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适冷菌Pseudoalteromonas sp. Bsi590嘌呤核苷磷酸化酶基因克隆及酶学性质研究
中文摘要

在地球这个大生态系统中存在着广泛的低温环境,如占地球表面14%的两极地区及海洋深处(90%的海水其平均温度为5℃或更低)等,在这些特殊的环境中生活着一类微生物即低温微生物。20世纪90年代以来,随着海洋生物技术的兴起,国内外学者相继从海冰中分离得到多种新的海洋极端微生物。利用基因重组技术,克隆极端酶的编码基因,并进行序列和结构分析,从而探讨极端酶的酶的学性质研究蛋白结构与功能的关系,是目前研究极端酶的主要方法之一。 嘌呤核苷磷酸化酶(EC 2.4.2.1 purine nucleoside phosphorylase,简称PNP)是嘌呤核苷补救途径中的普遍存在的一种酶,广泛存在于哺乳动物、寄生虫和微生物中,催化如下可逆反应:嘌呤核苷+磷酸盐↔嘌呤碱基+核糖-1-磷酸。在药物设计、核苷合成、分子进化和基因治疗上具有重要研究意义。 本文首先研究了4个属的14株分离自北极海冰中的低温微生物的基本生长特性,并采用透明圈平板初筛的方法对其产酶的特性进行了研究,发现大部分菌株的最适生长温度在在15-20℃,多数菌株可在37℃下生长,但较为缓慢,生长需要有NaCl的弱碱性的环境,能够分泌多种胞外水解酶。属于适冷微生物。通过设计兼并引物PCR扩增得到了假交替单胞菌Pseudoalteromonas sp.Bsi590嘌呤核苷磷酸化酶(PiPNP)的全长基因,并对其序列进行了分析,该基因全长702bp,编码233个氨基酸残基,密码子分析发现存在6个稀有密码子,预测蛋白分子量为25,018.61,等电点为4.78。该基因与Pseudoalteromonas haloplanktis TAC125嘌呤核苷磷酸化酶基因相似性87.3%,蛋白相似性为93.6%。基因序列提交Genbank,登录号为EF222283。。根据对E.coli PNP(EcPNP)底物结合位点和酶催化活性中心的研究,EcPNP中与底物结合的氨基酸残基His4,Gly20,Arg24, Arg43,Arg89,Asp112,Leu158,Phe159,Met180,Glu181和形成催化部位的氨基酸残基Asp204在PiPNP中高度保守。对三种代表高温、中温和低温来源微生物中PNP氨基酸比较分析,发现高温菌中Met,Asp的含量(1.69,3.39)明显比中温菌(5.02,7.95)和低温菌(3.86,7.73)的低,PiPNP中同功能的Asp+Asn/Glu+Gln比例(0.67)高于中温(0.49)和高温PNP(0.54)。三种酶所含有的疏水性氨基酸比例一致(38%)。根据PNP系统进化树分析,同源三聚体PNP和同源六聚体分属在两个类群,不同底物催化特性的酶与按照蛋白晶体结构进行的分类一致,说明蛋白的结构决定催化专一性。来自大肠杆菌的两个PNP(PNP-Ⅰ和PNP-Ⅱ)亲源性低,分布在比较远。假交替单胞菌属的PNP没有形成种属特异性的分支,说明该基因在进化过程中十分保守,对新物种的形成不起决定作用。本文以X衍射获得的EcPNP蛋白三维空间结构为模板,将PiPNP的氨基酸序列进行同源模建,获得三维空间结构,两种蛋白的空间结构十分相似,均呈现αβ结构。 为了研究适冷菌PiPNP的催化特性和温度稳定性,以及探讨与EcPNP的结构和功能关系,本文克隆了大肠杆菌PNP基因,利用pET表达系统构建了N段含有6-His-Tag的PiPNP和EcPNP重组质粒,经过一步快速亲和纯化得到活性蛋白。根据黄嘌呤氧化酶能将肌苷进行磷酸化反应生成的次黄嘌呤转变成尿酸,在293 nm有最大吸收峰的原理,建立了PNP酶活力测定的方法,对PiPNP和EcPNP的结构和功能进行了研究。比较PiPNP和EcPNP的CD分析,发现PiPNP具有较低的α-helix,较高的β-turn,PiPNP结构较松散,尿素SHSDS可使PiPNP蛋白的二级结构发生迅速改变。温度微扰实验发现PiPNP和EcPNP在一段很窄的温度变化下结构迅速改变,它们的温度转变点是58℃和62℃,PiPNP的热稳定性比EcPNP低。比较不同肌苷浓度对酶最适催化温度发现,催化低浓度肌苷PiPNP最适温度30-35℃,EcPNP最适温度50℃,催化高浓度肌苷PiPNP最适温度50-60℃,EcPNP最适温度60-70℃,底物浓度对PiPNP最适酶活温度影响较大。温度稳定性研究发现P/PNP保温30min,酶的活性在37-42℃时发生急剧变化,50℃完全失活,EcPNP酶在50-55℃发生急剧变化,65℃时酶活性已完全丧失。将PiPNP放置在50 mM磷酸盐缓冲液中,能显著增加酶的稳定性,80℃放置30 min,剩余酶活38%。 PiPNP和EcPNP动力学研究发现,磷酸盐浓度在0.1-5 mM变化时,PiPNP和EcPNP酶催化反应速度表现出非米氏方程特性,在1.2-5 mM之间PiPNP和EcPNP酶符合米氏方程线形双曲线特征,Km分别是0.21 mM和0.34 mM,最大催化反应速度分别为24.63 U/㎎和10.04 U/㎎。PiPNP和EcPNP不同温度下酶催化反应速度表现出高浓度肌坩抑制酶活的特性,PiPNP在37℃的Km是EcPNP两倍,催化效率比EcPNP高20%。PiPNP和EcPNP都能催化各种6-位替代的嘌呤核苷,具有广泛的底物专一性, PiPNP的最适催化底物为肌苷,PiPNP催化6-氧(鸟苷,肌苷)效率和EcPNP相近,但催化6-氨基嘌呤(腺苷)和6-甲基嘌呤的效率比EcPNP低,表明PiPNP和EcPNP在与底物结合的部位存在差异。 关键词:大肠杆菌;假交替单胞菌;嘌呤核苷磷酸化酶;低温微生物;酶活性;圆二色谱;温度微扰;稳定性;腺苷;肌苷

英文摘要

Extremely cold environments widely exist in the great ecosystem of the earth. For example, the polar regions which cover 14% of earth's surface and the deep sea where 90% of seawater has an average temperature at 5℃ or lower. There are cold-adapted microorganisms living in these special environments. The research on cold-adapted bacteria has substantial development since 1990s, with a better knowledge to biotechnology, many methods and skills has been developed to clone and express gene and their important use products in different fields-food industry. It has become a new hot field of research to the research of protein structure and functions. We conducted the studied of the growth characteristics and extracellular hydrolase activities of 14 strains of cold adapted bacteria isolated from the Artic sea ice and screening several strains that had high amylase, protease, cellulase and lipase activities. This research built the base for the development and utilization of cold adapted bacteria. The results showed that optimal growth temperature for strains was 15℃ or 20℃. The optimal pH value was about 8.0, yet they hardly grow at acid condition, 3% NaCl was necessary for better growth. These strains have different abilities in producing amylase, protease, cellulase and lipase .These results can provide a basis for further developing and exploiting the cold adapted marine microbes resources. According to the conserved N-terminal sequence of PNP proteins and C-terminal sequence of Pseudoalteromonas haloplanktis TAC125, the complete gene sequence was successfully amplified from Pseudoalteromonas sp Bsi590 and deposited in Genbank (accession number EF222283). The deduced amino acid sequence displayed 96 and 60% of identity with P. haloplanktis TAC125 PNP and E.coli PNP, respEctively. The proposed active-site residues in EcPNP i.e., His4, Gly20, Arg24, Arg43, Arg89, Asp112, Leu158, Phe159, Met180, Glu181, Asp204 are conserved in the Pseudoalteromona PNPs sequence. Alignment with other PNP proteins showed significant homology surrounding phosphate binding site and catalysis site, forming the consensus amino sequences GPDLRA and TVSDH, respectively. Analysis of three different PNP from high-,mid- and low-temperature microbiology, a high ratio of Met and Asp was found in high- temperature microbiology, both are 1.69 and 3.39, the corresponding the content for the mid- and low-temperature microbiology was (5.02, 7.95 ) , (3.86,7.73) respectively. PiPNP was found a high ratio of Asp+Asn/Glu+Gln(0.67) compared with mid-temperature microbiology (0.49) and high-PNP temperature microbiology (0.54) . They both share an identity of hydrophobic amino acid content. According to the phylogeny, PNPs are tightly clustered into two different clusters in agreement with the protein structure, showing little change during evolution. PiPNP was overexpressed in Escherichia coli Rosetta (DE3) pLysS at 25, 30 and 37℃ with the induction of 1 mM IPTG. Under the experimental conditions, different induction temperatures had no significant influence on the PNPs protein folding, as PNPs were mainly in soluble form in the cell lysate after sonication. N-terminal his-tagged PiPNP and EcPNP were purified to apparent homogeneity using Ni²⁺ -chelating column to the research of their catalytic activity. By HPLC, they share high degree of purity, only one main peak can be seen, EcPNP has a calculated molecular weight of 126.262 Da, while PiPNP has a calculated molecular weight of 111,208 Da. Both sharing a similar folding pucker by homology modeling. CD analysis show they both share α-helix and β-sheet, PiPNP has little content of α-helix, but a littleβ-turn, which shows a more flexible conformation in structure. Compared with EcPNP, PiPNP possessed a lower temperature optimum and thermal stability. As for PNP enzymes in general, PiPNP and EcPNP displayed complicated kinetic properties, Pi PNP possessed higher K〓 and catalytic efficiency (k〓/K〓) compared to PcPNP at 37℃. Substrate specificity results showed PiPNP catalyzed the phosphorolysis of various 6-position substitutions of purine ribonucleosides and deoxyribonucleosides, a better activity with inosine, while no activity towards pyrimidine nucleosides. The protein conformation was analyzed by temperature perturbation difference spectrum.Results showed that PiPNP had lower conformation transition point temperature than EcPNP, phosphate buffer and KCl had significant influence on PiPNP protein conformation stability and thermostability. The effect of pH on PiPNP enzymatic activity was investigated in the pH range of 4-11, the optimum pH corresponded to the pH-dependent stability profile of reaction, with an optimum between pH 8-10 and stable for 2.5 h at pH 10, only about half of the maximal activity at acid condition. The PiPNP and EcPNP assay were performed in 25 mM sodium phosphate buffer (pH 7.5) with 0.5 mM inosine as substrate, temperatures varied from 0 to 65℃. A broad optimal temperature for PiPNP was 30~35℃ .At 0℃ (on ice-water), 35% of the maximal activity was observed, while EcPNP showed optimum temperature at 50-60℃. At high inosine concentration, PiPNP and EcPNP has a higher catalytic temperature: PiPNP 50-60℃, EcPNP 60-70℃. In order to determine thermostability, the enzymes were incubated for 30min in pH 7.5 20mM Tris-HCl at a particular temperature before measuring the residual activity under standard conditions. The PiPNP was stable up to 37℃ and a little fall was observed above 37℃, retaining only 74% of its original activity at 42℃, reaching total inactivation at 50℃. EcPNP remained 85% activity at 50℃, but the activity decreased drastically at 55℃, totally inactivated at 65℃. Meat-induced unfolding of PiPNP and EcPNP protein was analyzed by 277nm UV spectroscopy between 20 and 70℃, showing PiPNP and EcPNP thermal unfolding process were irreversible. The beginning point of the transition of the PiPNP was 58℃ which were lower than that of the EcPNP enzyme (62℃) in 5mM Tris-HCl buffer. PiPNP was more stable in 50mM phosphate buffer than in Tris-HCl buffer since the UV absorption change was little. PiPNP conformation transition was rEcorded at different KCl concentrations in Tris-HCl buffer, the KCl induced strong enzyme conformation stabilization, 0.5 M KCl markedly increases the conformation transition point temperature from 58℃ to 63℃, 1 M KCl induced least changes of the UV absorption indicated high ionic strength promoted the PiPNP conformation stability. Direct evidence that KCl and phosphate affected enzyme stability had been provide by the comparison of the residual activity of PiPNP in the absent and present of KCl and phosphate. The PiPNP was incubated at a defined temperature from 35 to 50℃. After 1 h of incubation at 50℃ with 0.5 M KCl and 1 M KCl, the enzyme activities still retained 81% and 38%, respectively, whereas it was inactivated without KCl. phosphate exerts a protEction toward temperature inactivation of the enzyme, after 1 h incubation at 45℃, it retained 84% and 57% of its catalytic activity in the presence of 50 mM and 100 mM sodium phosphate comparing with 45% activity when the enzyme was incubated alone. The enzyme remained higher residual activity at high concentration of substrates. This result indicated that the binding of this substrate raised the conformational stability of the enzyme, thus reducing its susceptibility to thermal denaturation. The kinetic parameters for inosine and phosphate are compared. When the variable substrate was inosine, the reaction kinetics displayed a mixture of negative and positive cooperativity, as well as substrate inhibition at high concentration. But within low inosine concentration ranging from 50 μM to 600 μM at 37℃ for PiPNP and EcPNP, the linear Michaelis-Menten kinetics plots were observed. K〓 for inosine was about two times for PiPNP than EcPNP, the catalytic efficiency, K〓/K〓, was 1.2-fold higher compared to EcPNP at 37℃. Like the homologous hexameric PNP, PiPNP was characterized by a broad substrate specificity that recognized purine nucleosides with substitutions in the 6-position served as substrates, such as adenosine, inosine, guanosine and 6-methyl purine nucleosides, while inosine was the preferred substrate for PiPNP. The rate of phosphorylasis of ribonucleosides showed more efficiently than deoxyribonucleosides. PiPNP was unable to metabolize the pyrimidine nucleosides and at very low rate to arabinofuranosyladenine. Keywords: Escherichia coli; Pseudoalteromonas; purine nucleoside phosphorylase; low temperature microbiology; enzyme activity ; circular dichroism; Heat-induced unfolding; stability; adenosine; inosine

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