近平滑假丝酵母细胞催化乙酰基三甲基硅烷不对称还原反应

 采用近平滑假丝酵母细胞用于催化乙酰基三甲基硅烷不对称还原反应, 可高选择性地生成 (R)-1-三甲基硅乙醇. 结果表明, 固定化于海藻酸钙的细胞催化该反应的产物收率比游离细胞的高. 不同辅底物对该反应的影响显著, 以葡萄糖为辅底物时, 反应的初速率较快, 产物收率较高. 该反应的最适条件为: 辅底物 (葡萄糖) 浓度 110 mmol/L, 振荡速度 180 r/min, 缓冲液 pH 值 6.0, 反应温度 30 oC, 底物浓度 20 mmol/L. 在此反应条件下反应的初速率、产物收率和产物的 ee 值分别为 11.4 μmol/h, 96.5% 和 99.9%.     

活性酵母细胞不对称催化苯乙酮还原及树脂吸附对反应的促进作用

 以苯乙酮为模型底物,研究了水相体系中酵母细胞催化前手性芳香酮不对称还原生成相应手性醇的反应特性. 实验发现,酵母细胞催化苯乙酮不对称还原的产物以(S)-α-苯乙醇为主,反应的立体选择性很高,(S)-α-苯乙醇的对映体过量值可达99%左右. 在pH为7～8, 酵母细胞用量为0.2 g/ml的条件下能获得较高的产物收率(可达35%左右). 酵母细胞能选择性地氧化(S)-α-苯乙醇,而留下(R)-α-苯乙醇. 在反应体系中加入合适的吸附树脂,可以降低底物和产物对细胞的毒害作用,显著提高反应底物的初始浓度,从而提高产物收率.     

酵母细胞催化芳香酮的不对称还原反应

 研究了酵母细胞催化芳香酮的不对称还原反应,采用正交试验综合考察了反应温度、反应时间、底物浓度和酵母浓度等因素对底物苯乙酮转化率和产物(S)-1-苯基乙醇对映选择性的影响. 结果表明,影响苯乙酮转化率的因素依次为底物浓度、反应时间、反应温度和酵母浓度,影响(S)-1-苯基乙醇对映选择性的因素依次为反应温度、底物浓度、酵母浓度和反应时间. 同时考察了芳香酮结构对产物对映选择性的影响,发现对映选择性的变化规律符合Prelog规则,与羰基相连的两个基团体积差异越大,对映选择性越好,最高的对映体过量值达到了96.4%.     

双层固定化酵母催化还原4-氯-乙酰乙酸乙酯的研究

以海藻酸钠为内层载体,利用机械强度良好的聚乙烯醇(PVA)改性海藻酸钠混合物作为外层载体,双层固定化高活性酵母,得到固载的生物催化剂,用于由磷酸缓冲溶液和环己烷组成的双相体系,对4-氯-乙酰乙酸乙酯(COBE,ethyl 4-cloro-3-oxobutyrate)进行不对称催化加氢合成(S)-4-氯-3-羟基丁酸乙酯[(S)-CHBE],ethyl[(S)-4-chloro-3-hydro butanoate].在最适条件下,双层固定化酵母的不对称催化还原反应优于游离细胞和单层固定化细胞.在底物COBE的最佳浓度和进料速率分别为170mmol/L,125mL/h的连续操作条件下,最高(S)-CHBE转化率为94.2%,最高对映体过量值为98%.与游离细胞催化COBE相比,反应转化率提高3%,最佳底物浓度比游离细胞增加1倍;与单层固定化细胞催化还原相比,对映体过量值提高8%.连续性实验表明,双层固定化酵母细胞在连续使用5d,其转化率和对映选择性没有明显下降.     

底物中硅原子对苦杏仁醇腈酶催化不对称转氰反应的影响

 对比研究了不同条件下苦杏仁醇腈酶催化乙酰基三甲基硅烷及其碳结构类似物3,3-二甲基-2-丁酮的转氰反应. 结果表明,苦杏仁醇腈酶催化乙酰基三甲基硅烷转氰反应的初速率及对映体选择性均高于其碳结构类似物在同一条件下的对应值. 动力学研究结果表明,苦杏仁醇腈酶催化乙酰基三甲基硅烷转氰反应的表观动力学参数为Km=27.12 mmol/L和vmax=7.05 mmol/(L·h),活化能为51.92 kJ/mol. 苦杏仁醇腈酶催化3,3-二甲基-2-丁酮转氰反应的表观动力学参数为Km=146.58 mmol/L和vmax=2.52 mmol/(L·h),活化能为75.04 kJ/mol. 根据硅原子的特性及酶反应机理合理解释了实验结果.     

呼吸缺陷型面包酵母催化β-羰基酯的不对称还原

以4-氯-3-羰基丁酸乙酯为模型底物,葡萄糖为碳源,对呼吸缺陷型酵母催化不对称还原合成(S)-4-氯-3-羟基丁酸乙酯的反应进行了研究.通过在254nm下紫外诱变,经2,3,5-氯化三苯四氮唑平板和非发酵型碳源培养基鉴定,得到6株面包酵母的呼吸缺陷型变异株.并对呼吸缺陷型酵母催化特性和线粒体复合体Ⅰ,Ⅰ Ⅲ,Ⅱ Ⅲ和Ⅳ的活性分别进行了测定.结果表明,呼吸缺陷型酵母催化底物转化率在49%～75%,产物收率在31%～69%,对映体过量值最高可达72%,比正常酵母提高了18%.突变株线粒体内复合体Ⅰ～Ⅳ的活性与正常酵母相比都有不同程度的下降.     

高压CO2条件下粗状假丝酵母催化苯乙酮不对称还原合成α-苯乙醇

 研究了在高压CO2条件下利用粗状假丝酵母Candida valida CICC1444活性细胞催化苯乙酮不对称还原合成(R)-α-苯乙醇. 高压CO2条件可对粗状假丝酵母细胞的活性产生一定的影响,但反应的选择性反而有所提高,证实了高压CO2作为该反应环境是可行的. 考察了底物浓度、细胞用量、反应温度和压力等因素对反应的影响. 结果表明,这些因素对反应产率有很大的影响,在最佳反应条件下,反应底物苯乙酮的转化率和产物α-苯乙醇的产率均可达90%以上,对映体过量值(ee)保持在80%左右. 与常压静置条件下的反应相比,高压CO2条件下粗状假丝酵母催化苯乙酮不对称还原反应的转化率和产率分别提高了20%和40%左右,对映体选择性也提高了5%～10%.     

双芳烃桥连salen配体的合成及其钛配合物在硅腈对醛的不对称加成反应中的应用

新型双芳烃桥连的salen钛配合物是一种高活性的催化三甲基硅腈对醛不对称加成反应的催化剂,底物与催化剂用量比率可达1 000:1.由(R,R)-1,2.环已二胺和3,5-二叔丁基水杨醛合成的催化剂4在催化三甲基硅腈对醛不对称加成反应中达到87%的对映选择性.     

N-亚磺酰基脯胺酰胺催化N-芳基亚胺的不对称硅氢化还原反应

利用N-亚磺酰基脯胺酰胺类催化剂催化N-芳基亚胺的不对称硅氢化还原反应,收率24%～83%,对映体选择性75%～99%.     

卡宾催化对亚甲基苯醌的不对称硼化反应的研究

使用商业化的手性三氮唑盐和联硼酸频哪醇酯[B₂(pin)₂],实现了N-杂环卡宾催化的对亚甲基苯醌的1,6-不对称硼加成反应.该反应条件温和,底物适用范围广,官能团耐受性好,且有良好的反应收率及对映选择性.值得注意的是,在无过渡金属存在下,即使催化剂用量降至底物的0.1 mol%,反应仍能保持高收率和高对映选择性.     

Asymmetric reduction of ketones by employing Rhodotorula sp. AS2.2241 and synthesis of the β-blocker (R)-nifenalol

A broad range of prochiral ketones were efficiently reduced to the corresponding optically active secondary alcohols using resting cells of Rhodotorula sp. AS2.2241. The microbial reduction system exhibited high activity and enantioselectivity in the reduction of various aromatic ketones and acetylpyridines (>97% ee), but moderate to high enantioselectivity in the reduction of α- and β-keto esters. (R)-Nifenalol, a β-adrenergic blocker, was also synthesized using 2-bromo-1(R)-(4-nitrophenyl)ethanol (97% ee) which was prepared through the asymmetric reduction of 2-bromo-1-(4-nitrophenyl)ethanone employing Rhodotorula sp. AS2.2241. The simple preparation and the high activity of the biocatalyst turned this system into a versatile tool for organic synthesis.     

有机溶剂/缓冲液双相体系中固定化Acetobacter sp.CCTCC M209061细胞催化1-(4-甲氧基)-苯基乙醇不对称氧化反应

在有机溶剂/缓冲液双相体系中, 利用固定化醋酸杆菌Acetobacter sp.CCTCC M209061细胞高对映体选择性地催化1-(4-甲氧基)-苯基乙醇(MOPE)的不对称氧化反应, 成功地拆分外消旋MOPE得到对映体纯(S)-MOPE. 与游离细胞相比, 固定化细胞催化反应速度有所降低, 但其稳定性(包括操作稳定性、 热稳定性和储藏稳定性)明显提高. 固定化细胞连续使用10批次(每批次12 h)后, 仍能保留其初始催化活性的58%以上, 而游离细胞仅保留约20%的相对活性. 在所考察的不同有机溶剂中, 正己烷不仅能较好地溶解底物, 而且对细胞的生物相容性相对较好, 因而提高了反应底物浓度、 反应初速度、 对映体回收率及残留底物e.e.值, 是反应体系中最适宜的有机相. 该反应的最适宜正己烷体积分数为60%, 辅底物为50 mmol/L丙酮, 底物浓度为40 mmol/L, 缓冲液pH=6.5, 反应温度为30 ℃; 在此条件下, 反应初速度为80.4 μmol/min, 反应12 h后, 对映体回收率和残留底物e.e.值分别为51.0%和99.9%, 明显好于水单相反应体系.     

树脂吸附对红酵母催化苯乙酮不对称还原反应的促进作用

以红酵母静息细胞催化苯乙酮不对称还原合成(S)-α-苯乙醇为模型反应, 研究了在反应体系中加入吸附树脂对反应的影响. 结果表明, 水相中较高浓度(＞50 mmol/L)的苯乙酮对红酵母细胞具有显著毒性; 加入适量的吸附树脂可以降低水相中苯乙酮的浓度从而减小对细胞的毒害作用. 考察了3种不同极性的吸附树脂(HZ-801, HZ-806和HZ-816)对底物和产物的吸附能力, 发现在3种树脂中疏水性最强的HZ-816树脂对反应底物和产物具有相对较高的吸附能力. 进一步研究结果表明, 当底物浓度为100 mmol/L, 细胞湿重为500 g/L时, 与不加树脂的对照实验相比, 加入100 g/L HZ-816树脂后, 使得48 h的反应转化率从54%提高到91%, 与此同时细胞的死亡率从99%下降为10%. 含有产物的树脂经乙醇解吸后即可循环使用.     

A biosensor for L-proline determination by use of immobilized microbial cells

A biosensor to quantify L-proline within 10(-5)-10(-3) mole/L concentration is described. Immobilized Pseudomonas sp. cells grown in a medium containing L-proline as the only source of carbon and nitrogen were used to create the biosensor. The cells oxidized L-proline specifically consuming O2 and did not react with other amino acids and sugars. The change in oxygen concentration was detected with a Clark oxygen membrane electrode. The cells were immobilized by entrapment in polyvinyl alcohol (PVA) cryogel. The resultant biocatalyst had a high mechanical strength and retained its L-proline-oxidizing ability for at least two months.     

光控生物不对称还原苯乙酮的研究

本文以类球红杆菌(Rhodobacter sphaeroides)为生物催化剂, 将苯乙酮催化还原成具有光学活性的手性醇. 研究了反应条件对该反应的影响.     

Preparation and preliminary characterization of exopolysaccharides by yeast Rhodotorula acheniorum MC

The effects of various carbon and nitrogen sources on the synthesis of exopolysaccharides by Rhodotorula acheniorum MC were studied. The dynamic viscosity of cell-free culture broths during exopolysaccharide synthesis were measured. The highest values for the viscosity (10.14 MPa.s) and crude polysaccharide productivity (6.6 g/L) were obtained in a medium supplied with 5% sucrose. Ammonium sulfate was the most favorable nitrogen source for exopolysaccharide synthesis. The value of pH played a determinant role, and the obligatory condition for exopolysaccharide production was low (pH 1.7-2.0) during the fermentation. The chemical composition and sugar constituents of the crude exopolysaccharides were determined. Mannose was the main monosaccharide component, and its concentration was the highest (69.13%) in the crude exopolysaccharide synthesized in the medium that included 5% sucrose as a carbon source.     

Highly enantioselective and regioselective nickel-catalyzed coupling of allenes, aldehydes, and silanes

A complex derived from Ni(cod)2 and NHC-IPr catalyzes a three-component coupling reaction involving allenes, aldehydes, and organosilanes and transfers the axial chirality of the allene to a stereogenic center in the product with very high fidelity. An unexpected regioselectivity is observed; favored are allylic rather than homoallylic alcohol derivatives, corresponding to the unusual process of coupling two electrophilic atoms: the allene sp and aldehyde carbon atoms. In all cases, high enantioselectivity, high Z/E selectivity, and, with differentially substituted allenes, high site selectivity are observed. This transformation represents the first enantioselective multicomponent coupling process of allenes.     

Influence of carbon and nitrogen sources and temperature on hyperproduction of a thermotolerant β-glucosidase from synthetic medium by Kluyveromyces marxianus

The effect of carbon source and its concentration, inoculum size, yeast extract concentration, nitrogen source, pH of the fermentation medium, and fermentation temperature on β-glucosidase production by Kluyveromyces marxianus in shake-flask culture was investigated. These were the independent variables that directly regulated the specific growth and β-glucosidase production rate. The highest product yield, specific product yield, and productivity of β-glucosidase occurred in the medium (pH 5.5) inoculated with 10% (v/v) inoculum of the culture. Cellobiose (20 g/L) significantly improved β-glucosidase production measured as product yield (Y _P/S ) and volumetric productivity (Q _P ) followed by sucrose, lactose, and xylose. The highest levels of productivity (144 IU/[L·h]) of β-glucosidase occurred on cellobiose in the presence of CSL at 35°C and are significantly higher than the values reported by other researchers on almost all other organisms. The thermodynamics and kinetics of β-glucosidase production and its deactivation are also reported. The enzyme was substantially stable at 60°C and may find application in some industrial processes.     

Biohydrogen Production from Cheese Whey Wastewater in a Two-Step Anaerobic Process

Cheese whey-based biohydrogen production was seen in batch experiments via dark fermentation by free and immobilized Enterobacter aerogenes MTCC 2822 followed by photofermentation of VFAs (mainly acetic and butyric acid) in the spent medium by Rhodopseudomonas BHU 01 strain. E. aerogenes free cells grown on cheese whey diluted to 10 g lactose/L, had maximum lactose consumption (～79%), high production of acetic acid (1,900 mg/L), butyric acid (537.2 mg/L) and H(2) yield (2.04 mol/mol lactose; rate,1.09 mmol/L/h). The immobilized cells improved lactose consumption (84%), production of acetic acid (2,100 mg/L), butyric acid (718 mg/L) and also H(2) yield (3.50 mol/mol lactose; rate, 1.91 mmol/L/h). E. aerogenes spent medium (10 g lactose/L) when subjected to photofermentation by free Rhodopseudomonas BHU 01 cells, the H(2) yield reached 1.63 mol/mol acetic acid (rate, 0.49 mmol/L/h). By contrast, immobilized Rhodopseudomonas cells improved H(2) yield to 2.69 mol/mol acetic acid (rate, 1.87 mmol/L/h). The cumulative H(2) yield for free and immobilized bacterial cells was 3.40 and 5.88 mol/mol lactose, respectively. Bacterial cells entrapped in alginate, had a sluggish start of H(2) production but outperformed the free cells subsequently. Also, the concomitant COD reduction for free cells (29.5%) could be raised to 36.08% by immobilized cells. The data suggest that two-step fermentative H(2) production from cheese whey involving immobilized bacterial cells, offers greater substrate to- hydrogen conversion efficiency, and the effective removal of organic load from the wastewater in the long-term.