适冷菌Pseudoal teromonas sp.Bsi590嘌呤核苷磷酸化酶基因克隆及酶学性质研究
|
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,存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 haloplanktisTAC125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,尿素和SDS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℃,催化高浓度肌甘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PiPNP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/mg和10.04 U/mg。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-位替代的嘌呤核甘,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。
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,cellutase 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 Ni~(2+)-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 shareα-helix andβ-sheet,PiPNP has little content ofα-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,PiPNP possessed higher K_m and catalytic efficiency (k_(cat)/K_m) compared to EcPNP 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℃.
Heat-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 50μM to 600μM at 37℃for PiPNP and EcPNP,the linear Michaelis-Menten kinetics plots were observed.K_m for inosine was about two times for PiPNP than EcPNP,the catalytic efficiency,K_(cat)/K_m,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.
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 haloplanktisTAC125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,尿素和SDS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℃,催化高浓度肌甘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PiPNP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/mg和10.04 U/mg。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-位替代的嘌呤核甘,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。
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,cellutase 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 Ni~(2+)-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 shareα-helix andβ-sheet,PiPNP has little content ofα-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,PiPNP possessed higher K_m and catalytic efficiency (k_(cat)/K_m) compared to EcPNP 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℃.
Heat-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 50μM to 600μM at 37℃for PiPNP and EcPNP,the linear Michaelis-Menten kinetics plots were observed.K_m for inosine was about two times for PiPNP than EcPNP,the catalytic efficiency,K_(cat)/K_m,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.
引文
[1]范光南,傅世宗.极端环境微生物研究概况[J].福建热作科技,2000,(2):12-15.
[2]林影,凌晨晖.海洋微生物极端酶的研究[J].海洋科学,1999,(2):19-21.
[3]Morita RY.Psychrophilic bacteria[J].Bacteriol Rev,1975,39:144-167
[4]Margesin R,et al.Psychrophiles:from Biodiversity to Biotechnology[M].Berlin Heidelberg:Springer-Verlag,2008:31-47.
[5]俞勇,李会荣,陈波,曾胤新,何剑锋.海冰细菌研究进展[J].极地研究,2005,17(1):75-85.
[6]Breezee J,Cady N,Staley JT.Subfreezing growth of the sea ice bacterium "Psychrornonas ingrahamii"[J].Microb Ecol,2004,47:300-304.
[7]Yao TD,Xiang SR,Zhang XJ,Pu JC.Microbiological characteristics recorded by Malan and Puruogangri ice core[J].Quat Sci,2003,23:193-199.
[8]Price PB.A habitat for psychrophiles in deep Antarctic ice[J].Proc Natl Acad Sci USA,2000,97:1247-1251.
[9]曾胤新,陈波,俞勇,李会荣.极地微生物及其低温酶[C].见:第三届海洋高科技论坛,厦门,2005.
[10]Nicholas JR.Toward a molecular understanding of cold activity of enzyme from psychrophiles[J].Extremophiles,2000,4:83-90.
[11]曾胤新,俞勇,李会荣,陈波.北极海洋细菌BSw20353的胞外蛋白酶性质[J].极地研究,2006,18(3):206-214.
[12]Feller G,Payan F,Theys F,et al.Stability and structural analysis of α-amylase from the antarctic psychrophile Alteromonas haloplanctis A23[J].European Journal of Biochemistry,1994,222(2):441-447.
[13]Tatiana G,Margarita K,Hoda Y,Maryna R,Ralf G,Hauke T,Garabed A.Diversity and cold-active hydrolytic enzymes of culturable bacteria associated with Arctic sea ice,Spitzbergen[J].Extremophiles,2004,8:475-488
[14]Huston AL,Krieger-Brockett BB,Deming JW.Remarkably low temperature optima for extracellular enzyme activity from Arctic bacteria and sea ice[J].Environmeutal microbiology,2000,2(4):383-388.
[15]林永成.海洋微生物及其代谢产物[M].北京:化学工业出版社,2003.
[16]张树证.酶制剂工业(下册)[M].北京:轻工业出版社,1984.
[17]张刚,汪天虹,张臻峰,等.产低温淀粉酶的海洋真菌筛选及研究[J].海洋科学,2002,26(2):35-39.
[18]俞勇,李会荣,陈波等.低温脂肪酶产生菌的筛选、鉴定及其部分酶学性质[J].高技术通讯,2003,13(10):89-95.
[19]张占海.中国第二次北极科学考察报告[R].北京:海洋出版社.2004.
[20]席宇,朱大恒等.假替单胞菌及其胞外生物活性物质研究进展[J].微生物学通报,2005,32(3):108-112.
[21]Feller G,Narinx E.et al.Temperature dependence of growth,enzyme secretion and activity of psychrophilic Antarctic bacteria[J].Appl Microbiol Biotechnol,1994,7:479-483.
[22]Hellio FC,Orange N,el al.Growth temperature controls the production of a single extracellular protease by Pseudomonas flurescens MFO in the presence of various inducers[J].Res Microbiol,1993,144(8):617-625.
[1]Gautier,G.,Gauthier,M.and Christen,R.Phlylogenetic analysis of the genera Alteromonas,Shewanella,and Moritella using genes coding for small-subunit rRNA sequences and division of the genus Alteromonas into two genera,Alteromonas(emended) and Pseudoalteromonas gen.nov.,and proposal of twelve new species combinations[J]..Int J Syst Bacteriol,1995,45:755-761.
[2]席宇,朱大恒,刘红涛,等.假交替单胞菌及其胞外尘物活性物质研究进展[J].微生物学通报,2005,32(3):108-110.
[3]Giudice A L,Michaud L,Gentile Get al.Ecophysiological characterization of cultivable Antarctic psychrotolerant marine bacteria able to degrade hydrocarbons [J].Classe Di Scienze Fisiche Matematiche Naturali,2005,1:1-11.
[4]Se'bastien Violot et al.Structure of a full length psychrophilic cellulase from Pseudoalteromonas haloplanktis revealed by X-ray diffraction and small angle X-ray scattering[J].J Mol Biol,2005,348:1211-1224.
[5]Fernandes S,et al.β-Galactosidase from a cold-adapted bacterium:purification,characterization and application for lactose hydrolysis[J].Appl Microbiol Biotechnol,2002,58:313-321.
[6]张锐,曾涧颖.极端微生物产碱性蛋白酶菌株的筛选及发酵条件研究[J].微生物学通报,2001,28(4):5-9.
[7]Angela Maria Cusano,et al.Secretion ofpsychrophilic a-amylase deletion mutants in Pseudoalteromonas haloplanktis TAC125[J].Appl Microbiol Biotechnol,2002,5:295-302.
[8]连明珠,张金伟,曾涧颖.南极产低温几丁质酶菌株的16S rDNA序列分析及其发酵条件及酶学性质研究[J].高技术通报,2005,15(9):58-61.
[9]Feller G,Narinx E,Arpigny JL,et al.Temperature dependence of growth,enzyme secretion and activity of psychrophilic Antarctic bacteria[J].Appl Microbiol Biotechnol,1994,41:477-479.
[10]曾胤新,俞勇,蔡明宏等.低温微生物及其酶类的研究概况[J].微生物学杂志,2004,24(5):83-88.
[11]Feller G,Gerday C.Psychrophilic enzymes:molecular basis of cold adaptation cell[J].Mol Life Sci,1997,53:830-841.
[12]Bzowska A,Kulikowska E,Shugar D.Purine nucleoside phosphorylases:properties,functions,and clinical aspects[J].Pharmacol Ther,2000,88:349-425.
[13]Hanrahan,JR,Hutchinson,DW.The enzymatic synthesis of antiviral agents[J]. J Biotechnol,1992,23(1):95-212.
[14]闫蓬勃,丁庆豹,邱蔚然,等.酶法合成抗癌新药氟铁龙前体—5-FUR[J].华东理工大学学报,2001,27(3):143-146.
[15]Suresh P,Nau RV.Enzymatic synthesis of thymidine using bacterial whole cells and isolated purine nucleoside phosphorylase[J].Biocatal Biotransform,1997,15:147-158.
[16]Rogert MC,Trelles JA,Porro S.Microbial synthesis of antiviral nucleosides using Escherichia coli BL21 as biocatalyst[J].Biocatalysis and Biotransformation,2002,20(5):347-351.
[17]邱蔚然,唐沽宇,高媛,唐孝宣.由鸟苷经酶法生产利巴韦林[J].中国医药工业杂志,1997,28(1):14-17.
[18]Shirae H,Yodozeki K,Uchiyama M,et al.Enzymatic production of ribavirin from purine nucleosides by Brevibacterium acetylicum ATCC954[J].Agric Biol Chem,1998,52(4):1777-1783.
[19]Pugmire MJ,Ealick SE.Structural analysis reveals two distinct families of nucleoside phosphorylases[J].Biochem J,2002,361:1-25.
[20]Thompson JD,Gibson TJ,Plewniak F,Jeanmougin F,Higgins DG.The CLUSTAL_X windows interface:flexible strategies for multiple sequence alignment aided by quality analysis tools[J].Nucleic Acids Res,1997,25:4876-4882.
[21]Schwede T,Kopp J,Guex N,and Peitsch MC.SWISS-MODEL:an automatedprotein homology-modeling server[J].Nucleic Acids Research,2003,31:3381-3385.
[22]史兆兴.兼并PCR及其应用[J].生物技术通报,2004,15(2):172-175.
[23]陈静,倪坚,陈江野.简并PCR法用于白色念珠菌MAPK新基因的筛选[J].生物化学与生物物理学报,2000,32(3):305-311.
[24]王洪振,宋朝霞,刘文广,等.简并PCR技术及其在基因克隆中的应用[J].遗传,2003,25(2):201-204.
[25]Olayiwola A.Comparative sequence and structure analysis reveal features of cold adaptation of an enzyme in the thennolysin family[J].Protein:Structure Function and Bioinformatics,2006,62:435-449.
[1]Krenitzky T,Koszalka G.Purine nucleoside synthesis,an efficient method employing nucleoside phosphorylases[J].Biochemistry,1981,20:3615-3621.
[2]Ling F,Inuoe Y,Kimura A.Induction,purification and utilization ofpurine nucleoside phosphorylase and uridine phosphorylase from Klebsiella sp.[J].Process Biochem,1994,29:355-361.
[3]Cacciapuoti G,Porcelli M.et al.Purification and characterization of extremely thermophilic and thennostable 5-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus:purine nucleoside phosphorylase activity and evidence for intersubunit disulfide bonds[J].J Biol chem,1994,269:24762-24769.
[4]Hamamoto T,Noguchi T,Midorikawa Y.Purification and characterization of purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase from Bacillus stearothermophihts TH 6-2[J].Biosci Biotechnol Biochem,1996,60:11790-1180.
[5]Porter DJ.Purine nucleoside phosphorylase.Kinetic mechanism of the enzyme from calf spleen[J].J Biol Chem,1992,267:7342-7351.
[6]Tebbe J,Wielgus KB,Schroeder W,Luic M,Shugar D,Saenger W,Koellner G,Bzowska A.Purine nucleoside phosphorylase(PNP) from Celhtlomonas sp.,a different class of PNP different from both "low-molecular weight" mammalian and "high molecular weight" bacterial PNPs[J].Protein Eng(suppl.)1997,10:90-95.
[7]Jensen,KF,Nygaard P.Purine nucleoside phosphorylase from Salmonella typhimurium and Escherichia coli.Purification and some properties[J].Eur J Biochem,1975,51:253-265.
[8]Gianese G,Bossa F and Pascarella S.Comparative structural analysis of psychrophilic and meso- and thennophilic enzymes[J].Proteins,2002,47:236-249.
[9]Shoichet BK,et al.A relationship between protein stability and protein function [J].Proc Natl Acad Sci U.S.A.,1995,92:452-456.
[10]阎隆飞,孙之荣.蛋白质分子结构[M].北京:清华大学出版社,1999:150-160.
[11]Luo Y,Baldwin R,Trifluoroethanol stabilizes the pH 4 folding intermediate of sperm whale apomyoglobin[J].J Mol Biol,1998,27(9):49-57.
[12]Lecoq K,Belloc C,Konrad M.et al.YLR209c encodes Saccharomyces cerevisiae purine nucleoside phosphorylase[J].J bacteriology,2001,183(16):4910-4913.
[13]唐上华,熊坤,冯喆等.产气肠杆菌W8401嘌呤核菅磷酸化酶基因的克隆及其在大肠杆菌中的表达[J].工业微生物,1989,19(4):1-5.
[14]Bennett E,Li C,Allan P,Parker W,Ealick S.Structural basis for substrate specificity ofEscherichia coli purine nucleoside phosphorylase[J].J Biol Chem,2003,278:47110-47118.
[15]Feller G,Narinx E,Arpigny J,Aittaleb M,Baise E,Genicot S.Enzymes from psychrophilic organisms[J].FEMS Microbiol Rev,1996,18:189-202.
[16]Pugmire M,Ealick S.Structural analysis reveals two distinct families of nucleoside phosphorylases[J].Biochem J,2002,361:1-25.
[17]Bzowska A,Kulikowska E,Shugar D.Purine nucleoside phosphorylases:properties,functions,and clinical aspects[J].Pharmacol Ther,2000,88:349-425.
[18]Ying X,Georges F,Charles G,Nicolas G.Metabolic Enzymes from Psychrophilic Bacteria:Challenge of adaptation to low temperatures in ornithine carbamoyltransferase from Moritella abyssi[J].J biology,2003,185(7):216 I-2168.
[19]Kalckar HM.Differential spectrophotometry of purine compounds by means of specific enzymes:I.Deten-nination of hydroxypurines[J].J Biol Chem,1947,167:429-443.
[20]John L,Serena F,Julie B,et al.Expression,purification,and characterization of recombinant purine nucleoside phosphorylase from Escherichia coli[J].Protein Expression and Purification,2001,22:180-188.
[21]Feller G,Arpigny JL,Narinx E,Gerday C.Molecular adaptations of enzymes from psychrophilic organisms[J].Comparative Biochemisry and Physiology,1997,118(3):495-499.
[22]Xu Y,Glansdorff N.Was our ancestor a hyperthermophilic procaryote?[J].Comp Biochem Physiol A,2002,133:677-688.
[1] Friedkin M. Enzymatic synthesis of deoxy-xanthosine by the action of xanthosine phosphorylase in mammalian tissues [J]. J Am Chem Soc, 1952, 74:112-115.
[2] Kalckar HM. Differential spectrophotometry of purine compounds by means of specific enzymes: I. Determination of hydroxypurines [J]. J Biol Chem, 1947,167:429-443.
[3] Parks RE, Agarwal RP. Purine nucleoside phosphorylase [J]. Enzymes, 1972, 7:483-514.
[4] Bzowska A, Kulikowska E, Shugar D. Purine nucleoside phosphorylases: properties, functions, and clinical aspects [J]. Pharmacol Ther, 2000, 88: 349-425.
[5] Bzowska A, Kulikowska E, Shugar D. Properties of purine nucleoside phosphorylase (PNP) of mammalian and bacterial origin [J]. Z Naturforsch C 1990,45:59-70.
[6] Jensen, KF. Purine nucleoside phosphorylase from Salmonella typhimurium and Escherichia coli. Initial velocity kinetics, ligand binding and reaction mechanism [J]. Eur J Biochem, 1976, 67:377-386.
[7] Robertson CR, Hoffee PA. Purification and properties of purine nucleoside phosphorylase from Salmonella typhimurium [J]. J Biol Chem, 1977,248:2040-2043.
[8] Ling F, Inuoe Y, Kimura A. Purification and characterization of novel nucleoside phosphorylase from Klebsiella sp., its use in the enzymatic production of adenine arabinoside [J]. Appl Environ Microbiol, 1990, 56:3830-3834.
[9] Appleby,TC, Mathews II, Porcelli M, Cacciapuoti G, Ealick SE. Three dimensional structure of a hyperthermophilic 5'-Deoxy-5'- methylthioadenosine phosphorylase from Sulfolobus Solfataricus [J]. J Biol Chem, 2001, 276:39232-39242.
[10] Cacciapuoti G, Forte S, Moretti MA et al.A novel hyperthermostable 5'-deoxy- 5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus [J]. FEBS J, 2005, 272:1886-1899.
[11] Cercignani, G. Substrate specificities of adenosine deaminase and adenosine phosphorylase from Bacillus cereus [J]. Ital J Biochem, 1982, 32:243-252.
[12] Hamamoto T, Noguchi T, Midorikawa Y. Purification and characterization of purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase from Bacillus stearothermophilus TH 6-2 [J]. Biosci Biotechnol Biochem, 1996,60:11790-1180.
[13] Pugmire MJ, Ealick SE. Structural analysis reveals two distinct families of nucleoside phosphorylases [J]. Biochem J, 2002, 361:1-25.
[14] Yang Z, Parker WB, Sorscher EJ, Ealick SE. PNP Anticancer gene therapy [J].Current Topics in Medicinal Chemistry, 2005, 5:1259-1274.
[15] Seeger C, Poulsen C, Dandanell G. Identification and characterization of genes (xapA, xapB, and xapR) involved in xanthosine catabolism in Escherichia coli [J]. J Bacteriol, 1995,177:5506-5516.
[16] Bennett EM, Li C, Allan PW, Parker WB, Ealick SE. Structural basis for substrate specificity of Escherichia coli purine nucleoside phosphorylase [J]. J Biol Chem, 2003, 278:47110-47118.
[17] Shugar D, Saenger W, Koellner G. Crystal structure of the purine nucleoside phosphorylase (PNP) from Cellulomonas sp. and its implication for the mechanism of trimeric PNPs [J]. J Mol Biol, 1999,294:1239-1255.
[18] Cacciapuoti G, Fusco S, Caiazzo N, et al. Heterologous expression of 5'- methyl thioadenosine phosphorylase from the archaeon Sulfolobus solfataricus: characterization of the recombinant protein and involvement of disulfide bonds in thermophilicity and thermostability [J]. Protein Expression Purification, 1999,16:125-135.
[19] Cacciapuoti G, Porcelli M, et al. Purification and characterization of extremely thermophilic and thermostable 5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus: purine nucleoside phosphorylase activity and evidence for intersubunit disulfide bonds [J]. J Biol Chem, 1994, 269: 24762-24769.
[20] Hori N, Watanabe M, et al. Purification and characterization of thermostable purine nucleoside phosphorylase of Bacillus stearothermophilus JTS 859 [J].Agric Biol Chem, 1989, 53:2205-2210.
[21] Krenitsky TA, Koszalka GW, Tuttle JV. Purine nucleoside synthesis, an efficient method employing nucleoside phosphorylases [J]. Biochemistry, 1981, 20:3615-3621.
[22] Tebbe J, Wielgus-Kutrowska B. et al. Purine nucleoside phosphorylase (PNP) from Cellulomonas sp., a different class of PNP different from both "low-molecular weight" mammalian and "high-molecular weight" bacterial PNPs [J]. Protein Eng (suppl.), 1997, 10:90.
[23] Krenitsky TA., Tuttle JV. Correlation of substrate stabilization patterns with proposed mechanisms for three nucleoside phosphorylases [J]. Biochim Biophys Acta, 1982, 703:247-249.
[24] Cacciapuoti G, Servillo L, Moretti M, Porcelli M. Conformational changes and stabilization induced by phosphate binding to 5'-methylthioadenosine phosphorylase from the thermophilic archaeon Sulfolobus solfataricus [J]. Extremophiles, 2001:5:295-302.
[25] Porter DJ. Purine nucleoside phosphorylase. Kinetic mechanism of the enzyme from calf spleen [J]. J Biol Chem, 1992, 267:7342-7351.
[26] Accorsi A, Piacentini MP, et al. Purine nucleoside phosphorylase from human erythrocytes: a kinetic study of the fully separated isoenzymes [J]. Biochem Int,1991,24:23-31.
[27] Jensen KF. Purine nucleoside phosphorylase from Salmonella typhimuriwn and Escherichia coli. Initial velocity kinetics, ligand binding and reaction mechanism [J]. Eur J Biochem, 1976, 67:377-386.
[28] Erion M D, Stoeckler JD, Guida WC, Walter RL, Ealick SE. Purine nucleoside phosphorylase. 2. Catalytic mechanism [J]. Biochemistry, 1997, 36:11735-11748.
[29] Fedorov A, Shi W, Kicska G, Transition state structure of purine nucleoside phosphorylase and principles of atomic motion in enzymatic catalysis [J]. Biochemistry, 2001,40:853-860.
[30] Kim BK, Cha S, Parks RE. Purine nucleoside phosphorylase from human erythrocytes: II. Kinetic analysis and substrate-binding studies [J].J Biol Chem, 1968,243:1771-1776.
[31] Koszalka GW, Vanhooke J, Short SA, Hall WW. Purification and properties of inosine-guanosine phosphorylase form Escherichia coli K-12 [J]. J Bacteriol,1988, 170:3493-3498.
[32] Erion MD, Takabayashi K, Smith HB, et al. Purine nucleoside phosphorylase. 1.Structure-function studies [J]. Biochemistry, 1997, 36: 11725-11734.
[33] Mao C, Cook WJ, Zhou M, et al. The crystal structure of Escherichia coli purine nucleoside phosphorylase: a comparison with the human enzyme reveals a conserved topology [J]. Structure, 1997, 5:1373-1383.
[34] Stoeckler JD, Poirot AF, Smith RM, et al. Purine nucleoside phosphorylase: 3. Reversal of purine base specificity by site- directed mutagenesis[J].Biochemistry,1997,36:11749-11756.
[35]Hanrahan JR,Hutchinson DW.The enzymatic synthesis of antiviral agents[J].J Biotechnol,1992,23(1):95-212.
[36]闫蓬勃,丁庆豹,邱蔚然,等.酶法合成抗癌新药氟铁龙前体—5-FUR[J].华东理工大学学报,2001,27(3):143-146.
[37]林珏,蒋红,唐上华,等.酶法生产抗艾滋病药物AZT的前体—5-甲基尿嘧啶核苷的研究[J].工业微生物,1993,23(1):1-4.
[38]Shirae H,Yodozeki K,Uchiyama M,et al.Enzymatic production of ribavirin from purine nucleosides by Brevibacterium acetylicum ATCC954[J].Agric Biol Chem,1998,52(4):1777-1783.
[39]邱蔚然,唐洁宇,高媛,唐孝宣.山鸟苷经酶法生产利巴韦林[J].中国医药工业杂志,1997,28(1):14-17.
[40]Suresh P,Naur V.Enzymatic synthesis of thymidine using bacterial whole cells and isolated purine nucleoside phosphorylase[J].Biocatalysis and Biotransformation,1997,15:147-158.
[41]Ishige T,Honda K,Shimizu S.Whole organism biocatalysis[J].Curt opin Chem Biol,2005,9:I74-80.
[42]Rogert MC,Trelles JA,Porto S.Microbial synthesis of antiviral nucleosides using Escherichia coli BL21 as biocatalyst[J].Biocatalysis and Biotransfonnation,2002,20(5):347-351.
[43]Trelles JA,Fernandez J,Condezo LA,et al.Nucleoside synthesis by immobilised bacterial whole cell[J].Journal of Molecular Catalysis B:Enzymatic,2004,30:219-227.
[44]Deharvengt S;Wack S;Uhring M,Aprahamian M,Haiti A.Suicide gene/prodrug therapy for pancreatic adenocarcinoma by E.coli purine nucleoside phosphorylase and 6-methylpurine 2'-deoxyriboside[J].Pancreas,2004,28:54-64.
[45]Hassan AE,Shortnacy AT,Montgomery JA,Secrist JA.A convenient synthesis of 2'-deoxy-2-fluoroadenosine:a potential prodrug for sucide gene therapy[J].Nucleosides Nucelotides Nucleic Acids,2000,19:559-565.
[46]Elizabeth SL,Martinez N,Rogert MC.An improved microbial synthesis of purine nucleosides[J].Biotechnology Letters,2000,22:1277-1280.
[47]Kicska GA,Tyler PC,Evans,GB,et al.Purine-less death in Plasmodium falciparum induced by immucillin H, a transition state analogue of purine nucleoside phosphorylase [J]. J Biol Chem, 2002,277:3226- 3231.
[48] Giblett ER, Ammann AJ, Wara DW, et al. Nucleoside-phosphorylase deficiency in a child with severely defective T-cell immunity and normal B-cell immunity [J]. Lancet, 1975, 1: 10101-1013.
[49] Bzowska A, Kulikowska E, Darzynkiewcz E, Shugar D. Purine nucleoside phosphorylase. Structure activity relationships for substrate and inhibitor properties of N-1-, N-7-, and C-8-substituted analogues: differentiation of mammalian and bacterial enzymes with N-1-methy- linosine and guanosine [J]. J Biol Chem, 1988,263:9212-9217.
[50] Sorscher EJ, Peng S, Bebok Z, Allan PA, Bennett LL, Parker J, WB Tumour bystander killing in colonic carcinoma utilizing the Escherichia coli deo D gene to generate toxic purines [J]. Gene Ther, 1994,1: 233-238.
[51] Mohr L, Shankara S, Yoon SK, et al. Gene therapy of hepatocellular carcinoma in vitro and in vivo in nude mice by adenoviral transfer of the Escherichia coli purine nucleoside phosphorylase gene [J]. Hepatology, 2000, 31: 606-614.
[52] Hughes BW, King SA, Allen PW, Parker WB, Sorscher EJ. Cell to cell contact is not required for bystander cell killing by Escherichia coli purine nucleoside phosphorylase [J]. J Biol Chem, 1998, 273: 2322-2328.
[53] Sorscher EJ, Townes TM, Waud WR, Ealick SE. Designer gene therapy using an Escherichia coli purine nucleosidephosphorylase/prodrug system [J]. Chem Biol, 2003, 10:1173-1181.
[1]范光南,傅世宗,蔡海洋.极端环境微生物研究概况[J].福建热作科技,2000.2:12-15.
[2]Forster J.Uber einige Eigenschaften leuchtender Bakterien[J].Centr Bakteriol Parasitenk,1887,2:337-340.
[3]Schmidt-Nielsen,S.Ueber einige psychrophile Mikroorganismen und ihr Vorkommen[J].Centralbl.Bakteriol.Parasitenkd.Ⅱ Abt,1902,9:145-147.
[4]Ingraham JL and Stokes JL.Psychrophilic bacteria[J].Microbiol Mol Biol Rev,1959,23(3):97-108.
[5]Morita RY.Psychrophilic bacteria[J].Bacteriol Rev,1975,39:144-167.
[6]Deming JW.Psychrophiles and polar regions[J].Current opinion in Microbiology,2002,5:301-309.
[7]Friedmann EI.Endolithic microorganisms in the Antarctic cold desert[J].Science,1982,215:1045-1053.
[8]Rivkina EM,Friedmann El,Mckay CP,et al.Metabolic activity of permafrost bacteria below the freezing point[J].Appl Environ Microbiol,2000,66:3230-3233.
[9]Christner BC,Mosley-Thompson E,Thompson LG,et al.Isolation of bacteria and 16S rDNAs from Lake Vostok accretion ice[J].Environ Microbiol,2001,3:570-577.
[10]Breezee J,Cady N,Staley JT.Subfreezing growth of the sea ice bacterium "Psychromonas ingrahamii"[J].Microb Ecol,2004,47:300-304.
[11]Panikov NS,Flanaganb PW,Oechelc WC,Mastepanovd MA,Christensend TR.Microbial activity in soils frozen to below -39℃[J].Soil Biol Biochem,2006,38:785-794.
[12]辛明秀,马延和.嗜冷菌和耐冷菌[J].微生物学通报,1999(2):109-155.
[13]Price PB.Microbial life in glacier ice and implications for a cold origin of life.FEMS Microbiol Ecol,2007,59:217-231.
[14]Miteva VI,Sheridan PP,Brenchley JE.Phylogenetic and physiological diversity of microorganisms isolated from a deep greenland glacier ice core[J].Appl Environ Microbial,2004,70(1):202-213.
[15]张国华,罗会颖,陈劲春,姚斌.新疆天山雪莲根部冻土产酶微生物的初步研究[J].中国农业科技导报,2008,10(2):82-87.
[16]Tanner AC.and Herbert RA.A numerical taxonomic study of Gram-negative bacteria isolated from the Antarctic marine environment[J].CNEXO(Actes Colloq.),1982,14:31-38.
[17]李会荣,俞勇,曾胤新,陈波,任大明.北极太平洋扇区海洋沉积物细菌多样性的系统发育分析[J].微生物学报,2006,46(2):177-183.
[18]曾胤新,陈波.低温微生物适冷特性及其在食品工业中的潜在用途[J].2000,10(2):31-37.
[19]Nichols PD,McMeekin TA.Polyunsaturated fatty acids in Antarctic bacteria[J].Antarctic Science,1993,5:149-160.
[20]Volkman JK,Burton HR,Everitt DA,Allen DI.Pigment and lipid compositions of algal and bacterial communities in Ace Lake,Vestfold Hills,Antarctica[J].Hydrobiologia,1988,165(1):41-57.
[21]David SN,June O,Horacio G,Rodolfo RB,Tom AM.Effect of temperature and salinity stress on growth and lipid composition of Shewanella gelidimarina[J].Applied and Environmental Microbiology,2000,66(6):2422-2429.
[22]Ray MK,Kumar GS,Shivaji S.Phosphorylation of lipopolysaccharides in the Antarctic psychrotroph Pseudomonas syringae:a possible role in temperature adaptation[J].J Bacteriol.,1994,176(14):4243-4249.
[23]Phadtare S.Recent developments in bacterial cold-shock response[J].Curr Issues Mol Biol,2004,6:125-136.
[24]Takayuki H,Hitoshi I,Yasuhiko K.Yeast gene expression during growth at low temperature[J].Cryobiology,2003,46(3):230-237.
[25]Kawamoto J,Kurihara T,Kitagawa M,Kato I,Esaki N.Proteomic studies of an Antarctic cold-adapted bacterium,Shewanella livingstonensis Ac10,for global identification of cold-inducible proteins[J].Extremophiles,2007,11(6):819-826.
[26]Jiang W,Hou Y,Inouye M.CspA,the major cold-shock protein of Escherichia coli,is an RNA chaperone[J].J Biol Chem,1997,272:196-202.
[27]Thomas T.Archaeal cold-adapted proteins:structural and evolutionary analysis of the elongation factor 2 proteins from psychrophilic,mesophilic and thennophilic methanogens[J].FEBS Letters,439(3):281-286.
[28]Yutaka S,Mitsuru H,Kazufumi T,Masaaki M,Shigenori K.Possible involvement of an FKBP family member protein from a psychrotrophic bacterium Shewanella sp.SIB1 in cold-adaptation[J].European Journal of Biochemistry,2004,271(7):1372-1381.
[29]Julianne L,Torsten T,Ricardo C.Low temperature regulated DEAD-box RNA helicase from the antarctic archaeon,Methanococcoides burtoniil[J].Journal of Molecular Biology,2000,297(3):553-567.
[30]Michel V,Lehoux I,Depret G,Anglade P,Labadie J,Hebraud M.The cold shock response of the psychrotrophic bacterium Pseudomonas fragi involves four low-molecular-mass nucleic acid-binding proteins[J].J Bacteriol,1997,179:7331-7342.
[31]李芳,王博,艾秀莲,王志方,付建红.抗冻蛋白研究进展[J].新疆农业科学,2003,40(6):349-352.
[32]Julseth CR,Inniss WE.Induction of protein synthesis in response to cold shock in the psychrotrophic yeast Trichosporon pullans[J].Can J Microbiol 1990,36:519-524.
[33]唐赟.嗜冷菌适应低温的分子机制及其应用[J].西华师范大学学报(自然科学版),2004,25(4):388-393.
[34]辛明秀,周培瑾.冷活性琥珀酸脱氢酶的特性[J].北京师范大学学报(自然科学版),2004,40(1):108-113.
[35]Margesin R,Schinner F.Biotechnological applications of cold-adapted organisms[M].Heidelberg:Springer-Verlag,1999.
[36]辛明秀,周培瑾.温度对嗜冷酵母糖代谢途径某些关键酶的活性效应[J].微生物学报,2000,40(5):518-522.
[37]曾胤新,蔡明宏,陈波,等.一株产蛋白酶南极耐冷细菌的筛选及研究[J].生物技术,2001,11(2):17-20.
[38]Zeng YX,Chen B.Isolation and characteristics of one marine psychrotrophic celulase-generating bacterium Ar/w/b/75/10/5 from Chuckchi Sea,Arctic[J].Chinese Journal of Polar Science,2002,13(2):157-168.
[39]Nicholas JR.Toward a molecular understanding of cold activity of enzyme from psychrophiles[J].Extremophiles,2000,4:83-90.
[40]曾胤新,俞勇,蔡明宏,何剑锋,陈波.低温微生物及其酶类的研究概况[J].微生物学杂志,2004,24(5):83-88.
[41]Huston AL,Krieger-Brocket BB,Deming JW.Remarkable low temperature optima for extracelular enzyme activity from Arctic bacteria and sea ice[J].Environ Microbiol,2000,2(4):383-388.
[42]张锐,曾润颖.极端微生物产碱性蛋白酶菌株的筛选及发酵条件研究[J].微生物学通报,2001,28(4):5-9.
[43]Sawabe T,Takahasi H,Saeki H,Niwa K,Aonob H.Enhanced expression of active recombinant alginate lyase AlyPEEC cloned from a marine bacterium Pseudoalteromonas elyakovii in Escherichia coli by calcium compounds[J].Enzyme and Microbial Technology,2007,40:285-291.
[44]Ochiai T,Fukunaga N,Sasaki S.P urification and some properties of two NADP~+-specific isocitrate dehydrogenases from an obligately psychrophilic marine bacterium,Vibrio sp.strain ABE-I[J].J Biochem(Tokyo),1979,86(2):377-38.
[45]D'Amico S,Marx JC,Gerday C,Feller G.Activity stability relationships in extremophilic enzymes[J].J Biol Chem,2003,278:7891-7896.
[46]Feller G,Narinx E,Arpigny JL,Aittaleb M,Baise E,Genicot S,Gerday C.Enzymes from psychrophilic organisms[J].FEMS Microbiol Rev,1996,18:189-202.
[47]Feller G,Gerday C.Psychrophilic enzymes:hot topics in cold adaptation[J].Nature Rev Microbiol,2003,1:200-208.
[48]Feller G,et al.Cloning and expression in Escherichia coli of three lipaseencoding genes from the psychrotrophic antarctic strain Moraxella TA44[J].Gene,1991,102:111-115.
[49]Arpigny JL,et al.Cloning,sequence and structural features of a lipase from the antarctic facultative psychrophile Psychrobacter immobilis B10[J].Biochim.Biophys Acta.1993,1171:331-333.
[50]Rentier-Delrue F et al.Cloning and overexpression of the triose phosphate isomerase genes from psychrophilic and thermophilic bacteria structural comparison of the predicted protein sequence[J].J Mol Biol,1993,229:85-93.
[51]Garsoux G,Lamotte J,Gerday C,Feller G.Kinetic and structural optimization to catalysis at low temperatures in a psychrophilic cellulase from the Antarctic bacterium Pseudoalteromonas haloplanktis[J].Biochem J,2004,384:247-253.
[52]Feller G,Gerday C.Psychrophilic enzymes:molecular basis of cold adaptation [J].Cell Mol Life Sci,1997,53:830-841.
[53]孙谧,洪义国,李勃生,修朝阳.海洋微生物低温酶特性及其在工业中的潜在用途[J].海洋水产研究,2002,23(3):44-49.
[54]朱非,王珊,周培瑾.低温酶冷适应的分子机制及其在生物技术中的应用[J].微生物学报,2002,42(5):640-644.
[55]Welander U.Microbial degradation of organic pollutants in soil in a cold climate [J].Soil and Sediment Contamination,2005,14(3):281-291.
[56]Arianna T,Józef K,Aneta B,Hubert C,Halina K,Stanis B.Antarctic marine bacterium Pseudoalteromonas sp.22b as a source of cold- adapted β-galactosidase[J].Biomolecular Engineering,2003,20(5):317-324.
[57]Collins T,Hoyoux A,Dutron A,Georis J,Genot B,Dauvrin T,Arnaut F,Gerday C,Feller G.Use of glycoside family 8 xylanases in baking[J].J Cereal Sci,2006,43:79-84.
[58]Margesin R,et al.Psychrophiles:from Biodiversity to Biotechnology[M].Berlin Heidelberg:Springer-Verlag,2008:347-360.
[59]Rina M,Pozidis C,Mavromatis K,Tzanodaskalaki M,Kokkinidis M,Bouriotis V.Alkaline hosphatase from the Antarctic strain TAB5[J].Eur J Biochem,2000,67:1230-1238.
[60]Van den Burg B.Extremophiles as a source for novel enzymes[J].Current Opin Microbiol,2003,6:213-218.
[61]Asenjo JA,Andrews BA,Reyes F,Salamanca M,Burzio L.Protein and nucleic acid sequence encoding a krill-derived cold adapted trypsin-like activity enzyme [P].Patent No.WO2006022947,2006.
[62]李丽娟,张殿昌,龚世园.宏基因组技术在丌发未培养微生物资源中的应用[J].水利渔业,2007,3:13-15.
[63]Amann RI,Ludwig W,Schleifer KH.Phylogenetic identification and in situ detection of individual microbial cells without cultivation[J].Microbiol Rev,1995,59(1):143-169.
[64]张金伟,曾润颖.南极深海沉积物宏基因组DNA中低温脂肪酶基因的克隆,表达及性质分析[J].生物化学与生物物理进展,2006,33(12):1207-1214.
[65]Rees H,Grant S,Jones B,Grant W,I4eaply S.Detecting cellulase and esterase enzyme activities encoded by novel genes present in environmental DNA libraries[J].Extremophiles,2003,7:415-421.
[66]钱小红,贺福初.蛋白质组学:理论与方法[M]北京:科学出版社,2003:10-25.
[67]张德超.我国新疆一号冰川地区和北极地区低温菌的鉴定及多相分类学研究[D].北京:中国农业大学,2007.
[68]朱非.低温菌的多相分类学及系统发育学研究低温蛋白酶的分离纯化及性质[D].北京:中科院研究生院,2002.
[69]王玢.海洋适冷细菌MB-1产纤维素酶的研究[D].济南:山东大学,2003.
[70]陈秀兰.海洋适冷蛋白酶产生菌的筛选、酶的分离纯化及适冷机制研究[D].济南:山东大学,2002.
[71]连明珠.南极Pseudoalteromoflas sp.AC444低温几丁质酶性质分析与基因克隆及普早兹湾深海沉积物中几丁质酶基因多样性研究[D].厦门:厦门大学,2006.
[72]Hu JM,Li Hu H,Cao LX,et al.Molecular cloning and characterization of the gene encoding cold-active β-Galactosidase from a psychrotrophic and halotolerant Planococcus sp.L4[J].J Agric Food Chem,2007,55,2217-2224.
[73]孙谧.一种新型低温碱性蛋白酶、制造方法、应用和产生该蛋白酶的微生物[P].中国专利:00123404.8,2001-1-17.
[74]孙谧.一种新型海洋溶菌酶膜剂及其制备方法[P].中国专利:200510000033.7,2005-8-31.
[2]林影,凌晨晖.海洋微生物极端酶的研究[J].海洋科学,1999,(2):19-21.
[3]Morita RY.Psychrophilic bacteria[J].Bacteriol Rev,1975,39:144-167
[4]Margesin R,et al.Psychrophiles:from Biodiversity to Biotechnology[M].Berlin Heidelberg:Springer-Verlag,2008:31-47.
[5]俞勇,李会荣,陈波,曾胤新,何剑锋.海冰细菌研究进展[J].极地研究,2005,17(1):75-85.
[6]Breezee J,Cady N,Staley JT.Subfreezing growth of the sea ice bacterium "Psychrornonas ingrahamii"[J].Microb Ecol,2004,47:300-304.
[7]Yao TD,Xiang SR,Zhang XJ,Pu JC.Microbiological characteristics recorded by Malan and Puruogangri ice core[J].Quat Sci,2003,23:193-199.
[8]Price PB.A habitat for psychrophiles in deep Antarctic ice[J].Proc Natl Acad Sci USA,2000,97:1247-1251.
[9]曾胤新,陈波,俞勇,李会荣.极地微生物及其低温酶[C].见:第三届海洋高科技论坛,厦门,2005.
[10]Nicholas JR.Toward a molecular understanding of cold activity of enzyme from psychrophiles[J].Extremophiles,2000,4:83-90.
[11]曾胤新,俞勇,李会荣,陈波.北极海洋细菌BSw20353的胞外蛋白酶性质[J].极地研究,2006,18(3):206-214.
[12]Feller G,Payan F,Theys F,et al.Stability and structural analysis of α-amylase from the antarctic psychrophile Alteromonas haloplanctis A23[J].European Journal of Biochemistry,1994,222(2):441-447.
[13]Tatiana G,Margarita K,Hoda Y,Maryna R,Ralf G,Hauke T,Garabed A.Diversity and cold-active hydrolytic enzymes of culturable bacteria associated with Arctic sea ice,Spitzbergen[J].Extremophiles,2004,8:475-488
[14]Huston AL,Krieger-Brockett BB,Deming JW.Remarkably low temperature optima for extracellular enzyme activity from Arctic bacteria and sea ice[J].Environmeutal microbiology,2000,2(4):383-388.
[15]林永成.海洋微生物及其代谢产物[M].北京:化学工业出版社,2003.
[16]张树证.酶制剂工业(下册)[M].北京:轻工业出版社,1984.
[17]张刚,汪天虹,张臻峰,等.产低温淀粉酶的海洋真菌筛选及研究[J].海洋科学,2002,26(2):35-39.
[18]俞勇,李会荣,陈波等.低温脂肪酶产生菌的筛选、鉴定及其部分酶学性质[J].高技术通讯,2003,13(10):89-95.
[19]张占海.中国第二次北极科学考察报告[R].北京:海洋出版社.2004.
[20]席宇,朱大恒等.假替单胞菌及其胞外生物活性物质研究进展[J].微生物学通报,2005,32(3):108-112.
[21]Feller G,Narinx E.et al.Temperature dependence of growth,enzyme secretion and activity of psychrophilic Antarctic bacteria[J].Appl Microbiol Biotechnol,1994,7:479-483.
[22]Hellio FC,Orange N,el al.Growth temperature controls the production of a single extracellular protease by Pseudomonas flurescens MFO in the presence of various inducers[J].Res Microbiol,1993,144(8):617-625.
[1]Gautier,G.,Gauthier,M.and Christen,R.Phlylogenetic analysis of the genera Alteromonas,Shewanella,and Moritella using genes coding for small-subunit rRNA sequences and division of the genus Alteromonas into two genera,Alteromonas(emended) and Pseudoalteromonas gen.nov.,and proposal of twelve new species combinations[J]..Int J Syst Bacteriol,1995,45:755-761.
[2]席宇,朱大恒,刘红涛,等.假交替单胞菌及其胞外尘物活性物质研究进展[J].微生物学通报,2005,32(3):108-110.
[3]Giudice A L,Michaud L,Gentile Get al.Ecophysiological characterization of cultivable Antarctic psychrotolerant marine bacteria able to degrade hydrocarbons [J].Classe Di Scienze Fisiche Matematiche Naturali,2005,1:1-11.
[4]Se'bastien Violot et al.Structure of a full length psychrophilic cellulase from Pseudoalteromonas haloplanktis revealed by X-ray diffraction and small angle X-ray scattering[J].J Mol Biol,2005,348:1211-1224.
[5]Fernandes S,et al.β-Galactosidase from a cold-adapted bacterium:purification,characterization and application for lactose hydrolysis[J].Appl Microbiol Biotechnol,2002,58:313-321.
[6]张锐,曾涧颖.极端微生物产碱性蛋白酶菌株的筛选及发酵条件研究[J].微生物学通报,2001,28(4):5-9.
[7]Angela Maria Cusano,et al.Secretion ofpsychrophilic a-amylase deletion mutants in Pseudoalteromonas haloplanktis TAC125[J].Appl Microbiol Biotechnol,2002,5:295-302.
[8]连明珠,张金伟,曾涧颖.南极产低温几丁质酶菌株的16S rDNA序列分析及其发酵条件及酶学性质研究[J].高技术通报,2005,15(9):58-61.
[9]Feller G,Narinx E,Arpigny JL,et al.Temperature dependence of growth,enzyme secretion and activity of psychrophilic Antarctic bacteria[J].Appl Microbiol Biotechnol,1994,41:477-479.
[10]曾胤新,俞勇,蔡明宏等.低温微生物及其酶类的研究概况[J].微生物学杂志,2004,24(5):83-88.
[11]Feller G,Gerday C.Psychrophilic enzymes:molecular basis of cold adaptation cell[J].Mol Life Sci,1997,53:830-841.
[12]Bzowska A,Kulikowska E,Shugar D.Purine nucleoside phosphorylases:properties,functions,and clinical aspects[J].Pharmacol Ther,2000,88:349-425.
[13]Hanrahan,JR,Hutchinson,DW.The enzymatic synthesis of antiviral agents[J]. J Biotechnol,1992,23(1):95-212.
[14]闫蓬勃,丁庆豹,邱蔚然,等.酶法合成抗癌新药氟铁龙前体—5-FUR[J].华东理工大学学报,2001,27(3):143-146.
[15]Suresh P,Nau RV.Enzymatic synthesis of thymidine using bacterial whole cells and isolated purine nucleoside phosphorylase[J].Biocatal Biotransform,1997,15:147-158.
[16]Rogert MC,Trelles JA,Porro S.Microbial synthesis of antiviral nucleosides using Escherichia coli BL21 as biocatalyst[J].Biocatalysis and Biotransformation,2002,20(5):347-351.
[17]邱蔚然,唐沽宇,高媛,唐孝宣.由鸟苷经酶法生产利巴韦林[J].中国医药工业杂志,1997,28(1):14-17.
[18]Shirae H,Yodozeki K,Uchiyama M,et al.Enzymatic production of ribavirin from purine nucleosides by Brevibacterium acetylicum ATCC954[J].Agric Biol Chem,1998,52(4):1777-1783.
[19]Pugmire MJ,Ealick SE.Structural analysis reveals two distinct families of nucleoside phosphorylases[J].Biochem J,2002,361:1-25.
[20]Thompson JD,Gibson TJ,Plewniak F,Jeanmougin F,Higgins DG.The CLUSTAL_X windows interface:flexible strategies for multiple sequence alignment aided by quality analysis tools[J].Nucleic Acids Res,1997,25:4876-4882.
[21]Schwede T,Kopp J,Guex N,and Peitsch MC.SWISS-MODEL:an automatedprotein homology-modeling server[J].Nucleic Acids Research,2003,31:3381-3385.
[22]史兆兴.兼并PCR及其应用[J].生物技术通报,2004,15(2):172-175.
[23]陈静,倪坚,陈江野.简并PCR法用于白色念珠菌MAPK新基因的筛选[J].生物化学与生物物理学报,2000,32(3):305-311.
[24]王洪振,宋朝霞,刘文广,等.简并PCR技术及其在基因克隆中的应用[J].遗传,2003,25(2):201-204.
[25]Olayiwola A.Comparative sequence and structure analysis reveal features of cold adaptation of an enzyme in the thennolysin family[J].Protein:Structure Function and Bioinformatics,2006,62:435-449.
[1]Krenitzky T,Koszalka G.Purine nucleoside synthesis,an efficient method employing nucleoside phosphorylases[J].Biochemistry,1981,20:3615-3621.
[2]Ling F,Inuoe Y,Kimura A.Induction,purification and utilization ofpurine nucleoside phosphorylase and uridine phosphorylase from Klebsiella sp.[J].Process Biochem,1994,29:355-361.
[3]Cacciapuoti G,Porcelli M.et al.Purification and characterization of extremely thermophilic and thennostable 5-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus:purine nucleoside phosphorylase activity and evidence for intersubunit disulfide bonds[J].J Biol chem,1994,269:24762-24769.
[4]Hamamoto T,Noguchi T,Midorikawa Y.Purification and characterization of purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase from Bacillus stearothermophihts TH 6-2[J].Biosci Biotechnol Biochem,1996,60:11790-1180.
[5]Porter DJ.Purine nucleoside phosphorylase.Kinetic mechanism of the enzyme from calf spleen[J].J Biol Chem,1992,267:7342-7351.
[6]Tebbe J,Wielgus KB,Schroeder W,Luic M,Shugar D,Saenger W,Koellner G,Bzowska A.Purine nucleoside phosphorylase(PNP) from Celhtlomonas sp.,a different class of PNP different from both "low-molecular weight" mammalian and "high molecular weight" bacterial PNPs[J].Protein Eng(suppl.)1997,10:90-95.
[7]Jensen,KF,Nygaard P.Purine nucleoside phosphorylase from Salmonella typhimurium and Escherichia coli.Purification and some properties[J].Eur J Biochem,1975,51:253-265.
[8]Gianese G,Bossa F and Pascarella S.Comparative structural analysis of psychrophilic and meso- and thennophilic enzymes[J].Proteins,2002,47:236-249.
[9]Shoichet BK,et al.A relationship between protein stability and protein function [J].Proc Natl Acad Sci U.S.A.,1995,92:452-456.
[10]阎隆飞,孙之荣.蛋白质分子结构[M].北京:清华大学出版社,1999:150-160.
[11]Luo Y,Baldwin R,Trifluoroethanol stabilizes the pH 4 folding intermediate of sperm whale apomyoglobin[J].J Mol Biol,1998,27(9):49-57.
[12]Lecoq K,Belloc C,Konrad M.et al.YLR209c encodes Saccharomyces cerevisiae purine nucleoside phosphorylase[J].J bacteriology,2001,183(16):4910-4913.
[13]唐上华,熊坤,冯喆等.产气肠杆菌W8401嘌呤核菅磷酸化酶基因的克隆及其在大肠杆菌中的表达[J].工业微生物,1989,19(4):1-5.
[14]Bennett E,Li C,Allan P,Parker W,Ealick S.Structural basis for substrate specificity ofEscherichia coli purine nucleoside phosphorylase[J].J Biol Chem,2003,278:47110-47118.
[15]Feller G,Narinx E,Arpigny J,Aittaleb M,Baise E,Genicot S.Enzymes from psychrophilic organisms[J].FEMS Microbiol Rev,1996,18:189-202.
[16]Pugmire M,Ealick S.Structural analysis reveals two distinct families of nucleoside phosphorylases[J].Biochem J,2002,361:1-25.
[17]Bzowska A,Kulikowska E,Shugar D.Purine nucleoside phosphorylases:properties,functions,and clinical aspects[J].Pharmacol Ther,2000,88:349-425.
[18]Ying X,Georges F,Charles G,Nicolas G.Metabolic Enzymes from Psychrophilic Bacteria:Challenge of adaptation to low temperatures in ornithine carbamoyltransferase from Moritella abyssi[J].J biology,2003,185(7):216 I-2168.
[19]Kalckar HM.Differential spectrophotometry of purine compounds by means of specific enzymes:I.Deten-nination of hydroxypurines[J].J Biol Chem,1947,167:429-443.
[20]John L,Serena F,Julie B,et al.Expression,purification,and characterization of recombinant purine nucleoside phosphorylase from Escherichia coli[J].Protein Expression and Purification,2001,22:180-188.
[21]Feller G,Arpigny JL,Narinx E,Gerday C.Molecular adaptations of enzymes from psychrophilic organisms[J].Comparative Biochemisry and Physiology,1997,118(3):495-499.
[22]Xu Y,Glansdorff N.Was our ancestor a hyperthermophilic procaryote?[J].Comp Biochem Physiol A,2002,133:677-688.
[1] Friedkin M. Enzymatic synthesis of deoxy-xanthosine by the action of xanthosine phosphorylase in mammalian tissues [J]. J Am Chem Soc, 1952, 74:112-115.
[2] Kalckar HM. Differential spectrophotometry of purine compounds by means of specific enzymes: I. Determination of hydroxypurines [J]. J Biol Chem, 1947,167:429-443.
[3] Parks RE, Agarwal RP. Purine nucleoside phosphorylase [J]. Enzymes, 1972, 7:483-514.
[4] Bzowska A, Kulikowska E, Shugar D. Purine nucleoside phosphorylases: properties, functions, and clinical aspects [J]. Pharmacol Ther, 2000, 88: 349-425.
[5] Bzowska A, Kulikowska E, Shugar D. Properties of purine nucleoside phosphorylase (PNP) of mammalian and bacterial origin [J]. Z Naturforsch C 1990,45:59-70.
[6] Jensen, KF. Purine nucleoside phosphorylase from Salmonella typhimurium and Escherichia coli. Initial velocity kinetics, ligand binding and reaction mechanism [J]. Eur J Biochem, 1976, 67:377-386.
[7] Robertson CR, Hoffee PA. Purification and properties of purine nucleoside phosphorylase from Salmonella typhimurium [J]. J Biol Chem, 1977,248:2040-2043.
[8] Ling F, Inuoe Y, Kimura A. Purification and characterization of novel nucleoside phosphorylase from Klebsiella sp., its use in the enzymatic production of adenine arabinoside [J]. Appl Environ Microbiol, 1990, 56:3830-3834.
[9] Appleby,TC, Mathews II, Porcelli M, Cacciapuoti G, Ealick SE. Three dimensional structure of a hyperthermophilic 5'-Deoxy-5'- methylthioadenosine phosphorylase from Sulfolobus Solfataricus [J]. J Biol Chem, 2001, 276:39232-39242.
[10] Cacciapuoti G, Forte S, Moretti MA et al.A novel hyperthermostable 5'-deoxy- 5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus [J]. FEBS J, 2005, 272:1886-1899.
[11] Cercignani, G. Substrate specificities of adenosine deaminase and adenosine phosphorylase from Bacillus cereus [J]. Ital J Biochem, 1982, 32:243-252.
[12] Hamamoto T, Noguchi T, Midorikawa Y. Purification and characterization of purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase from Bacillus stearothermophilus TH 6-2 [J]. Biosci Biotechnol Biochem, 1996,60:11790-1180.
[13] Pugmire MJ, Ealick SE. Structural analysis reveals two distinct families of nucleoside phosphorylases [J]. Biochem J, 2002, 361:1-25.
[14] Yang Z, Parker WB, Sorscher EJ, Ealick SE. PNP Anticancer gene therapy [J].Current Topics in Medicinal Chemistry, 2005, 5:1259-1274.
[15] Seeger C, Poulsen C, Dandanell G. Identification and characterization of genes (xapA, xapB, and xapR) involved in xanthosine catabolism in Escherichia coli [J]. J Bacteriol, 1995,177:5506-5516.
[16] Bennett EM, Li C, Allan PW, Parker WB, Ealick SE. Structural basis for substrate specificity of Escherichia coli purine nucleoside phosphorylase [J]. J Biol Chem, 2003, 278:47110-47118.
[17] Shugar D, Saenger W, Koellner G. Crystal structure of the purine nucleoside phosphorylase (PNP) from Cellulomonas sp. and its implication for the mechanism of trimeric PNPs [J]. J Mol Biol, 1999,294:1239-1255.
[18] Cacciapuoti G, Fusco S, Caiazzo N, et al. Heterologous expression of 5'- methyl thioadenosine phosphorylase from the archaeon Sulfolobus solfataricus: characterization of the recombinant protein and involvement of disulfide bonds in thermophilicity and thermostability [J]. Protein Expression Purification, 1999,16:125-135.
[19] Cacciapuoti G, Porcelli M, et al. Purification and characterization of extremely thermophilic and thermostable 5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus: purine nucleoside phosphorylase activity and evidence for intersubunit disulfide bonds [J]. J Biol Chem, 1994, 269: 24762-24769.
[20] Hori N, Watanabe M, et al. Purification and characterization of thermostable purine nucleoside phosphorylase of Bacillus stearothermophilus JTS 859 [J].Agric Biol Chem, 1989, 53:2205-2210.
[21] Krenitsky TA, Koszalka GW, Tuttle JV. Purine nucleoside synthesis, an efficient method employing nucleoside phosphorylases [J]. Biochemistry, 1981, 20:3615-3621.
[22] Tebbe J, Wielgus-Kutrowska B. et al. Purine nucleoside phosphorylase (PNP) from Cellulomonas sp., a different class of PNP different from both "low-molecular weight" mammalian and "high-molecular weight" bacterial PNPs [J]. Protein Eng (suppl.), 1997, 10:90.
[23] Krenitsky TA., Tuttle JV. Correlation of substrate stabilization patterns with proposed mechanisms for three nucleoside phosphorylases [J]. Biochim Biophys Acta, 1982, 703:247-249.
[24] Cacciapuoti G, Servillo L, Moretti M, Porcelli M. Conformational changes and stabilization induced by phosphate binding to 5'-methylthioadenosine phosphorylase from the thermophilic archaeon Sulfolobus solfataricus [J]. Extremophiles, 2001:5:295-302.
[25] Porter DJ. Purine nucleoside phosphorylase. Kinetic mechanism of the enzyme from calf spleen [J]. J Biol Chem, 1992, 267:7342-7351.
[26] Accorsi A, Piacentini MP, et al. Purine nucleoside phosphorylase from human erythrocytes: a kinetic study of the fully separated isoenzymes [J]. Biochem Int,1991,24:23-31.
[27] Jensen KF. Purine nucleoside phosphorylase from Salmonella typhimuriwn and Escherichia coli. Initial velocity kinetics, ligand binding and reaction mechanism [J]. Eur J Biochem, 1976, 67:377-386.
[28] Erion M D, Stoeckler JD, Guida WC, Walter RL, Ealick SE. Purine nucleoside phosphorylase. 2. Catalytic mechanism [J]. Biochemistry, 1997, 36:11735-11748.
[29] Fedorov A, Shi W, Kicska G, Transition state structure of purine nucleoside phosphorylase and principles of atomic motion in enzymatic catalysis [J]. Biochemistry, 2001,40:853-860.
[30] Kim BK, Cha S, Parks RE. Purine nucleoside phosphorylase from human erythrocytes: II. Kinetic analysis and substrate-binding studies [J].J Biol Chem, 1968,243:1771-1776.
[31] Koszalka GW, Vanhooke J, Short SA, Hall WW. Purification and properties of inosine-guanosine phosphorylase form Escherichia coli K-12 [J]. J Bacteriol,1988, 170:3493-3498.
[32] Erion MD, Takabayashi K, Smith HB, et al. Purine nucleoside phosphorylase. 1.Structure-function studies [J]. Biochemistry, 1997, 36: 11725-11734.
[33] Mao C, Cook WJ, Zhou M, et al. The crystal structure of Escherichia coli purine nucleoside phosphorylase: a comparison with the human enzyme reveals a conserved topology [J]. Structure, 1997, 5:1373-1383.
[34] Stoeckler JD, Poirot AF, Smith RM, et al. Purine nucleoside phosphorylase: 3. Reversal of purine base specificity by site- directed mutagenesis[J].Biochemistry,1997,36:11749-11756.
[35]Hanrahan JR,Hutchinson DW.The enzymatic synthesis of antiviral agents[J].J Biotechnol,1992,23(1):95-212.
[36]闫蓬勃,丁庆豹,邱蔚然,等.酶法合成抗癌新药氟铁龙前体—5-FUR[J].华东理工大学学报,2001,27(3):143-146.
[37]林珏,蒋红,唐上华,等.酶法生产抗艾滋病药物AZT的前体—5-甲基尿嘧啶核苷的研究[J].工业微生物,1993,23(1):1-4.
[38]Shirae H,Yodozeki K,Uchiyama M,et al.Enzymatic production of ribavirin from purine nucleosides by Brevibacterium acetylicum ATCC954[J].Agric Biol Chem,1998,52(4):1777-1783.
[39]邱蔚然,唐洁宇,高媛,唐孝宣.山鸟苷经酶法生产利巴韦林[J].中国医药工业杂志,1997,28(1):14-17.
[40]Suresh P,Naur V.Enzymatic synthesis of thymidine using bacterial whole cells and isolated purine nucleoside phosphorylase[J].Biocatalysis and Biotransformation,1997,15:147-158.
[41]Ishige T,Honda K,Shimizu S.Whole organism biocatalysis[J].Curt opin Chem Biol,2005,9:I74-80.
[42]Rogert MC,Trelles JA,Porto S.Microbial synthesis of antiviral nucleosides using Escherichia coli BL21 as biocatalyst[J].Biocatalysis and Biotransfonnation,2002,20(5):347-351.
[43]Trelles JA,Fernandez J,Condezo LA,et al.Nucleoside synthesis by immobilised bacterial whole cell[J].Journal of Molecular Catalysis B:Enzymatic,2004,30:219-227.
[44]Deharvengt S;Wack S;Uhring M,Aprahamian M,Haiti A.Suicide gene/prodrug therapy for pancreatic adenocarcinoma by E.coli purine nucleoside phosphorylase and 6-methylpurine 2'-deoxyriboside[J].Pancreas,2004,28:54-64.
[45]Hassan AE,Shortnacy AT,Montgomery JA,Secrist JA.A convenient synthesis of 2'-deoxy-2-fluoroadenosine:a potential prodrug for sucide gene therapy[J].Nucleosides Nucelotides Nucleic Acids,2000,19:559-565.
[46]Elizabeth SL,Martinez N,Rogert MC.An improved microbial synthesis of purine nucleosides[J].Biotechnology Letters,2000,22:1277-1280.
[47]Kicska GA,Tyler PC,Evans,GB,et al.Purine-less death in Plasmodium falciparum induced by immucillin H, a transition state analogue of purine nucleoside phosphorylase [J]. J Biol Chem, 2002,277:3226- 3231.
[48] Giblett ER, Ammann AJ, Wara DW, et al. Nucleoside-phosphorylase deficiency in a child with severely defective T-cell immunity and normal B-cell immunity [J]. Lancet, 1975, 1: 10101-1013.
[49] Bzowska A, Kulikowska E, Darzynkiewcz E, Shugar D. Purine nucleoside phosphorylase. Structure activity relationships for substrate and inhibitor properties of N-1-, N-7-, and C-8-substituted analogues: differentiation of mammalian and bacterial enzymes with N-1-methy- linosine and guanosine [J]. J Biol Chem, 1988,263:9212-9217.
[50] Sorscher EJ, Peng S, Bebok Z, Allan PA, Bennett LL, Parker J, WB Tumour bystander killing in colonic carcinoma utilizing the Escherichia coli deo D gene to generate toxic purines [J]. Gene Ther, 1994,1: 233-238.
[51] Mohr L, Shankara S, Yoon SK, et al. Gene therapy of hepatocellular carcinoma in vitro and in vivo in nude mice by adenoviral transfer of the Escherichia coli purine nucleoside phosphorylase gene [J]. Hepatology, 2000, 31: 606-614.
[52] Hughes BW, King SA, Allen PW, Parker WB, Sorscher EJ. Cell to cell contact is not required for bystander cell killing by Escherichia coli purine nucleoside phosphorylase [J]. J Biol Chem, 1998, 273: 2322-2328.
[53] Sorscher EJ, Townes TM, Waud WR, Ealick SE. Designer gene therapy using an Escherichia coli purine nucleosidephosphorylase/prodrug system [J]. Chem Biol, 2003, 10:1173-1181.
[1]范光南,傅世宗,蔡海洋.极端环境微生物研究概况[J].福建热作科技,2000.2:12-15.
[2]Forster J.Uber einige Eigenschaften leuchtender Bakterien[J].Centr Bakteriol Parasitenk,1887,2:337-340.
[3]Schmidt-Nielsen,S.Ueber einige psychrophile Mikroorganismen und ihr Vorkommen[J].Centralbl.Bakteriol.Parasitenkd.Ⅱ Abt,1902,9:145-147.
[4]Ingraham JL and Stokes JL.Psychrophilic bacteria[J].Microbiol Mol Biol Rev,1959,23(3):97-108.
[5]Morita RY.Psychrophilic bacteria[J].Bacteriol Rev,1975,39:144-167.
[6]Deming JW.Psychrophiles and polar regions[J].Current opinion in Microbiology,2002,5:301-309.
[7]Friedmann EI.Endolithic microorganisms in the Antarctic cold desert[J].Science,1982,215:1045-1053.
[8]Rivkina EM,Friedmann El,Mckay CP,et al.Metabolic activity of permafrost bacteria below the freezing point[J].Appl Environ Microbiol,2000,66:3230-3233.
[9]Christner BC,Mosley-Thompson E,Thompson LG,et al.Isolation of bacteria and 16S rDNAs from Lake Vostok accretion ice[J].Environ Microbiol,2001,3:570-577.
[10]Breezee J,Cady N,Staley JT.Subfreezing growth of the sea ice bacterium "Psychromonas ingrahamii"[J].Microb Ecol,2004,47:300-304.
[11]Panikov NS,Flanaganb PW,Oechelc WC,Mastepanovd MA,Christensend TR.Microbial activity in soils frozen to below -39℃[J].Soil Biol Biochem,2006,38:785-794.
[12]辛明秀,马延和.嗜冷菌和耐冷菌[J].微生物学通报,1999(2):109-155.
[13]Price PB.Microbial life in glacier ice and implications for a cold origin of life.FEMS Microbiol Ecol,2007,59:217-231.
[14]Miteva VI,Sheridan PP,Brenchley JE.Phylogenetic and physiological diversity of microorganisms isolated from a deep greenland glacier ice core[J].Appl Environ Microbial,2004,70(1):202-213.
[15]张国华,罗会颖,陈劲春,姚斌.新疆天山雪莲根部冻土产酶微生物的初步研究[J].中国农业科技导报,2008,10(2):82-87.
[16]Tanner AC.and Herbert RA.A numerical taxonomic study of Gram-negative bacteria isolated from the Antarctic marine environment[J].CNEXO(Actes Colloq.),1982,14:31-38.
[17]李会荣,俞勇,曾胤新,陈波,任大明.北极太平洋扇区海洋沉积物细菌多样性的系统发育分析[J].微生物学报,2006,46(2):177-183.
[18]曾胤新,陈波.低温微生物适冷特性及其在食品工业中的潜在用途[J].2000,10(2):31-37.
[19]Nichols PD,McMeekin TA.Polyunsaturated fatty acids in Antarctic bacteria[J].Antarctic Science,1993,5:149-160.
[20]Volkman JK,Burton HR,Everitt DA,Allen DI.Pigment and lipid compositions of algal and bacterial communities in Ace Lake,Vestfold Hills,Antarctica[J].Hydrobiologia,1988,165(1):41-57.
[21]David SN,June O,Horacio G,Rodolfo RB,Tom AM.Effect of temperature and salinity stress on growth and lipid composition of Shewanella gelidimarina[J].Applied and Environmental Microbiology,2000,66(6):2422-2429.
[22]Ray MK,Kumar GS,Shivaji S.Phosphorylation of lipopolysaccharides in the Antarctic psychrotroph Pseudomonas syringae:a possible role in temperature adaptation[J].J Bacteriol.,1994,176(14):4243-4249.
[23]Phadtare S.Recent developments in bacterial cold-shock response[J].Curr Issues Mol Biol,2004,6:125-136.
[24]Takayuki H,Hitoshi I,Yasuhiko K.Yeast gene expression during growth at low temperature[J].Cryobiology,2003,46(3):230-237.
[25]Kawamoto J,Kurihara T,Kitagawa M,Kato I,Esaki N.Proteomic studies of an Antarctic cold-adapted bacterium,Shewanella livingstonensis Ac10,for global identification of cold-inducible proteins[J].Extremophiles,2007,11(6):819-826.
[26]Jiang W,Hou Y,Inouye M.CspA,the major cold-shock protein of Escherichia coli,is an RNA chaperone[J].J Biol Chem,1997,272:196-202.
[27]Thomas T.Archaeal cold-adapted proteins:structural and evolutionary analysis of the elongation factor 2 proteins from psychrophilic,mesophilic and thennophilic methanogens[J].FEBS Letters,439(3):281-286.
[28]Yutaka S,Mitsuru H,Kazufumi T,Masaaki M,Shigenori K.Possible involvement of an FKBP family member protein from a psychrotrophic bacterium Shewanella sp.SIB1 in cold-adaptation[J].European Journal of Biochemistry,2004,271(7):1372-1381.
[29]Julianne L,Torsten T,Ricardo C.Low temperature regulated DEAD-box RNA helicase from the antarctic archaeon,Methanococcoides burtoniil[J].Journal of Molecular Biology,2000,297(3):553-567.
[30]Michel V,Lehoux I,Depret G,Anglade P,Labadie J,Hebraud M.The cold shock response of the psychrotrophic bacterium Pseudomonas fragi involves four low-molecular-mass nucleic acid-binding proteins[J].J Bacteriol,1997,179:7331-7342.
[31]李芳,王博,艾秀莲,王志方,付建红.抗冻蛋白研究进展[J].新疆农业科学,2003,40(6):349-352.
[32]Julseth CR,Inniss WE.Induction of protein synthesis in response to cold shock in the psychrotrophic yeast Trichosporon pullans[J].Can J Microbiol 1990,36:519-524.
[33]唐赟.嗜冷菌适应低温的分子机制及其应用[J].西华师范大学学报(自然科学版),2004,25(4):388-393.
[34]辛明秀,周培瑾.冷活性琥珀酸脱氢酶的特性[J].北京师范大学学报(自然科学版),2004,40(1):108-113.
[35]Margesin R,Schinner F.Biotechnological applications of cold-adapted organisms[M].Heidelberg:Springer-Verlag,1999.
[36]辛明秀,周培瑾.温度对嗜冷酵母糖代谢途径某些关键酶的活性效应[J].微生物学报,2000,40(5):518-522.
[37]曾胤新,蔡明宏,陈波,等.一株产蛋白酶南极耐冷细菌的筛选及研究[J].生物技术,2001,11(2):17-20.
[38]Zeng YX,Chen B.Isolation and characteristics of one marine psychrotrophic celulase-generating bacterium Ar/w/b/75/10/5 from Chuckchi Sea,Arctic[J].Chinese Journal of Polar Science,2002,13(2):157-168.
[39]Nicholas JR.Toward a molecular understanding of cold activity of enzyme from psychrophiles[J].Extremophiles,2000,4:83-90.
[40]曾胤新,俞勇,蔡明宏,何剑锋,陈波.低温微生物及其酶类的研究概况[J].微生物学杂志,2004,24(5):83-88.
[41]Huston AL,Krieger-Brocket BB,Deming JW.Remarkable low temperature optima for extracelular enzyme activity from Arctic bacteria and sea ice[J].Environ Microbiol,2000,2(4):383-388.
[42]张锐,曾润颖.极端微生物产碱性蛋白酶菌株的筛选及发酵条件研究[J].微生物学通报,2001,28(4):5-9.
[43]Sawabe T,Takahasi H,Saeki H,Niwa K,Aonob H.Enhanced expression of active recombinant alginate lyase AlyPEEC cloned from a marine bacterium Pseudoalteromonas elyakovii in Escherichia coli by calcium compounds[J].Enzyme and Microbial Technology,2007,40:285-291.
[44]Ochiai T,Fukunaga N,Sasaki S.P urification and some properties of two NADP~+-specific isocitrate dehydrogenases from an obligately psychrophilic marine bacterium,Vibrio sp.strain ABE-I[J].J Biochem(Tokyo),1979,86(2):377-38.
[45]D'Amico S,Marx JC,Gerday C,Feller G.Activity stability relationships in extremophilic enzymes[J].J Biol Chem,2003,278:7891-7896.
[46]Feller G,Narinx E,Arpigny JL,Aittaleb M,Baise E,Genicot S,Gerday C.Enzymes from psychrophilic organisms[J].FEMS Microbiol Rev,1996,18:189-202.
[47]Feller G,Gerday C.Psychrophilic enzymes:hot topics in cold adaptation[J].Nature Rev Microbiol,2003,1:200-208.
[48]Feller G,et al.Cloning and expression in Escherichia coli of three lipaseencoding genes from the psychrotrophic antarctic strain Moraxella TA44[J].Gene,1991,102:111-115.
[49]Arpigny JL,et al.Cloning,sequence and structural features of a lipase from the antarctic facultative psychrophile Psychrobacter immobilis B10[J].Biochim.Biophys Acta.1993,1171:331-333.
[50]Rentier-Delrue F et al.Cloning and overexpression of the triose phosphate isomerase genes from psychrophilic and thermophilic bacteria structural comparison of the predicted protein sequence[J].J Mol Biol,1993,229:85-93.
[51]Garsoux G,Lamotte J,Gerday C,Feller G.Kinetic and structural optimization to catalysis at low temperatures in a psychrophilic cellulase from the Antarctic bacterium Pseudoalteromonas haloplanktis[J].Biochem J,2004,384:247-253.
[52]Feller G,Gerday C.Psychrophilic enzymes:molecular basis of cold adaptation [J].Cell Mol Life Sci,1997,53:830-841.
[53]孙谧,洪义国,李勃生,修朝阳.海洋微生物低温酶特性及其在工业中的潜在用途[J].海洋水产研究,2002,23(3):44-49.
[54]朱非,王珊,周培瑾.低温酶冷适应的分子机制及其在生物技术中的应用[J].微生物学报,2002,42(5):640-644.
[55]Welander U.Microbial degradation of organic pollutants in soil in a cold climate [J].Soil and Sediment Contamination,2005,14(3):281-291.
[56]Arianna T,Józef K,Aneta B,Hubert C,Halina K,Stanis B.Antarctic marine bacterium Pseudoalteromonas sp.22b as a source of cold- adapted β-galactosidase[J].Biomolecular Engineering,2003,20(5):317-324.
[57]Collins T,Hoyoux A,Dutron A,Georis J,Genot B,Dauvrin T,Arnaut F,Gerday C,Feller G.Use of glycoside family 8 xylanases in baking[J].J Cereal Sci,2006,43:79-84.
[58]Margesin R,et al.Psychrophiles:from Biodiversity to Biotechnology[M].Berlin Heidelberg:Springer-Verlag,2008:347-360.
[59]Rina M,Pozidis C,Mavromatis K,Tzanodaskalaki M,Kokkinidis M,Bouriotis V.Alkaline hosphatase from the Antarctic strain TAB5[J].Eur J Biochem,2000,67:1230-1238.
[60]Van den Burg B.Extremophiles as a source for novel enzymes[J].Current Opin Microbiol,2003,6:213-218.
[61]Asenjo JA,Andrews BA,Reyes F,Salamanca M,Burzio L.Protein and nucleic acid sequence encoding a krill-derived cold adapted trypsin-like activity enzyme [P].Patent No.WO2006022947,2006.
[62]李丽娟,张殿昌,龚世园.宏基因组技术在丌发未培养微生物资源中的应用[J].水利渔业,2007,3:13-15.
[63]Amann RI,Ludwig W,Schleifer KH.Phylogenetic identification and in situ detection of individual microbial cells without cultivation[J].Microbiol Rev,1995,59(1):143-169.
[64]张金伟,曾润颖.南极深海沉积物宏基因组DNA中低温脂肪酶基因的克隆,表达及性质分析[J].生物化学与生物物理进展,2006,33(12):1207-1214.
[65]Rees H,Grant S,Jones B,Grant W,I4eaply S.Detecting cellulase and esterase enzyme activities encoded by novel genes present in environmental DNA libraries[J].Extremophiles,2003,7:415-421.
[66]钱小红,贺福初.蛋白质组学:理论与方法[M]北京:科学出版社,2003:10-25.
[67]张德超.我国新疆一号冰川地区和北极地区低温菌的鉴定及多相分类学研究[D].北京:中国农业大学,2007.
[68]朱非.低温菌的多相分类学及系统发育学研究低温蛋白酶的分离纯化及性质[D].北京:中科院研究生院,2002.
[69]王玢.海洋适冷细菌MB-1产纤维素酶的研究[D].济南:山东大学,2003.
[70]陈秀兰.海洋适冷蛋白酶产生菌的筛选、酶的分离纯化及适冷机制研究[D].济南:山东大学,2002.
[71]连明珠.南极Pseudoalteromoflas sp.AC444低温几丁质酶性质分析与基因克隆及普早兹湾深海沉积物中几丁质酶基因多样性研究[D].厦门:厦门大学,2006.
[72]Hu JM,Li Hu H,Cao LX,et al.Molecular cloning and characterization of the gene encoding cold-active β-Galactosidase from a psychrotrophic and halotolerant Planococcus sp.L4[J].J Agric Food Chem,2007,55,2217-2224.
[73]孙谧.一种新型低温碱性蛋白酶、制造方法、应用和产生该蛋白酶的微生物[P].中国专利:00123404.8,2001-1-17.
[74]孙谧.一种新型海洋溶菌酶膜剂及其制备方法[P].中国专利:200510000033.7,2005-8-31.