伴有辅酶原位再生的胞外酶耦合法制备（R）-苯基乙二醇

经5轮诱变筛选,从近平滑假丝酵母（Candida parapsilosis CICC1676）中分离得到产NADH依赖型羰基还原酶（Carbonyl reductase,CR）菌株CP-9。所产羰基还原酶（CRCp-9）经两步快速纯化获得纯化倍数为11．5倍,比活力为1．84 U/mg的酶液,其还原反应的最适pH值为6．5,最适温度为40℃。该酶转化β-羟基苯乙酮制备手性化合物（R）-苯基乙二醇,因此是（R）-专一性羰基还原酶。该酶与NADH普适性再生酶-甲酸脱氢酶（For-mate dehydrogenase,FDH）在胞外相耦联,构建伴有辅酶再生与反复利用的CR/FDH双酶催化制备立体醇体系,底物β-羟基苯乙酮转化率达95．4％,产物（R）-苯基乙二醇得率为93％,辅酶的总转化数（Total turn number, TTN）达267,产物e．e．值为98．6％,批次耦合反应生产能力达0．8 g/L/h,较单酶催化有较大提高,与细胞转化法相比也具有较好的生产能力。因此,伴有辅酶再生的胞外酶耦合催化具有潜在的制备手性醇化合物的工业应用价值。     

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。     

乙醇脱氢酶与葡萄糖脱氢酶偶联催化制备(S)-1-(2,6-二氯-3-氟苯基)乙醇

(S)-1-(2,6-二氯-3-氟苯基)乙醇是抗癌药物克唑替尼的手性合成前体,可由2,6-二氯-3-氟苯乙酮经乙醇脱氢酶催化还原制备,还原中所需的还原型辅酶Ⅱ再生是该反应的技术瓶颈.本研究构建重组大肠杆菌E.coli BL21-ADH和E.coli BL21-GDH,实现了葡萄糖脱氢酶和乙醇脱氢酶的共表达,并进行偶联转化.结果表明,当在反应温度为30℃,pH为7的条件下,(S)-l-(2,6-二氯-3-氟苯基)乙醇的产量达到最高,在投料量为6％时,该体系转化率为93.75％.     

基于金属有机框架的固定化酰胺酶制备及催化合成(S)-4-氟苯甘氨酸

以微水溶剂热法快速制备的稳定锆基金属有机框架为载体,戊二醛为交联剂,采用交联法对酰胺酶进行固定化,考察了不同条件对酰胺酶固定化效率的影响.结果 表明,戊二醛浓度为1.0％、交联时间为180 min、载体与酶的质量比为8∶1,固定化效率最佳,固定化酶活力回收率达86.4％,蛋白负载量达115.3 mg/g.固定化酶最适温...     

固定化细胞拆分(R,S)-α-乙基-2-氧-1-吡咯烷乙酸甲酯的研究

【目的】筛选鉴定一株产酯酶用于选择性拆分(R,S)-α-乙基-2-氧-1-吡咯烷乙酸甲酯的菌株,利用该菌株固定化细胞催化拆分外消旋底物。【方法】通过富集培养、罗丹明B平板初筛及复筛培养获得一株选择性拆分(R,S)-α-乙基-2-氧-1-吡咯烷乙酸甲酯的菌株,通过对其形态、生理生化特征及16S r DNA序列分析,确立该菌株系统发育地位。优化了利用硅藻土-戊二醛吸附交联法对该菌体细胞固定化的条件,研究固定化细胞催化性质及操作稳定性。【结果】该菌为革兰氏阴性菌,鉴定其为甲基球状菌属(Methylopila)。固定化体系最优条件:聚乙烯亚胺0.15%(V/V),戊二醛0.2%(V/V),硅藻土6 g/L,菌体质量浓度100 g/L。与游离细胞相比,固定化细胞最适p H由8.0变为8.5,最适温度由35°C变为40°C,p H稳定性和温度稳定性都有所提高。Cu~(2+)、Mn~(2+)、Ca~(2+)能促进酶活,Zn~(2+)、Fe~(2+)抑制酶活。固定化细胞的有机溶剂耐受性较游离细胞有所提高。动力学分析细胞固定化后Km值变大,底物亲和力降低。利用固定化细胞水解(R,S)-α-乙基-2-氧-1-吡咯烷乙酸甲酯,底物浓度200 g/L,反应20 h,保留构型为S型,得率47.8%,对映体过量值ees为99.4%,重复使用12次后仍保留初始酶活的80%以上。【结论】开发了利用Methylopila sp.cxzy-L013固定化细胞择性拆分(R,S)-α-乙基-2-氧-1-吡咯烷乙酸甲酯的工艺,该工艺具有良好的工业应用前景。     

(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。     

聚氨酯泡沫固定产脂肪酶粗状假丝酵母(Candida valida T2)细胞的研究

对聚氨酯泡沫固定产脂肪酶粗状假丝酵母(Candida valida T2)细胞的固定化条件进行了研究.实验结果表明,聚氨酯泡沫颗粒经密度和粒径筛选,酸碱处理,以及固液比优化,载体固定细胞干质量比达到1571 mg/g,细胞脂肪酶的酶活为每克干细胞1415 U.电镜图片显示粗状假丝酵母菌(Candida valida T2)在载体孔隙内和脊壁上缠绕充盈,生长良好,固定结构稳定.固定化细胞连续催化水解桐籽油5批次,细胞相对水解酶活保持率达73%,固定细胞的损失率为12.5%.固定化细胞颗粒显示出良好的操作稳定性和酶活保持率,为进一步的应用研究提供了实验基础.     

生物催化3-(4-氯苯基)-戊二腈去对称性水解合成光学纯巴氯芬的关键前体

研究了利用生物催化剂制备(S)-4-氰基-3-(4-氯苯基)-丁酸.以3-(4-氯苯基)-戊二腈为底物,采用苯酚-次氯酸钠法对实验室保藏的菌株进行筛选,得到一株产物立体选择性较高的菌株赤霉菌Gibberella intermedia WX12,并对其催化特性和发酵条件进行了初步研究.以30 g/L的乳糖和20 g/L的蛋白胨分别为碳、氮源,发酵培养96 h,收集的菌体在50 mmol/L磷酸缓冲液(pH 8.0)中30℃催化反应24 h,将3-(4-氯苯基)-戊二腈转化为4-氰基-3-(4-氯苯基)-丁酸,产率为90％.将产物化学转化为巴氯芬,手性HPLC分析表明水解产物构型是(S),其对映异构体过量值ee＞ 99％.该产物可以用来合成光学纯的(R)-和(S)-巴氯芬.     

左旋麻黄素半生物合成的研究固定化酵母细胞生物合成L-PAC(L-苯基乙酰基甲醇)

本文对影响酵母菌生物合成L-PAC的主要因素进行了研究.结果表明酵母菌株Sc-5按2g鲜细胞/3.2%50ml海藻酸钠凝胶固定化后,所获固定化凝胶珠置于装有4倍体积反应液的容器中,在振荡频率220转/分、温度28-30℃、添加0.2%的Vc时,枇次生物合成L-PAC产量最高,达2.0g/L.     

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。     

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.     

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.     

Coexpression of a carbonyl reductase and glucose 6-phosphate dehydrogenase in Pichia pastoris improves the production of (S)-1-phenyl-1,2-ethanediol

Abstract

To develop an efficient biocatalyst to produce optically active (S)-phenyl ethanediol (PED), a carbonyl reductase SCRII and glucose 6-phosphate dehydrogenase were coexpressed intracellularly in Pichia pastoris. The recombinant enzyme PpSCRII was purified with a specific activity of 8.32 U mg^?1, over 36% higher than that of Escherichia coli SCRII. The recombinant cells P. pastoris/SCRIIG catalyzed the reduction of 2-hydroxyacetophenone to give (S)-PED with optical purity of >99% in a yield of 96.3%. The yield was improved by 19.9% and 25.7% over E. coli BL21/SCRII and Candida parapsilosis, respectively, when the reaction duration was shorted from 48 h to 24 h. When using glucose 50 g L^?1 as co-substrate, these P. pastoris/SCRIIG cells could be reused ten times and the optical purity and yield of (S)-PED kept at >99% enantiomeric excess and >85%, respectively.     

猪肝酯酶催化苯乙二醇环碳酸酯水解最佳反应条件的研究

猪肝酯酶是手性合成中重要的水解酶,在猪肝酯酶的催化下,苯乙二醇环碳酸酯发生水解,生成苯乙二醇。实验围绕影响猪肝酯酶催化反应活性的4个主要因素进行了系统研究,得到了最优的酶浓度（15g/L）、pH值（8.0）、温度（25~30℃）及有机溶剂种类和浓度（二氧六环,65%v/v）,为猪肝酯酶催化苯乙二醇环碳酸酯反应的进一步研究奠定了基础。     

（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。     

多酶融合表达体系以木聚糖为辅助底物高效合成(S)-苯乙二醇

[目的] 利用木聚糖为辅助底物加强手性催化反应中的辅酶循环,构建来源于近平滑假丝酵母（Candida parapsilosis）CCTCC M203011的（S）-羰基还原酶II（SCRII）、枯草芽孢杆菌（Bacillussp.）YX-1葡萄糖脱氢酶突变体Ala258Phe/GDH和里氏木霉（Trichoderma reesei）Rut C-30木聚糖酶（XYN2）在大肠杆菌（Escherichia coli）BL21（DE3）中的融合表达体系,高效合成（S）-苯乙二醇。[方法] 调节3种酶编码基因在pET-28a载体上的位置,运用重叠延伸PCR技术,构建了E.coli/pET-SCRII-A258F-XYN2和E.coli/pET-A258F-SCRII-XYN2两种重组菌,研究了其合成（S）-苯乙二醇的最适反应条件。[结果] 重组菌株E.coli/pET-SCRII-A258F-XYN2在底物2-羟基苯乙酮与辅助底物木聚糖的比例为1：1、35℃、pH为7.0条件下,（S）-苯乙二醇的产率达98.8%（W/W）;而重组菌株E.coli/pET-A258F-SCRII-XYN2在底物与辅助底物的比例为2：1、35℃、pH为7.0条件下,（S）-苯乙二醇的产率达95.6%（W/W）,两者合成产物的光学纯度均>99%。[结论] 通过构建3种酶的融合表达体系,成功将木聚糖酶和葡萄糖脱氢酶突变体介导的辅酶再生循环体系引入不对称生物合成反应,提高了手性转化效率,为将大自然中丰富的木聚糖用于手性催化奠定了较扎实的研究基础。     

Cloning and expression of the gene encoding (R)-specific carbonyl reductase from Candida parapsilosis CCTCC M203011

The gene which encodes (R)-specific carbonyl reductase (rCR) from Candida parapsilosis CCTCC M203011 was cloned, sequenced and compared with genes from the GenBank. The results indicated that rCR gene was 1011 bp, encoding a protein of 336 amino acids with a molecular weight of 35.9 kDa, and its nucleotide sequence showed 99% similarity to those of other members of the alcohol dehydrogenase superfamily. The rCR gene could express in recombinant strain Escherichia coli JM 109, and the expression plasmid could produce (R)-1-pheny-1,2-ethanediol (100% e.e., 80.14% yield) fromβ-hydroxyacetophenone without any additive to regenerate NAD+ from NADH.     

Steady-state kinetics of the oxidation of (S)-1-phenyl-1,2-ethanediol catalyzed by alcohol dehydrogenase from Candida parapsilosis CCTCC M203011

Kinetic studies of oxidation reaction of (S)-1-phenyl-1,2-ethanediol (PED) catalyzed by a NAD⁺-dependent alcohol dehydrogenase from Candida parapsilosis CCTCC M203011 obtained from China Center for Type Culture Collection (CPADH) were observed for getting insight into the deracemization redox reaction. The data of initial velocity experiments in the absence of product, product (β-hydroxy-hypnone) inhibition experiments and dead-end (pyrazole) inhibition experiments strongly suggest that the reaction follows Theorell-Chance BiBi mechanism in which the coenzymes bind to the free form of the enzyme firstly. The kinetic parameters of this model were estimated by using non-linear regression analysis software.