Mechanism of acetaldehyde-induced deactivation of microbial lipases
Tóm tắt
Microbial lipases represent the most important class of biocatalysts used for a wealth of applications in organic synthesis. An often applied reaction is the lipase-catalyzed transesterification of vinyl esters and alcohols resulting in the formation of acetaldehyde which is known to deactivate microbial lipases, presumably by structural changes caused by initial Schiff-base formation at solvent accessible lysine residues. Previous studies showed that several lipases were sensitive toward acetaldehyde deactivation whereas others were insensitive; however, a general explanation of the acetaldehyde-induced inactivation mechanism is missing. Based on five microbial lipases from Candida rugosa, Rhizopus oryzae, Pseudomonas fluorescens and Bacillus subtilis we demonstrate that the protonation state of lysine ε-amino groups is decisive for their sensitivity toward acetaldehyde. Analysis of the diverse modification products of Bacillus subtilis lipases in the presence of acetaldehyde revealed several stable products such as α,β-unsaturated polyenals, which result from base and/or amino acid catalyzed aldol condensation of acetaldehyde. Our studies indicate that these products induce the formation of stable Michael-adducts at solvent-accessible amino acids and thus lead to enzyme deactivation. Further, our results indicate Schiff-base formation with acetaldehyde to be involved in crosslinking of lipase molecules. Differences in stability observed with various commercially available microbial lipases most probably result from different purification procedures carried out by the respective manufacturers. We observed that the pH of the buffer used prior to lyophilization of the enzyme sample is of utmost importance. The mechanism of acetaldehyde-induced deactivation of microbial lipases involves the generation of α,β-unsaturated polyenals from acetaldehyde which subsequently form stable Michael-adducts with the enzymes. Lyophilization of the enzymes from buffer at pH 6.0 can provide an easy and effective way to stabilize lipases toward inactivation by acetaldehyde.
Tài liệu tham khảo
citation_journal_title=Curr Opin Struct Biol; citation_title=α/β hydrolase fold enzymes: the family keeps growing; citation_author=M Nardini, BW Dijkstra; citation_volume=9; citation_issue=6; citation_publication_date=1999; citation_pages=732-737; citation_doi=10.1016/S0959-440X(99)00037-8; citation_id=CR1
citation_journal_title=Protein Eng; citation_title=The α/β hydrolase fold; citation_author=DL Ollis, E Cheah, M Cygler, BW Dijkstra, F Frolow, SM Franken, M Harel, SJ Remington, I Silman, J Schrag, JL Sussmann, KHG Verschuren, A Goldmann; citation_volume=5; citation_issue=3; citation_publication_date=1992; citation_pages=197-211; citation_doi=10.1093/protein/5.3.197; citation_id=CR2
citation_journal_title=Annu Rev Microbiol; citation_title=Bacterial biocatalysts: molecular biology, three-dimensional structures, and biotechnological applications of lipases; citation_author=KE Jaeger, BW Dijkstra, MT Reetz; citation_volume=53; citation_publication_date=1999; citation_pages=315-351; citation_doi=10.1146/annurev.micro.53.1.315; citation_id=CR3
citation_journal_title=FEMS Microbiol Rev; citation_title=Bacterial lipases; citation_author=KE Jaeger, S Ransac, BW Dijkstra, C Colson, M van Heuvel, O Misset; citation_volume=15; citation_issue=1; citation_publication_date=1994; citation_pages=29-63; citation_doi=10.1111/j.1574-6976.1994.tb00121.x; citation_id=CR4
citation_journal_title=Catal Rev - Sci Eng; citation_title=Lipase-catalyzed esterification; citation_author=NN Gandhi, NS Patil, SB Sawant, JB Joshi; citation_volume=42; citation_issue=4; citation_publication_date=2000; citation_pages=439-480; citation_doi=10.1081/CR-100101953; citation_id=CR5
citation_journal_title=Trends Biotechnol; citation_title=Microbial lipases form versatile tools for biotechnology; citation_author=KE Jaeger, MT Reetz; citation_volume=16; citation_issue=9; citation_publication_date=1998; citation_pages=396-403; citation_doi=10.1016/S0167-7799(98)01195-0; citation_id=CR6
citation_journal_title=Catal Rev - Sci Eng; citation_title=Lipase and lipase-catalyzed esterification reactions in non-aqueous media; citation_author=SH Krishna, NG Karanth; citation_volume=44; citation_issue=4; citation_publication_date=2002; citation_pages=499-591; citation_doi=10.1081/CR-120015481; citation_id=CR7
citation_journal_title=Enzyme Microb Technol; citation_title=Ester synthesis in lipase-catalyzed reactions; citation_author=ARM Yahya, WA Anderson, M Moo-Young; citation_volume=23; citation_issue=7-8; citation_publication_date=1998; citation_pages=438-450; citation_doi=10.1016/S0141-0229(98)00065-9; citation_id=CR8
citation_journal_title=Tetrahedron Lett; citation_title=Enzymatic reactions in organic synthesis: 2-ester interchange of vinyl esters; citation_author=M Degueil-Castaing, B De Jeso, S Drouillard, B Maillard; citation_volume=28; citation_issue=9; citation_publication_date=1987; citation_pages=953-954; citation_doi=10.1016/S0040-4039(00)95884-2; citation_id=CR9
citation_journal_title=J Org Chem; citation_title=Convenient enzymatic resolution of alcohols using highly reactive, nonharmful acyl donors, 1-ethoxyvinyl esters; citation_author=Y Kita, Y Takebe, K Murata, T Naka, S Akai; citation_volume=65; citation_issue=1; citation_publication_date=2000; citation_pages=83-88; citation_doi=10.1021/jo9910785; citation_id=CR10
citation_journal_title=Curr Org Chem; citation_title=Stereoselective enzymatic acylations (transesterifications); citation_author=R Chenevert, N Pelchat, F Jacques; citation_volume=10; citation_publication_date=2006; citation_pages=1067-1094; citation_doi=10.2174/138527206777698093; citation_id=CR11
citation_journal_title=Angew Chem Int Ed Engl; citation_title=A high-throughput-screening method for determining the synthetic activity of hydrolases; citation_author=M Konarzycka-Bessler, UT Bornscheuer; citation_volume=42; citation_issue=12; citation_publication_date=2003; citation_pages=1418-1420; citation_doi=10.1002/anie.200390365; citation_id=CR12
citation_journal_title=Alcohol Clin Exp Res; citation_title=Reaction of acetaldehyde with proteins: formation of stable fluorescent adducts; citation_author=T Hoffmann, RJ Meyer, MF Sorrell, DJ Tuma; citation_volume=17; citation_issue=1; citation_publication_date=1993; citation_pages=69-74; citation_doi=10.1111/j.1530-0277.1993.tb00728.x; citation_id=CR13
citation_journal_title=Appl Environ Microbiol; citation_title=Enhancement of the stability of a prolipase from Rhizopus oryzae toward aldehydes by saturation mutagenesis; citation_author=M Di Lorenzo, A Hidalgo, R Molina, JA Hermoso, D Pirozzi, UT Bornscheuer; citation_volume=73; citation_issue=22; citation_publication_date=2007; citation_pages=7291-7299; citation_doi=10.1128/AEM.01176-07; citation_id=CR14
citation_journal_title=Biotechnol J; citation_title=Directed evolution of an industrial biocatalyst: 2-deoxy-D-ribose-5-phosphate aldolase; citation_author=S Jennewein, M Schurmann, M Wolberg, I Hilker, R Luiten, M Wubbolts, D Mink; citation_volume=1; citation_issue=5; citation_publication_date=2006; citation_pages=537-548; citation_doi=10.1002/biot.200600020; citation_id=CR15
citation_journal_title=Angew Chem; citation_title=New aspects of the Maillard reaction in foods and in the human-body; citation_author=F Ledl, E Schleicher; citation_volume=29; citation_issue=6; citation_publication_date=1990; citation_pages=537-548; citation_doi=10.1002/anie.199005653; citation_id=CR16
citation_journal_title=Chembiochem; citation_title=An activity, stability and selectivity comparison of propioin synthesis by thiamine diphosphate-dependent enzymes in a solid/gas bioreactor; citation_author=R Mikolajek, AC Spiess, M Pohl, S Lamare, J Büchs; citation_volume=8; citation_issue=9; citation_publication_date=2007; citation_pages=1063-1070; citation_doi=10.1002/cbic.200700095; citation_id=CR17
Berger B, Faber K: 'Immunization' of lipase against acetaldehyde emerging in acyl transfer reactions from vinyl acetate. J Chem Soc, Chem Commun. 1991, 1198-1200. 10.1039/c39910001198. 17
citation_journal_title=Biotechnol Lett; citation_title=Sensitivity of microbial lipases to acetaldehyde formed by acyl-transfer reactions from vinyl esters; citation_author=HK Weber, H Stecher, K Faber; citation_volume=17; citation_issue=8; citation_publication_date=1995; citation_pages=803-808; citation_doi=10.1007/BF00129008; citation_id=CR19
citation_journal_title=Biotechnol Tech; citation_title=Stabilization of Candida rugosa Lipase against acetaldehyde by adsorption onto Celite; citation_author=H Kaga, B Siegmund, E Neufellner, K Faber; citation_volume=8; citation_issue=6; citation_publication_date=1994; citation_pages=369-374; citation_doi=10.1007/BF00154305; citation_id=CR20
citation_inbook_title=Industrial Biotransformations; citation_publication_date=2006; citation_id=CR21; citation_author=A Liese; citation_author=K Seelbach; citation_author=C Wandrey; citation_publisher=Wiley-VCH
citation_journal_title=Bioresour Technol; citation_title=Stabilization of Candida rugosa lipase during transacetylation with vinyl acetate; citation_author=AB Majumder, MN Gupta; citation_volume=101; citation_publication_date=2010; citation_pages=2877-2879; citation_doi=10.1016/j.biortech.2009.09.088; citation_id=CR22
citation_journal_title=Angew Chem Int Ed; citation_title=Properties and synthetic applications of enzymes in organic solvents; citation_author=G Carrea, S Riva; citation_volume=39; citation_publication_date=2000; citation_pages=2226-2254; citation_doi=10.1002/1521-3773(20000703)39:13<2226::AID-ANIE2226>3.0.CO;2-L; citation_id=CR23
citation_journal_title=J Mol Catal B: Enzym; citation_title=Molecular reasons for lipase-sensitivity against acetaldehyde; citation_author=HK Weber, J Zuegg, K Faber, J Pleiss; citation_volume=3; citation_issue=1-4; citation_publication_date=1997; citation_pages=131-138; citation_doi=10.1016/S1381-1177(96)00045-8; citation_id=CR24
citation_journal_title=Proc Natl Acad Sci USA; citation_title=Enzyme-catalyzed processes in organic solvents; citation_author=A Zaks, AM Klibanov; citation_volume=82; citation_issue=10; citation_publication_date=1985; citation_pages=3192-3196; citation_doi=10.1073/pnas.82.10.3192; citation_id=CR25
citation_journal_title=Biotechnol Bioeng; citation_title=On the pH memory of lyophilized compounds containing protein functional groups; citation_author=HR Costantino, K Griebenow, R Langer, AM Klibanov; citation_volume=53; citation_issue=3; citation_publication_date=1997; citation_pages=345-348; citation_doi=10.1002/(SICI)1097-0290(19970205)53:3<345::AID-BIT14>3.0.CO;2-J; citation_id=CR26
citation_journal_title=Biocatal Biotrans; citation_title=Proteolytic activity in commercial triacylglycerol hydrolase preparations; citation_author=MA Bjurlin, S Bloomer; citation_volume=20; citation_issue=3; citation_publication_date=2002; citation_pages=179-188; citation_doi=10.1080/10242420290020705; citation_id=CR27
citation_journal_title=J Am Oil Chem Soc; citation_title=Composition and activity of commercial triacylglycerol acylhydrolase preparations; citation_author=MA Bjurlin, S Bloomer, MJ Haas; citation_volume=78; citation_issue=2; citation_publication_date=2001; citation_pages=153-160; citation_doi=10.1007/s11746-001-0236-9; citation_id=CR28
citation_journal_title=Eur J Lipid Sci Technol; citation_title=Identification of carboxylesterase activities of commercial triacylglycerol hydrolase (lipase) preparations; citation_author=MA Bjurlin, S Bloomer, MJ Haas; citation_volume=104; citation_publication_date=2002; citation_pages=143-155; citation_doi=10.1002/1438-9312(200203)104:3<143::AID-EJLT143>3.0.CO;2-N; citation_id=CR29
citation_journal_title=Org Process Res Dev; citation_title=What Is in a Biocatalyst?; citation_author=A Wells; citation_volume=10; citation_publication_date=2006; citation_pages=678-681; citation_doi=10.1021/op060006u; citation_id=CR30
citation_title=Biocatalyses in microaqueous organic media; citation_inbook_title=Handbook of Industrial Biocatalysis; citation_publication_date=2005; citation_id=CR31; citation_author=T Yamane
citation_journal_title=Tetrahedron; citation_title=Causes of unreproducibility of C. rugosa lipase-catalyzed reactions in slightly hydrated organic media; citation_author=P Dominguez de María, JVS Gago; citation_volume=55; citation_issue=28; citation_publication_date=1999; citation_pages=8555-8566; citation_doi=10.1016/S0040-4020(99)00473-1; citation_id=CR32
citation_journal_title=FEBS Lett; citation_title=Lipolytic enzymes LipA and LipB from Bacillus subtilis differ in regulation of gene expression, biochemical properties, and three-dimensional structure; citation_author=T Eggert, G van Pouderoyen, BW Dijkstra, KE Jaeger; citation_volume=502; citation_issue=3; citation_publication_date=2001; citation_pages=89-92; citation_doi=10.1016/S0014-5793(01)02665-5; citation_id=CR33
citation_journal_title=J Mol Biol; citation_title=The crystal structure of Bacillus subtilis lipase: a minimal α/β hydrolase fold enzyme; citation_author=G van Pouderoyen, T Eggert, KE Jaeger, BW Dijkstra; citation_volume=309; citation_issue=1; citation_publication_date=2001; citation_pages=215-226; citation_doi=10.1006/jmbi.2001.4659; citation_id=CR34
citation_journal_title=Proteins: Struct, Funct, Bioinf; citation_title=Very fast empirical prediction and interpretation of protein pKa values; citation_author=H Li, AD Robertson, JH Jensen; citation_volume=61; citation_issue=4; citation_publication_date=2005; citation_pages=704-721; citation_doi=10.1002/prot.20660; citation_id=CR35
citation_journal_title=J Biol Chem; citation_title=Identification of formaldehyde-induced modifications in proteins: reactions with model peptides; citation_author=B Metz, GF Kersten, P Hoogerhout, H Brugghe, H Timmermans, A de Jong, H Meiring, J ten Hove, WE Hennink, DJ Crommelin, W Jiskoot; citation_volume=297; citation_publication_date=2004; citation_pages=6235-6234; citation_id=CR36
citation_journal_title=J Biol Chem; citation_title=Identification of formaldehyde-induced modifications in proteins: reactions with model peptides; citation_author=B Metz, GFA Kersten, P Hoogerhout, HF Brugghe, HAM Timmermans, A de Jong, H Meiring, J ten Hove, WE Hennink, DJA Crommelin, W Jiskoot; citation_volume=279; citation_issue=8; citation_publication_date=2004; citation_pages=6235-6243; citation_doi=10.1074/jbc.M310752200; citation_id=CR37
citation_journal_title=Eur J Biochem; citation_title=A novel extracellular esterase from Bacillus subtilis and its conversion to a monoacylglycerol hydrolase; citation_author=T Eggert, G Pencreac'h, I Douchet, R Verger, KE Jaeger; citation_volume=267; citation_issue=21; citation_publication_date=2000; citation_pages=6459-6469; citation_doi=10.1046/j.1432-1327.2000.01736.x; citation_id=CR38
citation_journal_title=Biochem Biophys Res Commun; citation_title=Biophysical characterization of Gir2, a highly acidic protein of Saccharomyces cerevisiae with anomalous electrophoretic behavior; citation_author=VS Alves, DC Pimenta, E Sattlegger, BA Castilho; citation_volume=314; citation_issue=1; citation_publication_date=2004; citation_pages=229-234; citation_doi=10.1016/j.bbrc.2003.12.086; citation_id=CR39
citation_journal_title=Electrophoresis; citation_title=Anomalous electrophoretic behavior of a very acidic protein: Ribonuclease U2; citation_author=L Garcia-Ortega, VD Rios, A Martinez-Ruiz, M Onaderra, J Lacadena, AM del Pozo, JD Gavilanes; citation_volume=26; citation_issue=18; citation_publication_date=2005; citation_pages=3407-3413; citation_doi=10.1002/elps.200500261; citation_id=CR40
citation_journal_title=Biotechnol Prog; citation_title=Criteria to design green enzymatic processes in ionic liquid/supercritical carbon dioxide systems; citation_author=P Lozano, T De Diego, S Gmouh, M Vaultier, JL Iborra; citation_volume=20; citation_publication_date=2004; citation_pages=661-669; citation_doi=10.1021/bp0342497; citation_id=CR41
citation_journal_title=Food Res Int; citation_title=Aggregation behavior of Candida rugosa lipase; citation_author=YC Liou, AG Marangoni, RY Yada; citation_volume=31; citation_issue=3; citation_publication_date=1998; citation_pages=243-248; citation_doi=10.1016/S0963-9969(98)00099-4; citation_id=CR42
citation_journal_title=Biotechnol Bioeng; citation_title=Self-assembly of Pseudomonas fluorescens lipase into bimolecular aggregates dramatically affects functional properties; citation_author=G Fernández-Lorente, JS Palomo, M Fuentes, C Mateo, JM Guisán, R Fernández-Lafuente; citation_volume=82; citation_issue=2; citation_publication_date=2003; citation_pages=232-237; citation_doi=10.1002/bit.10560; citation_id=CR43
citation_journal_title=J Bacteriol; citation_title=Purification of extracellular lipase from Pseudomonas aeruginosa; citation_author=W Stuer, KE Jaeger, UK Winkler; citation_volume=168; citation_issue=3; citation_publication_date=1986; citation_pages=1070-1074; citation_id=CR44
citation_journal_title=Biochim Biophys Acta; citation_title=Purification and some properties of the staphylococcal extracellular lipase; citation_author=S Tyski, W Hryniewicz, J Jeljaszewicz; citation_volume=749; citation_issue=3; citation_publication_date=1983; citation_pages=312-317; citation_doi=10.1016/0167-4838(83)90241-8; citation_id=CR45
citation_journal_title=J Protein Chem; citation_title=Stability studies on a lipase from Bacillus subtilis in guanidinium chloride; citation_author=P Acharya, NM Rao; citation_volume=22; citation_issue=1; citation_publication_date=2003; citation_pages=51-60; citation_doi=10.1023/A:1023067827678; citation_id=CR46
citation_journal_title=J Am Chem Soc; citation_title=Acid-catalyzed enolization and aldol condensation of acetaldehyde; citation_author=LM Baigrie, RA Cox, H Slebocka-Tilk, M Tencer, TT Tidwell; citation_volume=107; citation_issue=12; citation_publication_date=1985; citation_pages=3640-3645; citation_doi=10.1021/ja00298a039; citation_id=CR47
citation_journal_title=J Phys Chem A; citation_title=Uptake and reaction kinetics of acetone, 2-butanone, 2,4-pentanedione, and acetaldehyde in sulfuric acid solutions; citation_author=W Esteve, B Nozière; citation_volume=109; citation_issue=48; citation_publication_date=2005; citation_pages=10920-10928; citation_doi=10.1021/jp051199a; citation_id=CR48
citation_journal_title=J Phys Chem A; citation_title=A kinetic and mechanistic study of the amino acid catalyzed aldol condensation of acetaldehyde in aqueous and salt solutions; citation_author=B Nozière, A Cordova; citation_volume=112; citation_issue=13; citation_publication_date=2008; citation_pages=2827-2837; citation_doi=10.1021/jp7096845; citation_id=CR49
citation_journal_title=Geophys Res Lett; citation_title=Formation of secondary light-absorbing "fulvic-like" oligomers: A common process in aqueous and ionic atmospheric particles?; citation_author=B Nozière, P Dziedzic, A Córdova; citation_volume=34; citation_publication_date=2007; citation_pages=L21812; citation_doi=10.1029/2007GL031300; citation_id=CR50
citation_journal_title=Drug Metab Rev; citation_title=Protein adducts generated from products of lipid oxidation: focus on HNE and one; citation_author=LM Sayre, D Lin, Q Yuan, X Zhu, X Tang; citation_volume=38; citation_issue=4; citation_publication_date=2006; citation_pages=651-675; citation_doi=10.1080/03602530600959508; citation_id=CR51
citation_journal_title=J Biol Chem; citation_title=Endogenous formation of protein adducts with carcinogenic aldehydes: implications for oxidative stress; citation_author=K Ichihashi, T Osawa, S Toyokuni, K Uchida; citation_volume=276; citation_issue=26; citation_publication_date=2001; citation_pages=23903-23913; citation_doi=10.1074/jbc.M101947200; citation_id=CR52
citation_journal_title=Appl Microbiol Biotechnol; citation_title=Biocatalytic production of enantiopure cyclohexane-trans-1,2-diol using extracellular lipases from Bacillus subtilis; citation_author=J Detry, T Rosenbaum, S Lütz, D Hahn, KE Jäger, M Müller, T Eggert; citation_volume=72; citation_issue=6; citation_publication_date=2006; citation_pages=1107-1116; citation_doi=10.1007/s00253-006-0391-9; citation_id=CR53
citation_journal_title=Appl Microbiol Biotechnol; citation_title=Investigating expression systems for the stable large-scale production of recombinant L-leucine-dehydrogenase from Bacillus cereus in Escherichia coli; citation_author=MB Ansorge, MR Kula; citation_volume=53; citation_issue=6; citation_publication_date=2000; citation_pages=668-673; citation_doi=10.1007/s002539900290; citation_id=CR54
citation_journal_title=J Biotechnol; citation_title=Simple fed-batch technique for high cell density cultivation of Escherichia coli; citation_author=DJ Korz, U Rinas, K Hellmuth, EA Sanders, WD Deckwer; citation_volume=39; citation_issue=1; citation_publication_date=1995; citation_pages=59-65; citation_doi=10.1016/0168-1656(94)00143-Z; citation_id=CR55
citation_journal_title=J Chromatogr; citation_title=New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues; citation_author=E Hochuli, H Dobeli, A Schacher; citation_volume=411; citation_publication_date=1987; citation_pages=177-184; citation_doi=10.1016/S0021-9673(00)93969-4; citation_id=CR56
citation_journal_title=Anal Biochem; citation_title=A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding; citation_author=MM Bradford; citation_volume=72; citation_publication_date=1976; citation_pages=248-254; citation_doi=10.1016/0003-2697(76)90527-3; citation_id=CR57
citation_inbook_title=Chemical reagents for protein modification; citation_publication_date=2004; citation_id=CR58; citation_author=RL Lundblad; citation_publisher=CRC Press
citation_title=Amidination of lysine side chains; citation_inbook_title=The Protein Protocols Handbook; citation_publication_date=2002; citation_pages=479-480; citation_id=CR59; citation_author=DS Tawfik; citation_publisher=Humana Press
citation_journal_title=Nature; citation_title=Membrane filtration for determining protein in the presence of interfering substances; citation_author=TP Bennett; citation_volume=213; citation_issue=5081; citation_publication_date=1967; citation_pages=1131-1132; citation_doi=10.1038/2131131a0; citation_id=CR60
citation_journal_title=J Gen Microbiol; citation_title=Growth-phase-dependent expression of the lipolytic system of Acinetobacter calcoaceticus BD413: cloning of a gene encoding one of the esterases; citation_author=RG Kok, VM Christoffels, B Vosman, KJ Hellingwerf; citation_volume=139; citation_issue=10; citation_publication_date=1993; citation_pages=2329-2342; citation_doi=10.1099/00221287-139-10-2329; citation_id=CR61
citation_inbook_title=Molecular Cloning: A Laboratory Manual; citation_publication_date=2001; citation_id=CR62; citation_author=J Sambrook; citation_author=P MacCallum; citation_author=D Russell
citation_journal_title=J Bacteriol; citation_title=Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens; citation_author=UK Winkler, M Stuckmann; citation_volume=138; citation_issue=3; citation_publication_date=1979; citation_pages=663-670; citation_id=CR63
citation_journal_title=Biochem J; citation_title=Micro method for determination of reactive carbonyl groups in proteins and peptides, using 2,4-dinitrophenylhydrazine; citation_author=R Fields, HB Dixon; citation_volume=121; citation_issue=4; citation_publication_date=1971; citation_pages=587-589; citation_doi=10.1042/bj1210587; citation_id=CR64
citation_journal_title=Proc Natl Acad Sci USA; citation_title=Rapid and efficient site-specific mutagenesis without phenotypic selection; citation_author=TA Kunkel; citation_volume=82; citation_issue=2; citation_publication_date=1985; citation_pages=488-492; citation_doi=10.1073/pnas.82.2.488; citation_id=CR65
citation_journal_title=J Mol Biol; citation_title=Studies on transformation of Escherichia coli with plasmids; citation_author=D Hanahan; citation_volume=166; citation_issue=4; citation_publication_date=1983; citation_pages=557-580; citation_doi=10.1016/S0022-2836(83)80284-8; citation_id=CR66
citation_journal_title=Nature; citation_title=Cleavage of structural proteins during the assembly of the head of bacteriophage T4; citation_author=UK Laemmli; citation_volume=227; citation_issue=5259; citation_publication_date=1970; citation_pages=680-685; citation_doi=10.1038/227680a0; citation_id=CR67
citation_journal_title=Bull Korean Chem Soc; citation_title=Highly sensitive and fast protein detection with Coomassie Brilliant Blue in sodium dodecyl sulfate-polyacrylamide gel electrophoresis; citation_author=D Kang, YS Gho, M Shu, C Kang; citation_volume=23; citation_issue=11; citation_publication_date=2002; citation_pages=1511-1512; citation_doi=10.5012/bkcs.2002.23.11.1511; citation_id=CR68
citation_journal_title=Biochim Biophys Acta; citation_title=Isoelectric focusing in polyacrylamide gels; citation_author=P Righetti, JW Drysdale; citation_volume=236; citation_issue=1; citation_publication_date=1971; citation_pages=17-28; citation_doi=10.1016/0005-2795(71)90144-9; citation_id=CR69