重组大肠杆菌不对称还原2-羟基苯乙酮合成(R)-苯基乙二醇

通过对近平滑假丝酵母(R)-专一性羰基还原酶（rCR）进行氨基酸序列分析并根据其序列的保守性设计PCR引物,以近平滑假丝酵母基因组为模板利用PCR技术得到目的基因rcr。该基因全长1011bp,共编码336个氨基酸。将该基因与表达载体pET21c连接后构建重组质粒pETRCR,并转化入Escherichia coli BL21（DE3）中进行表达。重组大肠杆菌具有(R)-专一性羰基还原酶活性,可催化不对称还原2-羟基苯乙酮合成(R)-苯基乙二醇。研究发现,在重组菌培养过程中,同添加IPTG诱导培养的情况相比,不添加IPTG诱导培养的酶活及不对称转化(R)-苯基乙二醇的效果较好。在反应过程中,转化48 h及在pH值 8～9的条件下更有利于不对称反应的进行。另外,较高的底物浓度会对反应产生抑制作用,通过提高重组菌细胞浓度可显著提高转化效果。在5 g/L的底物浓度条件下,利用0.3 g/mL重组菌细胞可还原2-羟基苯乙酮得到(R)-苯基乙二醇,产物光学纯度为95.5% e.e,产率为92.6%。     

树脂原位吸附促进高浓度底物不对称氧化还原合成（S）-苯基乙二醇

以近平滑假丝酵母(Candida parapsilosis)全细胞为催化剂,考察了添加吸附树脂对不对称氧化还原外消旋苯基乙二醇制备-(S)-苯基乙二醇(S-PED)的影响.通过树脂吸附性能考察,筛选出一种对底物有较快吸附速率和较大吸附量的树脂NKAⅡ,在反应体系中加入一定量NKAⅡ树脂,可以显著降低底物和产物对反应过程的抑制,提高反应的初始底物浓度.结合树脂对底物的吸附量和菌体反应的最适底物浓度,建立了树脂添加量随底物浓度变化的关系式,通过此公式添加树脂将溶液中底物产物总浓度控制在最适水平,从而实现高初始浓度底物,快速度反应.在40g/L初始底物浓度转化体系中加入0.74g树脂,平衡2h后,反应108h,产物S-PED的ee值和产率分别为98.1%和88.5%,产物浓度达到35.4 g/L.     

全细胞近平滑假丝酵母(Candida parapsilosis)SYB–1不对称转化苯基乙二醇

为了获得较高光学纯度和产率的光学纯苯基乙二醇，通过筛选得到不对称转化外消旋苯基乙二醇效果较好的菌株近平滑假丝酵母(Candida parapsilosis)SYB-1，其转化产物为S-苯基乙二醇，光学纯度为91% e.e.，产率为88%；通过对近平滑假丝酵母SYB-1拆分苯基乙二醇的反应条件进行优化研究，在细胞量5%、底物浓度8‰、pH值6.5、温度33℃、反应48 h条件下，产物光学纯度可以提高至e.e. 99%. 在证明中间体为b-羟基苯乙酮后推测全细胞近平滑假丝酵母SYB-1拆分外消旋苯基乙二醇的机理是通过将R-苯基乙二醇氧化为中间体后不对称还原为S-苯基乙二醇.     

离子液体/缓冲液两相体系中热带假丝酵母不对称还原制备(S)-1-[3,5-双(三氟甲基)苯基]乙醇

研究了1-丁基-3-甲基咪唑六氟磷酸盐[BMIM]PF6/缓冲液两相体系中,热带假丝酵母104细胞催化3,5-双三氟甲基苯乙酮不对称还原制备(S)-1-[3,5-双(三氟甲基)苯基]乙醇的反应过程。通过考察影响生物还原反应的主要因素,如离子液体浓度、辅助底物种类和浓度、底物浓度、菌体浓度和转化时间等,发现上述因素对产率影响较大,但基本不影响产物手性醇的光学纯度。优化得到的较佳还原反应条件为:[BMIM]PF6体积分数5%,辅助底物为60 g L 1异丙醇,底物3,5-双三氟甲基苯乙酮浓度70 mmol L 1,菌体浓度350 g L 1,转化时间24 h。在优化条件下,产率达82.5%,产物(S)-1-[3,5-双(三氟甲基)苯基]乙醇的对映体过量值大于99.9%。与水相转化相比,采用[BMIM]PF6/缓冲液两相体系进行3,5-双三氟甲基苯乙酮的生物不对称还原可有效提高底物浓度和产率,且反应时间缩短了6 h。     

有机酸对产丙二醇菌生长和生产的影响

为提高克雷伯氏肺炎杆菌厌氧发酵生产1,3-丙二醇的能力,在发酵过程中添加有机酸(OA),考察其对菌体生长、1,3-丙二醇及其他副产物合成的影响。实验表明:添加OA可促进菌体生长,加入质量浓度为0.2~0.5g/L的OA,菌体生长的菌液在590 nm处吸光度(OD)值比参照值高出30%;加入过量的OA(>1.0 g/L),则菌体生长受到抑制,OD值比参照值低37%;添加OA可促进单位细胞合成1,3-丙二醇,加入质量浓度为0.2~0.5g/L的OA,单位菌体的1,3-丙二醇质量浓度比参照高3.8%~17.5%;添加OA能提高甘油转化为1,3-丙二醇的转化速率,加入0.2~0.5 g/L的OA,甘油转化为1,3-丙二醇的最大转化速率达0.81 h-1。在15 L罐上批式发酵48 h,1,3-丙二醇最终质量浓度达42.9 g/L,甘油转化为1,3-丙二醇的平均转化率达64%。     

羰基还原酶与甲酸脱氢酶偶联催化制备(R)-苯基乙二醇

以重组菌E. coli Rosetta/pETDuet-rcr-fdh全细胞为催化剂,2-羟基苯乙酮和甲酸钠为双底物,研究了生物催化反应体系中温度、pH值、底物2-羟基苯乙酮的初始浓度、反应时间、细胞浓度和状态等对产物(R)-苯基乙二醇生成效率的影响规律. 结果表明,在温度35℃和pH 7.0、底物初始浓度6 g/L、时间36 h、湿细胞浓度10%(w)的条件下,产物的光学纯度高达98.37% e.e.,得率达79.14%. 在上述反应体系中添加5 mmol/L ZnSO4后,重组菌催化的不对称还原反应效率显著提高,产物(R)-苯基乙二醇光学纯度达到100% e.e.,得率高达86.3%.     

氮源影响酵母耐受超高浓度乙醇发酵胁迫条件

为考察蛋白胨和酵母浸出膏对酵母耐受超高浓度乙醇发酵胁迫条件的影响,以300g/L起始浓度葡萄糖开展实验。结果表明,与对照组(3g/L蛋白胨+5g/L酵母浸出膏作为氮源)相比,单独提高发酵培养基蛋白胨至6g/L或酵母浸出膏至12g/L,均可明显促进菌体生长和葡萄糖利用,终点乙醇体积分数由对照组的13.1%分别提高至14.4%和14.7%。研究表明,在发酵过程中,生长于提高蛋白胨浓度或酵母浸出膏浓度培养基的菌体,其质膜ATP酶活力和胞内海藻糖积累量明显高于对照组,而且发酵参数(如菌体生长、葡萄糖利用和终点乙醇体积分数)的提高与酶活力和海藻糖含量的增加密切相关,提示质膜ATP酶和胞内海藻糖在酵母耐受超高浓度乙醇发酵胁迫条件中的作用。     

乙醇对简单节杆菌TCCC11037生长和细胞性质的影响

研究了乙醇体积分数对简单节杆菌TCCC 11037 生长和细胞性质的影响.结果表明,随着乙醇体积分数的增大,菌体的延滞期延长,生物量和比生长速率下降,当乙醇体积分数为6%时几乎不能观察到明显的菌体生长;菌体形态随着乙醇体积分数的增大出现较明显的改变,当乙醇体积分数为8%时,细胞完整性明显破坏,胞浆大量外泄;菌体的耗糖能...     

代谢副产物对Klebsiella pneumoniae生长及合成1,3-丙二醇的影响

研究了Klebsiella pneumoniae在厌氧摇瓶中的生长代谢特性和基质消耗情况,发现主要副产物乙醇是抑制菌体持续生长及1,3-丙二醇合成的主要因素,外源添加实验表明,8 g/L乙醇可使K. pneumoniae比生长速率、1,3-丙二醇比合成速率、最大菌体浓度及1,3-丙二醇终浓度分别下降21.6%, 22.1%, 59.6%及33.5%;指数生长期加入乙醇对菌体生长代谢的抑制作用更加明显. 其他代谢副产物乙酸、乳酸、2,3-丁二醇对K. pneumoniae生长代谢也有不同程度影响,乙酸浓度仅2 g/L即可对菌体生长产生抑制,乙酸浓度达到5 g/     

E.coli M15 (pQTPL)高效发酵生产酪氨酸酚裂解酶的控制策略

在摇瓶和4 L发酵罐上研究了营养和环境条件对重组菌E. coli M15 (pQTPL)分批发酵生产酪氨酸酚裂解酶(TPL)的影响. 在培养基中添加20 g/L葡萄糖和1.0 g/L玉米浆使TPL酶活提高到63.1 U/g(干重). 在此基础上,维持发酵液中溶氧水平为30%,可使菌体浓度在8 h达到4.78 g/L,酶活为54.6 U/g,比对照组(不控制溶氧)分别提高了21%和31.6%. 采用溶氧反馈调节-限制性补料策略,可使菌体浓度提高到31.5 g/L. 采用两阶段温度和pH控制策略,在发酵前8 h控制pH 7.0、温度37℃,8 h 至发酵结束之间控制pH为8.0、温度为30℃,可使重组菌的TPL酶活达到154.4 U/g,并使TPL在细胞中过量表达,实现了高菌体浓度和高TPL酶活的统一.     

耦合透性化辅酶循环制备手性(R)-苯基乙醇

通过耦合透性化博伊丁假丝酵母(Candida boidinii,C.boidinii)加强了近平滑假丝酵母(Candida parapsilosis,C.parapsilosis)内羰基还原酶(Carbonyl reductase,CR)催化反应中的辅酶循环与反复利用,构建了有效的手性(R)-苯基乙醇透性化细胞耦合制备体系。实验结果如下:透性化的C.parapsilosis细胞底产物渗透效果增强,底物转化率由33.5%提升到43.8%;进一步外添加辅酶后,底物转化率由43.8%提高到了52.1%;耦合透性化C.boidinii细胞并同时外添加辅酶可大大增强辅酶循环效率,底物转化率可提高至71.8%。优化透性化细胞耦合体系反应条件,当以0.2 g近平滑假丝酵母(C.parapsilosis)为基准时,在C.parapsilosis与C.boidinii细胞质量比为1.5∶1,底物浓度为10 mmol/L,辅酶浓度为0.1 mmol/L时,底物转化率可达78.2%。离心法收集细胞进行多批次反应,当反应12批次时,耦合体系仍然保持有55%底物转化率,此时总的底物转化率达70.3%,产物经高效液相色谱测定为(R)-苯基乙醇,对映体过量值(e.e.值)达98.2%。     

生物不对称合成R-(-)-扁桃酸的影响因素

研究了酵母细胞(Saccharomyces cerevisiae)生物不对称还原苯乙酮酸合成R-(-)-扁桃酸的过程中,环境因子对底物的转化效率和产物对映体过量值的影响. 结果表明,高浓度的底物苯乙酮酸对酵母细胞的催化还原活性具有较显著的抑制效应. pH 6.5、温度32℃、严格厌氧为较适宜条件,底物苯乙酮酸的转化率和产物扁桃酸的得率分别可达97.0%和96.1%,R-(-)-扁桃酸的对映体过量值(ee)为95.1%.     

Asymmetric Borane Reduction of Prochiral Ketones Catalyzed by α,α-Disubstituted Aziridinemethanols

A series of novel α,α-disubstituted aziridinemethanols have been developed for the enantioselective reduction of ketones in refluxing tetrahydrofuran. The optically active secondary alcohol products were obtained in good enantiomeric excess (~96.8%) and excellent yields. The results showed that the substituent groups on the hydroxyl-bearing carbon of aziridinemethanols obviously affected the enantioselectivity.     

Yeast‐mediated enantioselective synthesis of chiral R‐(+)‐ and S‐(−)‐1‐phenyl‐1‐butanol from prochiral phenyl n‐propyl ketone in hexane–water biphasic culture

BACKGROUND: The hydrophobic phenyl n‐propyl ketone was used as a model compound to examine alcohol dehydrogenase activity in Saccharomyces cerevisiae mediated cell culture. Parameters such as pH, hexane‐to‐water volume percentage, and the amount of cofactor Zn²⁺ ion for either cell growth or reduction were studied to see their effect on the enantioselectivity toward the product R‐(+)‐ or S‐(?)‐1‐phenyl‐1‐butanol. RESULTS: The pH for cell growth in aqueous culture was 7.0, while the pH for reduction in the aqueous portion of the biphasic culture was 5.0. Without Zn²⁺ ion the biphasic cultures of middle to high hexane‐to‐water volume percentage exhibited an R‐(+)‐1‐phenyl‐1‐butanol enantiomeric excess of 53.7% to > 99%. Without Zn²⁺ ion the biphasic cultures at low hexane‐to‐water volume percentage possessed an S‐(?)‐1‐phenyl‐1‐butanol enantiomeric excess of 14.5–46.5%. Exclusively, the enantioselectivity for biphasic cultures containing Zn²⁺ ion was an S‐(?)‐1‐phenyl‐1‐butanol enantiomeric excess of 27.5% to > 99%. Reduction mediated in aqueous culture with varied amount of Zn²⁺ ion by the yeast Candida utilis also showed an S‐(?)‐1‐phenyl‐1‐butanol enantiomeric excess of 79.2–95.4%. CONCLUSION: The enantioselectivity of S. cerevisiae mediated biphasic culture reduction of phenyl n‐propyl ketone can be manipulated through the cofactor Zn²⁺ ion and the hexane volume percentage of the biphasic culture. Copyright © 2008 Society of Chemical Industry     

一种新的高立体选择性羰基还原酶的性质及分离

从近平滑假丝酵母（Candida parapsilosis CCTCC 203011）中分离得到了新的NADPH依赖型羰基还原酶。粗酶经硫铵分级沉淀、DEAE Sepharose离子交换层析、Phenyl-sepharose FF疏水层析、Blue Sepharose FF亲和层析后在SDS-PAGE上显示为单一条带,其酶蛋白的相对分子质量为30 kD。该酶还原反应的最适pH值为4.5,最适温度为35 ℃,Cu2+对羰基还原酶有强烈的抑制作用。该酶具有较高的底物专一性和立体选择性,对α-羟基苯乙酮和4-氯乙酰乙酸乙酯具有较高的不对称还原活力,其产物分别为（S）-苯基乙二醇和(R)-4-氯-3-羟基丁酸乙酯,e.e值分别为100%和94.3%。因此该酶蛋白是不对称合成手性醇有效的生物催化剂之一。经LC-MASS-MASS分析得到了酶蛋白中一个肽段的氨基酸序列,通过比对发现该酶与假定蛋白（hypothetical protein CaO19.10414）具有一定的同源性。     

Enantioselective hydrogenation of ethyl pyruvate. Influence of oxidative treatment of cinchonidine-modified platinum catalyst and hemiketal formation in alcoholic solvents

The reasons for the increase in the rate and enantiomeric excess after oxidative (aerobic) treatment of Pt/alumina in ethanol have been investigated. It is demonstrated that this treatment results in the formation of acetic acid and consequently in the protonation of the quinuclidine n₁ of cinchonidine. This favours the cinchonidine-pyruvate interaction and improves enantioselectivity. In addition, the reaction rate is enhanced due to acid catalysis of the carbonyl reduction. NMR and UV measurements indicate the rapid transformation of ethyl pyruvate to the corresponding hemiketal in primary alcohols as solvents. It is shown that the possible involvement of this hemiketal (and that formed between cinchonidine and ethyl pyruvate) as an intermediate in the pyruvate hydrogenation mechanism can be excluded.     

CALB脂肪酶的固定化及其拆分2-辛醇的研究

以AB-8、HZ-841、HZ-802三种大孔树脂做载体,采用物理吸附法,制备出固定化南极假丝酵母脂肪酶(CALB),并用其进行了拆分2-辛醇的研究。其中AB-8树脂做载体拆分效果最佳,其蛋白吸附量为37.94 mg/g树脂,吸附率94.86%,转酯化酶活3 000 U/g固定化酶,对映体选择性E=104。单因素优化实验得到的最佳拆分条件为:温度40℃,加酶量2.67 g/L,底物醇浓度3.76 mol/L。在该条件下,产物转化率可达50%,e.ep为97.8%。固定化pH在5.0~9.0内对拆分效果无显著影响。     

有机介质中脂肪酶促动力学拆发外消旋1-三甲基硅乙醇

The enantioselective esterification of racemic 1-trimethylsilylethanol with acids catalyzed by lipase in organic solvent was successfully performed. The influence of some factors on the reaction was investigated. Among the four lipases explored, Candlda rugosa lipase (CRL) showed the highest activity and enantioselectivity. Octanoic acid was the best acyl donor among the eleven acids studied and n-hexane was the most suitable medium for the reaction. The optimum shaking rate and temperature were found to be 150 r-rain-i and 20~(3 to 30~C, respectively.The enantiomeric excess of the remaining (S)-(-)-1-trimethylsilylethanol was 93% when substrate conversion was 53% upon incubation of the reaction mixture at 30~C, 150 r-rain-i for 12 h.     

Involvement of an Oxidation‐Reduction Equilibrium in Chromium‐Mediated Enantioselective Nozaki–Hiyama Reactions

Ligand induced enantioselective versions of the chromium(II)‐mediated Nozaki–Hiyama reaction to homoallyl alcohols proved to be very difficult to achieve, especially if any other nucleophile than the parent allylchromium(III) species was applied. Also, the reaction is frequently accompanied by the formation of oxidation side products, predominantly allyl ketones. This can be explained by an Oppenauer–(Meerwein–Ponndorf Verley) type mechanism (OMPV reaction). The addition of an enantiopure ligand to racemic chromium homoallyl alcoholate intermediates produced enantiomerically enriched homoallyl alcohols with an enantiomeric excess of up to 32%. This observation not only supports that the proposed OMPV oxidation‐reduction equilibrium plays a crucial role in Nozaki–Hiyama reactions, but also proves its involvement in enantioselective versions.