S-2-氯丙酸脱卤酶的定向进化及其应用

为提高S-2-氯丙酸脱卤酶的活力,通过易错PCR的方法将源于假单胞菌(Pseudomonas sp.CGMCC 3267)的脱卤酶(DehII)进行定向进化,进化酶DehII-B2的比活提高3.9倍。同源模建两者的三维结构,并与底物进行分子对接。结果表明,突变位点为A7I,进化酶DehII-B2的结合能比原始酶降低了1.4kcal/mol,活性中心Asp10与底物α碳原子的距离缩短了0.321 6nm,因而加快了酶反应速率、提高了酶比活。目前,该酶的比活高于实验室已得酶。与原始酶相比,其最适温度和热稳定性略有增加,最适pH和pH稳定性没有明显变化。对该酶的应用做了初步的探索,结果表明在40℃,60mmol/L底物浓度下反应10h,转化率达到49.6%,ees>99.9%,因此该酶有一定的应用价值。     

R-2-卤代酸脱卤酶的同源模建及底物对映体选择性研究

对来自假单胞菌ZJU26中的R-2-卤代酸脱卤酶(DehI-R)进行同源模建,分析其与底物的相互作用,为解析酶的底物对映体选择性提供理论依据.采用Sybyl中的APM模块首次构建并优化了R-2-卤代酸脱卤酶的三维结构,并用Procheck 验证结构模型的合理性.使用Suflex-Docking模块将结构模型与底物分别进行对接,分析相互之间的作用.序列比对结果显示,R-2-卤代酸脱卤酶与恶臭假单胞菌PP3中DehI的相似性达23.71％.Deh-R模建后的结构与模板很好的吻合.模型比对分析DehI-R中参与催化的残基,除Asn2.03外大部分都比较保守.分子对接结果表明,R-2-氯丙酸和S-2-氯丙酸都可以结合到活性位点上,决定其选择性的是值点Asn203,在RS-2-卤代酸脱卤酶所对应位点的残基为Ala,相比之下,Aan具备较大的空间位阻,从而阻止了S-2-氯丙酸的反应.利用Sybyl中的Biopolymer模块对R-2-卤代酸脱卤酶中的Asn203突变成具有不同空间位阻的Ala、Gly和Gln.突变酶与底物对接结果进一步证实了Asn203位点对R-2-卤代酸脱卤酶的底物对映体选择性作用.     

南海深海微生物酯酶EST12-7在制备(R)-2-氯丙酸乙酯中的应用研究

利用来源南海深海的微生物酯酶EST12-7不对称水解反应拆分制备（R）-2-氯丙酸乙酯。并探寻了温度、pH、底物浓度、有机溶剂和反应时间等因素对酯酶EST12-7催化制备（R）-2-氯丙酸乙酯的影响。结果表明,深海微生物酯酶EST12-7催化制备（R）-2-氯丙酸乙酯的最佳反应条件为：13.8 μg/ml酯酶EST12-7,50 mmol/L（±）-2-氯丙酸乙酯,2%正癸醇,pH8.5,30℃,0.05mol/L Tris-HCl,反应60 min。在最佳反应条件下,（±）-2-氯丙酸乙酯的转化率可达49%,所制备的（R）-2-氯丙酸乙酯的光学纯度为98%。通过对酯酶EST12-7拆分制备（R）-2-氯丙酸甲酯和（R）-2-氯丙酸乙酯进行比较,2-氯丙酸酯中的链长对酯酶EST12-7拆分反应有极大的影响。     

中华绒螯蟹（Eriocheir sinensis）N-乙酰-β-D-氨基葡萄糖苷酶的分离纯化及性质的初步研究

本研究以中华绒螯蟹内脏为材料,经过硫酸铵沉淀分级分离、两次DEAE-32离子交换柱层析和Sephadex G-100分子筛柱层析纯化,获得比活力为4490.79U/mg、纯化倍数为28.07倍的聚丙烯酰胺凝胶电泳纯的N-乙酰-β-D-氨基葡萄糖苷酶制剂。酶分子中各亚基的分子量分别为121.219、8.63和73.48 kD,等电点为4.5。以对-硝基苯-N-乙酰-β-D-氨基葡萄糖为底物,进行酶催化底物水解的反应动力学研究,结果表明：酶催化底物反应的最适pH为5.5,最适温度为45℃。该酶在pH4.9—9.3区域或40℃以下处理30min,酶活力保持稳定。酶促反应动力学符合米氏双曲线方程,测得米氏常数Km为0.357 mmol/L,最大反应速度Vm为10.41μmol/L.min。酶催化pNP-β-D-GlcNAc反应的活化能为76.50kJ/mol。金属离子对酶的效应试验表明：Mg^2＋、Ca^2＋和Ba^2＋对酶活力没有影响。Na＋对酶有激活作用,Li^＋、K^＋、Zn^2＋、Hg^2＋、Pb^2＋、Cu^2＋和Al3＋对酶活力表现出不同程度的抑制作用。     

拟南芥谷胱甘肽S-转移酶Zeta类进化酶的获得及其特性分析

拟南芥谷胱甘肽S-转移酶Zeta类(AtGSTZ)是一种与细胞代谢和环境净化密切相关的多功能酶.应用易错PCR和多轮DNA洗牌技术构建了AtGSTZ随机突变文库;再利用pH指示剂颜色改变法对突变文库进行筛选,获得了9个二氯乙酸脱氯活性提高的突变子.其中,NN23含25个氨基酸突变,比活力提高120%,NN20含24个氨基酸突变,比活力提高102%,EC1含2个氨基酸突变,比活力提高47%,其他6个为单点突变,比活力分别提高9%～60%.酶学分析显示,所有进化酶对底物二氯乙酸的催化效率和对谷胱甘肽的亲和力以及个别进化酶的复性能力都得到不同程度的提高,但热稳定性均没有明显改善.同时,对一系列与AtGSTZ空间折叠及催化活性相关位点进行了讨论.     

β-脱卤酶的基因克隆表达及其酶学性质

根据GenBank中的序列设计引物,克隆芽孢杆菌中的β-脱卤酶基因(命名为bhd)。以pET30a(+)为载体、Escherichia coli BL21(DE3)-CondonPlus为宿主菌,实现了bhd的高效表达。使用HisTrapTMFF亲和层析柱纯化重组β-脱卤酶,分子量约为23.1 kD。酶学性质研究表明,纯化的重组β-脱卤酶水解3-氯丙酸制备3-羟基丙酸的最适反应体系为30°C,100 mmol/L,pH 7.0的磷酸钠缓冲液。在最适反应条件下,重组β-脱卤酶的比活为16.2 U/mg,Km和Vmax分别为3.26μmol/L和17.86 mmol/(min.g protein)。在最适反应条件下,以10 mmol/L 3-氯丙酸为底物,反应36 h的转化率在93%以上。     

球孢白僵菌固定化生物转化合成D-HPPA

[目的]利用球孢白僵菌进行固定化生物转化,将底物R-(+)-2-苯氧基丙酸(D-PPA)转化合成产物R-(+)-2-(4-羟基苯氧基)丙酸(D-HPPA)。[方法]利用海藻酸钠和聚乙烯醇对球孢白僵菌进行包埋处理,并对包埋条件进行累积优化。[结果]4%海藻酸钠和4. 5%聚乙烯醇混合后,再加入2. 5%的氯化钙作为交联剂交联8 h。在此包埋条件下制备的白僵菌凝胶珠,置于30 g/L的D-PPA进行固定化生物转化。反应5 d后,产物浓度最终为29. 9 g/L,平均生产强度为5. 98 g/(L·d),底物转化率为99. 7%。[结论]海藻酸钠和聚乙烯醇可用于白僵菌的固定化,且较游离菌体的生物转化的反应周期缩短28. 6%,平均生产强度增加64. 7%,底物转化率提高17. 7%。     

来自假单胞菌ZJU26的R-2-氯丙酸脱卤酶的手性识别过程研究

立体选择性是2-卤代酸脱卤酶最重要的性质之一,但目前其手性识别过程尚不明确,对其进行研究和解析具有重要意义。以来自假单胞菌ZJU26的R-2-氯丙酸脱卤酶dehDIV-R为模型,研究了R-2-卤代酸脱卤酶的手性识别过程。首先通过测定反应产物的构型,确定dehDIV-R催化底物为S_N2反应。通过Discovery Studio 3.0对dehDIV-R进行同源模建及底物分子对接,由对接结果和序列比对确定dehDIV-R立体选择性的关键位点Asn236,预测dehDIV-R的立体选择性与反应时底物到达反应位置的空间位阻密切相关。对dehDIV-R进行虚拟突变,将Asn236位点突变成具有不同空间位阻的残基Ala和Ser,并分别与底物分子进行分子对接,预测突变酶的立体选择性。根据预测结果,对Asn236氨基酸残基进行定点突变,发现在Asn236突变为Ala后的A1酶显示出对RS底物的活力;在Asn236突变为Ser后的S1酶显示出与原始酶相反的立体选择性,实现了立体选择性的反转。与模型的预测结果相符,证明了模型的合理性。     

定点突变对酰胺水解酶DamH可溶性表达和酶活的影响

摘要:【目的】DamH是一种具有酯酶活性的酰胺水解酶,其非活性中心氨基酸残基的突变对重组酶可溶性表达和比酶活产生一定的影响。拟探索DamH的活性中心氨基酸残基构成,并对其非活性中心氨基酸残基突变对可溶性表达和比酶活的影响进行研究。【方法】通过重叠延伸的方法对DamH可能的活性中心氨基酸S149、E244和H274以及非活性中心氨基酸D165及N192进行定点突变,通过静息细胞测活验证了S149、E244和H274 在催化2-氯-N-(2’-甲基-6’-乙基苯基)乙酰胺(CMEPA)水解反应中的作用,通过Ni2+- NTA亲和层析对D165及N192突变子进行纯化,对突变株和野生型比酶活进行比较。【结果】研究表明S149A使DamH的CMEPA 水解酶活性下降为野生型的5%,E244A和H274A突变导致其失去活性;D165P和N192P突变影响到DamH的可溶性表达,表达量分别为野生型的28.2%和20.8%,突变子N192P、D165P比酶活分别为野生型比酶活的55.5%和49.7%。【结论】DamH催化酯类底物和芳基酰胺类底物可能共用同一活性中心S149、E244和H274,其两个α螺旋的转角处氨基酸侧链极性和刚性结构的改变对可溶性表达以及活性有很大的影响。     

棉铃虫N-乙酰-β-D-氨基葡萄糖苷酶的分离纯化及酶学性质

以棉铃虫Helicoverpa armigera蛹为材料,通过硫酸铵沉淀分级分离、Sephadex G-200分子筛柱层析和DEAE-32离子交换柱层析纯化,获得聚丙烯酰胺凝胶电泳纯的N-乙酰- β-D-氨基葡萄糖苷酶酶制剂。纯酶的比活力为2 678.79 U/mg。以对硝基苯-N-乙酰-β-D-氨基葡萄糖苷(pNP-β-D-GlcNAc)为底物,研究酶催化底物水解的反应动力学。结果表明：酶的最适pH为5.63,最适温度为55℃。该酶在pH 4～8区域较稳定,而在pH>8时能迅速失去活力;在50℃以下处理30 min,酶活力仍保持稳定,高于50℃,酶很快失去活力。酶促反应动力学符合米氏双曲线方程,测得米氏常数Km为0.16 mmol/L,最大反应速度Vm为10.73 μmol·L^-1·min^-1。酶催化pNP-β-D-GlcNAc反应的活化能为66.24 kJ/mol。     

A short-chain carbonyl reductase mutant is an efficient catalyst in the production of (R)-1,3-butanediol

R-1,3-butanediol (R-1,3-BDO) is an important chiral intermediate of penem and carbapenem synthesis. Among the different synthesis methods to obtain pure enantiomer R-1,3-BDO, oxidation–reduction cascades catalysed by enzymes are promising strategies for its production. Dehydrogenases have been used for the reduction step, but the enantio-selectivity is not high enough for further organic synthesis efforts. Here, a short-chain carbonyl reductase (LnRCR) was evaluated for the reduction step and developed via protein engineering. After docking result analysis with the substrate 4-hydroxy-2-butanone (4H2B), residues were selected for virtual mutagenesis, their substrate-binding energies were compared, and four sites were selected for saturation mutagenesis. High-throughput screening helped identify a Ser154Lys mutant which increased the catalytic efficiency by 115% compared to the parent enzyme. Computer-aided simulations indicated that after single residue replacement, movements in two flexible areas (VTDPAF and SVGFANK) facilitated the volumetric compression of the 4H2B-binding pocket. The number of hydrogen bonds between the stabilized 4H2B-binding pocket of the mutant enzyme and substrate was higher (from four to six) than the wild-type enzyme, while the substrate-binding energy was decreased (from −17.0 kJ/mol to −29.1 kJ/mol). Consequently, the catalytic efficiency increased by approximately 115% and enantio-selectivity increased from 95% to 99%. Our findings indicate that compact and stable substrate-binding pockets are critical for enzyme catalysis. Lastly, the utilization of a microbe expressing the Ser154Lys mutant enzyme was proven to be a robust process to conduct the oxidation–reduction cascade at larger scales.     

Cold adaptation of a mesophilic cellulase,EG Ⅲ from Trichoderma reesei,by directed evolution

Cold-active enzymes have received little research attention although they are very useful in industries. Since the structure bases of cold adaptation of enzymes are still unclear, it is also very difficult to obtain cold-adapted enzymes for industrial applications using routine protein engineering methods. In this work, we employed directed evolution method to randomly mutate a mesophilic cellulase, endoglucanase Ⅲ (EG Ⅲ) from Trichoderma reesei, and obtained a cold-adapted mutant, designated as w-3. DNA sequence analysis indicates that w-3 is a truncated form of native EG Ⅲ with a deletion of 25 consecutive amino acids at C-terminus. Further examination of enzymatic kinetics and thermal stability shows that mutant w-3 has a higher Kcat value and becomes more thermolabile than its parent. In addition, activation energies of w-3 and wild type EG Ⅲ calculated from Arrhenius equation are 13.3 kJ· mol-1 and 26.2 kJ ·mol-1, respectively. Therefore, the increased specific activity of w-3 at lower tempera     

Partial purification and properties of the common inherited forms of adenosine deaminase from human erythrocytes

1. The partial purification of adenosine deaminase, types 1, 2 and 2-1, from human erythrocytes is described. 2. The isoenzyme components characteristic of the three forms of the enzyme were partially resolved by chromatography on DEAE-Sephadex. 3. Gel chromatography of the various forms of the enzyme gave estimates of the molecular weights in the range 30000-35000. 4. Electrophoresis in starch gels containing increasing percentages of starch did not reveal any differences in molecular weight between the genetic variants or their isoenzyme components. 5. Analytical isoelectric-focusing experiments in polyacrylamide gels gave the following pI values for the four isoenzyme components present in type 2-1 erythrocytes: 4.70, 4.83, 4.94 and 5.06. 6. All forms of the enzyme gave K(m) values for adenosine of about 30mum and K(i) values of about 8mum for the competitive inhibitor purine riboside. 7. Reaction rates of the type 1 and 2 enzymes were measured at different temperatures. Both enzymes gave values for the energy of activation for hydrolysis of adenosine of about 33.4kJ/mol (8kcal/mol). 8. Heat inactivation of all forms of the enzyme was markedly dependent on ionic strength, the rate of inactivation increasing with increasing ionic strength. The type 1 and type 2 forms of the enzyme differed significantly in their susceptibility to heat inactivation. From the variation of rates of inactivation with temperature, values were obtained for the energies of activation for the heat inactivation of both enzymes as follows: type 1 enzyme 275.5kJ/mol (65.9kcal/mol) and type 2 enzyme 241.6kJ/mol (57.8kcal/mol.).     

A comparative study on the thermal decomposition of four cereal straws in an oxidizing atmosphere

The thermal decomposition characteristics of four types (wheat, barley, oats and rye) of cereal straws were studied. Two varieties from each type of straw were used. The thermal degradation behaviours and kinetic parameters (order of reaction, activation energy and preexponential factor) of the straws were compared. Two distinct reaction zones were observed for all types and varieties of straws. Thermal degradation rates in the first reaction zone were significantly higher than those in the second reaction zone. The activation energy was in the ranges of 80–102 kJ/mol and 34–75 kJ/mol, whereas the order of reaction was in the ranges of 1·3–2·3 and 0·1–0·7 for the first and second reaction zones, respectively. The Shaw variety of oats straw had the highest activation energies (102 and 75 kJ/mol) and reaction orders (2·3 and 0·7) in both the first and second reaction zones, respectively. The lowest activation energy (80 kJ/mol) and order of reaction (1·3), in the first reaction zone, corresponded to Absolvant and Monopol wheat straws. The activation energies and reaction orders of barley and rye straws were in the ranges of 85–94 kJ/mol and 1·9–2·3, respectively. There was not any significant difference between the rate constants of the straw varieties, in the first reaction zone. However, oats straws had significantly higher rate constants in the second reaction zone as compared to the rate constants of wheat, barley and rye straws.     

Kinetic and structural characterization of manganese(II)-loaded methionyl aminopeptidases

Manganese(II) activation of the methionyl aminopeptidases from Escherichia coli (EcMetAP-I) and the hyperthermophilic archaeon Pyrococcus furiosus (PfMetAP-II) was investigated. Maximum catalytic activity for both enzymes was obtained with 1 equiv of Mn(II), and the dissociation constants (K(d)) for the first metal binding site were found to be 6 +/- 0.5 and 1 +/- 0.5 microM for EcMetAP-I and PfMetAP-II, respectively. These K(d) values were verified by isothermal titration calorimetry (ITC) and found to be 3.0 +/- 0.2 and 1.4 +/- 0.2 microM for EcMetAP-I and PfMetAP-II, respectively. The hydrolysis of MGMM was measured in triplicate between 25 and 85 degrees C at eight substrate concentrations ranging from 2 to 20 mM for PfMetAP-II. Both specific activity and K(m) values increased with increasing temperature. An Arrhenius plot was constructed from the kcat values and was found to be linear over the temperature range 25-85 degrees C. The activation energy for the Mn(II)-loaded PfMetAP-II hydrolysis of MGMM was found to be 25.7 kJ/mol while the remaining thermodynamic parameters calculated at 25 degrees C are DeltaG+ = 50.1 kJ/mol, DeltaH+ = 23.2 kJ/mol, and DeltaS++ = -90.2 J x mol(-1) x K(-1).     

A defective proton pump, point-mutated bacteriorhodopsin Asp96----Asn is fully reactivated by azide.

Addition of azide fully restored the proton pump activity of defective bacteriorhodopsin (BR) mutant protein Asp96----Asn. The decay time of M of BR Asp96----Asn, the longest living intermediate, was decreased from 500 ms at pH 7.0 to approximately 1 ms under conditions of saturating azide concentrations. This decay was faster than the decay of M in the wild-type, where no such azide effect was detectable. Stationary photocurrents, measured with purple membranes immobilized and oriented in a polyacrylamide gel, increased upon addition of azide up to the level of the wild-type. Different small anions of weak acids restored the pump activity with decreasing affinity in the order: cyanate greater than azide greater than nitrite greater than formiate greater than acetate. The activation energy of the M decay in the mutant was higher in the presence (48 kJ/mol) than in the absence (27 kJ/mol) of 100 mM azide even though the absolute rate was dramatically increased by azide. This effect of azide is due to the substitution of a carboxamido group for a carboxylic group at position 96 which removes the internal proton donor and causes an increase in the entropy change of activation for proton transfer which is reversed by azide.     

The promotion of collagen polymerization by lanthanide and calcium ions.

Ca2+ (1-5 mM) and lanthanide (20-250 microM) ions enhance the rate of polymerization of purified calf skin collagen (1.5 mg/ml) at pH 7.0 in the presence of 30mM-Tris/HCl and 0.2 M-NaCl. Both the nucleation phase and the growth phase of polymerization are accelerated. The activation energy of the growth phase, 239.3 +/- 24.3 kJ/mol (57.2 +/- 5.8 kcal/mol), is decreased to 145.6 +/- 9.6 kJ/mol (34.8 +/- 2.3 kcal/mol) by 5 mM-Ca2+ and to 75.3 +/- 4.6 kJ/mol (18.0 +/- 1.1 kcal/mol) by 25 microM-Sm3+. In contrast, the activation energy of the nucleation phase, 191.6 +/- 23.4 kJ/mol (45.8 +/- 5.6 kcal/mol), is only slightly decreased by Ca2+ or Sm3+. Collagen fibrils formed in the presence of Sm3+ are thinner than control fibrils, and more thermoresistant.     

Channel-mediated permeation of ammonia gas through the peribacteroid membrane of soybean nodules

Ammonia permeability of the peribacteroid membrane (PBM) from N(2)-fixing soybean nodules was measured (8x10(-5) m/s) using isolated PBM in a stopped-flow spectrofluorimeter. Ammonia (NH(3)) uptake into PBM vesicles was inhibited by up to 42% by HgCl(2) (EC(50)=2.9 microM, mercaptoethanol-reversible) and reduced by ATP pre-incubation. The activation energy of NH(3) uptake (52 kJ/mol) increased (118 kJ/mol) with HgCl(2). Water transport was also HgCl(2)-sensitive (EC(50)=52.6 microM), but increased by ATP pre-incubation. NH(3) and H(2)O may permeate via different pathways through Nodulin 26 or there is another protein on the PBM that is permeable to NH(3).     

Kinetics and apparent activation energy of the reaction of the fluorogenic reagent 5-furoylquinoline-3-carboxaldehyde with ovalbumin

Incomplete labeling of proteins by a derivatizing reagent usually results in the formation of a large number of products, which can produce unacceptable band broadening during electrophoretic analysis. In this paper, we report on the reaction of the fluorogenic reagent 5-furoylquinoline-3-carboxaldehyde (FQ) with the lysine residues of ovalbumin. Mass spectrometry was first used to determine the distribution in the number of labels attached to the protein. At room temperature, 3.6+/-1.9 labels were attached after 30 min. The reaction rate and number of labels increased at elevated temperatures. At 65 degrees C, 6+/-2.5 labels were attached after 5 min. The apparent activation energy for this reaction is estimated as 48+/-17 kJ/mol. Based on the mass spectrometry study, the labeling reaction was assumed to consist of two steps. In the first, the protein unfolds to make lysine residues accessible. In the second, the reagents react with the epsilon -amine of the lysine residues. To test this hypothesis, submicellar capillary electrophoresis and laser-induced fluorescence were used to characterize the reaction mixture. The apparent activation energy was measured for the labeling reaction; the apparent activation energy was 57+/-12 kJ/mol for reaction performed in the separation buffer. Denaturing agents were added to the reaction mixture. The addition of 2 M thiourea with 6 M urea to the reaction resulted in a modest decrease in the apparent activation energy to 42+/-2 kJ/mol. The addition of 2.5 M or higher concentration of ethanol decreased the apparent activation energy to 32+/-2 kJ/mol. We conclude that the apparent activation energy for protein labeling is dominated by denaturation of the protein, and that the addition of suitable denaturing reagents can eliminate this contribution to the reaction chemistry.