Efficient Linear dsDNA Tagging Using Deoxyuridine Excision**

Site-specific strategies for exchanging segments of dsDNA are important for DNA library construction and molecular tagging. Deoxyuridine (dU) excision is an approach for generating 3’ ssDNA overhangs in gene assembly and molecular cloning procedures. Unlike approaches that use a multi-base pair motif to specify a DNA cut site, dU excision requires only a dT→dU substitution. Consequently, excision sites can be embedded in biologically active DNA sequences by placing dU substitutions at non-perturbative positions. In this work, I describe a molecular tagging method that uses dU excision to exchange a segment of a dsDNA strand with a long synthetic oligonucleotide. The core workflow of this method, called deoxyUridine eXcision-tagging (dUX-tagging), is an efficient one-pot reaction: strategically positioned dU nucleotides are excised from dsDNA to generate a 3’ overhang so that additional sequence can be appended by annealing and ligating a tagging oligonucleotide. The tagged DNA is then processed by one of two procedures to fill the 5’ overhang and remove excess tagging oligo. To facilitate its widespread use, all dUX-tagging procedures exclusively use commercially available reagents. As a result, dUX-tagging is a concise and easily implemented approach for high-efficiency linear dsDNA tagging.     

地下金属矿山斜坡道浆土路修筑工艺及应用研究

斜坡道是地下金属矿山的主要运输通道,其道路质量直接影响着矿山的安全运输和经济效益。针对斜坡道混凝土浇筑路面存在的筑路成本高、养护时间长、使用寿命短及维护困难等不足,引入浆土路修筑技术,开展了地下金属矿山斜坡道浆土路筑路材料配比试验、黏土搓条和崩解试验、水洗筛分试验,并在云南卡房分公司完成了浆土路试验路段的修筑。结果表明：浆土路筑路技术能够很好地应用于地下矿山道路修筑,具有施工工艺简单、筑路成本低、施工周期短、承载能力强、抑尘防滑和低碳环保等诸多优点,为地下金属矿山道路修筑提供了工程借鉴,对于矿山降本增效、节能减排及安全高效具有重要的意义。     

表面活性剂包衣脂肪酶在有机溶剂中催化酯合成

用合成的谷氨酸二烷基酯核糖醇对来源于Candidarugosa的脂肪酶进行了包衣 ,以月桂酸与月桂醇的酯化为模型反应 ,研究了各种操作条件对包衣酶活性的影响。结果表明 ,包衣酶制备过程中的缓冲溶液的最适 pH为 6.8,谷氨酸双十二烷基酯核糖醇的包衣效果最好 ,最适反应温度为 3 0℃ ,最佳溶剂为异辛烷。在 10h内底物转化率达 94%。     

新型功能高分子材料及其应用

功能高分子是一类具有特殊用途的高分子材料 ,印迹高分子、敏感性水凝胶和固定化酶是三种较有特色的功能高分子材料。该文将对上述三种功能高分子材料以及它们在生化分离、生物催化、物质分析与检测以及药物控制释放中的应用做一介绍 ,同时也对它们的不足和发展前景进行了评述     

非均相固定化酶促反应中“抗激发互交作用”的解析分析与定量计算

通过理论推导证明：在外扩散与化学抑制共存的非均相固定化酶促反应中，总有效系数η为外扩散有效系数ηｄｉｆ和化学抑制有效系数ηｃｈ的乘积，并且分别小于相应的ηｄｉｆ和ηｃｈ；从数学上解释了外扩散抑制和一般性化学抑制间所存在的“抗激发互交作用”。利用所得公式计算表明：底物浓度对外扩散与竞争性、非竞争性和反竞争性等不同化学抑制共存的非均相固定化酶促反应有明显不同的影响；扩散抑制均显著地影响非均相固定化酶促反应，使得反应体系的总有效系数随抑制作用的增加而明显减小，从而对这类反应有了更进一步的认识     

生物工程及其在化学工业中的应用

生物工程是近年来崛起的一门综合性的高新科学技术，也是当代新技术革命中最为活跃的领域。文章简要地介绍和评述了生物工程的主要内容及其在化学工业中应用的概况及发展趋势。     

Production of propylene glycol fatty acid monoesters by lipase-catalyzed reactions in organic solvents

Fatty acid monoesters of propylene glycol (1,2-propanediol) are good water-in-oil emulsifiers. These esters were synthesized enzymatically to overcome the problems associated with chemical processes. APseudomonas lipase was added to reaction mixtures containing propylene glycol and various acyl donors (fatty acids, fatty acid ethyl esters, fatty acid anhydrides and triglycerides) in organic solvents, and the mixtures were shaken at 30°C. The products were analyzed by gas chromatography. The yield of monoesters was affected by the acyl donors, organic solvents, temperature, water content, pH memory and reaction time. The anhydrous (lyophilized) enzyme and fatty acid anhydrides were best for monoester production. The optimum pH ranges were 4–5 and 8–10. The yields of propylene glycol monolaurate, monomyristate, monopalmitate, monostearate and monooleate with 50 mM fatty acid anhydrides as acyl donors were 97.2, 79.6, 83.7, 89.7 and 93.4 mM, respectively; those with 50 mM fatty acids as acyl donors were 37.3, 28.7, 28.7, 35.3 and 36.2 mM, respectively. The yields of propylene glycol monopalmitate, monostearate and monooleate with 50 mM triglycerides as acyl donors were 87.4, 65.1 and 83.2 mM, respectively.     

A lysine to arginine substitution at position 146 of rabbit aldolase A changes the rate-determining step to Schiff base formation

Lys146 of rabbit aldolase A [D-fructose-1,6-bis(phosphate):D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13 [EC] ] was changedto arginine by site-directed mutagenesis. The k_cat of the resultingmutant protein, K146R, was 500 times slower than wild-type insteady-state kinetic assays for both cleavage and condensationof fructose-1,6-bis(phosphate), while the Km for this substratewas unchanged. Analysis of the rate of formation of catalyticintermediates showed K146R was significantly different fromthe wild-type enzyme and other enzymes mutated at this site.Single-turnover experiments using acid precipitation to trapthe Schiff base intermediate on the wild-type enzyme failedto show a build-up of this intermediate on K146R. However, K146Rretained the ability to form the Schiff base intermediate asshown by the significant amounts of Schiff base intermediatetrapped with NaBH₄. In the single-turnover experiments it appearedthat the Schiff base intermediate was converted to productsmore rapidly than it was produced. This suggested a maximalrate of Schiff base formation of 0.022 s^–1, which wasclose to the value of k_cat for this enzyme. This observationis strikingly different from the wild-type enzyme in which Schiffbase formation is >100 times faster than k_cat. For K146Rit appears that steps up to and including Schiff base formationare rate limiting for the catalytic reaction. The carbanionintermediate derived from either substrate or product, and theequilibrium concentrations of covalent enzyme-substrate intermediates,were much lower on K146R than on the wild-type enzyme. The greaterbulk of the guanidino moiety may destabilize the covalent enzyme-substrateintermediates, thereby slowing the rate of Schiff base formationsuch that it becomes rate limiting. The K146R mutant enzymeis significantly more active than other enzymes mutated at thissite, perhaps because it maintains a positively charged groupat an essential position in the active site or perhaps the Argfunctionally substitutes as a general acid/base catalyst inboth Schiff base formation and in subsequent abstraction ofthe C4-hydroxyl proton.     

Theoretical examination of the mechanism of aldose-ketose isomerization

Two mechanisms for an aldose–ketose isomerization havebeen examined using high level ab initio and semiempirical molecularorbital methods. The proton transfer pathway via an enediolintermediate is shown to be favored in the absence of a metalion, while the hydride transfer pathway becomes favored in thepresence of a metal ion. Our calculations explain why the protontransfer pathway is operative in most aldose–ketose isomerizationreactions. These calculations also provide further support forthe previously proposed metal ion-mediated hydride transfermechanism for xylose isomerase.