Purification and Characterisation of the Enantiospecific Dioxygenases

purification翻译purification的中文翻译是“净化”或“纯化”。

这个词可以用于多种领域，包括环境科学、化学、生物学和心灵修炼等。

以下是一些关于purification的用法和中英文对照例句：1. Environmental Purification:- The purification of contaminated water is essential for maintaining a healthy ecosystem. (水的净化对于维护健康的生态系统至关重要。

)- Air purification systems are used to remove pollutants and improve indoor air quality. (空气净化系统用于去除污染物，改善室内空气质量。

)2. Chemical Purification:- The process of distillation is commonly used for the purification of liquids. (蒸馏过程常用于液体的纯化。

)- Crystallization is a technique used for the purification of solid substances. (结晶是一种用于固体物质纯化的技术。

)3. Biological Purification:- The liver plays a crucial role in the purification of toxins from the body. (肝脏在身体毒素的净化中起着关键作用。

)- The purification of DNA is necessary for many molecularbiology experiments. (DNA的纯化对于许多分子生物学实验是必要的。

)4. Spiritual Purification:- Meditation is a practice that allows for the purification of the mind and the attainment of inner peace. (冥想是一种允许净化心灵并达到内心平静的修炼。

Journal of Biotechnology 164 (2013) 123–129Contents lists available at SciVerse ScienceDirectJournal ofBiotechnologyj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /j b i o t ecImproved activity and thermostability of Bacillus pumilus lipase by directed evolutionNagihan Akbulut a ,∗,Merve Tuzlako˘g lu Öztürk a ,Tjaard Pijning b ,Saliha ˙I s ¸sever Öztürk a ,Füsun Gümüs ¸el a ,1a Department of Molecular Biology and Genetics,Gebze Institute of Technology (GIT),41400Kocaeli,TürkiyebLaboratory of Biophysical Chemistry,Groningen Biomolecular Sciences and Biotechnology Institute (GBB),University of Groningen,Nijenborgh 7,9747AG Groningen,The Netherlandsa r t i c l ei n f oArticle history:Received 11September 2012Received in revised form 20December 2012Accepted 21December 2012Available online 11 January 2013Keywords:Bacillus pumilus lipase BiocatalystDirected evolution DNA shuffling Thermostability3D homology modela b s t r a c tTo improve enzymatic activity of Bacillus pumilus lipases,DNA shuffling was applied to two lipase genes from local B.pumilus ing a high-throughput activity assay,the mutant with highest activity was selected.This chimeric mutant (L3-3),carrying two crossover positions and three point mutations,has a specific activity 6.4and 8.2times higher than the two parent enzymes.The mutant also is more tolerant to various detergents and organic solvents,and has a 9times longer half-life at 50◦C.Homology modeling of mutant L3-3,based on the highly homologous B.subtilis lipase A,shows that the increased thermostability is likely due to structural rigidification and reduced surface hydrophobicity.Increased specific activity may result from the location of mutations close to the active site.Together,our results show that it is possible to evolve,by DNA shuffling,B.pumilus lipase variants with improved applicability as biocatalysts,even if the two parent enzymes are highly similar.© 2013 Elsevier B.V. All rights reserved.1.IntroductionLipases (triacylglycerol acylhydrolases,EC 3.1.1.3)catalyze the hydrolysis and synthesis of esters of glycerol and long-chain fatty acids.Microbial lipases are of commercial interest for chemical,food,pharmaceutical,detergent and other industrial applications (Jaeger and Eggert,2002;Pandey et al.,1999;Sharma et al.,2001).Among them,Bacillus pumilus lipases have been classi-fied as members of subfamily I.4,sharing sequence identities of 74–77%(Arpigny and Jaeger,1999;Jaeger et al.,1999);also the well-characterized Bacillus subtilis lipases belong to this family.The subfamily I.4lipases are the smallest lipases known,having a minimal ␣/␤hydrolase fold (van Pouderoyen et al.,2001)and a solvent-exposed substrate-binding site.In order to use lipases as biocatalysts in industrial applications,it is often desirable to improve properties such as activity (in aque-ous or organic solvent environments),thermostability,substrate specificity and enantioselectivity (Arnold and Volkov,1999;Reetz,∗Corresponding author.Tel.:+902626052540;fax:+902626052505.E-mail addresses:nakbulut@.tr ,nagihanakbulut@ (N.Akbulut).1During this work,Prof.Dr.Füsun Gümüs ¸el passed away;we remember her with respect.2004).Directed evolution is a powerful approach to achieve such improvements.Rapid generation of molecular diversity is essential,and one of the best methods to achieve this is homologous recombi-nation through DNA shuffling (Crameri et al.,1998).When coupled with high-throughput screening,DNA shuffling and other directed evolution methods have often resulted in remarkable improve-ments of activity,thermostability or enantioselectivity (Crameri et al.,1998;Reetz,2004;Schmidt-Dannert and Arnold,1999).The lipase A from B.subtilis has been the subject of several such stud-ies (Acharya et al.,2004;Ahmad et al.,2008;Augustyniak et al.,2012;Dröge et al.,2006;Kamal et al.,2011).In contrast,a directed evolution approach for B.pumilus lipase has only been reported by Huang et al.(2008)who used error-prone PCR to evolve mutants with increased activity.We applied the DNA shuffling method,coupled with a high-throughput screening assay,to improve the activity of lipases of subfamily I.4produced by local isolates of B.pumilus .The lipase mutant (L3-3)showing the highest activity was sequenced and purified,and biochemically characterized.Obtained after a single round of DNA shuffling from two parents sharing 89%identity,this chimeric mutant has two cross-over positions and carries three point mutations.Its activity was 6.4and 8.2times higher than that of the two parent enzymes.Surprisingly,L3-3also displayed a remarkable increase in thermostability,with a 9times longer half-life (T 1/2)at 50◦C.Taking advantage of the high sequence similarity0168-1656/$–see front matter © 2013 Elsevier B.V. All rights reserved./10.1016/j.jbiotec.2012.12.016124N.Akbulut et al./Journal of Biotechnology164 (2013) 123–129with B.subtilis lipase A,a3D homology model was constructed for L3-3,and the role of sequence differences between the mutant and the parents on enzyme activity and thermostability is discussed.2.Materials and methods2.1.Isolation and identificationBacterial strains L5and L21had been isolated by traditional bacteriological methods from hot springs at Balıkesir and Bursa, Türkiye(Tuzlako˘g lu et al.,2003).Characterization of the strains was done using biochemical tests,microscopical observations (Sneath,1984)and16S rRNA gene sequencing(˙I s¸severÖztürk et al., 2008).Database homology searches were performed with BLAST (/Blast.cgi).2.2.Cloning and expression of parent lipase genesChromosomal DNA was isolated from B.pumilus strains L5and L21(Tuzlako˘g lu et al.,2003;˙I s¸severÖztürk et al.,2008)and used to amplify by PCR the lipase-encoding genes,using a pair of degen-erate primers(forward:21F,reverse:22R).After30amplification cycles,a0.65-kb PCR product was recovered from an agarose gel. Cloning was carried out with InsTAclone TM PCR Cloning Kit(Fer-mentas).The purified PCR products were ligated in pTZ57R/T,and E.coli JM109cells were transformed with this ligation product.The resulting plasmids were named pTZ-L5and pTZ-L21.For expres-sion studies,Hind III–Eco RI fragments from the plasmids pTZ-L5and pTZ-L21were subcloned into the expression vector pUC19previ-ously digested with the same enzymes,separately.E.coli JM109 cells carrying recombinant vectors were grown for24h in the pres-ence of ampicillin(100␮g/ml)and gene expression was induced with afinal concentration of0.1mM IPTG.2.3.DNA shuffling library constructionA library of random fragments was constructed using modi-fied DNA shuffling methods(Lorimer and Pastan,1995;Stemmer, 1994;Zhao and Arnold,1997).Two0.65kb DNA fragments contain-ing lipase genes from L5and L21were amplified by using primers 21F and22R.Fragments of0.65kb were purified from1%agarose gel.Parent DNA fragments were digested with bovine pancreas DNase I in the presence of Mn2+.A mixture of50␮l(containing 1.5␮g of each parent DNA)and5␮l10×digestion buffer(50mM Tris(tris(hydroxymethyl)aminomethane)–HCl,10mM MnCl2)was equilibrated at25◦C for5min;0.45U of DNase I(diluted in1×digestion buffer)was added.Digestion was performed at25◦C and terminated after11min by heating at90◦C for10min.The digested fragments were separated by1.5%agarose gel electrophoresis;frag-ments of<70bp were isolated and purified from the gel.PCR without primers.The reaction volume(50␮l)contained 20␮l purified fragments,0.4mM dNTP mix,2.5U Pfu DNA poly-merase,1×Pfu DNA polymerase reaction buffer.The following PCR protocol was applied:3min at96◦C,40cycles of1min at94◦C, 1min at55◦C,1min+5s/cycle at72◦C,10min at72◦C.PCR with primers.The reaction mixture containing reassembled DNA-fragments(1␮l)along with primers21F and22R was used to amplify the full-length genes,using the same PCR cycling program as described in PCR without primers.PCR conditions(50␮lfinal vol-ume):80pmol of each primer,1×Taq polymerase reaction buffer, 0.2mM dNTP mix(Roche)and2.5U Taq/Pfu(1:1)polymerase mix-ture.The purified PCR product was digested with Hind III–Eco RI and ligated into plasmid pUC19,which had been digested with corre-sponding restriction enzymes to create the recombination library. Freshly prepared E.coli JM109cells were transformed with the resulting DNA mixture.Cells were plated on LB-agar containing1.5%agar and1%ampicillin,and incubated overnight.2.4.Enzyme expression and library screeningActive transformants were assessed by a three-step screening protocol.First,transformant colonies were replicated on tributyrin-agar plates containing0.15%Gum Arabic and1.5%tributyrin in LB-agar(Liebeton et al.,2000),supplemented with1%ampicillin.After incubation for16h,enzyme secretion into the medium was induced by incubation for6h at4◦C.Transformants showing lipase activity (resulting in clear halos surrounding the colonies)were selected.In the second step,selected variants were inoculated into the individual wells of96-well plates containing250␮l LB with1% ampicillin.After overnight growth(37◦C),lipase activity in the cul-ture supernatant was assayed quantitatively using p NP-palmitate as substrate,according to the method of Eom et al.(2005)with slight modifications;absorbance at405nm was measured with a Fluostar Omega Microplate Reader(BMG Labtech).Measured activ-ities were normalized for culture density;variants showing a higher normalized activity than parent strains were selected.Selected variants were further confirmed and analyzed in a third step by growing them in shakeflask cultures at37◦C.Ten milliliters of LB medium containing1%ampicillin were inoculated with0.1ml pre-culture;gene expression was induced with0.1mM IPTG.Nor-malized lipase activity was assayed according to the method of Winkler and Stuckmann(1979).2.5.DNA sequencing,purification and characterization of parents and mutant lipaseThe plasmid DNA of parents L5and L21and of the trans-formant with highest activity was isolated and sequenced (see Supplementary Material,Section1.3).The expressed lipase enzymes were subjected to a single-step purification;their purity was determined from an SDS-PAGE gel.Purification details and characterization of the purified enzymes by determination of temperature and pH profiles and stability,the effect of various detergents,organic solvents,metal ions and inhibitors on activity, and analysis of substrate specificity are described in Supplementary Material(Section1.4).2.6.Modeling studiesAfter analysis of the B.pumilus L3-3mutant sequence by the FFAS03server(Jaroszewski et al.,2011)the structure with the high-est sequence identity(78%),B.subtilis lipase A(PDB ID:1I6W(van Pouderoyen et al.,2001)),was used as a template in the“One-to-one threading”protocol of the Phyre2server(Kelley and Sternberg, 2009)to obtain3D models of mutant L3-3and parents L5and L21.Differences and mutation positions of the models were eval-uated in PyMOL(Schrödinger,LLC,version1.2r1)by looking at interaction possibilities and clash problems.Secondary structure assignment was calculated with DSSP(Kabsch and Sander,1983); hydrogen-bonding was assessed within PyMOL.Structuralfigures were prepared with PyMOL.3.Results3.1.Isolation and identification of lipase-producing strainsCharacterization of the bacterial strains previously isolated from hot springs near Balıkesir and Bursa(Türkiye)showed that they are Gram-positive,rod shaped,aerobic,catalase-positive and sporeN.Akbulut et al./Journal of Biotechnology164 (2013) 123–129125Fig.1.Schematic representation of the sequences of the two Bacillus pumilus parents(L5,light gray,and L21,dark gray)and mutant L3-3.Dark gray and light gray colors in the L3-3mutant indicate from which parent the L3-3mutant derived its sequence.Because of local homology at the DNA level,the crossover positions in L3-3cannot be determined exactly;thefirst crossover position is between residues20/21and23/24,and the second crossover position is between residues149/150and168/169,as indicated by the shaded parts.Chimeric differences between the two parents are indicated with black triangles;the3point mutations in the L3-3mutant are indicated with black bars.A more detailed alignment is given in Supplementary Material Fig.S2.forming.Biochemical tests and16S rDNA gene analysis identified the strains as B.pumilus,and they were designated as L5and L21.3.2.Cloning,sequencing and expression of the parent lipasesThe0.65kbp lipase open reading frames(ORFs)of the two B.pumilus strains L5and L21were amplified from the chro-mosomal DNA(Supplementary Material Fig.S1).Cloning into pTZ57R/T and subsequent DNA isolation and sequencing con-firmed the presence of ORFs of645bp,encoding precursor lipases of215amino acid residues.DNA translation showed that the encoded enzymes contain a34-residue signal peptide(SignalP 4.0,http://www.cbs.dtu.dk/services/SignalP);after cleavage,the mature enzymes thus contain181amino acid residues.Sequence analysis revealed that the L5and L21parent lipases share89%iden-tity with each other(at the protein level),and78%identity with B. subtilis lipase A(Supplementary Material Fig.S2).The parent lipase gene sequences have been deposited in GenBank with accession numbers JX163855(L5)and JX163856(L21).The L5and L21lipase genes were successfully subcloned into a pUC19vector,as confirmed by digestion of the recombinant plasmid and identification of the645bp DNA fragments.Trans-formation of E.coli JM109cells with the recombinant plasmids resulted in active expression of the lipases,as was confirmed by lipase activity assays.3.3.DNA shuffling and screening of the libraryA random B.pumilus lipase library was generated by DNA shuf-fling,using<70bp fragments obtained from the two B.pumilus lipase parent genes L5and L21.Reassembled products ran as sin-gle bands with the correct size on agarose gels(Supplementary Material Fig.S1).These were used to transform E.coli JM109cells; 5500transformants(55%)expressed a functional lipase,forming clear halos due to the hydrolysis of tributyrin.The350transfor-mants with highest activity(as judged by eye)were selected for the second screening step.From these,the16transformants showing a higher activity than the parent strains were selected for a third screening step,in which more favorable conditions for bacterial growth were applied.The transformant showing the highest nor-malized activity was further characterized by comparison with the two parent lipases.Sequencing revealed that the lipase expressed by this transformant(L3-3)is a chimeric mutant with2crossover positions,resulting in a large middle fragment originating from the L5parent,and shorter N-and C-terminal fragments derived from the L21parent(Fig.1and Supplementary Material Fig.S2).There are11“chimeric differences”(residues that differ between the two parent enzymes)in the middle fragment and3such differences in the terminal fragments.In addition,L3-3carries3point muta-tions(G14S,A15G and V109S);they do not stem from either parent, nor are they present in B.subtilis lipase A.Consequently,L3-3is Table1Specific activity and half-life of the partially purified parent(L5,L21)and mutant (L3-3)enzymes.The specific activity is given before(raw)and after correction for purity(40,25and60%for L5,L21and L3-3,respectively).L5L21L3-3Raw specific activity(U/mg)1150±3558±411,012±4 Corrected specific activity(U/mg)2878±82238±1618,332±7T1/2,50◦C(min) 4.20±0.12 4.40±0.0338.5±0.7 different from parent L5at6positions,and different from parent L21at14positions.3.4.Purification and characterization of parent and mutant B. pumilus lipasesResults of the purification of the two parent B.pumilus lipases and mutant L3-3are summarized in Supplementary Material Table S1.Typically,thefinal yield of enzyme was about50%of the ini-tial activity,with a9-fold increase in specific activity compared to the culture lysate supernatant.On SDS-PAGE,the purified enzymes were observed at about19kDa(Supplementary Material Fig.S3), with purities of about40,25and60%for L5,L21and L3-3,respec-tively.We did not succeed in purifying the enzymes further.For both parent and mutant lipases,the optimum temperature was37◦C(Supplementary Material Fig.S4),but,after correction for the differences in purity,the specific activity of mutant L3-3 was about6.4and8.2times higher than that of the parents L5 and L21,respectively(Table1).At higher temperatures,activity decreased fast to near-zero values at55◦C,but the L3-3mutant clearly retained more activity than the parent enzymes(Fig.2).TheFig.2.Relative residual activity of parents(L5,L21)and L3-3after pre-incubation at different temperatures for30min.126N.Akbulut et al./Journal of Biotechnology 164 (2013) 123–129Fig.3.3D homology model of the B.pumilus L3-3mutant,generated with Phyre2(Kelley and Sternberg,2009)based on the crystal structure of B.subtilis lipase A (van Pouderoyen et al.,2001).The N-and C-terminal polypeptide segments derived from parent L21,containing the 3chimeric differences (M12,A20and V169)are shown in blue;the middle segment derived from parent L5is shown in gray.The three point mutations G14S,A15G and V109S are shown with yellow carbon atoms.The catalytic residue S77in the active site is also shown.(For interpretation of the references to color in figure legend,the reader is referred to the web version of the article.)half-life (at 50◦C)of mutant L3-3was 9times longer than that of the parent enzymes (Table 1).The pH-activity profiles of both parents and mutant L3-3were very similar (Supplementary Material Fig.S5a ),with an optimum pH of 8.0.The residual activity profiles after 1week of incubation at 4◦C were also similar,with 80–100%activity retained between pH 6.5and 10.0(Supplementary Material Fig.S5b ).Metal ions (10mM)in general had modest effects (Supplementary Material Fig.S6,left panel );relative activi-ties were in the range of 50–163%.The most prominent effect was observed for CuCl 2,which showed an increased activity for L3-3while the parent lipases were inhibited.In addition,CoCl 2and FeCl 2increased activity of L3-3significantly.The presence of CaCl 2slightly inhibited the mutant,while the presence of EDTA (ethylene diamine tetraacetic acid)(1or 10mM)hardly affected activity;PMSF (phenylmethylsulfonyl fluoride)strongly inhibited activity of both parent enzymes and the mutant (Supplementary Material Fig.S6,right panel ).All tested detergents,except for Na-deoxycholate,inhibited the activity of parent and mutant lipases at the highest tested concen-tration;CTAB (cetyl trimethylammonium bromide)(1%)and SDS (sodium dodecyl sulfate)(1%)almost completely inactivated the enzymes (Supplementary Material Fig.S7a ).However,in several cases mutant L3-3retained a significantly higher activity than the parents,or was even stimulated.Most of the tested organic solvents had a slightly inhibiting effect on the activity of parent enzymes at 10%concentration;this effect was stronger at higher concentration (30%)(Supplementary Material Fig.S7b ).Notably,the L3-3mutant showed a tolerance to all tested organic solvents at 10%concentration except isoamyl alcohol.Analysis of the substrate specificity of parent and mutant lipases revealed only small variations (Supplementary Material Fig.S8);mutant L3-3showed a slightly higher activity toward long chain triacylglycerol fatty acids than the parent enzymes.3.5.Structural observationsThe 3D models generated for the B.pumilus lipase L3-3mutant (Fig.3)and its L5and L21parents showed high Phyre confidence values.Of the 40sequence differences between the B.subtilis lipase A and the B.pumilus L3-3mutant (Supplementary Material Fig.S2),almost half are homologous substitutions.For 35of these,the sidechains are at the surface and exposed to the solvent;the remaining differences are located in the hydrophobic core,and comprise at most one methylene or methyl group.About half (19)of the differ-ences occur in non-regular secondary structure elements such as loops and 310helices.For the G14S mutation,a different side chain rotamer was chosen to avoid a close contact with the side chain of N18.For all other changed residues,no severe clash problems were observed.The three chimeric differences of L3-3with parent L5and the three point mutations in L3-3are described below.The chimeric differences (M12,A20and V169).Residue 12is located at the tip of a 6-residue loop (residues 10–15)connect-ing strand ␤3and helix ␩1/␣A (Fig.4a).Its side chain is exposed to the solvent,and the chimeric change from isoleucine to methionine may increase hydrophobic and van der Waals interactions with thesubstrate.Residue 20,at the start of helix ␣A,is located about 16˚Afrom the active site,and has a solvent-exposed side chain.Changing a phenylalanine to alanine at this position will considerably reduce the hydrophobicity at the surface,and the tendency to aggregate at higher temperatures.Residue 169is in helix ␣F;its side chain is located in the hydrophobic interior of the enzyme (Fig.3),far from the active site.The change from isoleucine to valine at this posi-tion (one methyl group)may slightly change local packing in the enzyme’s interior.The point mutations (G14S ,A15G and V109S ).Residue 14is located in the ␤3-␩1/␣A loop (residues 10–15),adjacent to the sub-strate binding cleft (Fig.4a).The introduction of the serine side chain has no effect on the main chain torsion angles (ϕ=−86◦, =−171◦),but it increases the local surface polarity.In addition,it provides the possibility of the formation of two additional hydro-gen bonds within the loop.The ␤-turn hydrogen bond interaction between the main chain oxygen atom of G11and the main chain nitrogen atom of S14is preserved.Mutation of residue 15intro-duces a third glycine residue in the ␤3-␩1/␣A loop (Fig.4a).In the parent B.pumilus enzymes (like in B.subtilis LipA),the alanine side chain at position 15points into the solvent,forming a hydropho-bic surface patch together with the side chain of Y17,at the rim of the substrate binding cleft.The absence of the methyl group in mutant L3-3mutant reduces local surface hydrophobicity.Residue 109is positioned at the surface,just after 310helix ␩4(Fig.4b).In both parent B.pumilus lipases L5and L21residue 109is a valine,and its mutation to serine reduces the local surface hydrophobic-ity.In mutant L3-3,this methyl group is absent,and local surface hydrophobicity is reduced.Moreover,the serine hydroxyl group isN.Akbulut et al./Journal of Biotechnology164 (2013) 123–129127Fig.4.Stereofigures of the L3-3mutant homology model.Point mutations are shown with yellow carbon atoms.Hydrogen bond interactions are shown as blue dashed lines.(a)Point mutations G14S and A15G located in the␤3-␩1/␣A loop(residues10–15)that connects strand␤1and helix␣A.The S14O␥atom has hydrogen bonding interactions with the N␦2atom of N18from helix␣A and the main chain nitrogen atom of G11.The␤-turn hydrogen bond interaction between G11O and G14N is also shown.The␤3-␩1/␣A loop is on one side of the substrate binding cleft,where the leaving group of a substrate would be bound.Some residues lining this part of the active site(I157,L160)are also shown in stick representation as well as the nucleophilic serine(S77).The main chain nitrogen atom of residue M12that forms part of the oxyanion hole is indicated with an asterisk(*).(b)The V109S point mutation;its side chain makes direct hydrogen bonds to the N␦2atom of N48,the N-terminal residue of helix ␣B,and to the main chain oxygen of A81(in helix␣C).The long␣B helix can make only one other direct hydrogen bond,between S56and D91.(For interpretation of the references to color infigure legend,the reader is referred to the web version of the article.)able to form two hydrogen bonds,similar to the equivalent thre-onine in B.subtilis lipase A.It can make one hydrogen bond to the main chain oxygen atom of residue A81(in helix␣C),and a second hydrogen bond to the N␦2atom of N48,the N-terminal residue of helix␣B.4.Discussion4.1.Activity and thermostability of mutant L3-3Mutant L3-3was selected from the shuffling library by screening for lipase activity.Our approach did not account for differences in lipase expression levels in the library and therefore may be biased.Nevertheless,our selection strategy resulted in a mutant with significantly improved specific activity.This mutant(L3-3) shows a6.4-and8.2-fold increase in specific activity,respectively when compared to its parent enzymes L5and L21.The usefulness of(mutant)lipases in industrial applications also depends on the effects of metal ions,detergents and organic solvents.For exam-ple,enzyme activity and stability in the presence of detergents is a requirement for laundry applications(Gaur et al.,2008).Further-more,tolerance to organic solvents facilitates the use of enzymes as biocatalysts in non-aqueous media,e.g.when it is necessary to dissolve or recover substrates or products in an organic phase, to decrease unwanted substrate or product inhibition(Hun et al., 2003),or when the product itself is an organic compound(e.g. methanol in the production of biodiesel)(Li et al.,2012).Our results indicate that for many of the compounds tested,mutant L3-3retains a comparable or higher relative activity than the parent enzymes,and therefore has improved characteristics as a possible biocatalyst.With respect to substrate specificity,L3-3remains a true lipase,with the highest activity observed for long chain tria-cylglycerol fatty acids,like the parent enzymes L5and L21.The significant stimulating effect of the presence of(10mM) FeCl2,CoCl2or CuCl2on L3-3is remarkable,since many lipases are inhibited by these metal salts(Gaur et al.,2008;Nthangeni et al.,2001;Sharma et al.,2001).The minimal effect of the metal-chelating agent EDTA on the activity of the parent enzymes and L3-3suggests that no metal binding sites exist,in agreement with previous studies.The strong inhibition by PMSF confirms that the enzymes under study are of the serine hydrolase class.Secondly, the tolerance of L3-3to detergents is comparable to or slightly bet-ter than that of the parent enzymes.Notably,the higher retained activity of L3-3in the presence of0.1%SDS compared to the parent enzymes,indicates that it is more resistant to unfolding.Thirdly,128N.Akbulut et al./Journal of Biotechnology164 (2013) 123–129the higher retained activity of L3-3for most organic solvents when compared to the parents indicates an increased tolerance to such compounds.Surprisingly,although we screened for activity as the desired property to be increased,L3-3also displays a remarkable increase in thermostability.Its half-life(T1/2)at50◦C of38.5min is a9.2-and8.8-fold improvement with respect to the parent enzymes. This is also reflected by a higher resistance to thermal inactivation: 70%of the initial activity of L3-3is retained after a30min incuba-tion at50◦C,a2.5-and3.7-fold increase compared to the parent enzymes(Fig.2).The fact that we obtained a mutant with both increased thermostability and activity indicates that it is possible to improve these properties at the same time.A similar case has been reported for Candida antarctica lipase B(Suen et al.,2004);there-fore a‘dual’screening approach involving both thermostability and activity may be generally beneficial for the directed evolution of lipase enzymes.In addition,further improvement of thermosta-bility and activity may be obtained by increasing the number of DNA-shuffling cycles.4.2.Structural implicationsIt has been proposed that several sequence/structural features contribute to the greater stability of thermophilic proteins(Kumar et al.,2000).These features include packing(of the core struc-ture),polar surface area,helical content/propensity,salt bridge and other hydrogen bond interactions,proline substitutions,insertions or deletions,loop stabilization and protein oligomerization.In the case of the B.pumilus L3-3mutant,the basis of enhanced thermosta-bility and activity(with respect to the parent enzymes)must lie, in one way or another,in the chimeric differences and point muta-tions.To study their structural effects,we constructed3D homology models for the B.pumilus L5and L21parent lipases and mutant L3-3(Figs.3and4),based on the crystal structure of B.subtilis lipase A(van Pouderoyen et al.,2001).Given the high sequence identity (78%)between the B.pumilus lipases and the B.subtili lipase A,and the nature and distribution of the about40differences between them,the homology models can be regarded as fairly reliable with an estimated root mean square deviation for backbone atoms of 0.6˚A(Chothia and Lesk,1986).Even in regions where differences are concentrated(mostly in loops and310helices),the main chain need hardly be affected because most of the differences are at the surface.In only one case(residue S14in L3-3)the model was man-ually adjusted to a more favorable side chain rotamer.Three of the six differences between mutant L3-3and parent L5occur in the␤3-␩1/␣A loop(residues10–15,Fig.4a),which forms one‘wall’of a narrow hydrophobic substrate binding cleft (Dröge et al.,2006).Both the chimeric I12M difference and A15G point mutation result in a reduced surface hydrophobicity,which may contribute to an increase in thermostability of L3-3.A similar proposal has been made for the A15S mutation in B.subtilis lipase A mutants3-3A9,4D3and6B(Ahmad et al.,2008;Kamal et al., 2011).The third difference,G14S,is thefirst reported mutation at this position for a family I.4lipase.The introduction of the serine side chain may increase thermostability by facilitating an addi-tional intra-loop hydrogen bond interaction with residue G11.This interaction would stabilize the conformation of the␤3-␩1/␣A loop, counteracting theflexibility-increasing effect of the introduction of a third glycine in this loop(mutation A15G).Together,the ther-mostability enhancing effects of mutations in the␤3-␩1/␣A loop may be attributed to a combination of reduced surface hydropho-bicity and stabilization of loop conformation.The observed enhanced activity of L3-3on p NP-palmitate as a substrate most likely stems from the G14S and I12M mutations in the substrate binding cleft.In the complexes of B.subtilis lipase A with different phosphonate inhibitors(Dröge et al.,2006)(PDB IDs:1R4Z,1R50),the IPG moiety of the inhibitor binds in a nar-row hydrophobic groove between residues I12-G13-G14(in the ␤3-␩1/␣A loop)and H156-I157.The importance of residues in the ␤3-␩1/␣A loop of B.subtilis lipase A has been shown previously in a loop-grafting study(Boersma et al.,2008),where enantioselectiv-ity toward IPG esters could be inversed by replacing this loop with loops originating from other␣/␤-hydrolases.A superposition(not shown)of L3-3with B.subtilis lipase A reveals that in L3-3the p NP moiety of the substrate would occupy the same space as the IPG moiety in B.subtilis lipase A.The side chain of a serine residue at position14would point into the binding groove and could interact with the substrate.The increased polarity of the environment of the scissile bond may favorably affect the hydrolysis of the cova-lent tetrahedral reaction intermediate.This reaction intermediate is stabilized by two peptide NH groups(the oxyanion hole)formed by residues12and78(Jaeger et al.,1999).Thus,the I12M and G14S mutations may affect both substrate affinity and reaction kinetics, apparently leading to a more active enzyme.On the other hand, substrate specificity is hardly affected(Supplementary Material Fig. S8).In contrast to the above mentioned mutations,point mutation V109S and the chimeric differences F20A and I169V,located at distances between12and16˚A from the active site serine,likely will not affect the activity of L3-3,at least not through short range effects.Instead,the F20A and V109S mutations appear to increase thermostability by reducing surface hydrophobicity,or by stabi-lizing interactions that are absent in the parent enzymes.Like in B.subtilis lipase A,two hydrogen bonds can link S109(T109in lipase A)to N48on helix␣B and to A81on helix␣C,thereby anchoring this part of the long109–123loop to the core secondary structure elements.The S109-N48hydrogen bond interaction also fixes the N-terminal end of the long␣B helix(Fig.4b),which has only one other hydrogen bond interaction(S56-D91).Likely, the stabilizing interactions due to the V109S mutation result in a more rigid enzyme structure,in agreement with the observa-tion that the L3-3mutant is more resistant to unfolding by SDS. Together with the removal of a solvent-exposed non-polar phenyl or methyl group of residues20and109,respectively,this results in enhanced thermostability.Finally,the I169V difference may affect thermostability by slightly changing the interior hydrophobic pack-ing of the enzyme.5.ConclusionsTo the best of our knowledge,our study describes thefirst application of DNA-shuffling to lipases from B.pumilus.From a single round of DNA-shuffling with two B.pumilus parent lipases, we have obtained a chimeric mutant(L3-3)with an up to8-fold increased specific activity and a9-fold increased half-life(at50◦C). The increased tolerance of L3-3to various detergents and organic solvents further enhances its application possibilities as a biocat-alyst.Based on a reliable homology model,we conclude that the observed enhancement of thermostability of L3-3is likely the con-sequence of(a)rigidification of enzyme structure by strengthening (hydrogen bonding)interactions between structural elements,and (b)the removal of hydrophobic patches on the enzyme surface (A15G,F20A,V109S).The same factors have been proposed to account for increased thermostability of evolved B.subtilis lipase A mutants(Ahmad et al.,2008;Kamal et al.,2011).The effect on enzyme activity is likely due to the fact that three of the six differences between mutant L3-3and parent L5(I12M,G14S,A15G) are in a loop adjacent to the substrate-binding site.These mutations may affect substrate binding and increase the reaction rate for the hydrolysis of the covalent reaction intermediate,but do not alter the substrate specificity.Although synergistic effects of mutations。

Materials Characterization Materials characterization is a crucial aspect of scientific research and development. It involves the study of the properties and behavior of different materials, and plays a significant role in various fields such as materials science, engineering, and manufacturing. By understanding the characteristics of materials, scientists and engineers can make informed decisions about their suitability for specific applications, design new materials with desired properties, and ensure the quality and reliability of products. One perspectiveon materials characterization is from the viewpoint of a materials scientist. For them, the process of characterization begins with the selection of appropriate techniques and instruments to analyze the material of interest. This could involve using techniques such as microscopy, spectroscopy, or diffraction to examine the structure, composition, and physical properties of the material. The scientist may also need to perform various tests, such as mechanical, thermal, or electrical tests, to assess the material's performance under different conditions. This comprehensive understanding of the material's properties is crucial for designing and optimizing materials for specific applications. From an engineer's perspective, materials characterization is essential for ensuring the reliability and performance of products. Engineers need to know how materials will behaveunder different operating conditions, such as temperature, pressure, or stress. By characterizing materials, engineers can make informed decisions about material selection, design components with appropriate dimensions and properties, andpredict the lifespan of products. For example, in the aerospace industry,materials characterization is critical for designing lightweight yet strong materials for aircraft structures, as well as understanding how these materialswill perform in extreme conditions. Another perspective on materials characterization comes from the manufacturing industry. Manufacturers rely on materials characterization to ensure the quality and consistency of their products. By characterizing raw materials and finished products, manufacturers can identify any variations or defects that may affect product performance or safety. For instance, in the pharmaceutical industry, materials characterization is used to analyze the composition and purity of drug substances and ensure that they meetregulatory standards. By doing so, manufacturers can guarantee the effectiveness and safety of their products. From a consumer's perspective, materials characterization may not be directly visible or apparent, but it greatly impacts the quality and performance of the products they use. For example, imagine buying a smartphone that claims to have a scratch-resistant screen. This claim is only possible because materials scientists and engineers have characterized the mechanical properties of the screen material and optimized it to resist scratches. Without materials characterization, consumers would not have access to products with the same level of performance and reliability. In conclusion, materials characterization is a vital aspect of scientific research, engineering, and manufacturing. It provides valuable insights into the properties and behavior of materials, enabling scientists, engineers, and manufacturers to make informed decisions about material selection, design, and quality control. From the perspective of a materials scientist, engineer, manufacturer, or consumer, materials characterization plays a crucial role in ensuring the performance, reliability, and quality of products.。

汽车发动机是为汽车提供动力的装置，是汽车的心脏，决定着汽车的动力性、经济性、稳定性和环保性。

下面是搜索整理的汽车发动机英文参考文献，欢迎借鉴参考。

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J.Serb.Chem.Soc.69(1)9–16(2004)UDC541.459:577.151.6 JSCS–3124Original scientific paperPurification and partial characterization of superoxidedismutase from the thermophilic bacteria Thermothrix sp.SVETLANA[EA TOVI]*,LJUBINKA GLIGI]*,@ELJKA RADULOVI]*and RA TKO M.JANKOV**#*Galenika a.d.,Institute,Batajni~ki drum b.b.,11080Belgrade,Serbia and Montenegro and**Faculty of Chemistry,University of Belgrade,Studentski trg16,11000Belgrade,Serbia and Montenegro(Received20June,revised8August2003)Abstract:Superoxide dismutase(SOD;EC1.15.1.1.),a high molecular weight compo-nent of the antioxidant defense system,provided promising results in the treatment ofoxidative damage.Thermothrix sp.,isolated from thermal spa water in Serbia,showedhigh superoxide dismutase activity.The SOD,from cell free extract,was purified tohomogenity by ammonium sulfate precipitation,Sephadex G75gel filtration chroma-tography and QAE Sephadex ion exchange chromatography.The specific activity of thepurified enzyme was9191U/mg.The purified enzyme was analyzed and partially char-acterized.SOD was localized in polyacrylamide gel by activity staining,based on thereduction of nitroblue tetrazolium(NBT)by superoxide.The enzyme molecular weightdetermined by gel chromatography is37kD.According to SDS PAGE it is composed oftwo subunits of equal size,joined by noncovalent interactions.The isoelectric point,as-sessed by isoelectric focusing is5.3.The optimum pH for enzyme activity was in therange of8to10.The optimum temperature for SOD activity was60ºC.After one hourof incubation at40,50and60ºC the SOD activity increases,but at80ºC,the SOD isdenaturated.After24hours of incubation at25ºC SOD activity only slightly decreases.Keywords:superoxide dismutase,Thermothrix sp.,isolation,purification,characterization.INTRODUCTIONThermophiles are a group of organisms characterized by their ability to live and flourish in unusually harsh conditions of high temperatures.Intrinsically stable and ac-tive at high temperatures,enzymes,products of thermophiles,offer major biotechno-logical advantages over enzymes of mesophilic or phychrophilic origin.Thermostable enzymes are easier to purify by heat treatment.Their thermostability is associated with a higher resistance to chemical denaturants and performing enzymatic reactions at high temperatures allows higher substrate concentrations,lower viscosity,fewer risks of microbial contamination and,often,higher reaction rates.SODs are a class of metalloproteins which catalyze the dismutation of superoxide radicals(O2·–)to oxygen and hydrogen peroxide.2#Serbian Chemical Society active member.910[EATOVI]et al.2O2·–+2H+®O2+H2O2SOD provides a vital defense mechanism against the formation of deleterious ox-ygen species.The presence of SOD in all aerobic organisms protects cells against oxi-dative stress.3Four common forms of the enzyme are known,differing in the metal ion cofactor in the active site.4Copper and zinc containing SODs have been found in the cytosol of eucaryotes,in chloroplasts,and in the periplasm of some prokaryotes.Fe-SOD is pres-ent in both aerobic and anaerobic bacteria,Archaea,and plants,whereas Mn-SOD is present in bacteria,Archaea,mitochondria and chloroplasts.Dismutases of the Fe-and Mn-type are closely related in sequence and structural homology.5A novel type of cytosolic SOD containing nickel as a cofactor has recently been discovered in several Streptomyces species.6This paper reports the isolation and partial characterization of a Mn-SOD from the thermophilic bacteria Thermothrix sp.EXPERIMENTALBacterial strain and growth conditionsThe thermophilic bacteria used in this work were isolated from thermal spa water in Serbia,purified and characterized.7The bacteria were grown in100ml of nutrition broth(1.5%peptone,0.5%meat extract, 0.5%NaCl,0.03%K2HPO4,pH7.2)in a1000ml Erlenmeyer flask,during20h at55ºC,and with120rpm agitation in a laboratory shaker.The cells were collected by centrifugation(15min,3000rpm,5ºC,Beckman J2-21centrifuge),and washed with0.067M phosphate buffer,pH7.8.Purification procedureThe pelleted cells were suspended in3ml of0.05M phosphate buffer,pH7.8containing1mM EDTA and ruptured with lysozyme(10mg ml-1,20min,24ºC).The suspension was centrifuged and the resulting clear supernatant was used.The cell-free extract was treated with(NH4)2SO4in two steps.First,solid(NH4)2SO4was added to the extract to50%saturation in an ice bath,then the mixture was stirred for15min,and left at4ºC for60min. The precipitate was removed by centrifugation.In the second step,the supernatant was treated with solid (NH4)2SO4to80%saturation,stirred for15min,and left at4ºC for60min.The precipitate with SOD activ-ity was centrifuged at4000rpm for30min(Hettich,Universal30RF centrifuge)and then dissolved in a min-imal volume of phosphate buffer saline(PBS),pH7.2and dialyzed at4ºC against the same buffer.Gel chromatography on Sephadex G75was carried out in PBS pH7.2on a column of dimensions 26´550mm.The fraction with SOD activity was eluted with55ml PBS and dialyzed against0.01M phos-phate buffer pH7.2over night.The determination of the molecular weight by gel filtration was carried out on Superose12HR10/30 column equilibrated with0.05M phosphate buffer pH7.2,and calibrated with the following molecular weight standards:immunoglobulin G(160000D),human serum albumin(67000D),b-lactoglobulin(35 000D),cytochrome c(12400D),vitamin B12(1355D),cytidine(246D).Ion exchange chromatography on QAE Sephadex was carried out in a20´250mm column,equili-brated with0.01M phosphate buffer pH7.2.The column was washed with0.1M phosphate buffer pH7.2 and the SOD was eluted with a concentration gradient of phosphate buffer pH7.2(0.2–0.3M).Protein concentrationProteins were determined by the method of Bradford,8using bovine serum albumin as the standard.Superoxide dismutase assaySOD was assayed by the method of Winterbourn et al .based on the ability of SOD to inhibit the reduc-tion of NBT by superoxide.9One unit was defined as the amount of enzyme causing one half of the maxi-mum inhibition of NBT reduction.9ElectrophoresisPolyacrylamide gel electrophoresis (P AGE)was performed as described by Laemmli 11using a 10%sep-arating gel,4%stacking gel,but without sodium dodecyl sulfate and b -mercaptoethanol.The protein bands were localized with Coomassie brilliant blue R250.The SOD was localized in situ by activity staining according to Beauchamp and Fridovich.10The glycoproteins were stained by specific staining with Schiff reagent.12SDS P AGE was performed using a 10%separating gel,4%stacking gel,with or without b -mercaptoethanol.The samples were heated for 5min at 100ºC in capped vials with 1%(w/v)SDS in the presence or absence of b -mercaptoethanol.The standards used to make a plot of log molecular weight versus mobility of the protein band were:phosphorylase B (94000D),bovine serum albumin (67000D),ovalbumin (43000D),trypsin inhibitor (20100D)and a -lactalbumin (14400D).The protein bands were lo-calized with Coomassie brilliant blue R250.Isoelectric focusing was performed on a Phast system (Pharmacia),500Vh.The pH gradient was 3.0–9.0,and the standards used to make a plot of pH versus mobility of the protein band were:trypsinogen (9.30);lentil lectin,basic (8.65);lentil lectin,middle (8.45);lentil lectin,acidic (8.15);myoglobin,basic band (7.35);myoglobin,acidic band (6.85);carbonic anhydrase B,human (6.55);carbonic anhydrase B,bovine (5.85);b -lactoglobulin A (5.20);soybean trypsin inhibitor (4.55);amyloglucosidase (3.50).RESUL TS AND DISCUSSIONPurification of SODThe results of a typical purification experiment are summarized in Table I.Aftereach purification step,the protein content and the enzyme activity were determined.SUPEROXIDE DISMUTASE 11Fig.1.Gel chromatography on a Sephadex G 75column;protein concentration (---),superoxide dismutaseactivity (—).12[EATOVI]et al.Fig.2.Ion-exchange chromatography on a QAE Sephadex column;protein concentration(´-´-´), superoxide dismutase activity(–l–),concentration of eluting buffer(—).According to our results,ammonium sulphate precipitation to50%saturation elimi-nated50%of the ballast protein,without loss in SOD activity,and the second precipi-tation step from50to80%ammonium sulfate saturation resulted in a SOD preparati-on of higher specific activity.After gel chromatography on Sephadex G75column,90 %of the ballast protein was discarded with a loss of SOD activity of about57%.Re-sults of this chromatographic procedure are shown in Fig.1.The SOD was eluted from a QAE Sephadex colum with a concentration gradient of phosphate buffer pH7.2(0.2–0.3M).Figure2presents the results of the ion exchange chromatography.The SOD fraction showed only one band of SDS PAGE after QAE Sephadex chromatography.It can be concluded that the SOD was purified to homogeneity after two-step ammonium sulphate precipitation,gel chromatography,and ion exchange chromatography.The total purification achieved by the procedure outlined in Table I was105fold over the first soluble extract,with a yield of15%.The specific activity of the purified enzyme was9191U/mg(in literature2500–10000U/mg14,16).The degree of purification of the SOD in every purification step was examined by SDS PAGE as shown in Fig.3a. TABLE I.Purification of SOD from Thermothrix sp.V olume/ml Total proteinmgTotal activityUSpecific activityU/mgYield/%Purification(fold)Cell-free exract17.55341328750%AS s.a18.2274224155102 1.8 80%AS p.b 5.72727239966 1.1 Dialysis8.52623118856 1.0 Sephadex G7550.6 2.599239224 4.5 QAE Sephadex20.70.07624919115105.4a ammonium sulfate supernatant,b ammonium sulfate pelletCharacterization of SODMolecular weight.The molecular weight of the SOD was determined by gel fil-tration on a Superose12HR10/30column equilibrated with0.05M phosphate buffer pH7.2.By this method,the molecular weight of the purified SOD was determined to be37kD.The subunit molecular weight was determined by SDS PAGE.In the pres-ence or absence of b-mercaptoethanol,the SOD showed only one band.The molecular weight in the absence and presence of b-mercaptoethanol was found to be18.6kD and 17.7kD,respectively.It can be concluded that the enzyme was composed of two sub-units of equal size,and that these subunits are not joined by interchain disulfide bonds. All known Mn-SODs(bacterial and mitochondrial)are either homodimers of homo-tetramers with subunit molecular weights of about20kD.13Specific staining for oligosaccharide-containing polypeptides indicated that the SOD from Thermothrix sp.is a glycoprotein.The only known SOD that is a glyco-protein is mammalian extracellular Cu/Zn SOD.14SUPEROXIDE DISMUTASE13Isoelectric point.The isoelectric point,determined by isoelectric focusing on a Phast system (pI gradient was 3.0–9.0)was found to be 5.3,as is shown in Fig.3b.Metal inhibition.Inhibition reactions with purified enzyme in the presence of some SOD inhibitors (cyanide,hydrogen peroxide and sodium azide)indicated that this enzyme is Mn-SOD.These results confirmed results with SOD eluted from polyacrylamide gel.8pH optimum.The SOD activity was determined over a wide pH range from 4to11.The SOD activity was the greatest in the pH range from 8–10(Fig.4a).It is clear that purified SOD is very active in alkaline solutions.For other Mn SODs,their activ-ity decreases at pH values greater than 7.8.15Temperature optimum and thermostability .The SOD activities were determined after preincubation of the enzyme for 30min over a wide temperature range (25–10014[EATOVI]et al.Fig.4.a)Effect of pH on the activity of the purified SOD.b)Effect of temperature on the activity of the pu-rified SOD.c)Effect of temperature on the stability of the SOD purified,after 15min (–n –),60min (–l –)and 24h (–s –)incubation.c)b)a)SUPEROXIDE DISMUTASE15ºC).The optimum temperature for the SOD activity was60ºC as is shown in Fig.4b. The SODs from mesophilic bacteria have a temperature optimum at lower tempera-tures(for example,SOD from alkaliphilic Bacillus have a temperature optimum at about35ºC),close to their growing temperature.16The influence of temperature on the SOD activity was determined after preincu-bation of the enzyme for different times(15min,1h,and24h),at different tempera-tures(25,40,50,60,70,80,and100ºC).After one hour incubation at40,50and60ºC,the SOD activity was increased,but at70ºC it was decreased.After one hour incu-bation at80ºC,the SOD was denatured(the activity was lost).After24hours incuba-tion at25ºC,the SOD activity was decreased by only7%,but at40ºC the loss was40 %,and at50ºC no activity remained(Fig.4c).SODs are thermostable enzymes,and after one hour incubation they are stable up to50ºC,as shown for Desulfovibrio gigas.17The activity of the SOD from the investigated bacteria was increased after one hour incubation at40,50and60ºC.CONCLUSIONSOD from the thermophilic bacteria Thermothrix sp.was purified to homogeneity and characterized for the first time.The enzyme was purified105times,with15%yield.It was estimated that SOD constituted0.86%of the protein of the crude soluble extract.The results demonstrate that Thermothrix sp.isolated from thermal spa water in Serbia is a potential producer of thermostable Mn SOD and that its large scale cultiva-tion for SOD production seems justified.I Z V O DPRE^I[]AVAWE I KARAKTERIZACIJA SUPEROKSID–DISMUTAZE IZTERMOFILNIH BAKTERIJA Thermothrix sp.SVETLANA[EATOVI]*,QUBINKA GLIGI]*,@EQKA RADULOVI]*i RATKO M.JANKOV***Galenika a.d.,Institut,Batajni~ki drum b.b.,11080Beograd i**Hemijski fakultet,Univerzitet u Beogradu,Studentski trg16,11000BeogradSuperoksid–dismutaza(SOD;EC1.15.1.1.)je komponenta antioksidativnog sistema koja daje zna~ajne rezultate u tretmanu oksidativnih o{te}ewa.Bakterije izolovane iz termal-nih bawskih voda Srbije,determinisane kao Thermothrix sp.,imaju visoku superoksid–dismu-taznu aktivnost.Superoksid–dismutaza iz}elijskog ekstrakta je pre~i{}ena dvostepenim talo`ewem amonijum–sulfatom,gel–hromatografijom na Sephadex G75koloni i jonoizme-wiva~kom hromatografijom na QAE Sephadex koloni.Specifi~na aktivnost pre~i{}enog enzima je9191U/mg.Superoksid–dismutaza je analizirana i okarakterisana.Molekulska masa pre~i{}enog enzima,odre|ena gel–hromatografijom,iznosi37kD.Enzim se sastoji iz dve identi~ne subjedinice spojene nekovalentnim interakcijama.Izoelektri~na ta~ka, odre|ena izoelektri~nim fokusirawem je5,3.Optimalna pH vrednost delovawa enzima predstavqa opseg pH od8do10.Maksimalna aktivnost enzima je na60ºC.Inkubacija na40,50 i60ºC(60min)pove}ava superoksid–dismutaznu aktivnost,dok se na80ºC enzim denaturi{e, dok inkubacija od24sata na sobnoj temperaturi neznatno smawuje aktivnost.(Primqeno20.juna,revidirano6.avgusta2003.)16[EATOVI]et al.REFERENCES1.Z.G.Zeikus,Enzyme.Microb.T echnol.1(1979)2432.M.McCord,I.Fridovich,Free Radical Biol.Med.5(1988)3633.I.Fridovich,Annu.Rev.Biochem.64(1995)974.I.Fridovich,J.Biol.Chem.272(1997)185155.E.Parcer,C.Blake,FEBS Lett.229(1988)3776.J.Chun,H.Y oun,Y.Yim,H.Lee,M.Kim,Y.Hah,S.Kang,J.Syst.Bacteriol.47(1997)4927.Lj.Gligi},@.Radulovi},G.Zavi{i},Enzyme Microbial T echnol.27(2000)7898.M.Bradford,Anal.Biochem.72(1976)2489.C.Winterbourn,E.Hawkins,M.Brain,W.Carrell,b.Clin.Med.85(1975)33710.C.Beauchamp,I.Fridovich,Anal.Biochem.28(1977)362emmli,Nature227(1970)68012.B.Leach,J.Collawn,W.Fish,Biochemistry19(1980)537413.R.Cannio,G.Fiorentino,A.Morana,M.Rossi,S.Bartiolucci,Frontiers in Bioscience5(2000)76814.T.Oury,J.Crapo,Z.V alnickova,J.Ehghild,Biochem.J.317(1996)5115.L.McCord,I.Fridovich,J.Biol.Chem.244(1969)604916.Y.Hakamada,K.Koike,T.Kobayashi,S.Ito,Extremophiles1(1997)7417.W.Dos Santos,I.Pacheco,M.Liu,M.Teixeira,A.Xavier,J.LeGall,J.Bacteriol.182(2000)796.。

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红芸豆红细胞凝集素单体的分离纯化和性质研究【摘要】目的研究一种从红芸豆中提取红细胞凝集素单体(PHA-E)的新方法。

方法红芸豆经过浸提，离子交换树脂分离，分子筛层析纯化后得到PHA-E样品。

采用电泳法测定其纯度、分子量和等电点。

用2%人细胞悬液测定样品凝集红细胞的能力和影响凝血的因素，使用硫酸苯酚法测定其糖含量。

结果经PAGE分析PHA-E样品为单带电泳纯，SDS-PAGE上显示亚基分子量为32 kD，等电点为6.5.样品使人红细胞50%凝集的蛋白质最低浓度为4 μg/ml左右。

单糖不影响PHA-E凝血活力，EDTA抑制其凝血活力，Zn2+促进其凝血。

糖含量为8.1%.【关键词】红细胞凝集素分离纯化凝血性质研究Purification and Partial Characterization of Erythrohemagglutinin from Phaseolus VulgarisHE Tao1，ZHANG Tao2*，WU Chang-ying1，WANG Liang1，SHAN Xiao-xue1，LUO Qin1(School of Preclinical Medicine，Chengdu Medical College，1.2006s Biotechnology，2.Biotechnology laboratory，Chengdu，610083，China)Abstract：Objective To study a new method for Erythrohemagglutinin（PHA-E）production from Phaseolus vulgaris.Methods PHA-E was purified from the excellent seeds of Phaseolus vulgaris by extraction，ion exchange chromatography and final gel filtration chromatography.The purity，the molecular weight and the isoelectric point of purified PHA-E were determined by electrophoresis.The ability and the influencing factor of agglutination were determined by 2% erythrocytes of human.Conclusion The purified PHA-E was single PAGE outline and had apparent subunit molecular weights of 32 kD by SDS-polyacrylamide gel electrophoresis.Isoelectric point of the PHA-E was 6.5.PHA-E can promote agglutination of human erythrocytes at 4 μg/ml，but its activity was not inhibited by any monosaccharide.EDTA caninhibit the ability of agglutination，and Zn2+ can accelerate it.The sugar content of PHA-E was 8.1%.Key words:erythrohemagglutinin；separation and purification；haemagglutination；characterization植物凝集素(Phytohemagglutinin PHA)是从豆科植物种子中提取的一种含糖蛋白质［1］。

Purification and Characterization of a NovelGalloyltransferase Involved in Catechin Galloylation in the Tea Plant (Camellia sinensis )*Received for publication,July 20,2012,and in revised form,October 31,2012Published,JBC Papers in Press,November 6,2012,DOI 10.1074/jbc.M112.403071Yajun Liu ‡1,Liping Gao ‡1,Li Liu §,Qin Yang ‡,Zhongwei Lu ‡,Zhiyin Nie ‡,Yunsheng Wang ‡,and Tao Xia §2From the ‡School of Life Science and §Key Laboratory of Tea Biochemistry and Biotechnology,Ministry of Education in China,Anhui Agricultural University,130West Changjiang Rd,Hefei,Anhui 230036,ChinaCatechins (flavan-3-ols),the most important secondary met-abolites in the tea plant,have positive effects on human health and are crucial in defense against pathogens of the tea plant.The aim of this study was to elucidate the biosynthetic pathway of galloylated catechins in the tea plant.The results suggested that galloylated catechins were biosynthesized via 1-O -glucose ester-dependent two-step reactions by acyltransferases,which involved two enzymes,UDP-glucose:galloyl-1-O -␤-D -glucosyl-transferase (UGGT)and a newly discovered enzyme,epicat-echin:1-O -galloyl-␤-D -glucose O -galloyltransferase (ECGT).In the first reaction,the galloylated acyl donor ␤-glucogallin was biosynthesized by UGGT from gallic acid and uridine diphos-phate glucose.In the second reaction,galloylated catechins were produced by ECGT catalysis from ␤-glucogallin and 2,3-cis -fla-van-3-ol.2,3-cis -Flavan-3-ol and 1-O -galloyl-␤-D -glucose were appropriate substrates of ECGT rather than 2,3-trans -flavan-3-ol and 1,2,3,4,6-pentagalloylglucose.Purification by more than 1641-fold to apparent homogeneity yielded ECGT with an estimated molecular mass of 241to 121kDa by gel filtration.Enzyme activity and SDS-PAGE analysis indicated that the native ECGT might be a dimer,trimer,or tetramer of 60-and/or 58-kDa monomers,and these monomers represent a het-erodimer consisting of pairs of 36-or 34-of and 28-kDa subunits.MALDI-TOF-TOF MS showed that the protein SCPL1199was identified.Epigallocatechin and epicatechin exhibited higher substrate affinities than ␤-glucogallin.ECGT had an optimum temperature of 30°C and maximal reaction rates between pH 4.0and 6.0.The enzyme reaction was inhib-ited dramatically by phenylmethylsulfonyl fluoride,HgCl 2,and sodium deoxycholate.Flavonoids,a major class of secondary metabolites in plants,have a number of important physiological roles as endogenous auxin transport regulators (1–3),root development (4,5),seed germination (6),allelopathy (7),plant-bacterium interaction (8,9),UV-B protection (10),and plant defense against pathogens and environmental stress (11).Flavonoids can be grouped into several subgroups including chalcone,flavone,flavonol,flavandiol,anthocyanin,proantho-cyanidin (oligomer or polymer of flavan-3-ols and flavan-3,4-diol units)and other specialized forms (12).Flavan-3-ols (cat-echins),which comprise ϳ70–80%of tea polyphenols,are rich in young leaves and shoots of the tea plant (Camellia sinensis (L.)O.Kuntze).Catechins,with a basic 2-phenylchromone structure,are characterized by the di-or tri-hydroxyl group substitution of the B ring,the 2,3-position isomer of the C ring,and presence of a galloyl group at the 3-postion of the C ring (Fig.1).On the basis of the classical definition proposed of galloyl group structural features,catechins are divided into gal-loylated and nongalloylated compounds.Galloylated catechins,including (Ϫ)-epigallocatechin gallate (EGCG)3and (Ϫ)-epi-catechin gallate (ECG),esterified often with gallic acid (GA)in the 3-hydroxyl group of the flavan-3-ol units are major catechin compounds that account for up to 76%of catechins in the tea plant (13,14).Catechins,especially EGCG,possess antioxidant activity,antimutagenic,anticarcinogenic,antidiabetic,antibacterial,and anti-inflammatory potential,antihypertensive and anticar-diovascular disease effects,solar UV protection,body weight control effects,and therapeutic properties for Parkinson dis-ease (15).The health-promoting effects of galloylated catechins are stronger than those of nongalloylated catechins (16,17).Flavonoid biosynthesis has been a major focus of investiga-tion in recent decades (12).As the building blocks of most pro-*Thiswork was supported by the Natural Science Foundation of China (30972401,31170647,and 31170282),the Natural Science Foundation of Anhui Province (11040606M73),the Collegiate Natural Science Founda-tion of Anhui Province (KJ2012A110),the Program for Changjiang Scholars and Innovative Research Team in University (IRT1101),and the Major Pro-ject of Chinese National Programmes for Fundamental Research and Development (2012CB722903).1Both authors contributed equally to this work.2To whom correspondence should be addressed.Tel.:86-551-5786003;Fax:86-551-5785729;E-mail:xiatao62@.3The abbreviations used are:EGCG,(Ϫ)-epigallocatechin gallate;ECG,(Ϫ)-epicatechin gallate;ECGT epicatechin:1-O -galloyl-␤-D -glucose O -gal-loyltransferase;GA,gallic acid;C,(ϩ)-catechin;EC,(Ϫ)-epicatechin;GC,(ϩ)-gallocatechin;EGC,(Ϫ)-epigallocatechin;␤G,␤-glucogallin,1-O -gal-loyl-␤-D -glucose;ConA,concanavalin A;GCG,(ϩ)-gallocatechin-3-gallate;SCPL,serine carboxypeptidase-like;UDPG,UDP glucose.THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL.287,NO.53,pp.44406–44417,December 28,2012©2012by The American Society for Biochemistry and Molecular Biology,Inc.Published in the U.S.A.F1Fn3anthocyanidins,the 2,3-trans -flavan-3-ol (ϩ)-catechin and 2,3-cis -flavan-3-ol (Ϫ)-epicatechin biosynthetic pathways have been investigated intensively at the biochemical and genetic levels (18,19).The biosynthetic pathway of nongalloylated cat-echins,which include (ϩ)-catechin (C),(Ϫ)-epicatechin (EC),(ϩ)-gallocatechin (GC),and (Ϫ)-epigallocatechin (EGC)is well documented.Some key genes and enzymes in the pathway include dihydroflavonol 4-reductase (EC 1.1.1.219),leucoan-thocyanidin reductase (EC 1.3.1.77),anthocyanidin synthase (EC 1.3.1.77),and anthocyanidin reductase (EC 1.3.1.77)(20–23).Despite recent progress in improving our understanding of flavan-3-ol synthesis,the mechanism involved in galloylation of catechins remains a mystery (19).In the 1980s,the biosynthesis of galloylated catechins and GA in the tea plant was investi-gated using radioactive tracer techniques.It was found that GA was presumably esterified with epigallocatechin and epicat-echin to form catechin gallates in young tea shoots,and the amount of GA might be a key limiting factor for the formation of EGCG and ECG (24).Understanding the galloylation of flavan-3-ols has been hin-dered by an absence of spontaneous genetic mutants for cate-chin biosynthesis.Niemetz and Gross (25)have done much research on the biogenetic pathways of hydrolyzable tannins.Their research confirmed that ␤-glucogallin (1-O -galloyl-␤-D -glucose (␤G))exerts a dual role functioning not only as an acyl acceptor but also as an efficient acyl donor.This work indicates ␤G plays the same role in biosynthesis of galloylated catechin (Fig.2).Galloylation of glucose with GA to yield ␤G,the first specific metabolite in the route to hydrolyzable tannins,was catalyzed by enzyme extracts from oak leaves with UDP-glu-cose serving as an activated substrate (26).This result indicated that,in galloylated catechin biosynthesis,␤G rather than GA might be a precursor of galloylated flavan-3-ols.In this study we sought to identify the enzymatic reactions and purify the key enzyme involved in galloylated catechin biosynthesis.Enzyme assays in vitro were designed to inves-tigate the biosynthesis of galloylated catechins,and the enzymes involved in galloylated catechin biosynthesis werepurified and identified.In addition,the enzyme properties were investigated.EXPERIMENTAL PROCEDURESPlant Materials —Leaves of the tea plant (C.sinensis (L.)O.Kuntze)were plucked from the experimental tea garden of Anhui Agricultural University during early summer.All of the samples were immediately frozen in liquid nitrogen and stored at Ϫ80°before analysis.Enzyme Extraction and Enzyme Assays —Enzyme extraction was performed in accordance with the method of Zhang et al.(27).All enzymes assays were conducted in phosphate buffer.In the multienzyme incorporative reaction system,theUGGT/FIGURE 1.Basic units of typicalcatechins.FIGURE 2.Reaction diagram of the galloylated catechin biosynthetic pathway.In the first reaction (I ),the galloylated acyl donor ␤G was biosyn-thesized by UGGT from the substrates GA and UDPG.In the second reaction (II),galloylated catechins ECG or EGCG were produced by ECGT from the substrates ␤G and nongalloylated catechins EC or EGC.Galloyltransferase Involved in Catechin GalloylationAQ:AAQ:BF2ECGT (UDP glucose:galloyl-1-O -␤-D -glucosyltransferase/epi-catechin:1-O -galloyl-␤-D -glucose O -galloyltransferase)assay solution was incubated at 30°C for 1.5h in a total volume of 1.5ml containing 50m M phosphate buffer (pH 6.0),0.4m M EGC or EC,1.4m M GA,2.3m M UDP glucose (UDPG),4m M ascorbic acid,and crude enzyme extract (0.55mg of total protein).The UGGT reaction solution was incubated at 30°C for 1.5h in a total volume of 1.5ml containing 50m M phosphate buffer (pH 6.0),2.3m M UDPG,1.4m M GA,4m M ascorbic acid,and crude enzyme extract (0.55mg of total protein).The ECGT assay solution was incubated at 30°C for 1h in a total volume of 1.5ml containing 50m M phosphate buffer (pH 6.0),0.4m M EGC or EC,0.96m M ␤G (Advanced Technology and Industrial Company,Hong Kong,China),4m M ascorbic acid,and crude enzyme extract (0.55mg of total protein).The above enzyme reactions were terminated by the addition of ethyl acetate.Each of the reaction products was extracted three times with 3ml of ethyl acetate.The ethyl acetate extract was evaporated and redissolved in 500␮l of methanol and then used directly for analyses of the enzymatic reaction products.For gel filtration-purified enzyme activity analysis,the ECGT assay solution was incubated at 30°C for 10min in a total vol-ume of 100␮l containing 50m M phosphate buffer (pH 6.0),0.4m M EGC or EC,0.96m M ␤G,and 5.6␮g ofenzyme.Enzymatic reactions were terminated by the addition of 10␮l of 5M HCl to the assay solution,and then the solution was used directly for analysis of the enzymatic reaction products.The protein concentration was determined by the Bradford method (28)using bovine serum albumin as a standard.In the control treatment,crude enzyme extract was heated to 100°C to inactivate enzyme activities.Analysis of UGGT and ECGT Enzyme Reaction Products —Extraction of enzyme reaction products was performed accord-ing to the method of Liu et al.(29).The solution (20␮l)of reaction products was spotted on a silica GF254TLC sheet (5ϫ20cm;HeFei BoMei Biotechnology Co.,Hefei,China)that was developed in chloroform:methanol:formic acid (28:10:1,v/v)and then sprayed with 1%vanillin-HCl (w/v)reagent.The spots of reaction products in the methanol extract were identified by R f values,and their visual color compared with those of cate-chin standards.The reaction products extract was analyzed by HPLC on a Phenomenex Synergi 4u Fusion-RP80column (5␮m,250ϫ4.6mm)with detection at 280nm.Ultraviolet spectra were recorded with a Waters 2487UV array detector (Waters Corp.,Milford,MA).For HPLC analysis,the solvent system consisted of 1%(v/v)acetic acid (A)and 100%acetonitrile (B).After injec-tion (5␮l),a linear gradient at a flow rate of 1.0ml/min was set as follows:B from 10to 13%(v/v)in 20min was initiated,then B from 13to 30%(v/v)between 20and 40min;B from 30to 10%(v/v)between 40and 41min.Peaks were identified by compar-ison of the retention times with those of standards.Analysis of enzyme reaction products by LC-MS followed the method of Miketova et al.(30).Enzymatic products were ana-lyzed by HPLC,and the area of the product peaks were collected and identified by LC-MS.Liquid chromatography electrospray-ionization-MS analyses were performed on a Thermo Finnigan LCQ Advantage instrument using the following conditions:negative ion detection mode,centroiding mode,multiplier at 1600keV,1000atomic mass units/s,source at 4.5kV,sheath gas at 70p.s.i.,auxiliary gas at 25p.s.i.,capillary temperature at 220°C,and UV detection at 220nm.Preparation and Identification of ␤-Glucogallin from Tea Plant —For ␤G analysis,1g of fresh leaves was crushed in liquid nitrogen and extracted with 5ml of methanol by sonication at room temperature for 10min followed by centrifugation at 4000ϫg for 15min,and the residues were re-extracted twice as above.The supernatants were pooled and evaporated and redissolved in 5ml of water.The pooled supernatants were then extracted three times with chloroform.The supernatant water phase was purified further with a SEP-PAK C 18cartridge,and after filtration the supernatant was separated by HPLC,and the ␤G peak was collected and freeze-dried.The powder was used for chemical identification by HPLC,MS,and 1H and 13C NMR spectroscopy.For HPLC analysis,the solvent system consisted of 1%acetic acid in water (A)and acetonitrile (B).After injection (5␮l),a linear gradient at a flow rate of 1.0ml/min was established as follows:B from 0to 10%(v/v)in 10min was initiated,then B from 10to 30%between 10and 30min.Peaks were identified by comparing the retention times with those of standards.The LC-MS analysis of ␤G employed the same method as that used for analysis of enzyme reaction products described above.1H and 13C NMR spectra were recorded in methanol-d 4on a Bruker Avance 400MHz spectrometer using TMS as an internal standard.Chemical shifts were expressed in ppm (␦).ECGT Isolation and Purification —ECGT purification con-sisted of acetone powder preparation,ammonium sulfate pre-cipitation,hydrophobic interaction chromatography,conca-navalin A (ConA)chromatography,and gel filtration.The first two steps were performed at 4°C,and the last three steps were conducted at room temperature.Gel filtration was performed to estimate the relative molecular mass of the enzyme.Step 1;Ammonium Sulfate Precipitation —The acetone pow-der was prepared by homogenization of 50g of tea leaves in cold acetone (Ϫ20°)with a Waring blender.The finely ground pre-cipitate was collected by vacuum filtration.The precipitate was washed several times with acetone until the washings were col-orless.This precipitate powder was used as a crude material for ECGT preparation.Precipitate powder (20g)was homogenized with 400ml of extraction buffer (50m M phosphate buffer (pH 7.0),4m M ␤-mercaptoethanol,1%(w/v)polyvinylpolypyrroli-done (Sigma))and filtered through cheesecloth.The homoge-nate was centrifuged at 15,000ϫg for 15min at 4°C,and the supernatant was fractionated with 20–40%ammonium sulfate.Step 2;Hydrophobic Interaction Chromatography —The pre-cipitate was dissolved in 20m M phosphate buffer (pH 7.0)con-taining 1M ammonium sulfate and loaded onto a butyl-Sephar-ose column (20cm ϫ2.5-cm inner diameter,Bio-Rad).For hydrophobic interaction chromatography,the solvent system consisted of 20m M phosphate buffer (pH 7.0)including 1M ammonium sulfate (A)and phosphate buffer (pH 7.0)(B).After application of the enzyme solution,the column was washed with five volumes of buffer A and subsequently eluted with a stepped gradient of 38,95,and 100%B at a flow rate 2.5ml/min.Step3;Affinity Chromatography—The active fractions were subjected to ConA-Sepharose4B chromatography(column10 cmϫ1.6cm inner diameter;GE Healthcare).For ConA chro-matography,the solvent system consisted of20m M Tris-HCl (pH7.0)containing0.5M NaCl(A)and20m M Tris-HCl(pH 7.0)containing0.5M␣-D-methylglucoside(B).After applying the enzyme solution,the column was washed with5volumes of buffer A and subsequently eluted with100%B at a flow rate of1 ml/min.Step4;Gel Filtration Chromatography—The active fractions were subjected to gel filtration on a Superdex200column(50 cmϫ1.6cm inner diameter;GE Healthcare)and eluted with20 m M phosphate buffer(pH7.0)containing0.15M NaCl at a flow rate of0.8ml/min.Step5:SDS-PAGE Assay—SDS-PAGE was performed in accordance with the method of Laemmli(31),after which the proteins were visualized with Coomassie Brilliant Blue using the methods of Oakley(32).Protein Identification by MALDI-TOF-TOF MS—Protein spots were cut from gels,destained for20min in50m MNH4HCO3solution containing30%acetonitrile,and washed inMilli-Q water until the gels were destained.The spots wereincubated in0.2M NH4HCO3for20min and then lyophilized.Each spot was digested overnight in12.5ng/ml trypsin in0.1MNH4HCO3.After trypsin digestion,the peptide mixtures wereextracted with8␮l of extraction solution(50%acetonitrile, 0.5%TFA)at37°for1h.Finally the extracts were dried underthe protection of N2.Samples were reconstituted in3␮l of50%acetonitrile containing0.1%TFA before MS analysis.A1-␮l drop of this peptide solution was applied to an Anchorchip target plate.After drying at room temperature,a0.1-␮l droplet of CHCA matrix was applied to the plate at the same position. Samples were analyzed with ultrafleXtreme(Bruker).All acquired spectra of samples were processed using flexControl-software(Bruker)in the default mode.Parent mass peaks with a mass range of500–3500Da were detected with a minimum S/N filter of10for precursor ion selection.The five most abun-dant MS peaks were selected for MS/MS analysis.The com-bined MS and MS/MS data from the MALDI-TOF-TOF anal-ysis were submitted to Mascot2.3.02for a search against the NCBI C.sinensis protein database(;827 sequences),C.sinensis Genome Database“cam.pep”to con-struct a protein data bank(40,551sequences,data not shown), and C.sinensis Genome Database“tie.pep”to construct a pro-tein data bank(49,413sequences,data not shown).The identi-fication was accepted based on results from three biological replicates.Properties of the ECGT Enzyme—For characterization of ECGT enzyme properties,gel filtration-purified enzyme was used.For determination of the optimum pH for the ECGT, citrate-phosphate buffer(pH4.0–5.5),phosphate buffer(pH 6.0–7.0),and Tris-HCl buffer(pH7.5–8.0)were used.The optimum temperature range for ECGT activity was tested from 0°to70°C at pH6.0.Other assay conditions were identical to those used in the routine assay.To test the effect of inhibitors on ECGT activity,the enzyme was incubated with the inhibitors for5min at30°C before the enzyme assay.Enzymatic activity was measured in the presenceof PMSF,ZnCl2,EDTA,and␤-mercaptoethanol at a final con-centration of0–50m M,and sodium deoxycholate and HgCl2 were used at a final concentration of0–5m M.Other assay con-ditions were identical to those used in the routine assay.For investigation of the effects of temperature and pH on enzyme stability,ECGT activity was tested after enzyme stor-age atϪ20,0,4,10,20,30,40,or50°for48h and after storage atpH4.0to9.0at4°for48h.In addition,the temporal stability of ECGT was determined after storage at4°C for0,2,7,20,or40 days.RESULTSEvidence for Biosynthetic Enzymes of Galloylated Catechins—Niemetz and Gross(25)confirmed that␤G acts not only as anacyl donor but as an acceptor in the biosynthesis of hydrolyz-able tannins.To determine whether catechin galloylation was similar to that of hydrolyzable tannin biosynthesis,a two-step enzyme assay incorporating the substrates GA,EC,or EGC and cosubstrate UDPG was designed,and the enzymatic products were analyzed via TLC and HPLC.The assay showed that UDPG was indispensable in the two-step enzymatic reaction,and a significant amount of␤G was detected in the enzymatic products by HPLC analysis(Fig.3).This result suggested a UDPG-dependent glucosyltransferase existed in the tea plant,and␤G was the enzymatic product.In addition,the galloylated catechins EGCG and ECG(Fig.3),but not(ϩ)-gallocatechin-3-gallate(GCG),were detected by the two-step enzyme assayvia TLC and HPLC(data not shown),which indicated that thecis-catechins EGC and EC were appropriate substrates of a gal-loyltransferase instead of the trans-catechin GC or C.Thesedata further confirmed that EGCG and ECG in the tea plant are biosynthesized via enzymatic galloylation of EGC and EC with␤G,whereas GCG in green tea beverages is derived from isomerization of EGCG during green tea production(33).To test the above assumptions further,two separate enzyme-reaction assays were performed.The first enzyme assay was designed to detect UDPG-glucosyltransferase activity with the substrates GA and UDPG,and the second assay was to detect galloyltransferase activity with the substrates␤G(or1,2,3,4,6-pentagalloylglucose)and EGC(or EC and GC).The enzymatic products were identified by TLC,HPLC,and LC-MS.In the UDPG-glucosyltransferase assay,the product␤G could not be analyzed effectively by TLC for lack of an appro-priate staining reagent.However,HPLC(Fig.4A)and LC-MS confirmed that the product was␤G(Fig.5A)and indicated the enzyme UGGT existed in the tea plant.In the galloyltransferase assay,TLC analysis of the enzyme reaction products showedtwo magenta spots with Rfvalues of0.43and0.28correspond-ing to the ECG and EGCG standards that were displayed in TLC sheets by staining with1%(w/v)vanillin-HCl reagent(Fig.6).This conclusion was confirmed by LC-MS.The parent ionswith m/z457and441corresponding to EGCG and ECG stand-ards,respectively,were observed(Fig.5,B and C).EGC,EC,and␤G were appropriate substrates of the galloyltransferase instead of GC(Fig.6)and1,2,3,4,6-pentagalloylglucose(PGG; Fig.4D).The deduced galloylated catechin biosynthetic path-way is depicted in Fig.2.Galloyltransferase Involved in Catechin GalloylationF3F4F5AQ:CF6Identification of ␤-Glucogallin in Tea Plant —To gain further evidence for the existence of ECGT and UGGT in the tea plant,␤G was extracted and identified from the leaves.An improved method for extraction and quantification of ␤G was estab-lished.A SEP-PAK C18cartridge was used in sample prepara-tion,and the purity of ␤G in the solvent was enhanced mark-edly.To prevent interference from noisy peaks,the linear gradient of the solvent system was optimized for ␤G analysis based on the method used for HPLC analysis of catechins.A single peak with retention time and spectral information con-sistent with those of the ␤G standard appeared at 11.75min in the chromatogram (Fig.7).The ␤G peak was collected largely via HPLC and identified by MS and NMR (Fig.8).The parent ion of the compound was detected at m /z 331,and majorfrag-FIGURE 3.HPLC analysis of UGGT/ECGT enzyme assay extracts.A and C ,the UGGT/ECGT assay solution was incubated at 30°C for 1.5h in a total volume of 1.5ml containing 50m M phosphate buffer (pH 6.0),0.4m M nongalloylated catechins (EGC or EC ),1.4m M GA,2.3m M UDPG,4m M ascorbic acid,1.5m M salicylic acid,and crude enzyme extract (0.55mg total of protein).The products ␤G and galloylated catechins EGCG or ECG were detected clearly in this two-step reaction enzyme assay.Peaks were identified by comparing the retention times with standards.B and D ,control treatments of the UGGT/ECGT assay extracts with the crude enzyme extract were heated to 100°C to inactivate enzyme activities.There were no enzymatic products in control treatments.AU ,absorbanceunits.FIGURE 4.HPLC analysis of UGGT and ECGT enzyme assay extracts.A ,the UGGT assay solution was incubated at 30°C for 1.5h in a total volume of 1.5ml containing 50m M phosphate buffer (pH 6.0),2.3m M UDPG,1.4m M GA,4m M ascorbic acid,1.5m M salicylic acid,and crude enzyme extract (0.55mg of total protein).The product ␤G was detected clearly in this assay.B and C ,the ECGT assay solution was incubated at 30°C for 1h in a total volume of 1.5ml containing 50m M phosphate buffer (pH 6.0),0.4m M nongalloylated catechins (EGC or EC ),0.96m M ␤G,4m M ascorbic acid,and crude enzyme extract (0.55mg of total protein).The galloylated catechins EGCG or ECG were detected clearly in this assay.D ,the ECGT assay solution was conducted with substrates 0.96m M 1,2,3,4,6-pentagalloylglucose (PGG ),0.4m M nongalloylated catechins (EGC ),and conditions otherwise identical to those of the ECGT assay.No galloylated catechins were produced effectively from the substrates 1,2,3,4,6-pentagalloylglucose and EGC.The product peaks of ␤G in A ,ECG in B ,and EGCG in C were collected and identified by LC-MS (see Fig.5).AU ,absorbance units.Galloyltransferase Involved in Catechin GalloylationF7F8ments were detected at m /z 271and 169,which were consistent with those of the ␤G standard.1H and 13C NMR spectra were recorded in methanol-d 4on a Bruker Avance 400MHz spec-trometer with TMS as an internal standard.Chemical shifts were expressed in ppm (␦).1H NMR ␦:7.086(2H,s,H-2Јand H-6Ј),5.615(1H,d,J ϭ8Hz,H-1),3.813(1H,dd,J ϭ12.0,1.6Hz,H-6a),3.658(1H,dd,J ϭ12.0,4.8Hz,H-6b),3.35to 3.45(4H,m,H-2-H-5).13C NMR ␦:167.06(C ϭO),146.53(C-5Ј),140.34(C-4Ј),120.78(C1Ј),110.54(C-2Ј,C-6Ј),95.97(C-1),78.84(C-5),78.23(C-3),74.15(C-2),71.11(C-4),62.36(C-6).These data were consistent with those of the standard com-pounds,and thus the compound was identified as ␤G.Purification of ECGT —Activity of ECGT was monitored throughout the purification.An ϳ1.64-fold purification was obtained by ammonium sulfate precipitation.The main active parts existed in the 20–60%ammonium sulfate fraction.About 10-fold specific activity was increased by hydrophobic interac-tion chromatography separation (Table 1).Monitoring of ECGT activity showed that ECGT enzyme was eluted from the column with a low ion eluent (Fig.9A ),which suggested ECGT was a highly hydrophobic protein.ConA-affinity chromatogra-phy was the most effective purification step for ECGT (Fig.9B ).Separation by ConA-affinity chromatography yielded an ϳ46-fold increase in purification (Table 1)and indicated that ECGT was a glycoprotein.An ϳ2-fold increase in specific activity was achieved by sep-aration on a Superdex 200column (Table 1).Obvious enzyme activities were detected approximately from 52min (fraction 2Ј)to 74min (fraction 6Ј,Fig.9C )with estimated molecular masses of 241to 121kDa based on a standard curve.To inves-tigate the subunit molecular masses of this enzyme,SDS-PAGE was routinely used for identification.The enzyme activity of lane 2was about double that of lane 6,whereas there were a large number of superfluous bands in lane 6from 40to 80kDa,so we speculated that three bands of estimated molecular masses of 36,34,and 28kDa were the subunits of this enzyme (Fig.9D ).One noteworthy phenomenon was that the SDS-PAGE Coomassie Brilliant Blue-stained bands changed with the degree of protein denaturation.SDS-PAGE analysis of lane 2showed that only two bands of 60and 58kDa remained after the enzyme was denatured in loading buffer containing 1%SDS,and three bands of 36,34,and 28kDa were present when the enzyme was denatured in loading buffer containing 5%SDS (Fig.10A).FIGURE 5.Mass spectra of products in the UGGT,ECGT,reaction assays.A ,shown are mass spectra of peaks corresponding to ␤G in the UGGT enzyme reaction assay (see Fig.4A ).The ions of full MS correspond to the ␤G standard.B and C ,MS assay of products peaks in the ECGT enzyme reaction assay (see Fig.4,B and C )are shown.The ions of 441and 457correspond to galloylated catechins ECG and EGCG standards,respectively.FIGURE 6.TLC assay of the ECGT reaction ne 1,ECG was produced with substrates ␤G and ne 3,EGCG was produced with substrates ␤G and ne 5,no product was produced with substrates ␤G and nes 2,4,and 6are catechin standards.Boxed band a ,ECG;boxed band b ,EGCG.FIGURE 7.␤-Glucogallin assay in tea leaves by HPLC.For HPLC analysis,the solvent system consisted of 1%acetic acid in water (A )and 100%acetonitrile (B ).After injection (5␮l),a linear gradient at a flow rate of 1.0ml min Ϫ1was established as follows:B from 0to 10%(v/v)in 10min was initiated,then B from 10to 30%(v/v)between 10min to 30min.Peaks were identified by comparing the retention times with the standard.mAU ,absorbance units.Galloyltransferase Involved in Catechin GalloylationAQ:QAQ:D,T1F9F10。