多酶级联催化合成（R）-β-酪氨酸

目前报道的β-酪氨酸生产方法需要较为复杂的底物而且大多依赖于贵金属催化剂。为了实现生物法绿色合成β-酪氨酸,通过人工设计的级联反应,将酪氨酸酚裂解酶和酪氨酸氨基变位酶进行级联,以苯酚、丙酮酸和铵盐等廉价化合物为底物合成(R)-β-酪氨酸。通过基因挖掘筛选了所需的酶元件,并采用蛋白质工程改造,提升了限速酶的催化效率。在大肠杆菌宿主中对所筛选的酶进行共同表达,经优化获得了(R)-β-酪氨酸合成菌株E. coli S10。在1 L规模反应中,菌株E. coli S10合成(R)-β-酪氨酸的转化率达到78%,ee值>99%。利用高分辨质谱(HRMS)和核磁共振图谱(NMR)分别鉴定了纯化产物的分子量和结构,结果表明通过该级联路径可成功合成目标产物(R)-β-酪氨酸。研究工作可为(R)-β-酪氨酸的绿色酶法生产提供理论指导。     

利用E.coli评价铁碳微电解处理印染废水的生物毒性变化

印染废水的成分复杂、色度高、毒性强，通过分析E.coli（大肠杆菌）的形态、抗氧化酶和生物标志物，研究铁碳微电解工艺处理前后印染废水生物毒性的变化。结果表明：E.coli在进水中呈破碎状态，而在铁碳微电解工艺出水中的E.coli大部分为正常形态；与进水的抗氧化酶系统相比，出水组中的丙二醛（MDA）、谷胱甘肽（GSH）、超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和总抗氧化能力（T-AOC）分别降低了80.85%、53.73%、67.74%、44.90%和43.38%，铁碳微电解工艺处理后的印染废水E.coli的抗氧化能力接近正常水平。进水和出水的葡萄糖消耗量抑制率分别为85%和47%；与进水的生物标志物相比，出水中热值升高21.95%，内源荧光蛋白升高112.96%，核酸含量降低44.04%。铁碳微电解工艺具有降低实际印染废水生物毒性的作用。     

排气管直径与深度对水力喷射空气旋流器传质性能的影响

对水力喷射空气旋流器(WSA)的中心排气管直径D_e和排气管插入深度S进行了设计优化研究,实验考察了不同D_e和S对WSA脱氨传质系数K_La和气相压降的影响,提出了综合评价传质过程效率的概念——单位压降传质效率η_p,讨论了D_e和S的设计取值。研究表明,当D_e增大时,K_La和气相压降均随之降低,较小D_e的WSA具有较高的气液传质性能,但气相压降比较大,而K_La和气相压降均随S增大而增大。综合能效关系表明,随着D_e的增大η_p增大,随着S的增大η_p出现先增大后降低的趋势,WSA设计中D_e与WSA直径D之比D_e/D宜为0.42～0.53,S与WSA筒体长度H之比S/H适宜取值约为0.70。     

环氧化物水解酶交联细胞聚集体催化合成(R)-环氧氯丙烷

利用聚乙烯亚胺(PEI) 絮凝、戊二醛(GA)交联对环氧化物水解酶全细胞进行了交联细胞聚集体(CLCAs)制备，考察了PEI浓度、GA浓度及硅藻土载体用量对CLCAs活力回收率的影响，结果表明PEI浓度、GA浓度及硅藻土载体用量最优值分别为3% (体积)和1%(体积)和6 g/L，此时CLCAs活力回收率可达88.4%。以CLCAs作为催化剂，以外消旋环氧氯丙烷((R,S)-ECH) 为底物，在异辛烷/磷酸盐缓冲液两相体系中催化合成(R)-环氧氯丙烷((R)-ECH)。结果表明，在异辛烷与缓冲液的体积比3∶7，底物浓度800 mmol/L，CLCAs加入量18 g /L，缓冲液pH 8.0，温度35℃条件下，(R)-环氧氯丙烷的摩尔产率达到45.2%，产物光学纯度为99.1% ee。考察了CLCAs在两相体系中的操作稳定性，重复使用9个批次活力基本保持不变，显示了良好的操作稳定性。     

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%。     

Aspergillus niger NL-1来源的木聚糖酶的耐热性能改造及其在水解木聚糖中的应用

为提高来源于Aspergillus niger NL-1的木聚糖酶MxynB的热稳定性，通过定点突变技术在木聚糖酶MxynB的相应位置引入二硫键（Cys¹¹⁶-Cys¹³⁵），获得突变酶MynxB-116-135；通过基因融合技术在木聚糖酶MxynB的N端融合了具有较高热稳定性的来源于嗜热菌Thermotoga thermarum DSM5069的纤维素结合结构域（CBD），获得突变酶CBD-MxynB和CBD-MxynB-116-135。分别将原酶及突变酶在E.coli BL21（DE3）中表达，经纯化后对比其酶学性质。结果显示，突变酶MxynB-116-135的最适温度为50℃，较原酶MxynB提高了5℃；突变酶CBD-MxynB和CBD-MxynB-116-135的温度稳定性明显提高，其中CBD-MxynB-116-135尤为突出，在70℃下保温2h仍能保持50%以上的酶活力，而原酶基本失活。研究表明，二硫键的引入和CBD的融合对提高MxynB的热稳定性具有重要的作用。此外，研究了突变酶CBD-MxynB-116-135水解玉米芯木聚糖的产物，结果显示玉米芯木聚糖质量浓度为2.5mg/mL，酶用量为40U/g，在50℃、pH 5.0条件下水解10h后，水解得率为39.6%，水解产物XOS_2-3含量占87.9%。结果表明，利用该突变木聚糖酶酶解碱提玉米芯木聚糖可产生以木二糖及木三糖为主要成分的低聚木糖。     

新能源汽车电池用无镁储氢合金的制备与性能研究

采用真空电弧熔炼和925 ℃/12 h退火的方法制备了Y_1-xLa_xNi_3.25Al_0.15Mn_0.15储氢合金（x=0~1）,研究了x值对储氢合金物相组成和电化学性能的影响。结果表明,x=0和0.15的储氢合金主要由LaNi₅和Ce₂Ni₇相组成,x=0.25、0.33和0.5储氢合金主要由Ce₅Co₁₉和Ce₂Ni₇相组成,x=0.75和1储氢合金主要由PuNi₃、LaNi₅和Ce₂Ni₇相组成;相同充放电循环周次下,x=0.15~1储氢合金的放电容量和抗氢致非晶化能力都高于x=0储氢合金,且随着x从0增加至1,储氢合金的最大放电容量（C_max）、容量保持率（S₁₀₀）、氢扩散系数（D₀）和高倍率放电性（HRD₉₀₀）都呈现先增加后减小趋势,在x=0.33时取得C_max、S₁₀₀、D₀和HRD₉₀₀最大值。Y_1-xLa_xNi_3.25Al_0.15Mn_0.15储氢合金的循环稳定性与合金电极的耐腐蚀性密切相关,高倍率放电性能取决于储氢合金的氢扩散速率。     

(1S,4R)-4,7,7-三甲基-6-氧杂二环[3.2.1]辛烷-1,4-二醇的一步催化合成及其除草活性

以过氧磷钼钨酸十六烷基吡啶盐（HPP）为氧化-酸双功能相转移催化剂,30%（质量分数）的H₂O₂为氧化剂,催化异松油烯一步转化合成（1S,4R）-4,7,7-三甲基-6-氧杂二环[3.2.1]辛烷-1,4-二醇（2）。优化后的反应条件为异松油烯用量4 mmol,催化剂HPP的用量7.35%（以异松油烯质量计）,溶剂氯仿用量0.8 mL,反应物料比（H₂O₂与异松油烯物质的量比）值为3,35℃反应6 h,此条件下异松油烯的转化率高达100%,化合物2的产率达到29%。红外光谱、核磁共振谱及单晶X衍射表征结果表明成功合成了化合物2。初步的除草活性实验结果表明：化合物2能抑制黑麦草根和芽的生长,在120 mmol/L浓度下,对黑麦草根和芽生长的抑制率分别高达91.8%和80.7%,与已报道的1,8-桉树脑相比,化合物2对黑麦草的抑制作用更好。     

乳糖诱导E.coli发酵生产FAD为辅基的葡萄糖脱氢酶

黄素腺嘌呤二核苷酸（FAD）为辅基的葡萄糖脱氢酶（FAD-GDH,EC1.1.5.9）,具有辅基结合紧密、催化效率高的优点,可替代目前诊断用葡萄糖氧化酶应用于血糖指标的临床生化检测。本文选取Burkholderia cepacia的葡萄糖脱氢酶基因（gdh）构建表达质粒pTrc99a-gdh,转化E. coli BL21(DE3)。异丙基硫代半乳糖苷（IPTG）诱导发酵,通过酶活测定及聚丙烯酰胺凝胶电泳分析,获得可溶性表达的FAD-GDH,分子量约为60000。利用乳糖替代IPTG作为诱导剂,摇瓶水平初步探索诱导条件,酶活达到994U/L。7.5L发酵罐放大培养该菌,采用梯度流加补料策略,分阶段温度控制,并采用不同的乳糖流加速率进行诱导。当采用0.3mL/min的乳糖流加速率时,酶活达22200U/L,菌体量达69.48g/L。经过镍柱层析,最终获得纯酶的比酶活104.5U/mg。为医用诊断原料用酶葡萄糖脱氢酶新酶种的工业化生产提供借鉴。     

GO/Al2O3复合纳滤膜的制备及其稳定性能研究

以平均孔径为20 nm的Al₂O₃管式超滤膜为载体，经多巴胺改性后，利用压力驱动沉积法成功制备出能在水溶液中长期稳定的GO/Al₂O₃复合纳滤膜，并通过改变负载量实现了对GO层厚的调控。结果表明，随错流时间的延长，不同GO负载量下GO/Al₂O₃复合纳滤膜的纯水渗透系数均呈现先降低后稳定的趋势。且随着GO负载量的增加，稳态纯水渗透系数逐渐降低；当GO负载量增加到90 mg/m²后，GO/Al₂O₃复合纳滤膜对一二价盐的渗透系数与截留率均无显著变化。同时，由于盐测试过程中残余的盐离子在GO片层间产生了交联作用，从而导致随着在纯水中存放时间的延长，不同GO负载量的GO/Al₂O₃复合纳滤膜对一二价盐的截留率均呈上升趋势。GO负载量为140 mg/m²的GO/Al₂O₃复合纳滤膜在水中浸泡680 h后对1 mmol/L Na₂SO₄的截留率可达到91.0%。GO/Al₂O₃复合纳滤膜对四种一二价盐的截留率满足：R(Na₂SO₄) > R(MgSO₄) > R(NaCl) > R(MgCl₂)。     

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.     

Diporphyrin tweezer for multichannel spectroscopic analysis of enantiomeric excess

Chiral 1,1’-binaphthyl-linked diporphyrin ‘tweezers’ (R)-1/(S)-1 and the corresponding zinc(II) complexes (R)-2/(S)-2 were prepared as chiral host molecules, and their utility for chiral analyses (especially enantiomeric excess (ee) determinations) were evaluated. Tris(1-n-dodecyl)porphyrins were used for the first time as the interacting units. Host capabilities of the diporphyrin tweezers were investigated by titrations with (R,R)- and (S,S)-cyclohexane-1,2-diamine (CHDA). The host molecules could be used as multichannel probes of ee by using UV-vis, circular dichroism (CD), fluorescence emission and ¹H nuclear magnetic resonance (¹H-NMR) methods. Chiral configurations could also be differentiated using CD or ¹H-NMR spectroscopy. All three optical techniques give good resolution of ee with reasonable sensitivity considering the low concentrations used (ca. 10⁻⁶ mol·L⁻¹). The ee determination of CHDA enantiomers using NMR spectroscopy is also possible because of the reasonably well separated resonances in the case of (R,R)- and (S,S)-CHDA. Non-metallated (R)-1/(S)-1 hosts could not be used to detect chiral information in a strongly acidic chiral guest. This work demonstrates the utility of 1,1’-binapthyl-linked chiral hosts for chiral analysis of ditopically interacting enantiomers.     

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. P3H1B, 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. The four DDHs were purified and biochemically characterized for oxidation activity toward (R)-1-phenyl-1,2-ethanediol, with the optimal reaction condition of pH9.0 (AnDDH), 10.0 (HcDDH) and 11.0 (GzDDH and LwDDH) and the temperatures at 40℃ (AnDDH), 50℃ (HcDDH) and 60℃ (GzDDH and LwDDH), respectively. The four enzymes were stable at the pH from 7.0 to 9.0 and below 40℃. Kinetic parameters of four DDHs showed that the HcDDH from Hazenella coriacea had high activity toward a broad range of vicinal diols. A series of racemic vicinal diols were successfully resolved by recombinant E. coli (HcDDH-NOX) resting cells co-expression of an NADH oxidase (NOX), affording (S)-diols and (1S, 2S)-trans-diols in ≥ 99% ee. The synthetic potential of HcDDH was proved by E. coli (HcDDH-NOX) via kinetic resolution of racemic trans-1,2-indandiol on a 100 ml scale reaction, (S, S)-trans-1,2-indandiol was prepared in 46.7% yield and >99% ee. In addition, asymmetric reduction of four α-hydroxy ketones (10-300 mmol·L^-1) by E. coli (HcDDH-GDH) resting cells resulted in >99% ee and 69-98% yields of (R)-vicinal diols. The current research expands the toolbox of DDHs to synthesize chiral vicinal diols and demonstrated that the mined HcDDH is a potential enzyme in the synthesis of a broad range of chiral vicinal diols.     

Photocatalytic inactivation of Escherischia coli: Effect of concentration of TiO2 and microorganism, nature, and intensity of UV irradiation

The efficiency of photocatalytic disinfection, used to inactivate Escherischia coli K12 under different physico-chemical parameters, was examined. The photocatalyst chosen was the semiconductor TiO₂ degussa P25 and the irradiation was produced by an HPK 125 lamp. The effect of titania concentration was investigated using two E. coli concentrations. The photocatalyst concentration ranged from 0.1 to 2.5 g/L. The evolution of E. coli inactivation as function of time was discussed depending on the E. coli and TiO₂ concentrations. The optimal concentration of the photocatalyst, 0.25 g/L, is lower than that necessary to absorb all photons and to degrade the organic compounds. Some hypotheses are presented to explain this behaviour. The effect of the different domains of UV light (UVA, UVB, and UVC) was also studied and modification of the light irradiation intensity is discussed. No bacteria photolysis was obtained with UVA but the use UVC had, on the contrary, a detrimental effect on bacteria survival. The addition of titania at a low concentration, 0.25 g/L, improved the inactivation of E. coli in the presence of UVA and UVB, but a detrimental effect was observed under UVC. The disinfection efficiency increases as a function of light intensity, whatever the photocatalytic conditions (different TiO₂ concentrations and different UV domains). No bacterial growth was observed after disinfection, whether the system contained titania or not.     

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.     

Use of coaxial photocatalytic reactor (CAPHORE) in the TiO2 photo-assisted treatment of mixed E. coli and Bacillus sp. and bacterial community present in wastewater

This paper reports the photocatalytic disinfection of water contaminated by a mixture of Escherichia coli and Bacillus sp. as well as that of wastewater containing a larger microbial community. The photocatalytic reactions were carried out in a coaxial photocatalytic reactor called CAPHORE, using TiO₂ P-25 of Degussa. E. coli is more sensitive than Bacillus sp. to photocatalytic treatment. Bacterial inactivation was dependent on organic matter and dissolved oxygen (DO) concentration.

Of the bacterial community present in partially treated wastewater, E. coli appears to be more sensitive to the treatment than Enterococcus sp., coliforms (other than E. coli), and Gram-negative (other than coliforms). After photocatalytic treatment, no bacterial recovery of previous groups was observed for 24 h in the dark. However a very low bacterial inactivation rate was observed for the whole bacterial population present in wastewater and detected by non-selective media. The effective disinfection time (EDT), the time necessary for total inactivation of bacteria without re-growth in a subsequent dark period referenced at 24 h (or 48 h), was reached only for Enterococcus sp., and coliform groups. EDT₂₄ was not reached for the whole population.