Characterization of a Tryptophan 6-Halogenase from Streptomyces albus and Its Regioselectivity Determinants

Tryptophan halogenases are found in diverse organisms and catalyze regiospecific halogenation. They play an important role in the biosynthesis of halogenated indole alkaloids, which are biologically active and of therapeutic importance. Here, a tryptophan 6-halogenase (SatH) from Streptomyces albus was characterized by using a whole-cell reaction system in Escherichia coli. SatH showed substrate specificity for chloride and bromide ions, leading to regiospecific halogenation at the C6-position of l -tryptophan. In addition, SatH exhibited higher performance in bromination than that of previously reported tryptophan halogenases in the whole-cell reaction system. Through structure-based protein mutagenesis, it has been revealed that two consecutive residues, A78/V79 in SatH and G77/I78 in PyrH, are key determinants in the regioselectivity difference between tryptophan 6- and 5-halogenases. Substituting the AV with GI residues switched the regioselectivity of SatH by moving the orientation of tryptophan. These data contribute to an understanding of the key residues that determine the regioselectivity of tryptophan halogenases.     

Crossing the Border: From Keto- to Imine Reduction in Short-Chain Dehydrogenases/Reductases

The family of NAD(P)H-dependent short-chain dehydrogenases/reductases (SDRs) comprises numerous biocatalysts capable of C=O or C=C reduction. The highly homologous noroxomaritidine reductase (NR) from Narcissus sp. aff. pseudonarcissus and Zt_SDR from Zephyranthes treatiae, however, are SDRs with an extended imine substrate scope. Comparison with a similar SDR from Asparagus officinalis (Ao_SDR) exhibiting keto-reducing activity, yet negligible imine-reducing capability, and mining the Short-Chain Dehydrogenase/Reductase Engineering Database indicated that NR and Zt_SDR possess a unique active-site composition among SDRs. Adapting the active site of Ao_SDR accordingly improved its imine-reducing capability. By applying the same strategy, an unrelated SDR from Methylobacterium sp. 77 (M77_SDR) with distinct keto-reducing activity was engineered into a promiscuous enzyme with imine-reducing activity, thereby confirming that the ability to reduce imines can be rationally introduced into members of the “classical” SDR enzyme family. Thus, members of the SDR family could be a promising starting point for protein approaches to generate new imine-reducing enzymes.     

抗真菌蛋白Rs-AFP2基因定点突变及其对大肠杆菌中表达的影响

为了研究遗传密码子对表达调控的影响,利用PCR重叠延伸法,对萝卜抗真菌蛋白Rs-AFP2基因编码序列区的部分核苷酸进行沉默突变,构建突变体Rs-AFPm.序列分析表明,PCR产物全长240bp,有一个阅读框,编码的蛋白由29个氨基酸的信号肽和51个氨基酸的抗真菌蛋白组成.突变体与突变前的Rs-AFP2基因相比,在编码区第3号氨基酸Lys相差一个碱基(TTG→TTA),第5号氨基酸Gln相差一个碱基(CAG→CAA),第6号稀有密码子Arg相差两个碱基(CAG→CGA).重新合成引物,将切除信号肽的Rs-AFP2基因和Rs-AFPm基因与原核表达载体pET-21b(+)分别重组到大肠杆菌BL21菌株.IPTG诱导后,二者均得到了表达.软件分析显示,突变前pETAFPo表达产物占全菌蛋白的3%,突变后pETAFPm的表达产物占全菌蛋白含量的8%;表达蛋白主要以包涵体的形式存在,包涵体经超声波破碎后,蛋白质复性,抑菌结果表明,pETAFPm表达产物的抑菌半径大于pETAFP2表达产物的抑菌半径.这些都说明改造后的Rs-AFPm基因与Rs-AFP2基因相比,已有效地提高表达量.     

弹性-粘弹性复合结构随机响应的各阶谱矩的计算方法

提出了一种新的计算弹性-粘弹性复合结构随机响应的各阶谱矩的计算方法，它是一种时域复模态分析方法。利用此方法获得了弹性-粘弹性复合结构在白噪声、滤过白噪声等典型平稳随机激励下随机响应的各阶谱矩的解析表达式，分析了粘弹性对各阶谱矩的影响。此计算方法简便、易用，无论单自由度或多自由度系统均适用，为进一步研究弹性-粘弹性复合结构在随机激励下的可靠性打下良好基础。     

组合结构体的控制爆破拆除

根据待拆构筑物的结构特点和周围环境，分析论述构件倒塌的可能性。因地制宜选择，制定出合理的爆破方案。并通过对爆破参数，起爆网络，安全防护等进行精心设计和施工，拆除爆破达到了预期的效果。     

80kt/a硫磺回收装置国产化设计

介绍了无在线炉硫回收工艺的基本原理、技术特点和催化剂使用情况。已投用的工业装置运行结果证明 ,用该工艺设计的硫磺回收装置 ,硫回收率和尾气净化度高 ,投资少 ,能耗低 ,流程简单 ,操作控制方便 ,社会效益和经济效益显著 ,适用于同类装置的新建和改扩建项目 ,市场前景广阔。     

挖金湾矿近距离煤层联合布置开采可行性研究

提出挖金湾矿近距离煤层联合布置开采方案，并对开采方案的技术难点进行了分析，找出了解决方法。     

多变量解耦极点配置组合自校正前馈控制器

本文提出了一种新的多变量解耦极点配置自校正前馈控制器,它具有使伺服跟踪性能和 随机调节性能两者均优的组合自校正特性.仿真例子说明了其有效性.     

18臂机械井径与磁法井径组合测井仪原理及应用

文章主要介绍了18臂机械井径与磁法井径组合测井仪的仪器结构、主要技术特性、测量原理及测井资料在油田中的应用。通过对测井资料的分析，阐述了该仪器在套损检测、补贴效果检查、射孔工程作业效果评价等方面的应用，为油田套管分析提供了可靠的保证。     

锚索锚杆联合支护在破碎顶板中的应用

结合工程工作面概况，就锚索和锚杆补强加固支护参数的选择进行了介绍，并指出锚索、锚杆的联合支护，保证了顶板的完整性，有效地控制了巷道的变形。