Appropriate Cold Bacteria Pseudoal Teromonas Sp.bsi590 Purine Nucleoside Phosphorylation Enzyme Gene Cloning And Enzymatic Properties

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 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 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.