全合成3α-羟类固醇脱氢酶基因密码子优化及酶性质研究

以NCBI报道的Comamonas testosteroni ATCC11996中3α-羟类固醇脱氢酶基因(3α-hydroxysteroid dehydrogenase gene,3α-hsd,AF092031.2)为模板,通过改变碱基序列但不影响酶的氨基酸序列,将该基因相对于大肠杆菌的密码子适用指数由0.78提高到0.87,GC含量由原来的63.17降低到56.46,大幅度减少了GC簇及寡聚A和T局域的存在,提高3α—hsd的可表达性。全合成基因定向克隆到pET28a载体中,并转化至大肠杆菌B121(DE3)中表达。采用乳糖诱导后,SDS—PAGE检测在约27 kD处有一高效表达的蛋白条带。采用Ni螯合柱分离纯化3α-HSD,获得了较高纯度及较高的产率。酶学性质初步分析表明,全基因合成表达的3α-HSD的性质与原始的3α-HSD基本相同。     

睾酮假单胞菌3α-羟类固醇脱氢酶基因的质粒载体构建及表达

从土壤中分离睾酮假单胞菌,提取其基因组DNA,PCR扩增3α-羟类固醇脱氢酶（3α-hsd）基因,将扩增产物用NdeⅠ/BamHⅠ消化,切下目的基因片段克隆到质粒pET-15b中构建重组pET-15b.将重组pET-15b转化入E.coli DH5α中,经酶谱分析和测序,鉴定出正确的重组质粒pET-15b.将重组pET-15b转化入宿主菌E.coli BL21(DE3) pLysS中,用硫代半乳糖苷(IPTG)进行诱导表达.提取细菌总蛋白质进行SDS-聚丙烯酰胺凝胶电泳(PAGE)分析并测定酶活性.粗提物中酶活性高达2.45×10⁵ U/L.利用重组蛋白中的6个组氨酸(His)组成的“标签”进行亲和层析,经一步金属螯合亲和层析纯化后,重组蛋白在SDS-PAGE上呈现出均一的单一条带,回收率达68%.活性和纯度均较高的目的蛋白3α-HSD的获得,为血清总胆汁酸酶循环法测定奠定了基础.     

大鼠20 α－羟类固醇脱氢酶在昆虫细胞中的表达

大鼠２０α羟类固醇脱氢酶（２０α－Ｈｙｄｒｏｘｙｓｔｅｒｏｉｄｄｅｈｙｄｒｏｇｅｎａｓｅ,２０αＨＳＤ）ｃＤＮＡ片段,被插入杆状病毒（ＢａｃｕＩｏｖｉｒｕｓ）的转移载体ｐＢｌｕｅＢａｃⅢ,经野生型病毒ＤＮＡ的共转染,从被转染的昆虫细胞中获得重组病毒。Ｎｏｒｔｈｅｒｎｂｌｏｔ分析,重组病毒感染细胞有２０αＨＳＤ基因表达。感染细胞裂解液的Ｗｅｓｔｅｒｎ印迹法分析,３７ｋＤ的蛋白带被２０αＨＳＤ抗体识别．体外酶活性测定发现,感染细胞裂解液中含有２０αＨＳＤ酶促活性以上结果提示,大鼠２０αＨＳＤ在杆状病毒昆虫表达系统成功地获得表达,为今后大量制备和纯化２０αＨＳＤ创造条件。     

高等植物葡萄糖-6- 磷酸脱氢酶与6- 磷酸葡萄糖酸脱氢酶基因的不同进化起源

葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶是植物戊糖磷酸途径中的两个关键酶。在克隆了水稻质体葡萄糖-6-磷酸脱氢酶基因OsG6PDH2和质体6-磷酸葡萄糖脱氢酶基因Os6PGDH2基础上,分析比较了水稻胞质和质体葡萄糖-6-磷酸脱氢酶基因和6-磷酸葡萄糖酸脱氢酶基因的基因结构、表达特性和进化地位。结合双子叶模式植物拟南芥两种酶基因的分析结果,认为高等植物葡萄糖-6-磷酸脱氢酶基因和6-磷酸葡萄糖酸脱氢酶基因在进化方式上截然不同,葡萄糖-6-磷酸脱氢酶的胞质基因与动物和真菌等真核生物具有共同的祖先;6-磷酸葡萄糖酸脱氢酶的胞质酶和质体酶基因都起源于原核生物的内共生。讨论了植物葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶基因可能的进化模式,为高等植物及质体的进化起源提供了新的资料。     

高等植物葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶基因的不同进化起源

葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶是植物戊糖磷酸途径中的两个酶.在克隆了水稻质体葡萄糖-6-磷酸脱氢酶基因OsG6PDH2和质体6-磷酸葡萄糖脱氢酶基因Os6PGDH2基础上,分析比较了水稻胞质和质体葡萄糖-6-磷酸脱氢酶基因和6-磷酸葡萄糖酸脱氢酶基因的基因结构、表达特性和进化地位.结合双子叶模式植物拟南芥两种酶基因的分析结果,认为高等植物葡萄糖-6-磷酸脱氢酶基因和6-磷酸葡萄糖酸脱氢酶基因在进化方式上截然不同,葡萄糖-6-磷酸脱氢酶的胞质基因与动物和真菌等真核生物具有共同的祖先;6-磷酸葡萄糖酸脱氢酶的胞质酶和质体酶基因都起源于原核生物的内共生.讨论了植物葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶基因可能的进化模式,为高等植物及质体的进化起源提供了新的资料.     

3β,20α-羟基甾体脱氢酶的动力学性质

3β,20α-羟基甾体脱氢酶(3β,20α-Hydroxysteroid dehydrogenase,3β,20α-HSD)是从胎羊血中分离得到的。分子量为35kD。该酶以NADPH为辅酶,有两种底物。以孕酮为底物时,Km=30.8μmol/L,Vmax=0.7nmol min~(-1)(nmol enzyme)~(-1);以5α-二氢睾酮(5α-Dihydrotestosterone,5α-DHT)为底物时,Km=74μmol/L,Vmax=1.3nmol min~(-1)(nmol enzyme)~(-1)。5α-DHT竞争性抑制20α-还原活性,Ki=102μmol/L。16α-溴代乙酰氧基(16α-Bromo acetoxyprogesterone,16α-BAP)是3β,20α-HSD不可逆竞争性抑制剂,t_(1/2)=75min。对3β和20α还原活性的抑制常数Ki分别为23μmol/L和58μmol/L。     

兔肾皮质-D氨基酸氧化酶和α-羟酸氧化酶的铈法定位及能谱分析

用铈作捕捉剂,在光镜和电镜下进行兔肾皮质的D-氨基酸氧化酶和α-羟酸氧化酶活性定位。光镜下用Ce-DAB法可显示这两种氧化酶的定位;电镜下,这两种酶主要定位在肾近端小管微绒毛和过氧物酶体上,用能谱仪对这些酶反应产物进行X射线微区元素分析显示铈峰,表明酶的铈法反应具特异性。     

Ru基催化剂选择性催化氧化5-羟甲基糠醛制备2,5-呋喃二甲酸研究进展

2,5-呋喃二甲酸(FDCA)是一种重要的生物质基单体,有望替代对苯二甲酸(PTA)生产可降解的生物质聚酯材料,缓解对化石资源的依赖以及环境的污染。如何经济、高效、绿色地合成FDCA是目前迫切需要解决的难题。5-羟甲基糠醛(HMF)作为典型的生物质平台化合物,来源广泛且绿色可持续,以其为原料催化氧化制备FDCA近年来备受关注。负载型Ru基催化剂由于其催化活性高、选择性好、成本相对合理,被认为是HMF催化氧化制备FDCA良好的催化材料。本文基于HMF不同氧化路线及反应机制,首先概述了不同活性组分Ru基催化剂的发展历程及其在HMF氧化反应中的应用,接着详细分析了碱添加剂、溶剂及载体对反应的影响,并阐释了相应的催化机制,最后对Ru基催化剂在HMF催化氧化制备FDCA中的工业化应用进行了总结和展望。     

S-扁桃酸脱氢酶基因的克隆及表达

S-扁桃酸脱氢酶能够选择性催化S-扁桃酸生成苯甲酰甲酸。通过PCR扩增获得Pseudomonas p utida NUST的S-扁桃酸脱氢酶全长基因(mdlA),并构建了表达载体pET30a(+)-mdlA,转化大肠杆菌E.coli BL21(DE3)后,经异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导获得表达,SDS-PAGE结果显示表达蛋白为43kDa。所以工程菌细胞具有转化S-扁桃酸生成苯甲酰甲酸能力。     

光滑球拟酵母中α-酮戊二酸脱氢酶系生理作用解析

[目的]研究α-酮戊二酸脱氢酶系在光滑球拟酵母碳代谢流、能量代谢和氨基酸代谢中的生理作用.[方法]通过敲除光滑球拟酵母中编码α-酮戊二酸脱氢酶系中E1酶的基因kgd1,构建α-酮戊二酸脱氢酶活性缺失菌株T.glabrata kgd1::kan,并考察KGDH缺失引起TCA循环关键酶活性,碳代谢流量以及胞内氨基酸和能荷水平等方面的变化.[结果]光滑球拟酵母中α-酮戊二酸脱氢酶活性的缺失导致:(1)细胞启动乙醛酸途径,通过形成TCA-乙醛酸循环实现TCA循环的正常代谢;(2)胞内NADH/NAD+水平下降33.7%,ATP/ADP水平下降31.8%,而与NADH代谢相关的丙酮酸脱氢酶、异柠檬酸脱氢酶和苹果酸脱氢酶的活性分别提高58.1%、33.3%和32.5%;(3)胞内丙酮酸含量下降50.1%,而胞内琥珀酸、苹果酸和α-酮戊二酸含量则分别增加了172.7%、66.1%和41.1%;(4)丙酮酸族氨基酸含量下降29.3%,而胞内谷氨酸族氨基酸和天冬氨酸族氨基酸含量则提高了34.7%和26.8%.[结论]上述研究结果表明,α-酮戊二酸脱氢酶系在微生物细胞中心碳代谢、能量代谢和氨基酸代谢中发挥着重要作用.     

Catalytic mechanism and application of formate dehydrogenase

NAD⁺-dependent formate dehydrogenase (FDH) is an abundant enzyme that plays an important role in energysupply of methylotrophic microorganisms and in response to stress in plants. FDH belongs to the superfamily of D-specific 2-hydroxy acid dehydrogenases. FDH is widely accepted as a model enzyme to study the mechanism of hydride ion transfer in the active center of dehydrogenases because the reaction catalyzed by the enzyme is devoid of proton transfer steps and implies a substrate with relatively simple structure. FDH is also widely used in enzymatic syntheses of optically active compounds as a versatile biocatalyst for NAD(P)H regeneration consumed in the main reaction. This review covers the late developments in cloning genes of FDH from various sources, studies of its catalytic mechanism and physiological role, and its application for new chiral syntheses.Translated from Biokhimiya, Vol. 69, No. 11, 2004, pp. 1537–1554.Original Russian Text Copyright © 2004 by Tishkov, Popov.     

Oxidation of 15-hydroxyeicosatetraenoic acid and other hydroxy fatty acids by lung prostaglandin dehydrogenase

The oxidation of the 15-hydroxy group of prostaglandins of the A, E, and F series by the NAD+-dependent prostaglandin dehydrogenase (PGDH) has been well documented. In addition to prostaglandins, we have observed that the purified lung PGDH also will oxidize 15-HETE to a novel metabolite that was isolated by reverse-phase HPLC and identified by gas chromatography-mass spectrometry as the 15-keto-5,8,11-cis-13-trans-eicosatetraenoic acid (15-KETE). The Km for 15-HETE was 16 microM, which was 2.5 times lower than the value obtained for PGE1. In addition to 15-HETE, 5,15-diHETE and 8,15-diHETE also were substrates for the lung PGDH with Km values of 138 and 178 microM, respectively. Other hydroxy derivatives of eicosatetraenoic acid that did not have a hydroxy group at carbon atom 15 did not support the PGDH-mediated reduction of NAD+. In addition to the 15-hydroxy derivatives of eicosatetraenoic acid, 12-HHT also was a substrate for the lung enzyme with a Km of 12 microM. These data indicate that omega 6-hydroxy fatty acids, in addition to prostaglandins, are also substrates of the lung NAD+-dependent PGDH and that the enzyme does not require the cyclopentane ring of prostaglandins.     

Transformation of some hydroxy amino acids to other amino acids

It has been observed that -hydroxy--amino acids are transformed into other amino acids, when heated in dilute solutions with phosphorous acid, phosphoric acid or their ammonium salts. It has been shown that as in the case of previously reported glycine-aldehyde reactions, glycine also reacts with acetone to give -hydroxyvaline under prebiologically feasible conditions. It is suggested, therefore, that the formation of -hydroxy--amino acids and their transformation to other amino acids may have been a pathway for the synthesis of amino acids under primitive earth conditions.     

Sugar-related hydroxy acids from Phaseolus and trifolium species

-Hydroxy acids isolated from leaves of French bean (Phaseolus vulgaris) and clover (Trifolium incarnatum) were analysed by GLC as trimethylsilyl derivatives and identified by MS. Large amounts of a 2-C-methyltetronic acid and appreciable amounts of gluconic acid and of a 2-C-(hydroxymethyl)pentonic acid were found from French bean. Glyceric acid was the predominant acid from clover but the presence of several other acids, e.g. threonic and malic acids, was also demonstrated.     

Transformation of some hydroxy amino acids to other amino acids.

It has been observed that beta-hydroxy-alpha-amino acids are transformed into other amino acids, when heated in dilute solutions with phosphorous acid, phosphoric acid or their ammonium salts. It has been shown that as in the case of previously reported glycine-aldehyde reactions, glycine also reacts with acetone to give beta-hydroxyvaline under prebiologically feasible conditions. It is suggested, therefore, that the formation of beta-hydroxy-alpha-amino acids and their transformation to other amino acids may have been a pathway for the synthesis of amino acids under primitive earth conditions.