融合双亲短肽提高腈水合酶的热稳定性研究
|
摘要
腈水合酶(Nitirle hydratase,NHase)催化腈类物质转化为酰胺类物质,目前用于工业生产丙烯酰胺。但在催化过程中释放的热量易导致酶分子失活。研究通过蛋白质融合技术对腈水合酶进行分子改造,提高热稳定性。将2种双亲自组装肽(self-assembling peptides,SAPs)EAK16和ELK16分别融合至恶臭假单胞菌Pseudomonas putida NRRL-18668来源NHase非催化亚基β的N末端,构建出2种融合型NHase:EAK16-NHase和ELK16-NHase。经过表达、纯化后测定酶活力,发现EAK16-NHase和ELK16-NHase的酶活力分别为(426±14)U/mg和(372±12)U/mg,保留野生型酶活力的97%和85%。在50℃条件下孵育0~60 min,每5 min取样后测定残存酶活力,EAK16-NHase和ELK16-NHase酶活力半衰期(T50)分别为35 min和40 min,野生型NHase为20 min。说明融合EAK16和ELK16均能提高NHase的热稳定性。研究表明融合SAPs能在不显著影响酶活力的条件下提高酶的热稳定性。
Nitrile hydratase( NHase) catalyzes nitriles to the amides,it is at present widely used in industrial production of acrylamide. However,the heat released during the catalysis process easily inactivated the enzyme molecule involved. In this study,protein engineering by translational fusion was employed to enhance the thermo-stability of the enzyme. Herein,two types of self-assembling peptides( SAPs) EAK16 and ELK16 was translationally fused to the non-catalytic subunit β of NHase from Pseudomonas putida NRRL-18668,yielding two chimeric fusions NHase EAK16-NHase and ELK16-NHase. Next,the two types of recombinant proteins were expressed in E. coli and purified before the enzyme activity were determined. It was found that the specific activity for EAK16-NHase and ELK16-NHase was( 426 ± 14) U / mg and( 372 ± 12) U / mg respectively,reserved 97% and 85% of that in wild-type NHase. Furthermore,after incubation of the wild-type NHase and chimeric NHase at 50 ℃ for a period of 0 min to 60 min and sampled at the interval of 5 min,the residual activity was determined. The data showed that the half-life of the enzymatic activity( T50) for EAK16-NHase and ELK16-NHase was 35 min and 40 min,respectively,while the wild-type enzyme was 20 min,indicating that translational fusion of SAPs was able to augment the thermo-stability of NHase. These results manifested that the thermo-stability was actually able to enhance that was not bound to at the cost of losing activity.
Nitrile hydratase( NHase) catalyzes nitriles to the amides,it is at present widely used in industrial production of acrylamide. However,the heat released during the catalysis process easily inactivated the enzyme molecule involved. In this study,protein engineering by translational fusion was employed to enhance the thermo-stability of the enzyme. Herein,two types of self-assembling peptides( SAPs) EAK16 and ELK16 was translationally fused to the non-catalytic subunit β of NHase from Pseudomonas putida NRRL-18668,yielding two chimeric fusions NHase EAK16-NHase and ELK16-NHase. Next,the two types of recombinant proteins were expressed in E. coli and purified before the enzyme activity were determined. It was found that the specific activity for EAK16-NHase and ELK16-NHase was( 426 ± 14) U / mg and( 372 ± 12) U / mg respectively,reserved 97% and 85% of that in wild-type NHase. Furthermore,after incubation of the wild-type NHase and chimeric NHase at 50 ℃ for a period of 0 min to 60 min and sampled at the interval of 5 min,the residual activity was determined. The data showed that the half-life of the enzymatic activity( T50) for EAK16-NHase and ELK16-NHase was 35 min and 40 min,respectively,while the wild-type enzyme was 20 min,indicating that translational fusion of SAPs was able to augment the thermo-stability of NHase. These results manifested that the thermo-stability was actually able to enhance that was not bound to at the cost of losing activity.
引文
[1]Prasad S,Bhalla TC.Nitrile hydratases(NHases):at the interface of academia and industry[J].Biotechnol Adv,2010,28(6):725-741.
[2]Wang YJ,Zheng YG,Xue JP,et al.Characterization of nitrile hydratation catalysed by Nocardia sp.108[J].World J Microb Biot,2007,23(3):355-362.
[3]Okamoto S,Eltis LD.Purification and characterization of a novel nitrile hydratase from Rhodococcus sp.RHA1[J].Mol Microbiol,2007,65(3):828-838.
[4]Alfani F,Cantarella M,Spera A,et al.Operational stability of Brevibacterium imperialis CBS 489-74 nitrile hydratase[J].J Mol Catal B-Enzym,2001,11(4-6):687-697.
[5]Miyanaga A,Fushinobu S,Ito K,et al.Crystal structure of cobalt-containing nitrile hydratase[J].Biochem Biophys Res Commun,2001,288(5):1169-1174.
[6]Petrillo KL,Wu S,Hann EC,et al.Over-expression in Escherichia coli of a thermally stable and regio-selective nitrile hydratase from Comamonas testosteroni 5-MGAM-4D[J].Appl Microbiol Biotechnol,2005,67(5):664-670.
[7]Foerstner KU,Doerks T,Muller J,et al.A nitrile hydratase in the eukaryote Monosiga brevicollis[J].PLo S One,2008,3(12):e3976.
[8]Nagashima S,Nakasako M,Dohmae N,et al.Novel non-heme iron center of nitrile hydratase with a claw setting of oxygen atoms[J].Nat Struct Biol,1998,5(5):347-351.
[9]Kobayashi M,Shimizu S.Cobalt proteins[J].Eur J Biochem,1999,261(1):1-9.
[10]Cui Y,Cui W,Liu Z,et al.Improvement of stability of nitrile hydratase via protein fragment swapping[J].Biochem Biophys Res Commun,2014,450(1):401-408.
[11]Zhou Z,Hashimoto Y,Kobayashi M.Self-subunit swapping chaperone needed for the maturation of multimeric metalloenzyme nitrile hydratase by a subunit exchange mechanism also carries out the oxidation of the metal ligand cysteine residues and insertion of cobalt[J].J Biol Chem,2009,284(22):14930-14938.
[12]Fung SY,Yang H,Bhola PT,et al.Self-Assembling Peptide as a Potential Carrier for Hydrophobic Anticancer Drug Ellipticine:Complexation,Release and In Vitro Delivery[J].Adv Funct Mater,2009,19(1):74-83.
[13]Ge J,Lu DN,Yang C,et al.A Lipase-Responsive Vehicle Using Amphipathic Polymer Synthesized with the Lipase as Catalyst[J].Macromol Rapid Comm,2011,32(6):546-550.
[14]Schoch GA,Attias R,Belghazi M,et al.Engineering of a water-soluble plant cytochrome P450,CYP73A1,and NMR-based orientation of natural and alternate substrates in the active site[J].Plant Physiol,2003,133(3):1198-1208.
[15]Lu X,Liu S,Zhang D,et al.Enhanced thermal stability and specific activity of Pseudomonas aeruginosa lipoxygenase by fusing with self-assembling amphipathic peptides[J].Appl Microbiol Biotechnol,2013,97(21):9419-9427.
[16]Wang Q,Yang Z,Gao Y,et al.Enzymatic hydrogelation to immobilize an enzyme for high activity and stability[J].Soft Matter,2008,4(3):550-553.
[17]余越春,崔文璟,刘义,等.Rhodococcus rhodochrous J1腈水合酶在大肠杆菌中的表达策略[J].工业微生物,2014,44(2):14-19.
[18]Liu Y,Cui W,Xia Y,et al.Self-subunit swapping occurs in another gene type of cobalt nitrile hydratase[J].PLo S One,2012,7(11):e50829.
[19]Nagasawa T,Takeuchi K,Yamada H.Characterization of a New Cobalt-Containing Nitrile Hydratase Purified from Urea-Induced Cells of Rhodococcus-Rhodochrous J1[J].Eur J Biochem,1991,196(3):581-589.
[20]沈嘉澍,沈标.嗜热酶的耐热机理[J].微生物学杂志,2010,30(2):80-85.
[21]Zhang S.Fabrication of novel biomaterials through molecular self-assembly[J].Nat Biotechnol,2003,21(10):1171-1178.
[22]Zhao XB,Pan F,Lu JR.Recent development of peptide selfassembly[J].Prog Nat Sci-Mater,2008,18(6):653-660.
[23]Davis ME,Hsieh PC,Takahashi T,et al.Local myocardial insulin-like growth factor 1(IGF-1)delivery with biotinylated peptide nanofibers improves cell therapy for myocardial infarction[J].Proc Natl Acad Sci U S A,2006,103(21):8155-8160.
[24]Bokhari MA,Akay G,Zhang S,et al.The enhancement of osteoblast growth and differentiation in vitro on a peptide hydrogelpoly HIPE polymer hybrid material[J].Biomaterials,2005,26(25):5198-5208.
[25]Fung SY,Yang H,Chen P.Formation of colloidal suspension of hydrophobic compounds with an amphiphilic self-assembling peptide[J].Colloid Surface B,2007,55(2):200-211.
[26]Wu W,Xing L,Zhou BH,et al.Active protein aggregates induced by terminally attached self-assembling peptide ELK16 in Escherichia coli[J].Microb Cell Fact,2011,10(1):9.
[2]Wang YJ,Zheng YG,Xue JP,et al.Characterization of nitrile hydratation catalysed by Nocardia sp.108[J].World J Microb Biot,2007,23(3):355-362.
[3]Okamoto S,Eltis LD.Purification and characterization of a novel nitrile hydratase from Rhodococcus sp.RHA1[J].Mol Microbiol,2007,65(3):828-838.
[4]Alfani F,Cantarella M,Spera A,et al.Operational stability of Brevibacterium imperialis CBS 489-74 nitrile hydratase[J].J Mol Catal B-Enzym,2001,11(4-6):687-697.
[5]Miyanaga A,Fushinobu S,Ito K,et al.Crystal structure of cobalt-containing nitrile hydratase[J].Biochem Biophys Res Commun,2001,288(5):1169-1174.
[6]Petrillo KL,Wu S,Hann EC,et al.Over-expression in Escherichia coli of a thermally stable and regio-selective nitrile hydratase from Comamonas testosteroni 5-MGAM-4D[J].Appl Microbiol Biotechnol,2005,67(5):664-670.
[7]Foerstner KU,Doerks T,Muller J,et al.A nitrile hydratase in the eukaryote Monosiga brevicollis[J].PLo S One,2008,3(12):e3976.
[8]Nagashima S,Nakasako M,Dohmae N,et al.Novel non-heme iron center of nitrile hydratase with a claw setting of oxygen atoms[J].Nat Struct Biol,1998,5(5):347-351.
[9]Kobayashi M,Shimizu S.Cobalt proteins[J].Eur J Biochem,1999,261(1):1-9.
[10]Cui Y,Cui W,Liu Z,et al.Improvement of stability of nitrile hydratase via protein fragment swapping[J].Biochem Biophys Res Commun,2014,450(1):401-408.
[11]Zhou Z,Hashimoto Y,Kobayashi M.Self-subunit swapping chaperone needed for the maturation of multimeric metalloenzyme nitrile hydratase by a subunit exchange mechanism also carries out the oxidation of the metal ligand cysteine residues and insertion of cobalt[J].J Biol Chem,2009,284(22):14930-14938.
[12]Fung SY,Yang H,Bhola PT,et al.Self-Assembling Peptide as a Potential Carrier for Hydrophobic Anticancer Drug Ellipticine:Complexation,Release and In Vitro Delivery[J].Adv Funct Mater,2009,19(1):74-83.
[13]Ge J,Lu DN,Yang C,et al.A Lipase-Responsive Vehicle Using Amphipathic Polymer Synthesized with the Lipase as Catalyst[J].Macromol Rapid Comm,2011,32(6):546-550.
[14]Schoch GA,Attias R,Belghazi M,et al.Engineering of a water-soluble plant cytochrome P450,CYP73A1,and NMR-based orientation of natural and alternate substrates in the active site[J].Plant Physiol,2003,133(3):1198-1208.
[15]Lu X,Liu S,Zhang D,et al.Enhanced thermal stability and specific activity of Pseudomonas aeruginosa lipoxygenase by fusing with self-assembling amphipathic peptides[J].Appl Microbiol Biotechnol,2013,97(21):9419-9427.
[16]Wang Q,Yang Z,Gao Y,et al.Enzymatic hydrogelation to immobilize an enzyme for high activity and stability[J].Soft Matter,2008,4(3):550-553.
[17]余越春,崔文璟,刘义,等.Rhodococcus rhodochrous J1腈水合酶在大肠杆菌中的表达策略[J].工业微生物,2014,44(2):14-19.
[18]Liu Y,Cui W,Xia Y,et al.Self-subunit swapping occurs in another gene type of cobalt nitrile hydratase[J].PLo S One,2012,7(11):e50829.
[19]Nagasawa T,Takeuchi K,Yamada H.Characterization of a New Cobalt-Containing Nitrile Hydratase Purified from Urea-Induced Cells of Rhodococcus-Rhodochrous J1[J].Eur J Biochem,1991,196(3):581-589.
[20]沈嘉澍,沈标.嗜热酶的耐热机理[J].微生物学杂志,2010,30(2):80-85.
[21]Zhang S.Fabrication of novel biomaterials through molecular self-assembly[J].Nat Biotechnol,2003,21(10):1171-1178.
[22]Zhao XB,Pan F,Lu JR.Recent development of peptide selfassembly[J].Prog Nat Sci-Mater,2008,18(6):653-660.
[23]Davis ME,Hsieh PC,Takahashi T,et al.Local myocardial insulin-like growth factor 1(IGF-1)delivery with biotinylated peptide nanofibers improves cell therapy for myocardial infarction[J].Proc Natl Acad Sci U S A,2006,103(21):8155-8160.
[24]Bokhari MA,Akay G,Zhang S,et al.The enhancement of osteoblast growth and differentiation in vitro on a peptide hydrogelpoly HIPE polymer hybrid material[J].Biomaterials,2005,26(25):5198-5208.
[25]Fung SY,Yang H,Chen P.Formation of colloidal suspension of hydrophobic compounds with an amphiphilic self-assembling peptide[J].Colloid Surface B,2007,55(2):200-211.
[26]Wu W,Xing L,Zhou BH,et al.Active protein aggregates induced by terminally attached self-assembling peptide ELK16 in Escherichia coli[J].Microb Cell Fact,2011,10(1):9.