盖子环替换及定点突变提高菜豆环氧化物水解酶对邻甲基苯基缩水甘油醚的催化性能

菜豆环氧化物水解酶1和2（PvEH1、PvEH2）能够动力学拆分外消旋邻甲基苯基缩水甘油醚（rac-oMGE）,从而保留(R)-oMGE。基于对PvEH1和PvEH2结构的同源模拟和分析,发现二者分子中的盖子环差异较大,故本文选择盖子环作为研究目标。经融合聚合酶链式反应（FPCR）,获得了PvEH2的盖子环区域被PvEH1对应区域替换的杂合酶Pv2Pv1。用全细胞酶E. coli/pv2pv1催化rac-oMGE,当(S)-oMGE刚好水解完全时,产物(S)-3-邻甲苯基-1,2-丙二醇（(S)-oTPD）的ee_p由PvEH2的58.3%提高至75.5%。为进一步提高酶的性质,在Pv2Pv1中选取11个氨基酸位点进行丙氨酸(A)突变,获得最优突变子E. coli/pv2pv1^K176A,活性为E. coli/pv2pv1（4.2U/g）的2.1倍,且当S构型的底物刚好完全水解时,(S)-oTPD的ee_p进一步提高为80.3%。分子对接分析发现,盖子环替换和K176位点突变为A,均使(R)-oMGE环氧环中的C_α更易受到酶中D101位点的攻击。利用E. coli/pv2pv1^K176A催化150mmol/L rac-oMGE水解制备(R)-oMGE（ee_s＞99%）和(S)-oTPD（ee_p=80.4%）,二者的产率Y_S和Y_P分别为32.7%和60.1%,时空产率STY_S和STY_P为1.6g/(L·h)和3.3g/(L·h)。本实验为改善EH的催化性质提供了一种有效策略。     

Asymmetric bioreduction of γ- and δ-keto acids by native carbonyl reductases from Saccharomyces cerevisiae

Optically pure (R)-γ- and (R)-δ-lactones can be prepared by intramolecular cyclization of chiral hydroxy acids/esters reduced asymmetrically from γ- and δ-keto acids/esters using Saccharomyces cerevisiae (S. cerevisiae) as a whole-cell biocatalyst. However, some of the enzymes catalyzing these reactions in S. cerevisiae are still unknown up to date. In this report, two carbonyl reductases, OdCR1 and OdCR2, were successfully discovered, and cloned from S. cerevisiae using a genome-mining approach, and overexpressed in Escherichia coli (E. coli). Compared with OdCR1, OdCR2 can reduce 4-oxodecanoic acid and 5-oxodecanoic acid asymmetrically with higher stereoselectivity, generating (R)-γ-decalactone (99% ee) and (R)-δ-decalactone (98% ee) in 85% and 92% yields, respectively. This is the first report of native enzymes from S. cerevisiae for the enzymatic synthesis of chiral γ- and δ-lactones which is of wide uses in food and cosmetic industries.     

Characterization of four diol dehydrogenases for enantioselective synthesis of chiral vicinal diols

Enantiopure vicinal diols are important building blocks used in the synthesis of fine chemicals and pharmaceutical compounds. Diol dehydrogenase(DDH) mediated stereoselective oxidation of racemic vicinal is an efficient way to prepare enantiopure vicinal diols. In this study, four new bacterial DDHs(AnDDH from Anoxybacillus sp. P3 H1 B, HcDDH from Hazenella coriacea, GzDDH from Geobacillus zalihae and LwDDH from Leptotrichia wadei) were mined from the GenBank database and expressed in E. coli T7...     

手性NAD类似物合成及其辅酶应用

烟酰胺腺嘌呤二核苷酸（NAD）是生命体中必不可少的氧化还原辅因子。NAD具有β-NAD和α-NAD两种差向异构体,而大多数NAD依赖酶偏好β-NAD。近年来,已合成大量结构简化的NAD类似物,并探索其作为氧化还原辅因子应用于生物催化反应。通常认为酶天然具有立体选择性,而很多结构简化的NAD类似物不含手性中心,暗示有必要合成手性NAD类似物以评估其性能。本研究拟设计合成手性NAD类似物,并初步探讨部分氧化还原酶利用它们作为辅酶的催化性能。以商品化光学纯的氨基-3-苯基丙酸为手性原料,合成了两对NAD类似物对映体,并用来源于巨芽孢杆菌的突变型细胞色素P450单加氧酶（P450 BM3 R966D/W1046S）和来源于硫化叶菌的突变型葡萄糖脱氢酶（SsGDH I192T/V306I）,测试这些NAD类似物作为辅酶的催化活性。结果发现,P450 BM3 R966D/W1046S利用还原型S构型NAD类似物的催化效率高于对应的R构型NAD类似物;而SsGDH I192T/V306I则对NAD类似物对映体均没有活性。研究初步表明,氨基酸可作为手性原料制备立体构型明确的NAD类似物,而氧化还原酶表现出偏好特定手性NAD类似物的现象,也提示需要合成更多的手性NAD类似物,并系统测试和匹配氧化还原酶及其突变体,以创建出基于辅酶依赖型氧化还原酶的新催化体系。     

卤醇脱卤酶催化合成(R)-1-氯-3-苯氧基-2-丙醇的工艺优化

以卤醇脱卤酶重组湿菌体E. coli BL21（pET28a-HHDH）为催化剂,催化外消旋的1-氯-3-苯氧基-2-丙醇的动力学拆分可以获得光学纯的(R)-1-氯-3-苯氧基-2-丙醇。本文系统地研究了卤醇脱卤酶催化合成光学纯(R)-1-氯-3-苯氧基-2-丙醇的影响因素,对反应pH、反应温度、菌体浓度、亲核试剂 {\mathrm{N}}_{\mathrm{3}}^{-} 浓度和底物浓度进行了探究。结果表明,卤醇脱卤酶催化合成(R)-1-氯-3-苯氧基-2-丙醇的最佳工艺条件为：pH为7.0,反应温度为28℃,菌体浓度为22.5g/L,亲核试剂NaN₃的浓度为50mmol/L,底物外消旋1-氯-3-苯氧基-2-丙醇的浓度为10mmol/L。在此工艺条件下,(R)-1-氯-3-苯氧基-2-丙醇的ee值和收率分别为100%和16.97%。     

Asymmetric reduction of aliphatic and cyclic ketones with secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus: effects of substrate

The reduction of aliphatic ketones catalyzed by a secondary alcohol dehydrogenase (SADH) from Thermoanaerobacter ethanolicus affords (S) -alcohols in high enantiomeric purities, when the chain has six or more carbons. With 2-pentanone, the reduction gives (S)-2-pentanol, and (R)-stereoselectivity is observed in the case of 2-butanone at 37°C. The rate of the reduction of aliphatic methyl ketones decreases about three-fold for each additional methylene increment. A temperature dependent reversal of enantiospeficity is observed in the oxidation of enantiomers of 2-butanol. A linear dependence of RTInE with temperature is observed for 2-butanol, 2-pentanol, and 2-hexanol. The cofactor analogues, thionicotinamide adenine dinucleotide phosphate (SNADP) and acetylpyridine adenine dinucleotide phosphate (APADP) gave higher enantioselectivity in the reduction of 2-butanone to (R) -2-butanol. For the reduction of cyclic ketones, SADH is enantiospecific for (S) isomers of cyclic alkyl ketones, and the transfer of hydrogen is stereoselective for the Re face to give (1S) cyclic alcohols. The facial stereospecificity of SADH for hydride transfer to NADP was determined by NMR, and it was found to be re-specific ( ‘A face’ ). In order to explain the stereoselectivity of SADH catalyzed reductions, a model is proposed that emphasizes the importance of the stability of substrate conformation and the steric interaction between substrate, enzyme and coenzyme.     

Racemization of (R)-(−)-10-methyl-Δ-octalin: Stereochemical requirements for double bond migration (isomerization) on metal catalysts

(R)-(−)-10-methyl-Δ¹⁽⁹⁾-octalin was synthesized in a mixture with cis- and trans-(R)-(−)-10-methyl-Δ¹⁽²⁾-octalin in a five step sequence. After removal of the cis- and trans-(R)-(−)-10-methyl-Δ¹⁽²⁾-octalins by hydrogenation over 5% Pt/C, 5% Rh/C, or 5% Pd/C, the remaining (R)-(−)-10-methyl-Δ¹⁽⁹⁾-octalin was monitored for racemization (double bond migration) during continued hydrogenation. No racemization occurs over these catalysts. However, slow racemization does occur in increasing amounts during hydrogenations over a series of 1% Pd/SiO₂ catalysts with dispersions increasing from 36%D to 84%D. The ratio of cis- to trans-9-methyldecalin changes from 1.8 over 5% Rh/C, 1.6 over 5% Pt/C, 1.1 over 5% Pd/C, to 1.0 over the 1% Pd/SiO₂ catalysts. Since double bond migration does not occur over the 5% catalysts, it cannot be used to account for differences in the ratio of cis- to trans- products. Congestion around the double bond inhibits addition, but double bond migration seems to require the allylic hydrogens to be nearly perpendicular to the planes of the double bond and the surface.     

Enantioseparation of 3-chlorophenylglycine enantiomers using Mandyphos-Pd as chiral extractant

Chiral extractant plays a key role in chiral extraction, and considerable efforts have been undertaken for the development of new and efficient chiral extractants in recent years. This work demonstrated for the first time that chiral ferrocenyl diphosphine ligand(Mandyphos-Pd) had considerable ability to enantioseparate 3-Chlorophenylglycine enantiomers with separation factor(α) of 2.64. Mandyphos-Pd concentration and p H had significant influences on enantioselectivity, while operating temperature showed less influence. The extraction experiments can be performed at room temperature(20 °C) which had the advantage of energy saving. After optimization, the highest performance factor(pf, 0.08376) was obtained at the condition of p H 7.8 and Mandyphos-Pd concentration 1.2 mmol·L~(-1). According to the experimental results, the possible recognition mechanism was discussed.     

藻渣热解特性及动力学分析

通过热重实验研究N₂气氛下升温速率对索氏提脂后的小球藻热解特性的影响,利用管式炉在N₂气氛下快速热解实验得出：在400℃时,小球藻热解转化率最高,生物油产率达57.6%,热解气为10%。采用等转化率方法FWO和KAS法对藻渣热解动力学进行分析和比较,结果表明：藻渣热解的主要热解阶段为25～800℃,可分为3个阶段,藻渣的DTG曲线存在两个失重峰,且随着升温速率提高,TG和DTG曲线都向高温区偏移,最大失重速率和残余固体质量都增加。N₂气氛条件下藻渣的主要热解阶段表观活化能和指前因子分别为228.46 kJ/mol和2.49×10²¹ min^-1,此阶段下FWO法和KAS法均能很好模拟藻渣热解数据,线性拟合相关系数(R²)均在0.96以上,最佳热解函数为dα/dT=2.49×10²¹/β exp(-228.46/(RT))(1-α)⁸。     

Enantioselective hydrogenation of isophorone and kinetic resolution of 3,3,5-trimethylcyclohexanone over Pd catalysts in the presence of (S)-proline

Enantioselective hydrogenation of isophorone and kinetic resolution of 3,3,5-trimethylcyclohexanone (TMCH) over Pd catalysts in the presence of (S)-proline revealed that the enantioselectivity for hydrogenation of isophorone was mainly originated from the kinetic resolution of TMCH. The rapid hydrogenation of isophorone primarily yielded racemic TMCH, and the followed kinetic resolution consumed the (R)-TMCH enantiomer, leaving the (S)-TMCH enantiomer in excess. The kinetic resolution of racemic TMCH is closely related to the acidic/basic properties of the support, and the addition of K₂CO₃ to Al₂O₃ provided more enantio-differentiating environment through the enhanced adsorption of (S)-proline on the catalyst surface. As a result, the Pd/Al₂O₃-K₂CO₃ catalyst with finely dispersed Pd particles and enhanced adsorption of (S)-proline gave very high enantioselectivities (e.e. value up to 98%) for the enantioselective hydrogenation of isophorone.     

Effects of enantiomeric solvents on the activity, thermostability and activation energy of lipoprotein lipase

The kinetic parameters (K_m and V_max) of lipoprotein lipase (LPL)-catalyzed transesterification were determined in (R)- and (S)-carvone, and in mixtures of the two. It was found that only V_max was significantly affected by solvent chirality. LPL thermostability was not influenced by solvent config- uration, whereas activation energy was twice as high in (R)-carvone as in (S)-carvone.     

薇甘菊粉末吸附亚甲基蓝和碱性品红的性能研究

以薇甘菊为原料,经粉碎制成不同粒径的粉末吸附剂。探究其对亚甲基蓝（MB）和碱性品红（BF）的吸附性能,研究发现薇甘菊粉末吸附亚甲基蓝和碱性品红的最佳投加量分别为20和10 g/L,最佳粒径为0.3~0.45 mm,pH值为5,温度40 ℃,振荡吸附时间40 min,此条件下,薇甘菊粉末对亚甲基蓝和碱性品红的吸附量分别为4.870和9.729 mg/g。利用准一级、准二级动力学方程模拟了薇甘菊粉末吸附亚甲基蓝和碱性品红的动力学过程,结果表明：薇甘菊粉末吸附亚甲基蓝和碱性品红的过程符合准二级动力学模型。计算了吸附过程中的焓变（ΔH^⊖）、吉布斯自由能（ΔG^⊖）、熵变（ΔS^⊖）等热力学参数,薇甘菊粉末吸附亚甲基蓝和碱性品红过程中ΔG^⊖在-2.13~-1.58和-4.66~-1.63 kJ/mol之间,ΔS^⊖分别为0.034和59.25 J/（mol·K）,ΔH^⊖分别为0.0099和14.75 kJ/mol,薇甘菊粉末吸附2种染料的ΔG^⊖均小于0,ΔH^⊖、ΔS^⊖均大于0,说明薇甘菊粉末吸附2种染料的过程是一个自发的趋于无序的吸热过程。     

CO2响应性L-脯氨酸基纳米反应器的构筑及其催化性能

采用可逆加成-断裂链转移（RAFT）分散聚合法制备了一系列固载L-脯氨酸、CO₂响应性结构单元甲基丙烯酸二乙氨基乙酯（DEA）含量不同的mPEG₂₂-b-P[BnMA _x -co-L-ProlA _n ]-b-PDEA _y PBL-b-PD-(DEA) _m （m=0,1,2,3）和单体序列结构不同的mPEG₂₂-b-P(L-ProlA)-b-P[BnMA-co-DEA] （PL-b-PBD）聚合物。利用核磁共振氢谱（¹H NMR）、X射线光电子能谱（XPS）和凝胶渗透色谱（GPC）对聚合物的化学结构及相对分子量分布进行了表征。采用动态光散射（DLS）对聚合物的自组装行为及形成纳米反应器后的CO₂响应性进行了研究。同时，结合耗散粒子动力学（DPD）模拟探究了聚合物亲疏水单体比例和序列结构对聚合物自组装结构的影响。最后，将制备的纳米反应器用于水相催化直接不对称Aldol反应，结果表明，PBL-b-PD-(DEA)₂具有最佳的催化性能[96%conv.，93/7(anti/syn)，94%ee]。该研究为探究聚合物自组装结构对其催化性能的影响提供了新思路。     

Quantitative Measurements of the Vinylacetate Content in High Pressure Ethylene Vinylacetate Copolymers

The pyrolysis-IR spectrometry method, described in a previous paper, ² is extended to EVA copolymers having a% wt VA content up to 55. The 3460 cm^-1 '/2678 cm^-1 absorption ratio v.% wt VA calibration curve for IR measurements, shows a positive deviation from linearity. All results obtained by pyrolysis, IR spectrometry and ¹H-NMR are comparable, showing that each method is equally valid and gives fast, reliable% wt VA values.     

Surface dynamics during latex film formation

Surface dynamics during latex film formation has been investigated theoretically and experimentally by atomic force microscopy. The peak-to-valley distance, y(t), of the latex particles in the surface plane of the latex film decayed exponentially with time during film formation. A theoretical relationship between y(t) and time, t, is given by y(t)=y(0) exp[−t/τ], where y(0) is the value of y(t) when t is zero. τ is a characteristic constant related to the nature of polymer, the particle radius, the surface diffusion coefficient and the temperature. The relationship between the surface diffusion coefficient, D_s, y(0), the radius of the latex particles, R, temperature, T, and τ is given approximately by D_s=1.2×10⁻²⁰y(0)²[2R−y(0)]²T/τ (cm²/s), where the units are manometers for y(0) and R, kelvin for temperature, and seconds for τ. By measuring the decay of y(t) with time, the surface diffusion coefficient can be obtained. The surface diffusion coefficient for a poly(methyl methacrylate-co-butylacrylate) (50:50) copolymer latex film was found to be A×10⁻¹³ cm²/s, A is temperature-dependent.     

A comparison of the attachment of the decay products of radon-220 and radon-222 to monodispersed aerosols

The adsorption of radioactive aerosols is of prime importance when assessing the hazard of inhalation of radioactive materials, especially in the uranium mining industry. In previous studies of the attachment rate, contradicting results on the dependence on particle size have been found. The attachment rate for the attachment of the decay products of Rn-222 to monodispersed polystyrene aerosols (of a diameter from 1.2 to 5.3 μm) in the concentration range 1–67 particles cm⁻³ was measured directly. This is compared with the attachment rate of radon-220 decay products which was measured in a previous experiment. As before, the attachment between the radioactive atoms and the aerosols was found to be directly proportional to the particle concentration N, as well as to the square of the particle radius R.

The attachment coefficient of 4.1 × 10⁵ R² cm³ min⁻¹ and the sticking probability of 0.13 for Rn-222 decay products were found to be larger than that for the decay products of Rn-220 (2.4 × 10⁵ R² cm³ min⁻¹ and 0.08, respectively). The mechanism for the attachment, however, is the same.     

UV/oxone降解水中甲氧苄啶的效能及模型评价

研究采用UV、oxone、UV/oxone共3种工艺降解甲氧苄啶（TMP）的效果及动力学。采用响应面曲线法研究含有HCO₃ ^-、Cl^-、NO₃ ^-和pH四因素、三水平的水体条件下UV/oxone对TMP的降解效果及模型，并选用4种实际水源水体为水质背景来评价模型预测值与实际降解值的差别，最后比较了3种工艺的同等降解率条件下的降解效能。结果发现UV和oxone单独降解TMP时仅降解了5.5%和62.0%，而UV/oxone可达到93.2%，且降解过程符合拟一级反应动力学，动力学常数为0.1768min^-1。采用相对速率法可得到?SO₄ ^–与TMP的反应速率常数为2.07×10⁸L/(mol·s)。通过响应面曲线法得到UV/oxone降解TMP的回归方程式，对应的p值小于0.0001，拟合缺失项P不显著（0.9726>0.05），校正决定系数R ²=0.82＞0.8，说明该模型可信度高，采用实际水体进行模型验证后发现实际降解值基本符合模型预测值。降解率一定的条件下UV/oxone耗时最短，是一种高效、快速、可行的降解工艺。     

Walden product, ion-solvent interactions and solvent structure in water-rich mixtures ionic conductances in water-sulfolane, water-acetonitrile and water-dimethylsulfoxide

Conductance measurements are reported for several salts in binary aqueous mixtures containing up to 60 mole % sulfolane, 20 mole % acetonitrile and 20 mole % dimethylsulfoxide. The variations of R = (λ^±₀η₀)_s/(λ^±₀η₀)_w with solvent composition have been compared with those observed in other water-rich mixtures. Alkali cations show R values greater than one with maxima in all the solvent mixtures. This behaviour has been discussed in terms of “sorting”, “averaging” and “steric” effects. Contrary to what happens to alkali cations, halide ions show R values greater or lesser than one according to whether the organic solvent respectively increases or decreases water structure. On these bases we suggest that conductometric behaviour of the halide ions may be indicative of the effect of the cosolvent on the water structure in water-rich mixtures and that DMSO is a water structure breaker.     

Using multiple site-directed modification of epoxide hydrolase to significantly improve its enantioselectivity in hydrolysis of rac-glycidyl phenyl ether

The epoxide hydrolase gene (SpEH) from Sphingomonas sp. HXN-200 was synthesized and expressed in robust Escherichia coli cells that had a dual protection system. The enantioselectivity (E-value) of the recombinant SpEH was 7.7 and the yield of the remaining (R)-PGE was 24.3% for the hydrolysis of racemic phenyl glycidyl ether (rac-PGE). To improve the catalytic properties of SpEH, the site-directed mutagenesis was carried out based on homology modeling, sequence alignment and molecular docking. Six residues (V195, V196, F218, N226, Q312, and M332) near the active site were mutated to hydrophobic amino acids and the positive mutations were selected for combinatorial mutation. The optimal mutant SpEH^{V196A/N226A/M332A} had an enhanced E-value of 21.2 and a specific activity of 4.57 U·mg^-1-wet cells, which were 2.8-, and 2.3-fold higher than those of wild-type SpEH. The optimal temperature and pH for purified SpEH^{V196A/N226A/M332A} to catalyze the hydrolysis of rac-PGE were 25 ℃ and 7.0 with 200 U·mg^-1. The enantioselectivity and yield of the remaining (R)-PGE of E. coli_SpEH^{V196A/N226A/M332A} increased from 7.7 to 21.2 and 24.3% to 40.9%, respectively. The molecular docking and kinetic parameter analyses showed that SpEH^{V196A/N226A/M332A} has a greater affinity toward (S)-PGE than (R) - PGE, and that it was more difficult for the O-atom of ASP170 to achieve the nucleophilic attack on the Cα of (R)-PGE, resulting in its improved enantioselectivity.     

(CH3CH[NH3]CH2NH3)1/2·ZnPO4, a zincophosphate analogue of aluminosilicate zeolite thomsonite

The synthesis and structure of (CH₃CH[NH₃]CH₂NH₃)_1/2·ZnPO₄, an organically templated zincophosphate (ZnPO) analogue of aluminosilicate zeolite thomsonite (THO), are described. The ZnPO framework is built up from an alternating, vertex-sharing, network of ZnO₄ and PO₄ groups (d_av(Zn–O)=1.944 (8) Å, d_av(P–O)=1.535 (9) Å, θ_av(Zn–O–P)=130.5°) involving distinctive 4=1 secondary building units. The 1,2-diammonium propane cations are highly disordered in the [0 0 1] 8-ring channels. Crystal data: (CH₃CH[NH₃]CH₂NH₃)_1/2·ZnPO₄, M_r=198.42, orthorhombic, space group Pncn (no. 52), a=14.119 (6) Å, b=14.136 (5) Å, c=12.985 (5) Å, V=2591 (3) ų, Z=10, R(F)=0.057, R_w(F)=0.061 (for a twinned crystal).