Carbonyl reductase SCRII from Candida parapsilosis catalyzes anti-Prelog reaction to (S)-1-phenyl-1,2-ethanediol with absolute stereochemical selectivity

An (S)-specific carbonyl reductase (SCRII) was purified to homogeneity from Candida parapsilosis by following an anti-Prelog reducing activity of 2-hydroxyacetophenone. Peptide mass fingerprinting analysis shows SCRII belongs to short-chain dehydrogenase/reductase family. Its coding gene was cloned and overexpressed in Escherichia coli. The recombinant SCRII displays the similar enzymatic characterization and catalytic properties to SCR. It catalyzes the enantioselective reduction of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol with excellent optical purity of 100% in higher yield than SCR. Based on the sequence-structure alignment, several single-point mutations inside or adjacent to the substrate-binding loop or active site were designed. With respect to recombinant native SCRII, the A220 and E228 mutations almost lost enantioselectivity towards 2-hydroxyacetophenone reduction. The catalytic efficiencies (k_cat/K_m) for the A220 or E228 variants are <7% that of the unmutated enzyme. This work provides an excellent catalyst for enantiopure alcohol preparation and the lethal mutations of A220 and E228 suggest their importance in substrate-binding and/or catalysis.     

小鼠卵子在不同条件下的体外受精

本实验比较了小鼠精、卵细胞在不同生理状态下体外的受精能力。结果表明,体内受精率明显地高于体外(p<0.05),自发排出的卵子比超数排出的卵子受精率高(p<0.05),体外获能的附睾精子比体内获能的子宫精子受精率高(p<0.01)。唯独用超数排出的卵子和体外获能的附辜精子体外受精时,其受精率和体内相似。     

Pseudomonas putida KT2440低特异性L-苏氨酸醛缩酶的表达、酶学性质及温度稳定性提高

【目的】从Pseudomonas putida KT2440基因组中,钓取低特异性L-苏氨酸醛缩酶基因(lta E),构建重组大肠杆菌。研究目标酶的酶学性质,和关键氨基酸位点突变对酶活和温度稳定性的影响。【方法】以P. putida KT2440基因组DNA为模板,PCR扩增出lta E基因,构建重组表达质粒p ET28a-KT2440并转化Escherichia coli BL21 (DE3),获得重组菌E. coli BL21 (DE3)/p ET-KT2440,利用Ni~(2+)柱亲和层析纯化低特异性L-苏氨酸醛缩酶(LTA),对关键氨基酸位点Thr206和Lys207实施定点突变。【结果】SDS-PAGE结果表明LTA在大肠杆菌中获得高效表达,分子量为40k Da左右,与理论值大小相符。Ni~(2+)柱亲和层析纯化LTA,获得单一条带。利用双酶耦联法测得LTA酶活为5577.3U/mg,最适反应温度为50°C,最适p H为8.0。在温度低于45°C,p H 5.0-9.0时,重组酶较稳定。LTA酶的Km和kcat值为23.95 mmol/L和19216.6 s–1。Mg~(2+)、Ca~(2+)金属离子对LTA有明显的促进作用,而Ni~(~(2+))、Cu~(2+)、Zn~(2+)、Fe~(2+)等对酶有明显的抑制作用。该酶在叔丁基甲基醚溶剂中具有良好的耐受性,在叔丁基甲基醚中保存1h后仍保留90%以上的酶活。Thr206Ser突变明显提高了酶对温度的稳定性。Lys207对酶催化功能是必需的,该位点突变对酶活都是致死的。【结论】克隆并表达P. putida KT2440的LTA酶,研究了酶学性质,通过定点改造提高了酶的温度稳定性,筛选获得一种酶耐受性好的有机溶剂,为LTA酶在有机溶剂中高效稳定催化β-羟基-α-氨基酸奠定了较坚实的研究基础。     

Ultra-high Sensitivity Plasmonic Nanosensor Based on Multiple Fano Resonance in the MDM Side-Coupled Cavities

We propose a compact plasmonic structure comprising a metal-dielectric-metal (MDM) waveguide coupled with a side cavity and groove resonators. The proposed system is investigated by the finite element method. Simulation results show that the side-coupled cavity supports a local discrete state and the groove provides a continuous spectrum, the interaction between them, gives rise to the Fano resonance. The asymmetrical line shape and the resonant wavelength can be easily tuned by changing the geometrical parameters of the structure. Moreover, we can extend this plasmonic structure by the double side-coupled cavities to gain the multiple Fano resonances. The proposed structure can serve as an excellent plasmonic sensor with a sensitivity of ～1900 nm/RIU and a figure of merit of about ～3.8?×?10⁴, which can find wide applications for nanosensors.     

基因芯片技术在微生物学研究中的应用

近年来,基因芯片技术的诞生使得在一个实验中就可以同时对成千上万个基因进行转录水平的表达和DNA同源性分析成为可能。该项技术已被应用于揭示许多微生物体的转录表达和基因组的差异,随着越来越多的微生物基因组全序列测定的完成,基因芯片正逐渐成为许多微生物学研究领域中的一项常规技术。归纳了该技术在微生物生理,致病性,流行病学,生态,进化,代谢工程及发酵优化等研究中的应用。     

地衣芽孢杆菌E7氨肽酶的异源表达及其在高效蛋白水解中的应用

【目的】将地衣芽孢杆菌(Bacilluslicheniformis)E7氨肽酶基因pepN克隆到大肠杆菌(Escherichia coli) BL21中,实现氨肽酶Ec PepN的异源表达,研究重组酶的酶学性质及其与碱性蛋白酶协同作用,高效水解大豆蛋白和酪蛋白,产生小分子活性肽和游离氨基酸。【方法】以地衣芽孢杆菌E7基因组DNA为模板,将氨肽酶基因pepN克隆到载体pET28a中,构建重组表达载体pET28-pepN,转化到大肠杆菌BL21感受态细胞中,经DNA测序验证,获得重组菌E. coli BL21/pET28-pepN。利用镍离子亲和层析柱对重组酶进行分离纯化,研究纯酶的pH和温度稳定性、半衰期和NaCl的耐受性等酶学性质。以商品化氨肽酶与碱性蛋白酶协同作用为对照,重组酶Ec PepN与碱性蛋白酶协同水解大豆蛋白和酪蛋白,测定水解产物中小分子活性肽和游离氨基酸的组成。【结果】Ec PepN在大肠杆菌BL21中可溶性表达,SDS-PAGE分析表明纯化的重组酶在52kDa左右显示单一条带。在7种测定底物中,Ec PepN的最适底物为Ala-pNA。在最适条件(pH 9.0和50°C...     

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。     

Effects of alkyl polyglycoside, a nonionic surfactant, and forage-to-concentrate ratio on rumen fermentation, amino acid composition of rumen content, bacteria and plasma in goats

In the present study, the effects of different forage-to-concentrate ratios (F:C) and an alkyl polyglycoside (APG) supplementation on parameters of rumen and blood metabolism were investigated in goats. A 2 x 2 factorial experiment was arranged within a 4 x 4 Latin square design (four 22-day periods), using four wether goats equipped with permanent ruminal cannulas. The experimental diets included two F:C levels (40:60 vs. 60:40), and two APG supplementation levels (None or 13 ml APG daily per animal). Rumen contents and blood samples were collected at the end of each period. Dietary F:C alteration affected plasma urea and influenced the proportions of leucine, histidine, arginine, glycine, proline, alanine, valine, phenylalanine, cysteine and tyrosine in rumen content, and the proportions of methionine, threonine and proline in solid-associated bacteria (SAB) significantly. Dietary APG decreased the proportions of valine and phenylalanine in rumen content, and the histidine content of liquid-associated bacteria. The interaction between dietary F:C and APG was significant for the proportions of glycine and alanine in rumen content, and the proportions of lysine and threonine in SAB. The proportion of lysine was greater, but the proportion of threonine was less in SAB for goats fed high F:C diet without APG supplementation. The proportions of plasma free amino acids and glucose concentration were not affected by experimental treatments. These results indicated that dietary APG addition affected the amino acid composition of the rumen content and ruminal bacteria, but this depended on the dietary F:C ratio. It is necessary to validate the effectiveness of dietary APG supplementation in further studies with more animals.     

(S)-羰基还原酶Ⅱ与葡萄糖脱氢酶共催化高效合成(S)-苯乙二醇北大核心CSCD

【目的】通过优化获得最佳酶活配比,设计近平滑假丝酵母(Candida parapsilosis)CCTCC M203011的(S)-羰基还原酶Ⅱ与枯草芽孢杆菌(Bacillus sp.)YX-1葡萄糖脱氢酶在大肠杆菌中的共表达体系,实现重组菌高效催化2-羟基苯乙酮,合成(S)-苯乙二醇。【方法】分别从重组大肠杆菌中纯化了(S)-羰基还原酶Ⅱ和葡萄糖脱氢酶,研究了2种酶共催化2-羟基苯乙酮的最佳酶活比例,最适催化温度和pH,由此构建(S)-羰基还原酶Ⅱ和葡萄糖脱氢酶的共表达体系。【结果】(S)-羰基还原酶Ⅱ的比酶活力为1.3 U/mg,葡萄糖脱氢酶的比酶活力为13.5 U/mg。在总酶活力为1 U时,(S)-羰基还原酶Ⅱ和葡萄糖脱氢酶共催化体系中,确定了2种酶的最佳比例在1∶1到5∶1(U/U)之间,最适反应温度为30℃,pH为7.0。在此基础上构建了(S)-羰基还原酶Ⅱ和葡萄糖脱氢酶基因比为1∶1的共表达体系,共表达重组菌破碎上清液中(S)-羰基还原酶Ⅱ和葡萄糖脱氢酶酶活分别为0.76 U/mg和0.73 U/mg,两者的酶活比例为1∶1。在上述确定的最适催化条件下,其催化10 g/L 2-羟基苯乙酮,产物(S)-苯乙二醇的光学纯度和得率均高达99%以上。与仅含有(S)-羰基还原酶Ⅱ的重组大肠杆菌相比,共表达体系转化产物(S)-苯乙二醇的得率明显提高,且转化时间由原来的24 h缩短为13 h。【结论】通过确定(S)-羰基还原酶Ⅱ和葡萄糖脱氢酶最佳酶活配比,为构建手性催化的靶酶和辅酶再生酶共表达体系,为实现手性化合物的高效制备提供了研究基础。