氨甲酰磷酸不同合成途径在大肠杆菌中的比较

【背景】氨甲酰磷酸是生物合成代谢中精氨酸与嘧啶的重要前体物质,在工业微生物生产精氨酸与嘧啶及其衍生物中发挥关键作用。【目的】在大肠杆菌Escherichia coli BW25113中比较氨甲酰磷酸不同合成途径的催化效率。【方法】在大肠杆菌Escherichia coli BW25113中过表达鸟氨酸氨甲酰基转移酶(OTC)的基础上,分别过表达大肠杆菌自身的氨基甲酸激酶(CK)和氨甲酰磷酸合酶(CPSⅡ)并表征其反应效果。通过优化底物供应(调整底物浓度与引入L-谷氨酰胺合成酶)对CK与CPSⅡ的催化反应进行优化。【结果】在大肠杆菌中过表达OTC,建立细胞水平氨甲酰磷酸检测体系。在此基础上比较不同来源的CK,发现大肠杆菌来源的CK效果最好,50mmol/LNH4HCO3条件下全细胞催化9h得到2.95±0.15mmol/LL-瓜氨酸;过表达CPSⅡ时,50mmol/LL-谷氨酰胺催化9h得到3.16±0.29 mmol/L L-瓜氨酸。通过改变底物NH4HCO3浓度和引入外源L-谷氨酰胺合成酶(GS)等方式对CK与CPSⅡ的催化反应分别进行优化后,100 mmol/L NH4HCO3条件下,L-瓜氨酸浓度分别提高至4.67±0.55mmol/L和6.12±0.38mmol/L,且过表达GS后CPSⅡ途径可以利用NH3,不需要额外添加L-谷氨酰胺。【结论】引入L-谷氨酰胺合成酶后的CPSⅡ途径合成氨甲酰磷酸的能力优于CK途径,为精氨酸、嘧啶及其衍生物的合成提供了一种更加高效的策略。     

游动放线菌Actinoplanes sp.SE50/110中阿卡波糖脱氧氨基糖单元的生物合成

【目的】解析Actinoplanes sp.SE50/110(简称SE50/110)中阿卡波糖脱氧氨基糖单元的生物合成机制。【方法】经过BLASTp分析,推测了Acb A、Acb B和Acb V负责阿卡波糖脱氧氨基糖单元的生物合成。首先,本研究在SE50/110中分别构建了acb A、acb B和acb V的同框缺失和回补突变株。然后,利用大肠杆菌BL21(DE3)/p Gro7分别对Acb A、Acb B和Acb V成功实现了可溶性表达。最后,以D-葡萄糖-1-磷酸为起始底物,通过体外催化反应,研究脱氧氨基糖单元的生物合成过程和相关蛋白的酶学性质。【结果】在SE50/110中分别缺失acb A、acb B和acb V基因后,相应突变株均丧失了阿卡波糖的合成能力,将acb A、acb B和acb V基因分别回补后,各菌株又恢复了阿卡波糖的合成能力,证明了它们均为阿卡波糖生物合成的必需基因。在体外酶促反应中,D-葡萄糖-1-磷酸-胸腺嘧啶转移酶Acb A催化D-葡萄糖-1-磷酸和d TTP合成d TDP-D-葡萄糖,对D-葡萄糖-1-磷酸的Km值为(0.185±0.053)mmol/L,Vmax为(2.366±0.217)μmol/(min·mg);对d TTP的Km值为(4.964±1.089)mmol/L,Vmax为(60.310±5.419)μmol/(min·mg)。d TDP-D-葡萄糖-4,6-脱水酶Acb B催化d TDP-D-葡萄糖转化为d TDP-4-酮基-6-脱氧-D-葡萄糖,Km值和Vmax分别为(0.353±0.089)mmol/L和(306.401±28.740)μmol/(min·mg)。氨基转移酶Acb V催化d TDP-4-酮基-6-脱氧-D-葡萄糖生成d TDP-4-氨基-4,6-双脱氧-D-葡萄糖,Km值和Vmax分别为(1.411±0.293)mmol/L和(3.447±0.279)μmol/(min·mg)。【结论】本研究阐明了阿卡波糖脱氧氨基糖单元的生物合成过程,为全面解析阿卡波糖生物合成途径奠定了基础。同时,测定了相关酶的动力学参数,为代谢工程改造SE50/110,提高阿卡波糖产量提供了重要的理论依据。     

基于己糖激酶与葡萄糖-6-磷酸脱氢酶共表达的辅酶NADPH高效再生

【目的】构建己糖激酶与葡萄糖-6-磷酸脱氢酶的大肠杆菌共表达体系,以葡萄糖为底物实现辅酶NADPH的高效再生。【方法】通过分子生物学方法,克隆己糖激酶HKgs、HKpp基因,并于Escherichia coli BL21(DE3)中表达,再将己糖激酶HKgs、HKpp分别与葡萄糖-6-磷酸脱氢酶Gpd PP共表达,实现NADPH的原位再生。比较两个共表达工程菌的辅酶再生效果,并针对催化活力较高的工程菌BL21(HKgs+Gpd PP)进行表达条件优化。【结果】NADPH再生活力达到856 U/L。该辅酶再生体系与醇脱氢酶Adh R联合催化,使不对称还原4-氯乙酰乙酸乙酯的催化活力提高至原始值的2.5倍。【结论】通过己糖激酶与葡萄糖-6-磷酸脱氢酶在大肠杆菌中的共表达,构建了一个新的NADPH高效再生体系,并用于醇脱氢酶催化的不对称还原反应。     

非核糖体肽Skyllamycin B生物合成中Ⅰ型硫酯酶底物杂泛性的初步研究

【背景】Skyllamycins是一类从链霉菌中发现的具有血小板生长因子抑制和生物膜抑制作用的非核糖体肽类,其环肽环合反应是由非核糖体肽合成酶中的硫酯酶功能域催化完成。【目的】克隆和表达Skyllamycin非核糖体肽合成酶最后一个模块中的硫酯酶(Skyxy-TE)基因,合成Skyxy-TE底物类似物,通过体外催化实验表征Skyxy-TE的底物杂泛性。【方法】采用Ligation Independent Cloning(LIC)方法,从一株含有Skyllamycin B生物合成基因簇的链霉菌Streptomycessp.PKU-MA01239中克隆和表达skyxy-TE,通过镍离子柱亲和层析纯化Skyxy-TE。运用固相多肽合成法合成2个底物类似物1和2,进行Skyxy-TE的体外催化实验。【结果】通过对Skyxy-TE的表达纯化,获得了纯度较好的可溶性蛋白;通过固相多肽合成,得到了能够模拟Skyllamycin B底物类似物的化合物1和2,硫酯酶蛋白体外催化化合物1和2得到了化合物3和4,化合物3和4通过核磁共振和高分辨质谱确认为环肽。【结论】Skyllamycin B生物合成中Skyxy-TE表现出一定的底物杂泛性,可以识别底物类似物催化环化反应,该研究为将来利用化学-酶联法制备更多环肽类似物提供了依据。     

南海中华小尖柳珊瑚中嘌呤和嘧啶类化合物的研究

对采自海南三亚的中华小尖柳珊瑚Muricella flexuosa的化学成分进行研究,采用反复硅胶柱色谱法、Sephadex LH-20柱色谱法及重结晶等手段对化合物进行分离和纯化,通过理化性质及光谱分析并结合文献对照,鉴定得到11个嘌呤、嘧啶类化合物:咖啡碱(1),1,7-二甲基次黄嘌呤(2),1-甲基次黄嘌呤(3),7,9-二甲基-6-氮甲基嘌呤-8-酮(4),7-甲基腺嘌呤(5),1,7-二甲基嘌呤-6,8-二酮(6),尿嘧啶(7),胸腺嘧啶(8)2,’-脱氧尿嘧啶核苷(9)2,’-脱氧胸腺嘧啶核苷(10),3-乙基-2’-脱氧尿嘧啶核苷(11)。其中化合物2～61,1为首次从该属中分离得到,化合物4和11为新的天然产物。     

鸭疫里默氏杆菌Mtan对底物SAH的催化活性

【目的】分析鸭疫里默氏杆菌(Riemerella anatipestifer,RA)不同血清型pfs基因的序列差异,并开展其编码蛋白S-腺苷高半胱氨酸核苷酶(Mtan,又称Pfs)的催化活性研究。【方法】PCR扩增9株不同血清型RA的pfs基因,分析其核苷酸序列的同源性;构建该基因的重组表达载体p Cold-RA-pfs,表达、纯化RA的重组蛋白Mtan(RA-Mtan);测定RA-Mtan对底物S-腺苷同型半胱氨酸(S-adenosylhomocysteine,SAH)的催化活性,运用哈维弧菌报告菌株BB170检测催化底物的自诱导物2(Autoinducer-2,AI-2)活性。【结果】对RA的pfs序列分析结果表明,不同血清型RA的核苷酸一致性在93.9%-100%之间;SDS-PAGE检测结果表明,RA-Mtan呈可溶性表达;酶活测定表明RA-Mtan和禽致病性大肠杆菌(Avain pathogenic Escherichia coli,APEC)的Lux S蛋白共同作用于底物时,可产生浓度为176.7μmol/L的同型半胱氨酸(Homocysteine,HCY);AI-2活性检测结果表明,产生的AI-2具有生物学活性。【结论】RA不同血清型的pfs高度保守,RA pfs基因的编码产物RA-Mtan在体外具有催化SAH的活性,RA-Mtan和禽致病性大肠杆菌的Lux S蛋白共同作用于底物SAH时,能产生有活性的AI-2,为进一步研究pfs对RA的调控作用提供参考。     

大肠杆菌来源的喹啉酸磷酸核糖转移酶和烟酸磷酸核糖转移酶的表达纯化及酶活性的初步检测

【目的】在原核表达体系中实现大肠杆菌来源的喹啉酸磷酸核糖转移酶(Quinolinic acid phosphoribosyl transferase,QPRT)和烟酸磷酸核糖转移酶(Nicotinic acidphosphoribosyl transferase,NaPPT)的表达与纯化,并利用酶的生物催化作用实现2,3-二羧酸喹啉的2位选择性脱羧得到烟酸【。方法】通过PCR扩增分别得到编码QPRT和NaPPT的基因片段,构建成原核表达质粒pET28a-NadC和pRSETB-PncB,在Escherichia coli(E.coli)中对其进行表达,在体外对目标蛋白进行纯化并利用高效液相色谱法(HPLC)检测酶催化反应的发生。【结果】成功表达纯化得到QPRT和NaPPT,检测结果表明在这两个酶的生物催化作用下可实现喹啉酸的2位选择性脱羧。     

基于酵母表面展示技术的胸苷磷酸化酶全细胞催化剂的构建

胸苷磷酸化酶(TP)在核苷类代谢通路中发挥重要作用,可催化生成多种核苷类似物。构建了TP的酵母表面展示系统作为全细胞催化剂。从大肠杆菌K12菌株中克隆编码TP的deo A基因,利用酵母表达质粒p KFS构建重组质粒,电击转化毕赤酵母GS115菌株。高拷贝阳性转化子经甲醇诱导96 h后,免疫荧光结果显示TP在酵母细胞表面成功展示。利用β-胸苷为底物,重组酵母细胞作为全细胞催化剂,经HPLC检测,结果表明展示在酵母表面的TP有催化活性,可以催化β-胸苷生成产物胸腺嘧啶。     

氧化还原对NDPK-A及突变体磷酸转移酶活性的影响

对核苷二磷酸激酶A(NDPK-A)及其4种半胱氨酸突变体进行诱导表达及纯化,测定它们在氧化还原条件及正常条件下的磷酸转移酶活性,研究氧化还原及二硫键异构对NDPK-A及突变体活性的影响。将实验室之前构建成功的野生型NDPK-A(PBV-NDPK-A)及4种突变型NDPK-A基因(PBV-NDPK-A C4S,PBV-NDPK-A C109S,PBV-NDPK-A C145S,PBV-NDPK-A C4/109/145S)在大肠杆菌中高效表达;以DEAE-sepharose Fast Flow离子交换层析与Cibacron Blue 3GA Sepharose CL-4B亲和层析技术纯化目的蛋白;HPLC法测定比较野生型NDPK-A及突变体在氧化还原和正常环境下磷酸转移酶活性。结果显示,NDPK-A及突变体在大肠杆菌中高效表达;经纯化分别获得了均一的NDPK-A蛋白及突变体蛋白,纯度均达到98%;在还原环境下NDPK-A及突变体的磷酸转移酶活性均高于正常环境下的活性,但是在氧化环境下的磷酸转移酶活性明显低于正常环境下。氧化还原环境对NDPK-A结构异构及磷酸转移酶活性有一定的影响,提示氧化还原环境可能调控NDPK-A二硫键的形成,影响蛋白的聚集状态,从而影响蛋白的磷酸转移酶活性,并且NDPK-A结构中可能有更为复杂的氧化还原调控酶活性机制。     

人组蛋白α-氨基乙酰基转移酶Nat11表达纯化、晶体生长及底物结合研究

α-氨基乙酰基转移酶11(Nat11)催化组蛋白H4和H2A氨基端乙酰化修饰,发挥着重要的表观遗传调控功能。将人Nat11基因构建到原核表达载体p SUMO中,转化入大肠杆菌BL21(DE3)进行重组表达。通过镍柱亲和层析等一系列体外纯化步骤,获得高纯度Nat11。利用等温滴定量热法(ITC),测得Nat11与底物多肽微摩尔量级结合常数。利用质谱技术,发现纯化后的Nat11结合有大肠杆菌内源产生的乙酰辅酶A或辅酶A,在ITC滴定过程中可以产生对多肽底物的乙酰化修饰,表明纯化获得的Nat11在溶液中具有酶活力。随后,对Nat11进行晶体生长研究,通过初筛优化获得蛋白截短体及底物-酶融合蛋白单晶。     

Cloning and expression of the glucose-1-phosphate thymidylyltransferase gene (gerD) from Streptomyces sp. GERI-155

GERI-155 is a macrolide antibiotic containing two deoxyhexose molecules, that has antimicrobial activity against Gram-positive bacteria. The deoxysugar biosynthetic gene cluster of GERI-155 was cloned from Streptomyces sp., GERI-155. One of the orfs, gerD, appeared to encode glucose-1-phosphate thymidylyltransferase (dTDP-glucose synthase), which converts dTTP and glucose-1-phosphate to dTDP-D-glucose and pyrophosphate. GerD was expressed in E. coli in vector pHJ2 and the expressed protein was purified to apparent homogeneity by ammonium sulfate precipitation and DEAE-Sepharose CL-6B and DEAE-Trisacryl column chromatography. The specific activity of the enzyme increased 16-fold with a recovery of 10%. It migrated as a single band on SDS-PAGE with a molecular mass of 30 kDa. The purified protein had glucose-1-phosphate thymidylyltransferase activity, catalyzing a reversible bimolecular group transfer reaction. In the forward reaction the highest activity was obtained with the combination of dTTP and alpha-D-glucose-1-phosphate, and only 5.5% of that activity was obtained with UTP in place of dTTP. In the opposite direction the purified protein was highly specific for dTDP-D-glucose and pyrophosphate.     

Cloning and expression of glucose-1-phosphate thymidylyltransferase gene (schS6) from Streptomyces sp. SCC-2136

The deoxysugar biosynthetic gene cluster of Sch 47554/Sch 47555 was cloned from Streptomyces sp. SCC-2136. One of the ORFs, schS6, appeared to encode glucose-1-phosphate thymidylyltransferase, which converts dTTP and glucose-1-phosphate to TDP-D-glucose and pyrophosphate. The dTDP-D-glucose is a key metabolite in prokaryotics as a precursor for a large number of modified deoxysugars, and these deoxysugars are a major part of various antibiotics, ranging from glycosides to macrolides. SchS6 was expressed in E. coli vector pSCHS6 and the expressed protein was purified to apparent homogeneity by ammonium sulfate precipitation and Ni-NTA affinity column chromatography. The specific activity of the purified enzyme increased 4.7-fold with 17.5% recovery. It migrated as a single band on SDS-PAGE with an apparent molecular mass of 56 kDa. The purified protein showed glucose-1-phosphate thymidylyltransferase activity, catalyzing a reversible bimolecular group transfer reaction. In the forward reaction, the highest activity was obtained with combination of dTTP and alpha-D-glucose-1-phosphate, and only 12% of that activity was obtained with the substrates UTP/alpha-D-glucose-1-phosphate. In the opposite direction, the purified protein was highly specific for dTDP-D-glucose and pyrophosphate.     

杀粉蝶菌素A1生物合成基因簇中甲基转移酶PieB2的功能

【目的】研究杀粉蝶菌素A1产生菌中甲基转移酶基因pieB2的功能。【方法】利用接合转移和同源重组双交换的方法,构建pieB2基因缺失突变株,以及利用接合转移的方法,构建回补菌株。通过高保真PCR克隆pieB2基因到表达载体pET28a上,构建质粒pJTU5997,转化入大肠杆菌E.coliBL21(DE3)/pLysE中诱导表达。利用高效液相色谱检测PieB2的体外酶活。【结果】获得了pieB2基因缺失的双交换突变株。发酵结果显示,该突变株不再产生杀粉蝶菌素A1,而是积累了一种脱甲基产物。N-末端融合组氨酸标签的PieB2在大肠杆菌中获得可溶性表达,通过体外催化证明了PieB2甲基转移酶的功能。【结论】体内遗传实验和体外生化实验证明了PieB2作为甲基转移酶在杀粉蝶菌素A1合成中的作用。     

Cell wall modifications during auxin-induced cell extension in monocotyledonous and dicotyledonous plants

There are several differences between monocotyledonous and dicotyledonous plants. The sensitivity towards added galactose which inhibits auxin-induced coleoptile elongation but not stem elongation is one of the conspicuous differences between the two types of plants. InAvena coleoptile segments, galactose, probably as galactose-1-phosphate, inhibits the formation of UDP-glucose from glucose-l-phosphate. The inhibition of UDP-glucose formation due to galactose is not found inPisum epicotyl segments. InAvena UTP: α-D-glucose-1-phosphate uridyltransferase (EC 2.7.7.9) which catalyzes the reaction from glucose-1-phosphate to UDP-glucose seems to be inhibited by galactose-1-phosphate.     

Galactose-1-Phosphatase in Rat Brain

A prominent galactose-1-phosphatase was isolated from rat brain and partially purified by chromatography on diethylaminoethyl-Sephacel, hydroxylapatite, and Sephacryl S-300 columns. The galactose-1-phosphatase was separated from alkaline phosphatase, and from two forms of glucose-1-phosphatase. The three columns gave a 10-fold increase in specific activity to 290 mol/min/mg of protein, with a yield of 15%. Of the eight sugar phosphates tested, galactose-1-phosphate was the best substrate for the purified enzyme, followed by glucose-1-phosphate, which was hydrolyzed 40% as rapidly as galactose-1-phosphate. Galactose-1-phosphatase had an optimum pH of 8.5 and a Km value of 2.5 mM for galactose-1-phosphate hydrolysis. Mg2+ was required for activity, and supported half-maximal activity at a concentration of 1.25 mM. Phosphate was the only potent inhibitor found ATP, arsenate, and vanadate caused moderate inhibition of 10 mM levels, whereas AMP, L-homoarginine, and L-phenylalanine stimulated enzyme activity. Galactose-1-phosphatase was determined to have a Stokes radius of 30 A and a sedimentation coefficient of 4.1S. These values were used to calculate a molecular weight of 50,200 and a frictional ratio showing the enzyme to be a globular protein. It is hypothesized that a similar phosphatase may play a role in reducing brain galactose-1-phosphate concentrations in patients with galactosemia.     

Cloning,expression, and purification of the His6-tagged hyper-thermostable dUTPase from Pyrococcus woesei in Escherichia coli: application in PCR

The gene encoding dUTPase from Pyrococcus woesei was cloned into Escherichia coli expression system. It shows 100% gene identity to homologous gene in Pyrococcus furiosus. The expression of N-terminal His(6)-tagged Pwo dUTPase was performed in E. coli BL21(DE3)pLysS and E. coli Rosetta(DE3)pLysS strain that contains plasmid encoding additional copies of rare E. coli tRNAs. E. coli Rosetta(pLysS) strain was found with two times higher expression yield of His(6)-tagged Pwo dUTPase than E. coli BL21(DE3)pLysS. The His(6)-tagged Pwo dUTPase was purified on Ni(2+)-IDA-Sepharose, dialyzed, and the enzyme activity was investigated. We found that His(6)-tag domain has no influence on dUTP hydrolytic activity. dUTP is generated during PCR from dCTP, which inhibits the polymerization of DNA catalyzed by DNA polymerase with 3(')-5(') exonuclease activity. We observed that the thermostable His(6)-tagged Pwo dUTPase used for the polymerase chain reaction with P. woesei DNA polymerase improves the efficiency of PCR and it allows for amplification of longer targets.     

High-level expression and single-step purification of leucyl-tRNA synthetase from Aquifex aeolicus

Aquifex aeolicus leucyl-tRNA synthetase is the only known heterodimeric LeuRS, consisting of two subunits with molecular masses of 74.0 and 33.5 kDa, and named alphabeta-LeuRS. The gene encoding alpha subunit was cloned into pSBET-b vector. Synthetic oligonucleotide encoding six histidine residues was also inserted in front of alpha subunit. PSBET-b vector contains argU gene, which encodes a rare Escherichia coli tRNA(Arg)(AGA/AGG). The argU gene helps A. aeolicus LeuRS, which contains AGA/AGG codons in exceptionally high frequency, express well in E. coli. The gene encoding beta subunit was inserted into pET-15b vector. E. coli BL21-CodonPlus (DE3) cells were transformed with the two recombinant plasmids to produce alphabeta-LeuRS with a His6 tag at the N-terminus of alpha subunit. The enzyme was purified by affinity chromatography on Ni-NTA Superflow. About 7 mg purified alphabeta-LeuRS was obtained from 250 ml culture. The His6-tag at the N-terminus did not affect the aminoacylation activity of the enzyme.     

Cloning and characterization of phosphoglucomutase and phosphomannomutase derived from Sphingomonas chungbukensis DJ77

The enzymes phosphoglucomutase (PGM) and phosphomannomutase (PMM) play an important role in the synthesis of extracellular polysaccharide. By colony hybridization of the fosmid library of Sphingomonas chungbukensis DJ77, an open reading frame (ORF-1) of 1,626 nucleotides, whose predicted product is highly homologous with other PGM proteins from several bacterial species, was identified. An additional open reading frame (ORF-2) of 1,437 nucleotides was identified, and its encoded protein shows a high level of similarity with the PGM/PMM protein family. The two genes were cloned into a bacterial expression vector pET-15b (+) and expressed in Escherichia coli as fusion proteins with (His)(6)-tag. Both recombinant proteins (designated as SP-1 and SP-2 for ORF-1 and ORF-2, respectively) exhibited PGM and PMM activities. The molecular masses of subunits of SP-1 and SP-2 were estimated to be around 58 and 51 kDa from SDS-PAGE, respectively. However, molecular masses of SP-1 and SP-2 in their native condition were determined to be approximately 59.5 and 105.4 kDa, according to non-denaturing PAGE, respectively. The SP-1 protein has a preference for glucose-1-phosphate rather than mannose-1-phosphate, while the preferred substrate of SP-2 is mannose-1-phosphate. Thus, the existence of two proteins with bifunctional PGM/PMM activities was first found S. chungbukensis DJ77.     

Catalytic properties of glucose-6-phosphate dehydrogenase from pea leaves.

A homogeneous preparation of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) with a specific activity of 3.88 U/mg protein was isolated from pea (Pisum sativum L.) leaves. The molecular mass of the G6PDH is 79 +/- 2 kD. According to SDS-PAGE, the molecular mass of the enzyme subunit is 40 +/- 3 kD. The Km values for glucose-6-phosphate and NADP are 2 and 0.5 mM, respectively. The enzyme has a pH optimum of 8.0. Mg2+, Mn2+, and Ca2+ activate the enzyme at concentrations above 1 mM. Galactose-6-phosphate and fructose-6-phosphate inhibit the G6PDH from pea leaves. Fructose-1, 6-bisphosphate and galactose-1-phosphate are enzyme activators. NADPH is a competitive inhibitor of the G6PDH with respect to glucose-6-phosphate (Ki = 0.027 mM). ATP, ADP, AMP, UTP, NAD, and NADH have no effect on the activity of the enzyme.