论文标题:生物催化合成UMP及乳清酸磷酸核糖转移酶特性的研究
论文作者
论文导师 许平,论文学位 博士,论文专业 微生物学
论文单位 山东大学,点击次数 135,论文页数 146页File Size9300K
2007-10-08论文网 http://www.lw23.com/lunwen_657676872/
UMP;; Corynebacterium ammoniagenes;; Orotate phosphoribosyltransferase;;Biocatalysis
核苷酸是一类在代谢上极为重要的生化物质,除了作为DNA、RNA的前体外,在细胞的生长代谢、能量的储存和转化、免疫反应,及信号传导中,都有核苷酸的参与。嘧啶核苷酸的用途广阔,在食品制造、农业、医药生产上都有极为重要的用途。近年来随着核酸工业的发展,其作为药物中间体、保健品和食品添加剂的重要性越来越突出。此外,它还是寡聚糖合成中的必需前体,而后者因其在生化识别过程中的重要作用成为众多研究领域的热点。尿嘧啶核苷酸(UMP)是嘧啶核苷酸生物合成中处于结点位置的重要核苷酸。在寡聚糖合成和抗癌药物生产中,UMP都是重要的前体物质。其市场需求很大,蕴含有良好的商业潜力。但与此同时,由于生产困难,嘧啶核苷酸价格昂贵已成为该领域进一步发展的主要限制因素。特别是在我国,嘧啶核苷酸产品缺乏限制了糖化学和糖生物学相关领域的研究。 微生物转化合成核苷酸,就是利用微生物作为酶源,催化核苷酸的前体物质转化为核苷酸。相对于其他生产方法,该方法不但缩短了生产周期,也使核苷酸的产量大大增加;反应体系简单,后提取工艺相对更简单容易;而且,该方法可以通过偶联不同的基因工程菌株,生产多种复杂核苷酸、核苷糖乃至寡聚糖。这在核苷酸工业、医药及糖化学、糖生物学合成工业中是极其重要的一个环节,也是其他方法不能替代的一个关键环节。本工作以UMP为模式核苷酸探讨了微生物催化合成核苷酸过程中的主要影响因素,分别从转化条件和菌株(催化关键酶)的角度对核苷酸催化合成过程进行了详细的描述。 首先根据核苷酸类物质的电荷及结构差异,分别建立了薄板层析和高压液相色谱(HPLC)的检测方法来分离检测核苷酸类物质。其中,薄板层析检测法可以分离乳清酸和UMP,定性分析转化液中的底物和产物;而建立在离子对色谱技术基础上的HPLC检测方法可以同时分离多种核苷酸类物质,并可根据标准曲线对反应中的产物定量,从而为后面的工作奠定了基础。 在建立了快捷的检测方法后,采用产氨棒杆菌模式菌株Cornebacteriumammoniagenes ATCC6872,以乳清酸为底物,建立了微生物催化合成UMP的方法。整个过程分两步进行;首先将C.ammoniagenes ATCC6872在高盐培养基中培养;在菌体细胞达到最高转化能力时收集细胞,作为催化反应体系的酶源。首先考察了培养条件对UMP生产的影响,在以玉米浆作氮源,pH7.0的培养基中培养15小时(处于对数生长中期)的菌体显示出最高的转化能力,在相同的反应条件下能生产更多的UMP。确定了培养条件以后,分别通过单因子实验和统计学实验对催化反应条件进行了优化。通过单因子实验,确定了催化反应液的最优组成为乳清酸,葡萄糖,磷酸离子(等摩尔的KH_2PO_4和K_2HPO_4),MgCl_2和Triton X-100。而统计学实验中的Plackett-Burman实验进一步明确了催化反应体系中最主要的影响因素;磷酸离子和作为酶原的C.ammoniagenes细胞。在此基础上,最后通过中心组合实验得到最优的反应物配比,使UMP的产量达到32 mM(10.4g l~(-1)),是优化前产量的3倍以上。 乳清酸磷酸核糖转移酶(OPRTase)催化乳清酸和5-磷酸核糖焦磷酸(PRPP)合成乳清苷酸,是嘧啶核苷酸合成中形成核苷酸的一步,也是生物催化法合成UMP的关键酶。利用巢式PCR和原位杂交的方法得到了C.ammoniagenesATCC6872中的一个623bp的DNA片断(p26)。序列分析显示该片断包含了555bp的编码OPRTase的基因pyrE和63bp的前导区。和已详细研究的Escherichiacoli和Salmonella typhimurium的OPRTase相比,其编码区编码的氨基酸序列的相似性只有28%。序列分析显示,OPRTase的大多数活性区和活性位点在克隆到的酶中都能找到,也有一些在S.typhimurium OPRTase中被认为是活性必需的位点在C.ammoniagenes的酶中被具有相似侧链或相似极性的其它氨基酸所代替。这些氨基酸被认为在催化中的作用并不会因此产生重大改变。但值得注意的是,S.typhimurium的OPRTase上的一个重要的活性位点,Lys 73,在C.ammoniagenes的OPRTase中却找不到。据报道,该位点和OPRTase的催化起始有关。该位点的缺失暗示C.ammoniagenes的OPRTase和之前报道的其它物种的OPRTase可能在性质和催化机理上有很大差异。因此,构建了pyrE基因的表达质粒,使该基因在E.coli中过量表达,并纯化了在E.coli中过量表达的C.ammoniagenes ATCC6872的OPRTase。 该酶和多数OPRTase一样,属于同源二聚体;催化的反应需要有二价金属离子的激活,Mg~(2+)是反应最适激活剂,其最适浓度为1-3 mM,Mn~(2+)和Co~(2+)也可以活化反应,而其他金属离子(Zn~(2+),Ca~(2+),Ba~(2+),Ni~(2+))都不能替代镁离子起到活化反应的作用;该OPRTase不耐热,50℃保温30 min就会造成50%的酶活损失,而最适反应温度出现在35℃;该酶催化的正反应(OMP合成反应)和逆反应(OMP焦磷酸解反应)的最适pH不同,正反应的最适pH为10.5-11.5,而逆反应的为5.5;对该酶催化的正逆反应的动力学参数也进行了研究,通过双倒数曲线得到乳清酸、PRPP、OMP和PPi的k_m值分别为33μM,64μM,45μM,和36μM。正反应的最大反应初速度(V_(max))为1,150 units mg~(-1)蛋白,逆反应的最大反应初速度(V_(max))为550 units mg~(-1)蛋白。 和其他已报道的OPRTase相比,C.ammoniagenes OPRTase的生化性质有几处明显的不同;(1)正逆反应pH不同。大多数OPRTase只检测了正反应的最适pH。(2)正反应最适pH偏高。已报道的OPRTase正反应最适pH一般在8-10,而C.ammoniagenes的OPRTase催化正反应的最适pH达到10.5-11.5;(3)OMP的K_m值偏高。比其他OPRTase报道的K_m值高了将近10倍。这些性质上的差异都指向一个催化上重要位点的缺失,Lys 73。该位点的缺失也暗示着C.ammoniagenes OPRTase的催化机理可能不同于以往的报道。 嘧啶合成基因的表达调控在多种生物中都有报道,本文也考察了C.ammoniagenes ATCC6872中编码OPRTase的pyrE基因的表达调控情况。对培养在含有尿嘧啶和不含有尿嘧啶的培养基中的原始菌株的OPRTase酶活的测定显示,尿嘧啶会抑制C.ammoniagenes ATCC6872 pyrE基因的表达。随后,带有63bp非编码前导序列的pyrE基因(p26)被克隆到一个表达载体上,并转化到E.coli宿主中。在E.coli中的实验证明p26对尿嘧啶敏感;培养在含有尿嘧啶的培养基中的p26转化子的OPRTase活力比不含尿嘧啶的培养基中低,而作为对照组的不含63 bp前导区的pyrE基因,在这两种培养基中显示了相差无几的活力。这说明在63 bp的前导区内包含了一个对尿嘧啶敏感的调控因子。 最常见的嘧啶合成基因的调控元件是衰减子。但是对p26的前导区的序列分析却显示在这63 bp前导区内,没有任何可能的衰减子结构。而随后在棒杆菌内的实验进一步表明,在C.ammoniagenes中这63 bp的前导区没有调控功能;含有p26的C.ammoniagenes转化子在尿嘧啶存在下表现出的OPRTase活力和不存在尿嘧啶时相差不大。这样的结果意味着63 bp的前导区中包含的调控因子只在E.coli中起作用,而在棒杆菌中不起作用。由于E.coli中已经发现有多种调控机制可以控制pyr基因的表达,那么,最有可能的情况或许是这63 bp中包含的E.coli的某一种调控机制在棒杆菌中是不存在的。 C.ammoniagenes是生物转化法生产嘧啶核苷酸的工业菌株,它提供了合成UMP的酶源和一个重要底物PRPP,是UMP合成的关键。OPRTase催化UMP催化合成中的第一步反应,它的表达受到体内核苷酸的反馈抑制。当UMP浓度过高时,会抑制该酶的表达,限制了UMP的产量。为了提高UMP产量,我们克隆了C.ammoniagenes ATCC6872的一个组成型启动子,IJ59,并将它与p26连接,构建了无需诱导即可过量表达合成中关键酶(OPRTase)的穿梭质粒pXMJIE。在E.coli中的实验表明该质粒在非诱导状态下培养12小时,其OPRTase活力能达到对照菌株的5倍以上。虽然由于研究时间有限,该表达质粒在C.ammoniagenes中表达的实验,现在还在进行当中,但根据Paik和Lee的报道,IJ59启动子在棒杆菌中的表达比在E.coli中还要略高,因此有理由相信,该质粒在C.ammoniagenes中一样可以得到高表达的OPRTase。通过构建能高效表达UMP合成中关键酶的C.ammoniagenes菌株,希望可以达到提高UMP产量的最终目的。
Nucleotides are central metabolites in the complex metabolic network of cell. They are the precursors of DNA and RNA,and carriers of reductive power as well as playing an essential role in the energy transduction and in the energy conservation of cell.They also play important functions in the coordination of metabolic processes and control.Pyrimidine nucleotides are wildly used in the agriculture,food industry and medical industry.They are directly involved in carbon and lipid metebolism providing energy-rich precursors for biosynthesis of oligosaccharides, which have been the focus of much research because of their roles in biochemical recognition processes.Uridine 5"-monophosphate(UMP)is an important nucleotide which can be easily converted to other pyrimidine nucleotides.It is an essencial material in production of oligosaccharides and some anti-cancer drugs. However,the synthesis of UMP usually needs a long time and results in a low yield. The separation and isolation of product are also generally difficult and expensive to perform. Biocatalytic process for nucleotides production use microorganism as enzyme source to convert relative precursor into nucleotides.Compared with other nucleotide production methods,this way can shorten the productive time,improve the yield and make the separation and isolation of product easilier.Furthermore, complex nucleotides,sugar nucleotide and even some oligosaccharides can be synthesized by this method using coupling metabolically engineered bacteria.This is important for the industry of nucleotide,medicine,glycochemistry and glycobiology. In this dissertation,two methods to assay nucleotides were established. Thin-layer chromatography(TLC)was used to assay orotate and UMP in the biocatalytic reaction mixture.While by using HPLC,several different nucleotides and bases could be separated and quantified.This part laid a foundation for the subsequent research of UMP production by biocatalytic process. Attempts were made with success to develop a two-step biocatalytic process for UMP production from orotic acid by Corynebacterium ammoniagenes ATCC 6872; the strain was first cultivated in a high salt mineral medium,and then cells were harvested and used as the catalyst in the UMP production reaction.Effects of cultivation and reaction conditions on UMP production were investigated.The cells exhibited the highest biocatalytic ability when cultivated in a medium containing corn steep liquor at pH 7.0 for 15 h in the exponential phase of growth.To optimize the reaction,both "one-factor-at-a-time" method and statistical method were performed. By "one-factor-at-a-time" optimization,orotic acid,glucose,phosphate ion (equimolar KH_2PO_4 and K_2HPO_4),MgCl_2,Triton X-100 were shown to be the optimum components for the biocatalytic reaction.Phosphate ion and C. ammoniagenes cell were furthermore demonstrated as the most important main effects on UMP production by Plackett-Burman design,indicating that 5-phosphoribosyl-1-pyrophosphate(PRPP)synthesis was the rate-limiting step for pyrimidine nucleotides production.Optimization by a central composition design (CCD)was then performed,and up to 32 mM(10.4 gl~(-1))UMP was accumulated in 24 h from 38.5 mM(6 gl~(-1))orotic acid.The yield was three-fold higher than the original UMP yield before optimization. Orotate phosphoribosyltransferase(OPRTase)catalyzes the reaction between orotate and the ribose-5-phosphate donor 5-phosphoribosyl-1-pyrophosphate(PRPP) to form orotidine-5-phosphate(OMP)and pyrophosphate.This is the first step in the biocatalytic process of UMP production.A 623-bp fragment was cloned by nested PCR and colony blotting method from C.ammoniagenes ATCC 6872,which was used in the biocatalytic production of UMP.Sequence analysis revealed that the fragment (p26)was consisted with a 555-nt-long coding region identical to the pyrE gene from C.glutamicum ATCC 13032,and a 63-nt region upstream the initiation trinucleotide, ATG The amino acid sequence of the cloned OPRTase,as deduced from the nucleotide sequence was compared with the reported OPRTase sequences from other organisms.Although the OPRTases from Escherichia coli and Salmonella typhimurium,which had been studied in detail,showed only 28% similarity with the cloned enzyme,a PRPP binding motif,a solvent-exposed catalytic loop,and some essential residues(Arg 99,Lys 100,and Lys 103)are well assigned in the cloned enzyme.However,it is noteworthy that several amino acid residues in active site of the S.typhimurium OPRTase can not be assigned in the C.ammoniagenes sequence by the alignment.Some of these residues are displaced by other residues with similar side chain or with similar polarity.But sequence analysis shows that there is a lack of an important conserved lysine(Lys 73 in Salmonella typhimurium OPRTase) in the C.ammoniagenes OPRTase.This lysine has been considered to contribute to the initiation of catalysis. The enzyme was overexpressed and purified from a recombinant E.coli.The molecular weight of the purified OPRTase was determined to be 45.4±1.5 kDa by gel filtration.Since the molecular mass of the enzyme was 21.3±0.6 kDa for the subunit,the native enzyme exists as a dimer.Divalent magnesium was necessary for the activity of the enzyme,and can be substituted by Mn~(2+)and Co~(2+).The optimal pH for forward(phosphoribosyl transfer)reaction is 10.5-11.5,which is higher than that of other reported OPRTase,and the optimal pH for reverse(pyrophosphorolysis) reaction is 5.5-6.5.The K_m values for the four substrates were determined to be 33μM for orotate,64μM for PRPP,45μM for OMP,and 36μM for pyrophosphate, respectively. Compared with other reported OPRTases,the OPRTase from C.ammoniagenes showed some different properties;(1)The dissimilar pH dependence between forward and reverse reactions.(2)The higher optimal pH for the forward reaction. (3)The K_m value for OMP is much larger than those of other organisms.These differences may be due to the absence of Lys 73 present in the active site of other OPRTases and known to interact with OMP and PRPP.And the catalytic biochemistry of the enzyme may be different from the known mehanisms. De novo synthesis of pyrimidines involves utilization of energy-rich labile intermediates such as carbamoyl phosphate,PRPP,and nucleoside triphosphates. The enzymes involved in UMP biosynthesis were strictly controlled by exogenous pyrimidines.In our research,the activity of OPRTase in C.ammoniagenes ATCC 6872 was showed repressed in the presence of exogenous uracil as well.Then the 63 nucleotides upstream the coding region were cloned together with the pyrE gene from C.ammoniagenes ATCC 6872.The expression experiments revealed that in E.coli, the specific activities of overexpressed OPRTase were depressed with exogenous uracil when the 63 nucleotides upstream exist,while the specific activities remained constant under the same conditions when the upstream region was removed.These indicate that pyrE expression is regulated in response to pyrimidines and the 63-nt upstream region participates in the regulation. The pyrimidine-sensitive transcriptional attenuation may be the most important and common mechanism in bacteria,which contributes to the regulation of the expression of pyrimidine synthetic(pyr)genes.The feature common to the mechanism is a Rho-independent terminator structure named attenuator in mRNA,whose key features are inverted repeats and a string of Ts in the nontemplate strand.However, sequence analysis showed the 63 nucleotides region immediately upstream of C. ammoniagenes pyrE contained neither a string of Ts nor a region of inverted repeats as the attenuator does.Moreover,the subsequent expression experiments performed in C.ammoniagenes showed that the expression of p26 was not sensitive to uracil, indicating that the mode of pyrimidine-specific regulation of the 63-bp fragment doesn"t exist in Coryneform bacteria,but only in E.coli. C.ammoniagenes is a Gram-positive non-pathogenic soil bacterium,which has strong ATP regeneration activity with sufficient PRPP supply,and has been widely used to produce UMP from orotic acid by either fermentative process or biocatalytic process.Functioning as the enzyme source,the cell is obviously a controlling factor in UMP production.OPRTase catalyzes the first step in the UMP production between orotate and the ribose-5-phosphate donor PRPP to form OMP.The expression of the enzyme has been shown to be strictly controlled by pyrimidines nucleotides.When the in vivo concentration of UMP is high,the expression of the enzyme will be repressed.This mechanism limits the yield of UMP.Therefore,we cloned a promoter IJ59,which could initiate transcription sufficiently without induction both in C.ammoniagenes ATCC6872 and E.coli.The promoter was ligated into a shuttle vector pXMJ19 with p26 forming a plasmid pXMJIE,which could overexpress OPRTase in both E.coli and C.ammoniagenes ATCC6872 at absence of IPTG.The expression experiments in E.coli showed that when cultivated for 12 h without induction,the specific activity of OPRTase from the E.coli harboring pXMJIE were over 5-fold higher than that from the E.coli without the plasmid. Although the expression of the plasmid in C.ammoniagenes is still under investigating,the promoter IJ59 has been reported to be more effient in Coryneform bacteria than in E.coli.Therefore,a recombinant C.ammoniagenes which can overexpress OPRTase would be constructed,and could be used to improve the UMP yield.
|
