NAD(P)H依赖型氧化还原酶不对称还原胺化制备手性胺的研究进展

不对称还原胺化反应是制备医药中间体手性胺结构单元的重要反应。目前已有许多不同种类的酶被应用于合成手性胺,其中NAD(P)H依赖型氧化还原酶催化的还原胺化反应最为引人注目,因为其能够一步将潜手性酮化合物完全转化为光学纯的手性胺化合物。文中以亚胺还原酶、氨基酸脱氢酶、冠瘿碱脱氢酶和还原性酮胺化酶为例,从NAD(P)H依赖型氧化还原酶的结构特征、作用机理、分子改造及催化应用等方面,综述了其在不对称还原胺化合成手性胺领域的研究进展。     

一种来源于毕赤酵母的高对映选择性羰基还原酶的性质及底物谱分析

手性醇是一类非常重要的化合物,羰基还原酶催化酮的不对称还原生成对应的手性醇.从毕赤酵母Pichia pastoris GS115基因组数据中找到一个潜在的NADPH依赖的羰基还原酶,研究毕赤酵母 P.pastoris GS115中的羰基还原酶.根据其核酸序列设计引物,从P.pastoris GS115基因组中扩增到目的基因ppcr,大肠杆菌BL21 (DE3)中表达,Ni-NTA纯化,对酶的性质和底物谱进行了研究.PPCR的最适反应温度为35℃,最适反应pH为6.0,低于45℃时有很好的稳定性.对3-甲基-2-羰基丁酸乙酯的Km和kcat分别为9.48 mmol/L和0.12 s-1. PPCR表现出广泛的底物谱和很高的对映选择性,对醛、α-酮酯、芳香族β-酮酯及芳香族酮都表现出了很好的活性,在测定的底物中,除极少数底物外,ee值均达到97％以上.因此,PPCR具有较好的应用前景.     

NQO1酶及其被氧环境诱导表达的研究进展

NAD（P）H：醌氧化还原酶1（NQO1）是真核细胞内普遍存在的一类黄素蛋白酶,它专性催化胞内双电子还原反应,能够解除醌类物质对细胞的毒害,从而起到保护细胞的作用。同时,它又能活化一些醌类抗肿瘤药物。本文综述了NQO1的基因结构、多态性、功能和活性调节,以有它在包内氧化还原环境和肿瘤治疗中的地位等方面的研究进展。     

羰基还原酶及其催化不对称合成大基团手性羟基类化合物的研究进展

手性羟基化合物以其独特的光、热和化学性质广泛应用于医药、农药、精细化工、功能材料等行业.立体专一性羰基还原酶能够直接针对关键手性位点催化不对称还原潜手性底物获得目的手性产物.基于羰基还原酶的底物多样性,具有不同化学结构和功能的醇类、酯类、氨基酸、环氧化合物等重要手性中间体能够通过不对称还原途径实现单一光学活性对映体的高效制备.然而,针对具有应用价值的含有大基团、结构复杂的潜手性羰基化合物,已知的羰基还原酶通常催化活性较低.本文综述了生物催化不对称氧化还原反应的特点和规律及其关键立体选择性羰基还原酶的性质和结构特征,并在此基础上,重点针对大基团手性羟基化合物的不对称合成,总结了羰基还原酶及其催化系统开发和应用的研究进展,并进一步提出解决该关键问题的主要发展策略.     

还原酶催化羰基不对称还原的应用进展

羰基不对称还原作为合成手性醇的重要方法,已成为近年来有机合成的研究热点。与传统化学法相比,利用还原酶催化前手性羰基化合物的不对称还原具有显著优势。介绍了还原酶的来源与形式,对完整细胞还原酶与游离还原酶在手性药物不对称合成中的应用进行了简要综述。     

伴有辅酶原位再生的胞外酶耦合法制备（R）-苯基乙二醇

经5轮诱变筛选,从近平滑假丝酵母（Candida parapsilosis CICC1676）中分离得到产NADH依赖型羰基还原酶（Carbonyl reductase,CR）菌株CP-9。所产羰基还原酶（CRCp-9）经两步快速纯化获得纯化倍数为11．5倍,比活力为1．84 U/mg的酶液,其还原反应的最适pH值为6．5,最适温度为40℃。该酶转化β-羟基苯乙酮制备手性化合物（R）-苯基乙二醇,因此是（R）-专一性羰基还原酶。该酶与NADH普适性再生酶-甲酸脱氢酶（For-mate dehydrogenase,FDH）在胞外相耦联,构建伴有辅酶再生与反复利用的CR/FDH双酶催化制备立体醇体系,底物β-羟基苯乙酮转化率达95．4％,产物（R）-苯基乙二醇得率为93％,辅酶的总转化数（Total turn number, TTN）达267,产物e．e．值为98．6％,批次耦合反应生产能力达0．8 g/L/h,较单酶催化有较大提高,与细胞转化法相比也具有较好的生产能力。因此,伴有辅酶再生的胞外酶耦合催化具有潜在的制备手性醇化合物的工业应用价值。     

羟胺氧化还原酶在生物脱氮中的研究进展

羟胺氧化还原酶(hydroxylamine oxidoreductase,HAO)属于多血红素蛋白酶家族,每个单体由7个电子转移血红素和1个催化血红素组成.HAO既可分别催化羟胺和肼的氧化反应,也可催化羟胺、一氧化氮及亚硝酸盐的还原反应.不同硝化细菌中,HAO的最适温度、pH、底物、产物特异性及酶抑制剂等存在差异.作为...     

生物催化立体选择性氧化还原中存在问题及其发展策略

以立体选择性氧化还原酶或其全细胞催化的不对称氧化还原反应已经成为转化光学活性手性醇及其他手性化合物的有效手段。然而,生物催化氧化还原反应体系存在着催化活性与专一性、反应体系与催化稳定性等生物催化剂所固有的局限性问题,而且,生物氧化还原反应必需辅酶及其再生问题也是限制该转化途径产业化应用的一个重要因素。围绕上述生物催化立体选择性氧化还原中存在的关键问题,现代分子生物技术及反应工程的不断突破和发展为改善生物催化立体选择性氧化还原在催化剂本身和反应工程方面的局限性提供了有效的发展策略,为其进一步大规模产业应用提供了发展基础。     

假单胞菌S-42对偶氮染料的脱色和降解代谢

假单胞菌S-42(Pseudomonas S-42)对活性艳橙KN-4R、直接深棕M、酸性媒介棕RH等偶氮染料具有良好的脱色作用,该菌株的细胞悬液、无细胞提取液和纯酶的脱色条件较一致,最适pH为7.0,最适温度为37℃。细胞悬液对上述三种染料的脱色率都可达90％。当细胞浓度为15mg／ml,反应5h,其脱色话力分别为1．75,2．4、0.95μg染料／mg细胞;无细胞提取液和纯酶要求厌氧和加有NADH时才具有话性;纯酶属于偶氮还原酶,分子量为34000士2000,对活性艳橙KN-4R的最大反应速度Vmax为13μmol.mg蛋白-1 min-1,米氏常数Km=54μmol。S-42菌株降解话性艳橙KN-4R的反应产物的吸收光谱和亚硝酸钢反应等方法检测结果表明,偶氮染料的生物降解开始于偶氮双键的还原裂解。本文还提出了S-42菌株降解活性艳橙KN-4R的假设反应式。     

高选择性不对称还原N,N-二甲基-3-酮-3-(2-噻吩)-1-丙胺的重组氧化还原酶催化性质及其酶促转化

【目的】通过表达多种重组立体选择性氧化还原酶,分析其催化不对称还原N,N-二甲基-3-酮-3-(2-噻吩)-1-丙胺(DKTP)的性质,从而构建酶促合成(S)-N,N-二甲基-3-羟基-3-(2-噻吩)-1-丙胺(DHTP)的反应体系。【方法】基于已有立体选择性氧化还原酶重组大肠杆菌,通过Ni离子亲和层析法纯化得到重组氧化还原酶,以DKTP为底物,考察不同重组氧化还原酶对DKTP的催化活性和选择性,进一步对高选择性酶促合成(S)-DHTP的重组酶CR2进行性质分析,并考察其在最适条件下不对称还原DKTP的过程。【结果】筛选获得产物构型为(S)-型的催化活性最高的酶为CR2,该酶米氏常数Km为0.135 mmol/L,kcat/Km为3.689 L/(mmol·s),最适p H 8.4(0.1 mol/L三乙醇胺缓冲液),最适反应温度为35°C,在10-45°C条件下和p H 7.5-8.5较为稳定,Zn2+离子对酶活有促进作用。CR2催化DKTP不对称还原反应6 h后,DHTP的产率达92.1%、光学纯度达99.9%。【结论】基于活性和选择性分析,获得不对称还原DKTP的目标酶CR2,其催化特性有利于高立体选择性还原DKTP生成度洛西汀中间体(S)-DHTP,从而为进一步提高酶促不对称还原DKTP的转化效率提供研究基础。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.