水／离子液体两相体系中出芽短梗霉催化4-氯-乙酰乙酸乙酯不对称还原合成（S）-4-氯-3-羟基-丁酸乙酯

考察了水/离子液体两相体系中出芽短梗霉(Aureobasidium pullulansCGMCC1244)催化4-氯乙酰乙酸乙酯(COBE)不对称还原生成光学活性(S)-4-氯-3-羟基丁酸乙酯((S)-CHBE)的性能,并对反应条件如摇床转速、相体积比、温度、初始底物浓度和pH值等进行了优化.结果表明,在水/1-丁基-3-甲基咪唑六氟磷酸盐体系中,出芽短梗霉催化COBE不对称还原生成(S)-CHBE,在30℃,pH6·6,摇床转速180r/min和不对称反应8h条件下,反应物的转化率、产物ee值和浓度分别达到95·6%,98·5%和47·1g/L.在控制pH值为6·6的情况下,通过分添加底物可有效提高产物(S)-CHBE浓度至75·1g/L.     

酵母发酵液直接催化4-氯-乙酰乙酸乙酯不对称还原生成4-氯-3-羟基-丁酸乙酯

 考察了面包酵母发酵液直接催化4-氯-乙酰乙酸乙酯(COBE)的不对称还原反应,并进行了手性添加物的筛选和反应条件的优化实验. 结果表明,以β-环糊精为手性添加物时,酵母发酵液催化COBE不对称还原生成光活性产物(S)-4-氯-3-羟基-丁酸乙酯((S)-CHBE)的产率和ee值分别高达76%和92%. 在一定条件下,增大β-环糊精浓度,有利于(S)-CHBE的生成. 最佳酵母菌培养时间为16～18 h, 最佳反应温度和pH值分别为29～31 ℃和7.2.     

水相中酵母细胞催化4-氯乙酰乙酸乙酯不对称还原反应

 利用活性酵母细胞催化4-氯乙酰乙酸乙酯(COBE)的不对称还原可以直接合成具有光学活性的4-氯-3-羟基丁酸乙酯(CHBE). 实验发现,在水相体系中主要生成D-(S)-型产物,COBE的转化率及CHBE的收率和光学选择性都比较高. 考察了底物和产物的浓度、辅助底物的种类和浓度、体系的pH和温度以及菌体培养条件等因素对反应的影响. 结果表明,在较低COBE浓度下R型产物占优,在高浓度下主要是S型产物; 较高的反应温度有利于S型产物的生成,pH≈8.0时可获得较高的S型产物选择性; 产物CHBE对COBE的还原反应具有一定的抑制作用. 在酵母催化COBE还原的同时需要一定的辅助底物来再生辅酶NAD(P)H,利用乙醇、异丙醇和仲丁醇作辅助底物可获得较高的立体选择性. 对数生长期和厌氧条件培养的细胞对生成S型产物较有利.     

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.     

重组大肠杆菌细胞不对称还原4-氯乙酰乙酸乙酯合成(R)-(+)-4-氯-3-羟基丁酸乙酯

 从赭色掷孢酵母(Sporobolomyces salmonicolor ZJU0105)中克隆出NADPH依赖型醛基还原酶基因,构建了重组大肠杆菌E.coli BL21(pET28-ALR0105), 该工程菌可以高效地表达醛基还原酶. 将重组细胞用于催化4-氯乙酰乙酸乙酯不对称还原,合成出具有光学活性的(R)-(+)-4-氯-3-羟基丁酸乙酯. 实验发现,在加入适量辅酶及辅酶再生酶的条件下,利用重组细胞催化还原反应可以获得比使用赭色掷孢酵母更高的转化率、产率和ee值,得到了几乎是光学纯的(R)-(+)-型产物,从而解决了酵母细胞催化此类反应ee值较低的问题. 考察了辅酶及共底物的添加、底物和产物的浓度、pH值、温度以及菌体密度等因素对还原反应的影响. 结果表明,不对称还原反应必须在辅酶NADPH和辅酶再生酶系及共底物葡萄糖的参与下进行; 底物和高浓度的产物对还原反应有一定的抑制作用; 当pH＞6.0时,反应的转化率及产率都显著降低; 高密度重组细胞可以减小底物的抑制作用.     

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%.     

磁性聚合物微球固定化青霉素G酰化酶制备光学纯(S)-2-氯苯甘氨酸

通过反相悬浮聚合法制备了超顺磁性环氧聚合物微球用于固定化青霉素G酰化酶,利用磁性固定化酶催化N-苯乙酰-(R,S)-2-氯苯甘氨酸进行不对称水解反应,制备出(S)-2-氯苯甘氨酸单一对映体。磁性固定化酶催化水解反应的适宜条件为:底物浓度100 mg·m L-1,反应温度和时间30℃和12 h,反应溶液p H 8.0。在此条件下,N-苯乙酰-(R,S)-2-氯苯甘氨酸的转化率为48.8%,产物(S)-2-氯苯甘氨酸的对映体过量值eep达99.4%。磁场下回收磁性固定化青霉素G酰化酶,重复使用6次,底物的转化率和产物的对映体过量值分别为47.8%和91.4%。     

不同反应体系中(S)-1-(4-甲氧基)-苯基乙醇的不对称生物合成

在不同反应体系中,研究了固定化红酵母细胞催化对甲氧基苯乙酮不对称还原合成(S)-1-(4-甲氧基)-苯基乙醇的反应.结果表明,在含离子液体的混合反应体系和双相反应体系中,该不对称还原反应的效率得到了不同程度的提高.此外,离子液体中阴阳离子类型对该反应的影响较大,特定阴阳离子间的匹配对同定化红酵母细胞发挥最佳催化效果至关...     

培养基碳源对固定化酵母细胞催化乙酰基三甲基硅烷不对称还原反应的影响

 尽管含芳香基等较大取代基团的底物能被生物转化为高对映体纯手性有机硅醇,但生物催化其他底物还原反应的产物收率及对映体选择性均较低. 作者筛选到一株能高效催化羰基/羟基不对称转化的酵母菌株,探讨了用固定化酵母细胞高效催化乙酰基三甲基硅烷不对称还原制1-三甲基硅乙醇的可能性,系统研究了培养基碳源及其浓度对该反应的影响. 发现在水/正己烷两相体系中,固定化酵母细胞能催化乙酰基三甲基硅烷不对称还原成1-三甲基硅乙醇. 可通过调节培养基碳源的种类及其浓度在一定程度上控制反应的产物收率及对映体选择性. 以最适碳源(3.0%麦芽汁)培养的酵母细胞催化该反应的产物收率和对映体过量值分别为91.3%和72.8%,远高于文献报道值.     

(3-氮-吗啉)-1-(4-氯)-苯基-1-丙酮不对称还原产物光学活性的理论计算

采用AM1方法对(S)-4-苄基-5,5-二苯基-1,3,2-噁唑硼烷催化(3-氮-吗啉)-1-(4-氯)-苯基-1-丙酮不对称还原反应的立体控制步骤进行了计算,获得了R和S型过渡态的优化构型及其热焓和熵等热力学参数,计算得到了该步反应中生成R和S型对映体的反应速率常数之比,据此得到的不对称还原反应最终产物的光学活性e.e.的理论计算值,与实验结果相近.     

Asymmetric Reduction of Ethyl 4-Chloro-3-oxobutanoate to Ethyl (S)-4-Chloro-3-hydroxybutanoate Catalyzed by Aureobasidium pullulans in an Aqueous/Ionic Liquid Biphase System

The asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE) catalyzed by Aureobasidium pullulans CGMCC 1244 was performed in an aqueous/ionic liquid biphase system to improve the productivity and optical purity of (S)-CHBE. The influences of reaction parameters such as shaking rate, phase volumetric ratio, reaction temperature, initial substrate concentration, and pH were studied. In the aqueous/[bmim]PF₆ biphase system and under the optimum reaction conditions of 30 °C, pH 6.6, rotation speed 180 r/min, and reaction time of 8 h, the conversion of COBE, the enantiomeric excess, and the concentration of (S)-CHBE were 95.6%, 98.5%, and 47.1 g/L, respectively. When the substrate was fed in batches under the condition of pH 6.6, the accumulated product concentration could reach up to 75.1 g/L.     

Activity of immobilized trypsin in the layer structure of polyelectrolyte microcapsules (PEMC)

Polyelectrolyte microcapsules composed by using the LbL technique on stabilized RBC as templates were coated with up to ten layer pairs of trypsin/PSS or trypsin/alginate. The trypsin layer growth was confirmed by particle electrophoresis, confocal laser scanning microscopy, flow cytometry, and protein determination according to Lowry. In the coating series with trypsin/PSS, the amount of immobilized enzyme was larger than that with trypsin/alginate. The enzyme immobilization led to activity reduction of up to 90% compared to that of the same enzyme amount in the solution. No significant differences between the activities of trypsin immobilized in combination with PSS and with alginate were found.     

Immobilization of <Emphasis Type="Italic">Escherichia coli</Emphasis> novablue γ-glutamyltranspeptidase in Ca-alginate-<Emphasis Type="Italic">k</Emphasis>-carrageenan beads

The recombinant Escherichia coli gamma-glutamyltranspeptidase (EcGGT) was immobilized in Ca-alginate-kappa-carrageenan beads. Effects of alginate concentration, amount of loading enzyme, and bead size on the entrapped activity were investigated. Optimum alginate concentration for EcGGT immobilization was found to be 2% (w/v). Using a loading enzyme concentration of 1.5 mg/g alginate, maximum enzyme activity was observed. With increase in bead size from 1.9 to 3.1 mm, the immobilization efficiency was decreased significantly because of mass transfer resistance. Thermal stability of the free EcGGT was increased as a result of the immobilization. Ca-alginate-kappa-carrageenan-EcGGT beads were suitable for up to six repeated uses, losing only 45% of their initial activity. Upon 30 days of storage the preserved activity of free and immobilized enzyme were found as 4% and 68%, respectively. The synthesis of L: -theanine was performed in 50 mM Tris-HCl buffer (pH 10) containing 25 mM L: -glutamine, 40 mM ethylamine, and 1.5 mg EcGGT/g alginate at 40 degrees C for 12 h, and a conversion rate of 27% was achieved.     

固定化酵母非水相催化羰基不对称还原反应的研究

用海藻酸钙包埋法对增殖培养的酵母细胞进行了固定化,并用于催化有机溶剂中乙酰乙酸乙酯的不对称还原反应。考察了固定化时所用缓冲溶液的pH、催化剂颗粒大小与用量、辅助底物种类、底物浓度、以及重复利用批次等因素对反应产物（S）-3-羟基丁酸乙酯的浓度和光学纯度的影响。结果表明,固定化时应采用pH为7的Tris-HCL缓冲溶液,颗粒的直径以2mm左右为较佳;反应时应以正已烷为溶剂,正已醇为辅助底物,固定化酵母颗粒的最适用量为6g/20ml反应液;底物的初始浓度以100mmol/L为佳,浓度过高对反应有一定的抑制作用;固定化细胞重复利用三次对映选择性基本保持不变。     

Production of 4-Deoxy-L-erythro-5-Hexoseulose Uronic Acid Using Two Free and Immobilized Alginate Lyases from Falsirhodobacter sp. Alg1

Falsirhodobacter sp. alg1 expresses two alginate lyases, AlyFRA and AlyFRB, to produce the linear monosaccharide 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH) from alginate, metabolizing it to pyruvate. In this study, we prepared recombinant AlyFRA and AlyFRB and their immobilized enzymes and investigated DEH production. Purified AlyFRA and AlyFRB reacted with sodium alginate and yielded approximately 96.8% DEH. Immobilized AlyFRA and AlyFRB were prepared using each crude enzyme solution and κ-carrageenan, and immobilized enzyme reuse in batch reactions and DEH yield were examined. Thus, DEH was produced in a relatively high yield of 79.6%, even after the immobilized enzyme was reused seven times. This method can produce DEH efficiently and at a low cost and can be used to mass produce the next generation of biofuels using brown algae.     

Immobilization of growing cells by polyethyleneimine-modified alginate

A unique polymer matrix that is suitable for immobilizing growing cells has been developed. Alginate was chemically modified with polyethyleneimine (PEI), and the resultant polymer aggregate was evaluated as a cell carrier. Our method of immobilization depends on reversible gelation of the PEI-modified alginate. Our hypothesis is that immobilized cells grow by dissolving the surrounding gel matrix; the dissolved polymer adduct is displaced peripherally and gelled again by the influx of calcium ion from the surrounding fermentation broth, retaining both cells and carrier polymer in the gel beads. Thus, the immobilized cells gain space for growth by expanding the carrier matrix. The PEI modification offers the following advantages: (1) improved mechanical strength; (2) improved cell retention; (3) increased catalyst life; (4) ease of pelletization; and (5) an apparent bacteriostatic capability. When immobilized yeast cells were applied to a continuous ethanol fermentation, 94% theoretical conversion of glucose to ethanol was observed, with a reactor productivity of 15–30 g/L/h in a nonsterile reactor. A 3-mo catalyst life and minimal cell washout were observed.     

Ethanol Production Using Immobilized <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in Lyophilized Cellulose Gel

A new lyophilization technique was used for immobilization of Saccharomyces cerevisiae cells in hydroxyethylcellulose (HEC) gels. The suitability of the lyophilized HEC gels to serve as immobilization matrices for the yeast cells was assessed by calculating the immobilization efficiency and the cell retention in three consecutive batches, each in duration of 72 h. Throughout the repeated batch fermentation, the immobilization efficiency was almost constant with an average value of 0.92 (12–216 h). The maximum value of cell retention was 0.24 g immobilized cells/g gel. Both parameters indicated that lyophilized gels are stable and capable of retaining the immobilized yeast cells. Showing the yeast cells propagation within the polymeric matrix, the scanning electron microscope images also confirmed that the lyophilization technique for immobilization of S. cerevisiae cells in the HEC gels was successful. The activity of the immobilized yeast cells was demonstrated by their capacity to convert glucose to ethanol. Ethanol yield of 0.40, 0.43 and 0.30 g ethanol/g glucose corresponding to 79%, 84% and 60% of the theoretical yield was attained in the first, second and third batches, respectively. The cell leakage was less than 10% of the average concentration of the immobilized cells.     

Comparison of Alcoholic Fermentation Performance of the Free and Immobilized Yeast on Water Hyacinth Stem Pieces in Medium with Different Glucose Contents

Ethanol fermentation with Saccharomyces cerevisiae cells was performed in medium with different glucose concentrations. As the glucose content augmented from 200 to 250 g/L, the growth of the immobilized cells did not change while that of the free cells was reduced. At higher glucose concentration (300, 350, and 400 g/L), the cell proliferation significantly decreased and the residual sugar level sharply augmented for both the immobilized and free yeast. The specific growth rate of the immobilized cells was 27–65 % higher than that of the free cells, and the final ethanol concentration in the immobilized yeast cultures was 9.7–18.5 % higher than that in the free yeast cultures. However, the immobilized yeast demonstrated similar or slightly lower ethanol yield in comparison with the free yeast. High fermentation rate of the immobilized yeast was associated with low unsaturation degree of fatty acids in cellular membrane. Adsorption of S. cerevisiae cells on water hyacinth stem pieces in the nutritional medium decreased the unsaturation degree of membrane lipid and the immobilized yeast always exhibited lower unsaturation degree of membrane lipid than the free yeast in ethanol fermentation.