新型羟基化酶基因的克隆及其洛伐他汀转化的应用

无锡他汀由洛伐他汀经拟无枝酸菌Amycolatopsis sp. CGMCC1149羟基化而来。为获得该转化过程中的关键酶——羟基化酶,运用简并PCR和SEFA PCR技术,从Amycolatopsis sp. CGMCC1149中克隆获得长度为1 212 bp的新型羟基化酶基因,并实现其在大肠杆菌中表达。BLAST序列分析表明该基因属于细胞色素P450基因超家族,并可编码一个含403氨基酸的蛋白,其分子量为44.8 kDa。二级结构预测结果表明：该蛋白包含有氧结合区、离子对结合区和血红素结合区等P450典型功能区。同时文中利用NADH、铁氧还蛋白和铁氧还蛋白还原酶建立了体外酶催化功能验证系统,首次实现无锡他汀转化底物洛伐他汀的体外羟基化。该结果为具有我国独立知识产权的无锡他汀大规模制备奠定了基础。     

一株对Monacolin K有转化作用菌株的鉴定

从1000多株放线菌中筛选出对Monacolin K有转化作用的菌株并对其进行初步鉴定。结果发现菌株ST2710的气丝交替或不规则分枝,不形成螺旋;可利用多种碳源;牛奶不凝固,不胨化;不利用纤维素,水解淀粉;通过对其细胞化学组分的分析,结果表明菌株ST2710细胞壁为Ⅳ型,糖型为A型,醌组分为MK-9(H4,H6),不含枝菌酸,G C含量为67.3%(摩尔比);将该菌株的16S rDNA序列与GenBank数据中已有序列进行比对,发现其序列与Amycolatopsis属菌株的16SrDNA序列相似性最高,达到99%以上。初步将菌株ST2710归类为Amycolatopsis sp.     

有机溶剂对黑根霉甾体11α-羟基化的影响

考察了有机溶剂对黑根霉甾体11α-羟基化反应中转化底物16α,17α-环氧孕甾酮生成11 α, 16α,17α-环氧孕甾酮的转化率和细胞色素P450酶浓度的影响.向培养28 h的培养液中添加终浓度0.035 mol/L的丙酮和1.75 g/L的底物,继续转化48 h.与未添加丙酮相比,添加丙酮后的底物转化率和细胞色素P450酶表达量分别提高了6.8%和30%.说明丙酮添加可明显提高黑根霉甾体11α-羟基化能力和细胞色素P450酶的表达.     

产绿原酸内生菌细胞色素P450基因的克隆和功能研究

以一株产绿原酸的内生枯草芽孢杆菌为基础,对其绿原酸途径的关键酶基因进行克隆和功能研究。克隆该内生菌的细胞色素P450基因并进行原核表达,对重组蛋白进行肉桂酸羟基化酶(C4H)酶活测定。结果显示,从内生菌中克隆了4个细胞色素P450酶系基因并进行了原核表达,其中P-4重组蛋白具有C4H活性,产生的香豆酸与LC-MS检测的结果一致,该酶的最适温度范围较宽,产物香豆酸对该酶有抑制作用。该内生菌可能利用其细胞色素P450酶系作为C4H的同工酶从而将产物导入绿原酸合成途径。     

微生物转化洛伐他汀条件的初步研究

以本实验室筛出的一株菌ST2710为出发菌,研究不同培养条件对转化率的影响。结果发现,菌株ST2710在种子培养基进行预培养,再转入转化培养基中培养2d后,加入洛伐他汀溶液1.0mL(10mg／mL)后再培养5d,产物的产率可达43．41％;就四种溶剂溶解底物条件进行了研究,结果发现用二甲基甲酰胺溶解洛伐他汀,产物产率为最高,并且转化作用不需要底物的诱导;以单独的菌丝体和上清液转化洛伐他汀时均低于培养液对洛伐他汀的转化作用。     

P450酶在植物三萜化合物生物合成中的催化与调控

植物三萜化合物是一类具有6个C₅异戊二烯单元的高附加值天然化合物,具有抗炎、护肝、抗肿瘤、抗氧化和降血压等重要药理活性。在三萜化合物生物合成过程中,细胞色素P450酶通过引入羟基、羧基、羰基以及环氧基等官能团,为丰富三萜结构的多样性起到了重要的作用。然而,目前P450酶底物催化特异性机制仍不清晰,异源底盘细胞中表达率低、与细胞色素氧化还原酶(CPR)的适配性差限制了其在植物三萜化合物微生物异源合成中的应用。本文系统地介绍了植物三萜化合物的合成途径、P450酶的催化系统组成和催化机制。通过P450酶的理性与非理性的分子改造,P450酶及其CPR的适应性匹配以及关键代谢途径的区室化研究,以期为P450酶在高效合成三萜化合物的应用提供研究思路。     

细胞色素P450酶系与除草剂代谢

细胞色素P450是广泛存在于动物、植物和微生物体内的一类具有混合功能的血红素氧化酶系。它不但能够催化苯丙烷类、萜类化合物和脂肪酸等内源性物质的生物合成 ,而且参与许多外源性物质包括除草剂等的生物氧化。综述了代谢除草剂的细菌、哺乳动物和植物细胞色素P450酶系 ,概述了细胞色素P450酶系参与除草剂代谢的作用方式 :脱烷基化作用、环甲基化羟基化作用和芳环的羟基化作用等。这些细胞色素P450酶系在培育除草剂抗性作物、生物安全和生物修复方面表现出了巨大的潜能     

VD_3羟基化酶的定向研究进展

维生素D3羟基化酶(Vdh)作为细胞色素酶P450s(CYP)蛋白家族成员,催化VD3形成有生物活性的1α,25(OH)2VD3。但是,由于VD3并不是Vdh的天然底物,Vdh羟基化活性较低。采用定向构建Vdh重组质粒的方法对Vdh催化活性位点给予优化,从而提高其羟基化能力。就目前对Vdh的结构特性和定向研究进行综述。     

利用正交法确定测定意大利蜜蜂幼虫细胞色素P4500-脱乙基活性最佳反应条件

荧光分光光度法检测意大利蜜蜂Apis mellifera ligustica Spinal细胞色素P450 O-脱乙基活性方便快捷、较灵敏,以7-乙氧基香豆素为底物.通过测定其产物7-羟基香豆素的荧光变化来确定细胞色素P450 O-脱乙基的活性.在利用荧光分光光度法检测意大利蜜蜂幼虫细胞色素P450 O-脱乙基活性过程...     

细胞色素P450 2B4的结构及其催化反应

细胞色素P450是广泛存在于动物、植物和微生物中的含亚铁血红素单加氧酶,参与致癌作用和药物代谢、类固醇激素合成、脂溶性维生素代谢、多不饱和脂肪酸转换为生物活性分子等生理过程。P450能够催化完成伯、仲碳氢键羟基化、烯烃和芳烃环氧化、碳碳键耦合和断裂、α羟基化(去烷基化和杂原子氧化)、还原、1,2-迁移(卤素、氢和苯)等有机反应。本文综述了P450 2B4的结构与功能,讨论了细胞色素P450 2B4的活性中心和底物识别位点、与底物反应和产物释放的机理,以及P450在有机合成中的应用。     

Advances in the bioconversion mechanism of lovastatin to wuxistatin by Amycolatopsis sp. CGMCC 1149

Wuxistatin, a novel statin and more potent than lovastatin, was converted from lovastatin by Amycolatopsis sp. (CGMCC 1149). Product I, an intermediate product, was found in the fermentation broth, and the structure analysis showed that product I had an additional hydroxyl group at the methyl group attached to C3 compared to lovastatin, which indicates that product I is one isomer of wuxistatin. Isotope tracing experiment proved that hydroxyl group of wuxistatin was provided by product I and the reaction from product I to wuxistatin was an intramolecular transfer. Hydroxylation reaction established in a cell-free system could be inhibited by CO and enhanced by ATP, Fe²⁺, and ascorbic acid, which were consistent with the presumption that the hydroxylase was an induced cytochrome P450. Study on proteomics of Amycolatopsis sp. CGMCC 1149 suggested that three identified proteins, including integral membrane protein, Fe-S oxidoreductase, and GTP-binding protein YchF, were induced by lovastatin and required during hydroxylation reaction. In conclusion, bioconversion mechanism of wuxistatin by Amycolatopsis sp. CGMCC 1149 was proposed: lovastatin is firstly hydroxylated to product I by a hydroxylase, namely cytochrome P450, and then product I is rearranged to wuxistatin by isomerases.     

Bioconversion of lovastatin to a novel statin by Amycolatopsis sp.

3-Hydroxy-3-methylglutaryl–coenzyme A (HMG–CoA) reductase catalyzes the conversion of HMG–CoA to mevalonic acid, which plays a significant role in cholesterol synthesis. Several statins, inhibitors of HMG–CoA reductase, can be synthesized and converted by microorganisms. Among 700 strains obtained from culture collections, one strain could convert lovastatin to a novel statin, wuxistatin. The strain was identified as a member of the genus Amycolatopsis based on 16S rRNA gene sequence, morphology analysis, and chemotaxonomic properties. Wuxistatin, a novel HMG–CoA reductase inhibitor, was purified by chromatography, and the structure was determined by electrospray ionization mass and nuclear magnetic resonance spectroscopy. The results show that wuxistatin was butanoic acid, 2-methyl-,1,2,3,5,8,8a-hexahydro-5-hydroxy-7-methyl-8-[2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl) ethy]-1-naphthalenyl ester. An additional hydroxyl group was added to lovastatin at the 5-position to yield wuxistatin. This modification enhanced the intrinsic inhibitory activity (IC50) of wuxistatin (41 ± 5 nM) for fourfold compared with lovastatin (160 ± 10 nM). A stoichiometric conversion of lovastatin to wuxistatin occurred.     

木质素合成酶C3H基因的生物信息学分析

应用生物信息学的方法对GenBank中拟南芥、火炬松、胡麻、紫花罗兰等植物香豆酸-3-羟基化酶（c3h）基因以及本实验室克隆的欧美杨107的c3h基因（AM920690）核苷酸序列以及翻译的氨基酸序列的组成成分、导肽、跨膜结构域、疏水性／亲水性、蛋白质二级结构及功能域等进行了初步分析预测。分析表明欧美杨107的C3H氨基酸序列不存在导肽,无跨膜结构域,肽链表现为亲水性;二级结构域与P450高度匹配,其高级结构中含有由含铁血红素和半胱氨酸组成的活性中心,并构建了c3h进化树,对其分子进化进行了探讨。     

Biotransformation of flavone by CYP105P2 from Streptomyces peucetius

Biocatalytic transfer of oxygen in isolated cytochrome P450 or whole microbial cells is an elegant and efficient way to achieve selective hydroxylation. Cytochrome P450 CYP105P2 was isolated from Streptomyces peucetius that showed a high degree of amino acid identity with hydroxylases. Previously performed homology modeling, and subsequent docking of the model with flavone, displayed a reasonable docked structure. Therefore, in this study, in a pursuit to hydroxylate the flavone ring, CYP105P2 was co-expressed in a two-vector system with putidaredoxin reductase (camA) and putidaredoxin (camB) from Pseudomonas putida for efficient electron transport. HPLC analysis of the isolated product, together with LCMS analysis, showed a monohydroxylated flavone, which was further established by subsequent ESI/MS-MS. A successful 10.35% yield was achieved with the whole-cell bioconversion reaction in Escherichia coli. We verified that CYP105P2 is a potential bacterial hydroxylase.     

Effect of purified cytochrome P450 incorporation into liposomal membranes on its properties]

The physico-chemical properties and hydroxylase activity of three forms of cytochrome P450, i. e. purified soluble hemoprotein, purified hemoprotein incorporated into the liposomal membrane and microsomal cytochrome P450, were studied. Soluble cytochrome P450 binds type I substrates in a lesser degree than does its microsomal form. The incorporation of hemoprotein into phosphatidyl choline liposomes restores the ability of purified cytochrome P450 to interact with these substrates. The soluble and lipid-bound forms of cytochrome P450 do not differ in their thermal stabilities and protease digestion. The liposome-bound cytochrome P450 has higher dimethylaniline, aniline and p-nitroanisol hydroxylase activities as compared to its soluble form. The aniline hydroxylase activity of microsomal, proteoliposomal and soluble forms of cytochrome P450 is inhibited by the tyrosinecopper complex with NADPH or cumole hydroperoxide as cosubstrates. The inhibiting effect of the complex on other hydroxylase activities depends on the type of cytochrome P450 and the cosubstrates and substrates used.     

Anti-P450lpr antiserum inhibits specific monooxygenase activities in LPR house fly microsomes.

Monooxygenase activity in microsomes from the LPR strain of house fly (Musca domestica L.) was inhibited by anti-P450lpr, and antiserum specific for house fly cytochrome P450lpr. Anti-P450lpr did not inhibit house fly cytochrome P450 reductase or rat cytochrome P450 monooxygenase assays, consistent with specific inhibition of P450lpr. Anti-P450lpr inhibited the ability of cytochrome P450 reductase to reduce carbon monoxide treated LPR microsomal cytochrome P450, up to 49% of the total, showing that inhibition of cytochrome P450 reduction is the major mechanism of inhibition. Anti-P450lpr inhibited 98% of methoxyresorufin-O-demethylase activity and all the benzo(a)pyrene hydroxylase activity in LPR microsomes, but none of the pentoxyresorufin-O-dealkylase activity. The antiserum partially inhibited ethoxyresorufin-O-dealkylase and ethoxycoumarin-O-dealkylase activity. These results demonstrate that methoxyresourfin-O-demethylase activity and benzo(a)pyrene hydroxylase activity are characteristic substrates for P450lpr activity in the LPR strain of house fly.