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.     

燕麦-绿豆间作效应及氮素转移特性

为探究燕麦(Avena sativa)-绿豆(Phaseolus radiatus)间作效应及氮素转移特性, 在不施氮肥的大田试验条件下, 设置3种种植模式(燕麦单作、绿豆单作和燕麦-绿豆间作), 采用传统挖根法和¹⁵N同位素标记法进行研究。结果表明, 间作系统中燕麦侵袭力强于绿豆, 绿豆生长受到抑制。整个生育期, 间作燕麦地上部干物质积累量比单作增加14.9%-33.1%, 2年成熟期间作燕麦的氮素积累量比单作分别提高53.1%和44.8%; 间作减少了开花结荚期绿豆氮素积累量和根瘤重量, 降低了绿豆的固氮效率, 绿豆的固氮效率2年平均降低23.7%, 生物固氮量平均减少11.66%。间作绿豆向燕麦的氮素转移率2年平均值达31.7%, 氮素转移量为212.16 kg∙hm^-2。燕麦-绿豆间作降低了开花结荚期绿豆的根瘤固氮酶活性和固氮效率, 但绿豆体内氮素转移增加了燕麦对氮素的吸收利用, 实现了地上部与地下部生长的相互调节和促进, 优化了农田生态系统的氮素管理。     

一例智力低下患者7q~ 标记染色体的来源鉴定

以人类染色体显微切割、PCR技术构建的现有人类染色体特异性和染色体区带特异性探针池作为绘画探针,采用正向染色体绘画技术,结合染色体筛查方法,查明了一例7q~ 标记染色体患者的染色体附加片段来源于3q26→3qter。确定该患者的核型为46,XX,-7, der(7)t(7;3)(7pter→7q32::3q26→3qter)。应用这个策略,能够快速有效地鉴定标记染色体的来源。     

Mutational studies of conserved residues in the dimer interface of nerve growth factor.

An understanding of the structure-function relationship of nerve growth factor (NGF) requires precise knowledge of all the residues and regions that participate in NGF receptor binding, receptor activation, and biological activity. Seven recombinant human NGF mutants having alanine substituted for residues located either in the NGF dimer interface or beta-strand region were studied to determine the role of each amino acid residue in NGF biological activity. F86A, T91A, R100A, and R103A remained nearly full active with 61, 120, 91, and 73% of wild-type activity, respectively, in the PC12 cell bioassay. Hydrophobic core and dimer interface residues Y52, F53, and F54 were studied in more detail. Y52A and F54A were expressed in very low levels, suggesting that these two residues may be important for protein stability. Y52A retained full biological activity (91%). F53A had a 20- and 70-fold reduction in biological activity and TrkA phosphorylation, respectively, with only a 5- to 10-fold effect on TrkA binding and no effect on low-affinity receptor binding. F54A had significantly decreased TrkA phosphorylation and biological activity (40-fold). The results suggest that F53 and F54 may play a structural role in TrkA receptor activation subsequent to binding.     

白细胞介素2-绿脓杆菌外毒素融合基因的克隆及高效表达

利用聚合酶链式反应和寡核苷酸介导的定向诱变技术构建了白细胞介素2-绿脓杆菌外毒素IL2-PE40、IL2-PE40KDEL、IL2-PE66~(4Glu)和IL2-PE66~(4Glu)KDEL融合基因的原核表达重组质粒,并实现了高效表达,表达产物占菌体总可溶蛋白的20％～30％。此外,由于这一表达质粒在IL-2cDNA与PE基因连接处引入了唯一的SmaⅠ位点,其5＇、3＇端分别含有唯一的EcoRⅠ、PstⅠ位点,因此可方便地用其它基因替换IL-2或PE基因而获得相应融合蛋白的表达质粒。     

冬小麦根表面氧化还原活力的研究

证实了两个不同品种的冬小麦根系表面存在着氧化ＮＡＤＨ和还原Ｋ３Ｆｅ（ＣＮ）６的氧化的活力。还原铁氰化物活力在ＰＨ５．５到８．５范围内随着ＰＨ值升高而增大,温度在１５℃到４５℃范围内随温度升高还原活力增强,４５℃达最高值,５５℃时活力急剧下降。     

嗜热蓝藻层理鞭枝藻（Mastigocladus laminosus）藻胆体在解离过程中荧光发射光谱和光能传递的研究

研究了层理鞭枝藻藻胆体在不同浓度磷酸缓冲溶液中解离过程中荧光发射光谱的变化和光能传递。完整藻胆体的７７Ｋ荧光光谱中只有一个峰,位于６８５ｎｍ它是末端发射体（核心－膜连接多肽和别藻蓝蛋白－Ｂ）的荧光峰。部分解离藻胆体的荧光光谱的主峰位移至６５２ｎｍ：次峰位于６８５ｎｍ;６６０ｎｍ为一弱荧光发射肩。它们依次为Ｃ－藻蓝蛋白,末端发射体和别藻蓝蛋白的荧光。严重解离藻胆体的荧光主峰移６４４ｎｍ;次峰由６８５ｎｍ移至６８２ｎｍ;６６０ｎｍ荧光发射肩消失。这表明Ｃ－藻蓝蛋白所捕获的光能已不能传递给别藻蓝蛋白,但可传递给末端发射体洞时又表明Ｃ－藻蓝蛋白不仅与别藻蓝蛋白相连接而且还与末端发射体相连接。提出该藻胆体光能传递链如下：核心－膜连接多肽藻红蓝蛋白→Ｃ－藻蓝蛋白→别藻蓝蛋白别藻蓝蛋白－Ｂ