一种新型(S)-羰基还原酶的克隆及其功能表达

【目的】从近平滑假丝酵母(Candida parapsilosis CCTCC M203011)基因组中钓取新型(S)-羰基还原酶基因(scrⅡ),对其生物转化手性醇的功能进行了验证。【方法】采用PCR的方法,从C.parapsilosis基因组中扩增出一段可能的羰基还原酶基因scrⅡ。以构建的重组菌Escherichia coli BL21/pET28a-scrⅡ为生物催化剂,2-羟基苯乙酮为底物进行催化反应,经HPLC分析,计算终产物的光学纯度和产率,确定了转化反应的最适温度和pH值。【结果】scrⅡ基因全长为840bp,编码279个氨基酸,与已报道的(S)-羰基还原酶基因scr的一致性为85%。氨基酸序列分析表明SCRⅡ具有典型短链醇脱氢酶的功能域:辅酶结合区域Thr40-Gly41-(X)3-Gly45-X-Gly47和催化三联体结构Ser172-(X)n-Tyr187-(X)3-Lys191。在30℃,0.1mmol/LIPTG的诱导下,(S)-羰基还原酶(SCRⅡ)在E.coli中过量表达。以10%(w/v)的重组菌为催化剂,高浓度(6g/L)2-羟基苯乙酮为底物,在最适反应温度35℃和pH5.5的条件下,转化产物(S)-苯基乙二醇的光学纯度高达99.1%e.e.,产率为89.6%。与(S)-羰基还原酶SCR相比较,底物浓度提高了一倍,产物的光学纯度和产率分别提高了10%和28%。【结论】采用分子克隆技术分离出新型羰基还原酶SCRⅡ的编码基因,该酶的发现为手性醇的高效制备奠定了坚实的研究基础。     

（R）与（S）-羰基还原酶偶联一步法制备（S）-苯乙二醇

【目的】通过 (R) - 和(S) -羰基还原酶在大肠杆菌中偶联,实现了一步法制备（S）-苯乙二醇的生物转化过程。【方法】将来源于近平滑假丝酵母(Candida parapsilosis CCTCC M203011)的(R)- 羰基还原酶基因（rcr）和(S) -羰基还原酶基因（scr）串联于共表达载体pETDuetTM-1上。重组质粒pETDuet-rcr-scr转化稀有密码子优化型菌株Escherichia coli Rosetta,获得酶偶联重组菌株E. coli Rosetta / pETDuet-rcr-scr。当重组菌体培养至OD600 0.6-0.8时,添加终浓度1 mmol/L IPTG,30℃诱导蛋白表达10 h。【结果】SDS-PAGE结果表明(R)- 和(S) -羰基还原酶均明显表达,它们的相对分子质量分别为37 kDa和30 kDa。重组菌生物转化结果表明：在pH7.0的磷酸缓冲液中,添加5 mmol/L Zn2+时,获得产物(S)-苯乙二醇,产物光学纯度为91.3% e.e.,产率为75.9%。【讨论】采用分子重组技术成功整合了两种氧化还原酶的催化功能,实现了(S)- 苯乙二醇的一步法转化,为简化手性醇制备途径提供了一条崭新的思路。     

(R)-专一性羰基还原酶与甲酸脱氢酶基因在大肠杆菌中的共表达

【目的】通过研究(R)-专一性羰基还原酶和甲酸脱氢酶基因在大肠杆菌中的共表达,解决较高底物浓度下不对称转化反应的辅酶限制性问题。【方法】分别以近平滑假丝酵母(Candida parapsilosis CCTCC M203011)和博伊丁假丝酵母(Candida boidinii)基因组为模板,采用PCR方法扩增得到(R)-专一性羰基还原酶基因（rcr）和甲酸脱氢酶基因（fdh）,克隆到共表达载体pETDuetTM-1中进行表达。共表达质粒pETDuet-rcr-fdh转化稀有密码子优化型菌株E. coli Rosetta,获得重组菌E. coli Rosetta/pETDuet-rcr-fdh。【结果】在30℃条件下,经1 mmol/L IPTG诱导表达8 h后,SDS-PAGE结果表明(R)-专一性羰基还原酶和甲酸脱氢酶均有明显的表达,其相对分子质量分别为37 kDa和 40 kDa。以高浓度(6 g/L)2-羟基苯乙酮为底物时,0.1 g重组菌细胞催化产生(R)-苯基乙二醇,产物光学纯度为100% e.e.,产率为85.9%。与无甲酸脱氢酶参与辅酶再生循环的重组菌E. coli Rosetta/pETDuet-rcr相比,产物光学纯度和产率分别提高了1.3和2.7倍。【讨论】该重组菌的构建为基因工程法生物合成(R)-苯基乙二醇的工业应用奠定了基础。     

(S)-羰基还原酶Ⅱ与葡萄糖脱氢酶共催化高效合成(S)-苯乙二醇北大核心CSCD

【目的】通过优化获得最佳酶活配比,设计近平滑假丝酵母(Candida parapsilosis)CCTCC M203011的(S)-羰基还原酶Ⅱ与枯草芽孢杆菌(Bacillus sp.)YX-1葡萄糖脱氢酶在大肠杆菌中的共表达体系,实现重组菌高效催化2-羟基苯乙酮,合成(S)-苯乙二醇。【方法】分别从重组大肠杆菌中纯化了(S)-羰基还原酶Ⅱ和葡萄糖脱氢酶,研究了2种酶共催化2-羟基苯乙酮的最佳酶活比例,最适催化温度和pH,由此构建(S)-羰基还原酶Ⅱ和葡萄糖脱氢酶的共表达体系。【结果】(S)-羰基还原酶Ⅱ的比酶活力为1.3 U/mg,葡萄糖脱氢酶的比酶活力为13.5 U/mg。在总酶活力为1 U时,(S)-羰基还原酶Ⅱ和葡萄糖脱氢酶共催化体系中,确定了2种酶的最佳比例在1∶1到5∶1(U/U)之间,最适反应温度为30℃,pH为7.0。在此基础上构建了(S)-羰基还原酶Ⅱ和葡萄糖脱氢酶基因比为1∶1的共表达体系,共表达重组菌破碎上清液中(S)-羰基还原酶Ⅱ和葡萄糖脱氢酶酶活分别为0.76 U/mg和0.73 U/mg,两者的酶活比例为1∶1。在上述确定的最适催化条件下,其催化10 g/L 2-羟基苯乙酮,产物(S)-苯乙二醇的光学纯度和得率均高达99%以上。与仅含有(S)-羰基还原酶Ⅱ的重组大肠杆菌相比,共表达体系转化产物(S)-苯乙二醇的得率明显提高,且转化时间由原来的24 h缩短为13 h。【结论】通过确定(S)-羰基还原酶Ⅱ和葡萄糖脱氢酶最佳酶活配比,为构建手性催化的靶酶和辅酶再生酶共表达体系,为实现手性化合物的高效制备提供了研究基础。     

Sortase A介导的(S)-羰基还原酶Ⅱ寡聚体高效立体选择性转化(S)-苯基乙二醇

【目的】以金黄色葡萄球菌(Staphylococcus aureus)的sortase A为"分子订书机",用于(S)-羰基还原酶Ⅱ分子之间的连接,获得催化功能与稳定性增强的氧化还原酶寡聚体,高效催化2-羟基苯乙酮,合成(S)-苯基乙二醇。【方法】从S.aureus基因组中克隆sortase A基因,在大肠杆菌中表达,通过镍柱和凝胶层析纯化重组酶,获得纯酶sortase A。通过基因工程手段在(S)-羰基还原酶Ⅱ的C末端添加GGGGSLPETGG序列,蛋白纯化获得(S)-羰基还原酶Ⅱ-GGGGSLPETGG,摸索了sortase A催化(S)-羰基还原酶Ⅱ-GGGGSLPETGG的分子连接,形成(S)-羰基还原酶Ⅱ寡聚体的最佳条件,并研究了寡聚体酶学性质及生物转化(S)-苯基乙二醇的效率。【结果】(S)-羰基还原酶Ⅱ寡聚体比酶活力为38.5 U/mg,比原始型(S)-羰基还原酶Ⅱ提高了6倍,最适反应温度为50°C,最适pH为6.0,在50°C放置1 h后酶活仍旧保持90%以上;蛋白质变性实验结果显示,(S)-羰基还原酶Ⅱ寡聚体的变性温度为60.1°C,比原始酶提高了10°C;生物转化结果显示(S)-羰基还原酶Ⅱ寡聚体在3 h内完全转化5 g/L 2-羟基苯乙酮,产生光学纯度为100%的(S)-苯基乙二醇,相比于重组大肠杆菌(S)-羰基还原酶Ⅱ全细胞催化时间缩短了16倍。【结论】本研究首次将sortase A应用于氧化还原酶的分子连接,显著提高了酶的催化效率和热稳定性,表明sortase在手性催化中有很大的潜在应用价值。     

马克斯克鲁维酵母GX-UN120糖转运蛋白基因的克隆及表征

【背景】马克斯克鲁维酵母(Kluyveromyces marxianus)具有完整的木糖代谢途径,可以高效利用木质纤维素中的木糖,因此对其糖转运蛋白基因的研究或可有效解决酵母木糖转运的相关问题。【目的】根据马克斯克鲁维酵母DMKU3-1042中KLMA＿70145和KLMA＿80101基因位点的功能预测,获得马克斯克鲁维酵母GX-UN120相应的糖转运蛋白基因序列并探究其功能。【方法】将转运蛋白基因分别克隆表达至酿酒酵母EBY.VW4000中考察重组菌株生长特性,以此间接评价对应转运蛋白的转运能力。【结果】Km＿SUT2基因编码的糖转运蛋白可有效提高宿主细胞转运木糖、阿拉伯糖、山梨糖、核糖、乳糖和葡萄糖的能力,但却不能转运甘露糖、果糖、蔗糖和半乳糖。类似地,Km＿SUT3基因编码的糖转运蛋白可提高细胞转运木糖、阿拉伯糖、山梨糖、半乳糖、核糖、乳糖和葡萄糖的能力,但却不能转运甘露糖和果糖。然而在葡萄糖存在的条件下,重组菌株对各种碳源的利用均受抑制,但Km＿SUT3转运木糖和核糖过程中受葡萄糖的抑制作用较小。【结论】马克斯克鲁维酵母GX-UN120中转运蛋白Km＿SUT2和Km＿SUT3可...     

海栖热袍菌极耐高温木聚糖酶基因xynB64在大肠杆菌中的融合表达

来源于极端嗜热菌海栖热袍菌(Thermotoga maritima MSB8)的木聚糖酶B具有极高的热稳定性,在饲料、造纸、能源和食品医药行业具有巨大应用潜力。携带酶基因xynB64的pET28a(+)重组载体在宿主大肠杆菌BL21(DE3)中诱导表达,重组酶活力较低。更换宿主为携带稀有tRNA基因的大肠杆菌:BL21-CodonPlus(DE3)-RIPL和Rosetta(DE3)后,酶活力分别提高了197%和277%,但是后者中的表达会形成部分包涵体。宿主菌为大肠杆菌Rosetta(DE3),更换载体为4种融合表达载体pET32a(+)、pET42a(+)、pET43.1a(+)和pMAL-c2X进行表达,重组酶分别融合了Trx、GST、Nus和MBP标签。其中Rosetta(DE3)/pMAL-c2X-xynB64表达酶活力最高,相当于Rosetta(DE3)/pET28a-xynB64表达酶的88%,而且目的酶表达量占全细胞蛋白的40%,几乎不形成包涵体。     

定点突变改变近平滑假丝酵母SCRⅡ催化苯乙酮衍生物底物谱

【目的】通过定点突变技术,改变近平滑假丝酵母短链羰基还原酶Ⅱ(SCRⅡ)催化苯乙酮衍生物的功能,为数种手性芳香醇的生产提供一种高效、安全的新型制备方法。【方法】通过氨基酸序列和蛋白结构比对的方法,选择SCRⅡ的底物结合域中关键氨基酸位点E228实施突变,构建相应的突变株Escherichia coliBL21/pET28a-E228S;以苯乙酮衍生物为底物,对突变株的酶活和生物转化功能进行了分析。【结果】酶活测定结果表明:突变株E.coli BL21/pET28a-E228S催化原始底物2-羟基苯乙酮的酶活仅为原始酶活的25%左右;而催化苯乙酮、4’-甲基苯乙酮、4’-氯苯乙酮的酶活是突变前的7-20倍。突变株E.coli BL21/pET28a-E228S生物转化2-羟基苯乙酮,获得产物(S)-苯基乙二醇的得率不超过10%,而以苯乙酮、4’-甲基苯乙酮、4’-氯苯乙酮为底物时,生物转化产物光学纯度维持在99%,得率高达80%以上。【结论】对底物结合域中的关键氨基酸实施突变,提高了SCRⅡ催化苯乙酮衍生物的底物广谱性,拓展了该酶的生物功能,为理性改造短链羰基还原酶的不对称还原催化功能和手性芳香醇的制备提供了新型途径。     

腐皮镰孢菌壳聚糖酶的酶学性质研究及其在酿酒酵母工业菌株中的表达

摘要：【目的】本研究旨在了解腐皮镰孢菌（Fusarium solani）壳聚糖酶的基本酶学性质及其在壳寡糖生产中的应用,构建能高效分泌表达壳聚糖酶的酿酒酵母工业菌株。【方法】采用RT-PCR扩增腐皮镰孢菌壳聚糖酶的cDNA序列;通过组氨酸标签,纯化得到E. coli表达的重组壳聚糖酶,并进行基本酶学性质研究;以薄层层析、高效液相色谱等技术对该酶的酶解产物进行分析;通过马克斯克鲁维酵母（Kluyveromyces marxianus）菊粉酶信号肽（INU1A）实现壳聚糖酶在酿酒酵母工业菌株N-27中的分泌表     

(S)-羰基还原酶II于酿酒酵母孢子中表达与功能

摘要:【目的】使近平滑假丝酵母(Candida parapsilosis CCTCC M203011)的(S) -羰基还原酶II 表达并包埋于酿酒酵母(Saccharomyces cerevisiae AN120)孢子中,实现了重组酶高效催化生产(S) -苯基乙二醇的转化过程。【方法】采用PCＲ 扩增技术,从近平滑假丝酵母基因组中克隆(S) -羰基还原酶II 基因,于酿酒酵母AN120中表达,以醋酸钾为唯一碳源诱导培养产生孢子,包埋(S)-羰基还原酶II。以该孢子为生物催化剂,2-羟基苯乙酮为底物进行生物转化反应,经HPLC分析,计算产物的光学纯度和得率。考察了孢子催化转化反应的最适温度和pH值,温度和pH 稳定性以及多批次使用性能。【结果】在最适反应温度40℃和pH6.0条件下,10%(W/V)子囊孢子催化6 g/L 2-羟基苯乙酮,产物(S) -苯基乙二醇的光学纯度和得率均高达99%以上。与重组大肠杆菌相比较,重组孢子合成(S)-苯基乙二醇的得率由89.7% 提高到99.0%,反应时间由48 h缩短为4 h;连续使用10批次后,其催化产物的光学纯度几乎不变,得率保持在85%以上。【结论】该研究首次实现了氧化还原酶在酵母孢子内的异源表达,为手性化合物的高效制备奠定了坚实的研究基础。     

近平滑假丝酵母(R)-羰基还原酶在大肠杆菌中的外泌表达及高效转化(R)-苯基乙二醇

克隆了近平滑假丝酵母(Candida parapsilosis)(R)-羰基还原酶基因rcr,构建胞外表达工程茵Escherichia coli BL21(DE3)/pET20b-rcr,实现了(R)-羰基还原酶在大肠杆菌中高效外泌表达,周质空间和发酵液酶的比活力分别达0.68 U/mg和0.26 U/mg,与大肠杆菌的胞内体系重组酶相比,酶的比活力提高了近两倍。为了更好地促进该重组酶的外分泌于大肠杆菌细胞外,通过添加温和型化学渗透剂甘氨酸,改善细胞壁的透性,(R)-羰基还原酶的活力提高至1.99 U,与添加甘氨酸前相比,酶活力提高了12.4倍,比活提高了4.3倍。浓缩后的发酵液催化2-羟基苯乙酮,产生(R)-苯基乙二醇,产率为88.1%,e.e.值为93.9%。与胞内重组酶相比,产率和光学纯度分别提高了44.4%和15.9%。本研究通过构建(R)-羰基还原酶的大肠杆菌分泌表达体系,大幅度提高了(R)-羰基还原酶的比活和生物转化手性醇的效率。     

Cloning and overexpression of the Exiguobacterium sp. F42 gene encoding a new short chain dehydrogenase, which catalyzes the stereoselective reduction of ethyl 3-oxo-3-(2-thienyl)propanoate to ethyl (S)-3-hydroxy-3-(2-thienyl)propanoate

Exiguobacterium sp. F42 was screened as a producer of an enzyme catalyzing the NADPH-dependent stereoselective reduction of ethyl 3-oxo-3-(2-thienyl)propanoate (KEES) to ethyl (S)-3-hydroxy-3-(2-thienyl)propanoate ((S)-HEES). (S)-HEES is a key intermediate for the synthesis of (S)-duloxetine, a potent inhibitor of the serotonin and norepinephrine uptake carriers. The responsible enzyme (KEES reductase) was partially purified, and the gene encoding KEES reductase was cloned and sequenced via an inverse PCR approach. Sequence analysis of the gene for KEES reductase revealed that the enzyme was a member of the short chain dehydrogenase/reductase family. The probable NADPH-interacting site and 3 catalytic residues (Ser-Tyr-Lys) were fully conserved. The gene was highly expressed in Escherichia coli, and the gene product was purified to homogeneity from the recombinant E. coli by simpler procedures than from the original host. The molecular mass of the purified enzyme was 27,500 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 55,000 as determined by gel filtration chromatography. Our results show that this enzyme can be used for the practical production of (S)-HEES.     

Molecular cloning and overexpression of the gene encoding an NADPH-dependent carbonyl reductase from Candida magnoliae, involved in stereoselective reduction of ethyl 4-chloro-3-oxobutanoate

An NADPH-dependent carbonyl reductase (S1) isolated from Candida magnoliae catalyzed the reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate (CHBE), with a 100% enantiomeric excess, which is a useful chiral building block for the synthesis of pharmaceuticals. The gene encoding the enzyme was cloned and sequenced. The S1 gene comprises 849 bp and encodes a polypeptide of 30,420 Da. The deduced amino acid sequence showed a high degree of similarity to those of the other members of the short-chain alcohol dehydrogenase superfamily. The S1 gene was overexpressed in Escherichia coli under the control of the lac promoter. The enzyme expressed in E. coli was purified to homogeneity and had the same catalytic properties as the enzyme from C. magnoliae did. An E. coli transformant reduced COBE to 125 g/l of (S)-CHBE, with an optical purity of 100% enantiomeric excess, in an organic solvent two-phase system.     

Biochemical characterisation of a NADPH-dependent carbonyl reductase from Neurospora crassa reducing α- and β-keto esters

A gene encoding an NADPH-dependent carbonyl reductase from Neurospora crassa (nccr) was cloned and heterologously expressed in Escherichia coli. The enzyme (NcCR) was purified and biochemically characterised. NcCR exhibited a restricted substrate spectrum towards various ketones, and the highest activity (468U/mg) was observed with dihydroxyacetone. However, NcCR proved to be very selective in the reduction of different α- and β-keto esters. Several compounds were converted to the corresponding hydroxy ester in high enantiomeric excess (ee) at high conversion rates. The enantioselectivity of NcCR for the reduction of ethyl 4-chloro-3-oxobutanoate showed a strong dependence on temperature. This effect was studied in detail, revealing that the ee could be substantially increased by decreasing the temperature from 40 °C (78.8%) to -3 °C (98.0%). When the experimental conditions were optimised to improve the optical purity of the product, (S)-4-chloro-3-hydroxybutanoate (ee 98.0%) was successfully produced on a 300 mg (1.8 mmol) scale using NcCR at -3 °C.     

对硝基苯酚降解菌Pseudomonas sp. PDS-7的降解特性及 其降解相关基因的克隆

[目的]本研究的目的是分离对硝基苯酚(PNP)降解菌,研究其对PNP的降解特性;克隆其降解相关基因,并进行表达.[方法]本研究通过富集培养法和系列稀释平板涂布法分离PNP降解菌株;采用形态观察、生理生化特征测定和16S rDNA分析对菌株进行初步鉴定;通过摇瓶试验研究菌株降解特性;利用SEFA-PCR技术克隆降解相关基因,并亚克隆到表达载体pET29a中,构建重组表达质粒pETpnpC,再转入受体菌E.coli BL21(DE3)中进行诱导表达;通过分光光度法测定表达产物的酶活力.[结果]分离到一株PNP降解菌PDS-7,将该菌株鉴定为假单胞菌属(Pseudomonassp.);该菌株能够以PNP作为唯一碳源、氮源和能源生长,菌株对PNP的最高耐受浓度为80 mg/L,最适降解温度为30℃,偏碱性条件有利于菌株对PNP的降解;克隆了PNP降解过程中的偏苯三酚1,2-双加氧酶基因pnpC及马来酰醋酸还原酶基因pnpD(GenBank登陆号EU233791);将pnpC在E.coli BL21(DE3)菌株进行了诱导表达,表达产物对偏苯三酚和邻苯二酚均有邻位开环活性,比活力分别为0.45 U/mg protein和0.37 U/mg protein,表明偏苯三酚1,2-双加氧酶基因pnpC得到了活性表达.[结论]分离鉴定了一株PNP降解菌Pseudomonas sp.PDS-7,研究了该菌株的降解特性,克隆和表达了降解相关基因.     

小鼠精囊自身抗原的原核表达和纯化

目的：在原核细胞中表达小鼠精囊自身抗原（SVA）,并对表达产物进行鉴定和纯化。方法：提取小鼠附睾组织总RNA,RT-PCR获得SVA的cDNA,设计并合成特异引物序列,进一步扩增出不含信号肽的SVA编码序列,连入原核表达载体pET28a中,经酶切和测序鉴定正确的重组质粒转化大肠杆菌Rosetta（DE3）感受态细胞,IPTG诱导表达,Western印迹分析表达产物His-SVA,采用Ni-NTA纯化融合蛋白His-SVA。结果：原核表达获得融合6个组氨酸的SVA,用抗His单克隆抗体进行Western印迹鉴定,检测到相对分子质量约18×10^3的目的蛋白,与理论值一致;经Ni-NTA纯化获得较高纯度的His-SVA融合蛋白。结论：获得了在大肠杆菌中表达的小鼠附睾蛋白SVA,为后续研究其对小鼠生殖的影响奠定了基础。     

米曲霉(Aspergillus oryzae)RIB40中烯酮/烯酯还原酶的异源表达及性质分析

以米曲霉(Aspergillus oryzae)RIB40基因组DNA为模版,通过PCR扩增其烯酮烯酯还原酶(AspER)基因(asper)后连接到表达载体pET32a(+)上,在大肠杆菌BL21(DE3)中以可溶形式表达。通过Ni-NTA亲和色谱层析纯化后,蛋白纯度提高1.9倍,回收率为60.62%。根据分子筛凝胶层析结果推算,AspER以二聚体形式存在。性质分析表明此酶为依赖于NADPH的氧化还原酶,最适pH为7.0～8.0,最适温度为40℃。对2-环己烯酮Km和kcat值分别为(2.45±0.36)mmol/L和(4.4±0.4)×103s-1。底物谱分析发现AspER对马来酰亚胺及其衍生物有较高的活性,其中对2-甲基马来酰亚胺的转化率和e.e.值均高于99%。     

Complementary selectivity to (S)-1-phenyl-1,2-ethanediol-forming Candida parapsilosis by expressing its carbonyl reductase in Escherichia coli for (R)-specific reduction of 2-hydroxyacetophenone

An (R)-specific carbonyl reductase from Candida parapsilosis CCTCCM203011 (CprCR) was shown to catalyze the asymmetric reduction of 2-hydroxyacetophenone to (R)-1-phenyl-1,2-ethanediol (PED), which is a critical chiral building block in organic synthesis. The gene (rcr) encoding CprCR was cloned based on the amino acid sequences of tryptic fragments of the enzyme. Sequence analysis revealed that rcr is comprised of 1008 nucleotides encoding a 35 977 Da polypeptide, and shares similarity to proteins of the medium-chain dehydrogenase/reductase (MDR) superfamily. Recombinant rcr expressed in Escherichia coli showed a specific 2-hydroxyacetophenone-reducing activity. Using rcr expressing cells, (R)-PED was obtained by asymmetric reduction, which is complementary in enantiomeric configuration to (S)-PED obtained by using whole cells of C. parapsilosis. After optimization of reaction conditions, (R)-PED was produced at 95.5% enantiomeric excess with a yield of 92.6% when isopropanol was used for cofactor regeneration.     

Biocatalytic synthesis of (S)-4-chloro-3-hydroxybutanoate ethyl ester using a recombinant whole-cell catalyst

A cofactor regeneration system for enzymatic biosynthesis was constructed by coexpressing a carbonyl reductase from Pichia stipitis and a glucose dehydrogenase from Bacillus megaterium in Escherichia coli Rosetta (DE3) PlySs. Transformants containing the polycistronic plasmid pET-PII-SD2-AS1-B exhibited an activity of 13.5 U/mg protein with 4-chloro-3-oxobutanoate ethyl ester (COBE) as the substrate and an activity of 14.4 U/mg protein with glucose as the substrate; NAD(H) was the coenzyme in both cases. Asymmetric reduction of COBE to (S)-4-chloro-3-hydroxybutanoate ethyl ester [(S)-CHBE] with more than 99% enantiomeric excess was demonstrated by transformants. Furthermore, the paper made a comparison of crude enzyme catalysis and whole-cell catalysis in an aqueous monophasic system and a water/organic solvent biphasic system. In the water/n-butyl acetate system, the coexpression system produced 1,398 mM CHBE in the organic phase, which is the highest yield ever reported for CHBE production by NADH-dependent reductases from yeasts. In this case, the molar yield of CHBE was 90.7%, and the total turnover number, defined as moles (S)-CHBE formed per mole NAD+, was 13,980.