华根霉脂肪酶有机相酯合成活性的重塑
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摘要
研究了具有较高酯合成活性的华根霉膜相关脂肪酶及其沉淀酶蛋白的重折叠处理过程,发现膜成分及表面活性剂可能是影响其活性的关键因素.进一步考察了影响异源表达的可溶性华根霉脂肪酶r27RCL酯合成活性重塑的关键因子及作用阶段.研究结果表明,表面活性剂对脂肪酶的酯合成活性具有关键影响,直接添加表面活性剂可使酯合成活性显著提高.在7种不同表面活性剂中,两性离子表面活性剂LPC14将r27RCL的酯合成活性提高了5. 75倍.分子动力学模拟结果表明,在有机相反应中,表面活性剂的添加使脂肪酶催化三联体之间的氢键作用力得到加强,从而提高了脂肪酶的有机相酯合成能力.
The regaining of ester synthesis catalytic ability of extracted and expressed Rhizopus chinensis lipase was investigated in this work. After de novo folding in a mimicking membrane environment,the maximum ester synthesis specific activity of extracted lipase precipitated protein( eRCL-P) was re-obtained and even increased for over 5 times. The refolding treatment of r27RCL,a Pichia pastoris expressed lipase with a high hydrolysis activity but low ester synthesis activity,with different membrane mimicking factors also increased the organic catalysis ability of the protein. The best enhancement of ester synthesis activity was shown when r27RCL was directly mixed with detergent,which indicated the interaction between lipase and regulation factor was established during mixing stage in water solution. Moreover,seven different detergents of three diversity types were introduced to confirm the result,and the zwitterionic detergent LPC14 increased the ester synthesis activity of r27RCL for the best effect as 5. 75 times. Although the circular dichroism spectrum showed that the secondary structure of r27RCL had no obviously changes,the molecular dynamics simulation demonstrated that,in organic reaction,the hydrogen bonds between the catalytic triad of r27RCL have been enhanced after mixing with detergent,which may therefore stimulate the ester synthesis activity of lipase. In addition,these results confirmed the regulation of lipase ester synthesis activity,which will surely establish the basics for further research and production.
The regaining of ester synthesis catalytic ability of extracted and expressed Rhizopus chinensis lipase was investigated in this work. After de novo folding in a mimicking membrane environment,the maximum ester synthesis specific activity of extracted lipase precipitated protein( eRCL-P) was re-obtained and even increased for over 5 times. The refolding treatment of r27RCL,a Pichia pastoris expressed lipase with a high hydrolysis activity but low ester synthesis activity,with different membrane mimicking factors also increased the organic catalysis ability of the protein. The best enhancement of ester synthesis activity was shown when r27RCL was directly mixed with detergent,which indicated the interaction between lipase and regulation factor was established during mixing stage in water solution. Moreover,seven different detergents of three diversity types were introduced to confirm the result,and the zwitterionic detergent LPC14 increased the ester synthesis activity of r27RCL for the best effect as 5. 75 times. Although the circular dichroism spectrum showed that the secondary structure of r27RCL had no obviously changes,the molecular dynamics simulation demonstrated that,in organic reaction,the hydrogen bonds between the catalytic triad of r27RCL have been enhanced after mixing with detergent,which may therefore stimulate the ester synthesis activity of lipase. In addition,these results confirmed the regulation of lipase ester synthesis activity,which will surely establish the basics for further research and production.
引文
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[33]Tang Q.Y.,Wang Y.H.,Mod Food Sci.&Technol.,2017,(3),179-183(唐庆芸,王永华.现代食品科技,2017,(3),179-183)
[34]Mingarro I.,Abad C.,Braco L.,Proc.Natl.Acad.Sci.USA,1995,92(8),3308-3312
[2]Jaeger K.E.,Eggert T.,Curr.Opin.Biotechnol.,2002,13(4),390-397
[3]Cui L.J.,Xu G.,Meng X.,Wu J.P.,Yang L.R.,Chem.Ind.Eng.Prog.,2014,33(8),2150-2154(崔丽娟,徐刚,孟枭,吴坚平,杨立荣.化工进展,2014,33(8),2150-2154)
[4]Jiang L.,Yu H.,Chem.Res.Chinese Universities,2014,30(3),396-399
[5]Stergiou P.Y.,Foukis A.,Filippou M.,Koukouritaki M.,Parapouli M.,Theodorou L.G.,Hatziloukas E.,Afendra A.,Pandey A.,Papamichael E.M.,Biotechnol.Adv.,2013,31(8),1846-1859
[6]Wang Z.,Luo W.,Fu J.,Li Z.,Wang Z.,LüP.,Chem.Res.Chinese Universities,2017,33(6),1-5
[7]Larios A.,García H.S.,Oliart R.M.,Valerio-Alfaro G.,Appl.Microbiol.Biotechnol.,2004,65(4),373-376
[8]Ghanem A.,Aboul-Enein H.Y.,Tetrahedron:Asymmetry,2004,15(21),3331-3351
[9]Monteiro J.B.,Nascimento M.G.,Ninow J.L.,Biotechnol.Lett.,2003,25(8),641-644
[10]Teng Y.,Xu Y.,Wang D.,J.Mol.Catal.B:Enzym.,2009,57(1-4),292-298
[11]Shah S.,Gupta M.N.,Process Biochem.,2007,42(3),409-414
[12]Karboune S.,Archelas A.,Baratti J.C.,Process Biochem.,2010,45(2),210-216
[13]Oda M.,Kaieda M.,Hama S.,Yamaji H.,Kondo A.,Izumoto E.,Fukuda H.,Biochem.Eng.J.,2005,23(1),45-51
[14]Aarthy M.,Saravanan P.,Gowthaman M.,Rose C.,Kamini N.,Chem.Eng.Res.Des.,2014,92(8),1591-1601
[15]Xu Y.,Wang D.,Mu X.Q.,Zhao G.A.,Zhang K.C.,J.Mol.Catal.B:Enzym.,2002,18(1),29-37
[16]Wang D.,Xu Y.,Teng Y.,Bioprocess Biosyst.Eng.,2007,30(3),147-155
[17]He Q.,Xu Y.,Teng Y.,Wang D.,Chin.J.Catal.,2008,29(1),41-46
[18]Di Lorenzo M.,Hidalgo A.,Haas M.,Bornscheuer U.T.,Appl.Environ.Microbiol.,2005,71(12),8974-8977
[19]Joerger R.D.,Haas M.J.,Lipids,1993,28(2),81-88
[20]Singh A.,Upadhyay V.,Upadhyay A.K.,Singh S.M.,Panda A.K.,Microb.Cell.Fact.,2015,14,41-51
[21]Singh S.M.,Sharma A.,Upadhyay A.K.,Singh A.,Garg L.C.,Panda A.K.,Protein Expr.Purif.,2012,81(1),75-82
[22]Satomura A.,Kuroda K.,Ueda M.,PLoS One,2015,10(5),e0124545
[23]Akbari N.,Khajeh K.,Rezaie S.,Mirdamadi S.,Shavandi M.,Ghaemi N.,Protein Expr.Purif.,2010,70(1),75-80
[24]Martini V.P.,Glogauer A.,Müller-Santos M.,Iulek J.,de Souza E.M.,Mitchell D.A.,Pedrosa F.O.,Krieger N.,Microb.Cell.Fact.,2014,13,171-185
[25]Contreras F.X.,Ernst A.M.,Wieland F.,Brugger B.,Cold Spring Harb.Perspect.Biol.,2011,3,1031-1033
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[27]Takada M.,Takeuchi H.,Shino M.,Hamano S.,Ohgoh T.,Biochem.Biophys.Res.Commun.,1989,164(2),653-663
[28]Yamaguchi H.,Miyazaki M.,Biomolecules,2014,4(1),235-251
[29]Kordel M.,Hofmann B.,Schomburg D.,Schmid R.D.,J.Bacteriol.,1991,173(15),4836-4841
[30]Seddon A.M.,Curnow P.,Booth P.J.,Biochim.Biophys.Acta,2004,1666(1/2),105-117
[31]Otzen D.E.,Biophys.J.,2002,83(4),2219-2230
[32]Carrasco-LóPez C.,Godoy C.,De B.L.R.,Fernández-Lorente G.,Palomo J.M.,Guisán J.M.,Fernández-Lafuente R.,Martínez-Ripoll M.,Hermoso J.A.,J.Biol.Chem.,2009,284(7),4365-4372
[33]Tang Q.Y.,Wang Y.H.,Mod Food Sci.&Technol.,2017,(3),179-183(唐庆芸,王永华.现代食品科技,2017,(3),179-183)
[34]Mingarro I.,Abad C.,Braco L.,Proc.Natl.Acad.Sci.USA,1995,92(8),3308-3312
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