不对称还原大位阻二芳香基甲酮的羰基还原酶的基因克隆及性质分析

由于二芳香基甲酮的位阻大及羰基两侧的取代基差异性较小,对其进行不对称还原是生物催化中具有挑战性的难题之一.文中通过对毕赤酵母GSl15基因组序列的分析,发现了一个潜在羰基还原酶基因pascr.将该基因克隆、表达在大肠杆菌Rosetta2 (DE3)中,通过Ni-NTA对重组蛋白进行了分离纯化,并对酶的性质进行了研究.PasCR专一性利用NADPH作为辅酶,其最适反应pH为6.5;最适反应温度为35℃;凝胶层析实验结合SDS-PAGE分析表明PasCR在溶液中以二聚体形式存在.PasCR能够不对称还原位阻较大的二芳香基甲酮类化合物,如4-甲基二苯甲酮、4-氯二苯甲酮、2-氯二苯甲酮等,对4-甲基二苯甲酮的还原产物的ee值达到了85％.     

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。     

一种新型醛酮还原酶的克隆表达、性质研究以及在不对称合成(R)-CHBE中的应用

为开发催化4-氯乙酰乙酸乙酯(COBE)制备(R)-4-氯-3-羟基丁酸乙酯((R)-CHBE)的新型催化剂,挖掘到了来自白色念珠菌SC5314中的一种NADPH辅酶依赖型醛酮还原酶CAK基因(cak),并将该基因在大肠杆菌中表达。将重组酶进行纯化后,测定其酶学性质,并构建了以葡萄糖为辅底物的双酶偶联辅酶再生系统,考察其不对称转化制备(R)-CHBE的能力。结果表明:CAK对多种醛酮类化合物有催化活性,其催化COBE的最适反应温度为40℃,最适p H为5。CAK在40℃下以及酸性条件中能保持较好的稳定性。Mg2+、Na+、K+对酶活有一定的激活作用,而Cu2+存在条件下酶会彻底失活。乙酸乙酯、邻苯二甲酸二丁酯对酶活的抑制作用较小。利用双酶偶联辅酶再生系统不对称转化制备(R)-CHBE。在合适的条件下,转化600 mmol/L的底物,产率达80.6%,产物对映体过量值(e.e.值)99%。     

产(R)-1,3-丁二醇的工程菌构建及转化条件研究

[目的]利用重组大肠杆菌实现(R)-1,3-丁二醇的生物合成。[方法]从脱硫球菌(Desulfococcus biacutus)中克隆得到羰基还原酶基因DbCR,构建pET28a-DbCR表达载体并在大肠杆菌E.coli BL21中表达,利用气相色谱对反应液进行检测。[结果]DbCR在pH 7.5、35℃的最适条件下的酶活力为4.5 U/mL。在50 mL全细胞反应体系中,重组工程菌在pH 7.5、30℃条件下,反应48 h时,对300 mmol/L底物4-羟基-2-丁酮的转化率 96%,产物(R)-1,3-丁二醇的e.e.值 99%。[结论]构建得到高效催化合成(R)-1,3-丁二醇的工程菌,工程菌对底物的转化率96%,产物纯度 99%。     

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。     

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。     

(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。     

拟南芥阿拉伯糖-5-磷酸异构酶的原核表达、纯化及酶催化特性

阿拉伯糖-5-磷酸异构酶(Kds D)是2-酮-3-脱氧辛糖酸(KDO)生物合成途径的第一个关键限速酶,通过无缝克隆技术将拟南芥Kds D基因构建至原核表达载体p ET-HTT,经过IPTG诱导,在大肠杆菌BL21(DE3)中获得了大量重组蛋白的可溶性表达;表达产物经Ni-NTA亲和层析和分子筛层析(SEC)方法进行酶蛋白的分离纯化步骤,得到纯度85%以上的高纯度酶;分子筛层析结果发现纯化后的目的蛋白Kds D在溶液中主要以多聚体、二聚体和单体形式存在,这同微生物来源Kds D酶在溶液中以四聚体形式存在很大差异;进一步使用Western blotting和MALDI-TOF MASS技术对纯化的蛋白进行鉴定;测定了拟南芥Kds D酶学性质,证明该酶催化反应的最适p H值为8.0,最适作用温度为37℃,各种金属离子在低浓度均对酶活性存在不同程度的抑制作用,其中以Co~(2+)、Cd~(2+)对酶活性的抑制作用最强,而5 mmol/L金属螯合剂EDTA对酶有激活作用。此外,以阿拉伯糖-5-磷酸(A5P)为底物时,拟南芥Kds D酶动力学常数Vmax和Km值分别为0.18 mmol/(L·min)、0.16 mmol/L,比较发现该酶与底物的亲和性高于大肠杆菌Kds D。以上研究结果为Kds D蛋白结构与功能及其在新型抗生素研制领域中的工业化应用奠定了基础。     

2,5-DKG还原酶I的分离纯化与性质研究

欧文氏菌ER97高效表达了从棒状杆菌SCB3058克隆的2,5-二酮基-D-葡萄糖酸(2,5-DKG)还原酶I基因,5 L罐发酵后,收集菌体破碎,将胞内可溶性的蛋白通过硫酸铵分级沉淀、DEAE-Sepharose CL-6B离子交换柱层析和Phenyl Sepharose CL-4B疏水柱层析后分离纯化到了2,5-DKG还原酶I,纯化了5倍,得率27%,比活力为3,418 U/mg。测定了该酶的一些特性参数:分子量为34 kD,等电点为6.0,它以NADPH为辅酶,将2,5-DKG还原为2-酮基-L-古龙酸(2-KLG),对NADPH和2,5-DKG底物的Km值分别是0.29mmol/L和14.7 mmol/L,1 mmol/L Cu2 、Zn2 等有强烈抑制作用,EDTA和巯基乙醇对该酶没有抑制作用,酶的最适pH为7.0,最适反应温度为40℃。     

毛壳霉内切菊粉酶的纯化与性质

毛壳霉 (Chaetomiumsp .)C34发酵液经硫酸铵分级沉淀、DEAE 纤维素 11离子交换层析、Q SepharoseFastFlow离子交换层析、SephacrylS 2 0 0凝胶过滤、PhenolSepharoseTM HP疏水层析 ,得到电泳纯的内切菊粉酶组分 ,纯化倍数为 30 8倍 ,活力回收率为 7 7%。用SDS PAGE测得该酶亚基的分子量为 6 6kD。菊粉酶的最适pH为 6 0 ,最适温度为 5 0～ 5 5℃。菊粉酶在 5 0℃以下 ,pH5 0～ 8 0时较稳定。Cu2 完全抑制酶的活性 ,Mn2 、Zn2 、Fe2 、EDTA以及NBS(N bromosuccinimide ,N 溴代丁二酰亚胺 )对该酶有很强的抑制作用。该酶对菊粉有较强底物专一性 ,产物主要为低聚果糖 ,也可作用于蔗糖 ,I S值为 2 0。以菊粉为底物时 ,Km 为 0 199mmol L ,Vmax为 115 μmol (mg·min)。     

Purification and properties of peroxidase from Pinus pinaster needles

Peroxidase (Ec 1.11.1.7) was purified from needles of Pinus pinaster to apparent homogeneity by DE-52 cellulose chromatography with a final recovery of enzyme activity of about 85%. The purified enzyme (A402/A275 = 1.05) had a specific activity of about 948 U/mg of protein and ran as a single protein band both on SDS-PAGE and native PAGE with Mr of 37,000 and 151,000, respectively. Both native PAGE and isoelectric focusing gels of the purified enzyme were stained for activity which coincided with the protein band. The pI of the purified enzyme was found to be 3.2 by isoelectric focusing on an ultrathin polyacrylamide gel. The enzyme has an optimum pH of activity of 5.0 and temperature optimum of 30 degrees C. Stability studies of the enzyme as a function of pH and temperature suggest that it is most stable at pH 5.0 and 0-40 degrees C, respectively.     

One-step purification and properties of catalase from leaves of Zantedeschia aethiopica

Catalase (E.C 1.11.1.6) was purified from leaves of Zandedeschia aethiopica to apparent homogeneity by a one-step hydrophobic interaction chromatography on a phenyl Sepharose CL-4B column. The purified enzyme preparation was obtained with a final recovery of enzyme activity of about 61% and a specific activity of 146 U/mg protein. The purified enzyme ran as a single protein band when analyzed both by native PAGE and SDS-PAGE corresponding to an Mr of 220,000 Da, which consists of 4 subunits with identical Mr of 54,000 Da. The pI of purified enzyme was found to be 5.2 by isoelectric focusing on ultrathin polyacrylamide gels. The purified catalase has an optimum temperature of activity at 40 degrees C, whereas it is stable between 0 degrees and 50 degrees C. As regards pH, the enzyme has an optimum activity at pH 7.0 and it is stable in the range pH 6-8. The absorption spectrum of the purified enzyme exhibited 2 peaks at 280 nm and 405 nm.     

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。     

Purification and characterization of an extracellular beta-1,4-mannanase from a marine bacterium, Vibrio sp. strain MA-138.

A beta-mannanase (EC 3.2.1.78) from Vibrio sp. strain MA-138 was purified by ammonium sulfate precipitation and several chromatographic procedures including gel filtration, adsorption, and ion-exchange chromatographies. The final ion-exchange chromatography Mono Q yielded one major active fraction and three minor active fractions. The major active fraction was purified to homogeneity on the basis of native polyacrylamide gel electrophoresis (PAGE). This purified enzyme was identified as a glycoprotein by periodic acid-Schiff staining and a monomeric protein with a molecular mass of 49 kDa by sodium dodecyl sulfate-PAGE. The pI of the enzyme was 3.8. The purified enzyme exhibited maximal activity at pH 6.5 and 40 degrees C and hydrolyzed at random the internal beta-1,4-mannosidic linkages in beta-mannan to give various sizes of oligosaccharides. The first 20 N-terminal amino acid sequence of the purified enzyme showed high homology with the N-terminal region of beta-mannanase from Streptomyces lividans 66.     

Cloning, expression, and characterization of Bacillus sp. snu-7 inulin fructotransferase

A gene encoding inulin fructotransferase (di-D-fructofuranose 1,2': 2,3' dianhydride [DFA III]-producing IFTase, EC 4.2.2.18) from Bacillus sp. snu-7 was cloned. This gene was composed of a single, 1,353-bp open reading frame encoding a protein composed of a 40-amino acid signal peptide and a 410-amino acid mature protein. The deduced amino acid sequence was 98% identical to Arthrobacter globiformis C11-1 IFTase (DFA III-producing). The enzyme was successfully expressed in E. coli as a functionally active, His-tagged protein, and it was purified in a single step using immobilized metal affinity chromatography. The purified enzyme showed much higher specific activity (1,276units/mg protein) than other DFA III-producing IFTases. The recombinant and native enzymes were optimally active in very similar pH and temperature conditions. With a 103-min half-life at 60 degrees C, the recombinant enzyme was as stable as the native enzyme. Acidic residues and cysteines potentially involved in the catalytic mechanism are proposed based on an alignment with other IFTases and a DFA IIIase.     

Structural studies on prolyl hydroxylase. Conformation from circular dichroism spectroscopy

The circular dichroism spectra of purified prolyl hydroxylase [EC 1.14.11.12] in the native and heat-denatured states were obtained at pH 7.8. Analysis of the far-uv spectrum of the native enzyme gave an estimate of 40% alpha-helix, 40% beta-structure and 20% random coil. The near-uv spectrum contained several negative dichroic bands that can be attributed to phenylalanyl, tyrosyl and tryptophyl residues situated in an asymmetric environment in the protein. These bands were not seen in the enzyme denatured by heat. The denaturation was monitored by changes in the alpha-helical content as well as the activity of the enzyme as a function of temperature. The normalized transition profiles with respect to the change in helical content and the loss of enzyme activity were coincidental, indicating the involvement of the alpha-helical segments in maintaining the enzyme activity.     

KPC-2型碳青霉烯酶的表达纯化及其催化抗生素水解的动力学

目的：表达纯化KPC-2型碳青霉烯酶,并研究其催化抗生素水解的酶动力学活性。方法：将KPC-2基因与pET-22b（＋）原核表达载体连接后转化大肠杆菌BL21（DE3）,IPTG诱导表达,表达产物经FF镍柱纯化后,SDS-PAGE检测蛋白的纯度;用BioTek酶联仪进一步检测其抗生素水解范围及动力学特性。结果：构建了高效表达KPC-2的工程菌,得到了纯度在90%以上的KPC-2蛋白,该蛋白能水解几乎所有β内酰胺类抗生素,其水解美罗培南等碳青霉烯类抗生素的能力较强,最适温度约为40℃,最适pH值约为6.5。结论：为进一步了解最常见的KPC功能与活性提供了重要信息。     

Extracellular phytase (E.C. 3.1.3.8) from Aspergillus ficuum NRRL 3135: purification and characterization

Extracellular phytase from Aspergillus ficuum, a glycoprotein, was purified to homogeneity in 3 column chromatographic steps using ion exchange and chromatofocusing. Results of gel filtration chromatography and SDS-polyacrylamide gel electrophoresis indicated the approximate molecular weight of the native protein to be 85-100-KDa. On the basis of a molecular weight of 85-KDa, the molar extinction coefficient of the enzyme at 280 nm was estimated to be 1.2 X 10(4) M-1 cm-1. The isoelectric point of the enzyme, as deduced by chromatofocusing, was about 4.5. The purified enzyme is remarkably stable at 0 degree C. Thermal inactivation studies have shown that the enzyme retained 40% of its activity after being subjected to 68 degrees C for 10 minutes, and the enzyme exhibited a broad temperature optimum with maximum catalytic activity at 58 degrees C. The Km of the enzyme for phytate and p-nitrophenylphosphate is about 40 uM and 265 uM, respectively, with an estimated turnover number of the enzyme for phytate of 220 per sec. Enzymatic deglycosylation of phytase by Endoglycosidase H lowered the molecular weight of native enzyme from 85-100-KDa to about 76-KDa; the digested phytase still retained some carbohydrate as judged by positive periodic acid-Schiff reagent staining of the electrophoresed protein. Immunoblotting of the phytase with monoclonal antibody 7H10 raised against purified native enzyme recognized not only native but also partially deglycosylated protein.     

麻疯树叶中过氧化物酶JCP-1的分离纯化和几种特性的检测

麻疯树叶片蛋白粗提液经硫酸铵分级沉淀,强阴离子琼脂糖、强阳离子琼脂糖和交联葡聚糖层析,得到一个比活为4499U·mg-1（蛋白）过氧化物酶,命名为JCP-1。其分子量为49kDa,等电点为pH3.3,最适pH为5.0-6.0。以H2O2为底物的Km为2.14mmol·L-1。JCP-1具有宽泛的最适保存pH（7.0-11.0）和较高的耐热性（80℃高温处理15min,活性保持在90%以上）。30%PEG6000处理模拟干旱胁迫及50℃高温胁迫麻疯树苗,其叶片中JCP-1活性分别提高121%和155%。     

Purification and characterization of polygalacturonase from banana fruit

Polygalacturonase isoenzyme 3 (PG-3) was purified to homogeneity with a specific activity of 0.7 mu katal mg-1 protein from banana fruit pulp. The purified enzyme was a glycoprotein with ca. 8% carbohydrate. The molecular weight of the native enzyme was found to be 90 +/- 10 kDa with a subunit molecular weight of 29 +/- 2 kDa. The enzyme exhibited optimum activity at pH 4.3 and temperature 40 degrees C with activation energy 35.4 kJ mol-1. A unique property of the enzyme was the requirement of -SH groups for the enzyme activity. The enzyme was inhibited by p-CMB and activated by 2-ME and DTT. The inhibition of p-CMB could be reversed by DTT. The enzyme contained eight free -SH groups. The Km of the enzyme was 0.15% for polygalacturonic acid.