.     

圆红冬孢酵母发酵菊芋块茎产油脂的研究

研究了圆红冬孢酵母Y4发酵菊芋块茎,菊芋品种及其处理方法对发酵产油的影响。结果表明,菊芋浸提汁、酸水解液或菊芋浆均可直接被圆红冬孢酵母Y4利用,发酵积累油脂,但白皮菊芋比紫皮菊芋更有利于油脂发酵。发酵菊芋浸提汁或酸水解液时,无需添加外源营养物,干菌体油脂含量可达到40%（w/w）;发酵菊芋浆时,白皮菊芋转化率达到12.1 g油/100 g去皮干菊芋。菊芋油脂发酵产品主要以16碳和18碳系脂肪酸为主,与常规植物油的脂肪酸组成相似,可作为制备生物柴油的新型替代原料。     

Production of d-arabitol from d-xylose by the oleaginous yeast Rhodosporidium toruloides IFO0880

Applied Microbiology and Biotechnology - The sugar alcohol d-arabitol is one of the Department of Energy’s top twelve bio-based building block chemicals. In this study, we found that the...     

D-Amino acid oxidase activity form Rhodosporiduim toruloides

The D-amino acid oxidase activity of Rhodosporidium toruloides CCRC 20306 was studied. The enzyme could be induced by D-alanine, and had pH and temperature optima of 8.5 and 60C, respectively. D-Amino acids with polar uncharged and/or nonpolar side chain were good substrates for the D-amino acid oxidase of CCRC 20306, whereas those with polar charged side chain were poor substrates. Benzoic acid and its derivatives were inhibitory to the enzyme activity.     

圆红冬孢酵母利用生物乙醇废水-木薯粉水解液发酵产油

【目的】获得能够高效降解生物乙醇废水化学需氧量(COD)的圆红冬孢酵母菌株,评估废水初始COD浓度对驯化菌株生长的影响,将木薯粉生产微生物油脂和高浓度有机废水降解过程整合,以生物乙醇废水为水源制备生物乙醇废水-木薯粉水解液培养基,明确产油效率高、生物乙醇废水COD降解率高的初始还原糖浓度。【方法】采用在高浓度的生物乙醇废水中进行多次驯化的方法,获得能够适应废水环境的圆红冬孢酵母菌株;采用双酶水解法对加入乙醇废水中的木薯粉进行水解;采用重量法监测生物量浓度变化,采用酸热法提取油脂,重铬酸钾法监测COD,DNS法测定废水还原糖浓度,凯氏定氮法测定总氮,钼酸铵比色法测定总磷。【结果】通过驯化筛选得到一株能耐受高浓度生物乙醇废水的优势菌株Rhodosporidium toruloides D5。以未稀释的废水为培养基,驯化菌株的最终生物量浓度和COD降解率分别为3.8 g/L和75.0%。采用生物乙醇废水-木薯粉水解液发酵时,控制初始还原糖浓度低于30 g/L时,生物量浓度和油脂浓度随初始还原糖浓度的升高而升高,均在120 h时达到最高COD降解率,初始还原糖浓度对达到的最大COD降解率无明显影响,废水N、P去除率分别达到99%和92%以上。【结论】在未经稀释的高浓度生物乙醇废水中可获得较高的生物量浓度;采用高浓度生物乙醇废水-木薯粉水解液培养基发酵产油,初始还原糖浓度为30 g/L,可在保证高油脂产量的同时,实现废水COD的高效降解,有效回收利用废水中残余的N、P源,从而降低微生物油脂生产和废水处理成本,研究结果可为开发廉价微生物油脂生产技术提供有用的参考。