Acta Cryst.(2010).F 66,1041–1044
doi:10.1107/S1744309110026886
1041
Acta Crystallographica Section F
Structural Biology and Crystallization Communications
ISSN 1744-3091嗤之以鼻什么意思
Purification,crystallization and preliminary
crystallographic analysis of the catalytic domain of the extracellular cellula CBHI from Trichoderma harzianum
高一家长会pptFrancieli Colussi,a Larissa C.Textor,a Viviane Serpa,a
Roberto Nobuyuki Maeda,b Nei Pereira Jr b and Igor Polikarpov a *
a
Instituto de Fı´sica de Sa ˜o Carlos,Universidade
de Sa ˜o Paulo,Avenida Trabalhador Saocarlen 400,13560-970Sa ˜o Carlos-SP,Brazil,and b
Universidade Federal do Rio de Janeiro,Centro
de Tecnologia,Escola de Quı
´mica,Laborato ´rio de Denvolvimento de Bioprocessos (LaDeBio),21949-900Rio de Janeiro-RJ,Brazil
Correspondence e-mail:ipolikarpov@ifsc.usp.br
Received 28April 2010Accepted 7July 2010
The filamentous fungus Trichoderma harzianum has a considerable cellulolytic activity that is mediated by a complex of enzymes which are esntial for the hydrolysis of microcrystalline cellulo.The enzymes were produced by the induction of T.harzianum with microcrystalline cellulo (Avicel)under submerged fermentation in a bioreactor.The catalytic core domain (CCD)of cellobiohydrola I (CBHI)was purified from the extracellular extracts and submitted to robotic crystal
lization.Diffraction-quality CBHI CCD crystals were grown and an X-ray diffraction data t was collected under cryogenic conditions using a synchrotron-radiation source.
1.Introduction
A variety of microorganisms degrade cellulo in nature.The enzy-matic process that is involved in the hydrolysis of cellulosic materials has been studied in depth for veral filamentous fungi,such as,for example,Trichoderma reei .It is widely accepted that this hydrolytic process is accomplished by veral cellulas through a ries of reactions that are mediated by exoglucanas (cellobiohydrolas I and II),endoglucanas and -glucosidas (Kubicek et al.,1993).The enzymes are of considerable biotechnological interest,while the reaction conditions and the production costs of the enzymes significantly influence their range of application (Zhou et al.,2008).Cellulas have been ud in waste recycling and in the processing of cellulo-rich raw materials in the food,detergent,paper and textile industries (Bhat,2000).Recently,the application of cellulas that have potential in the production of biofuels has gained significant importance.
Of the cellulas creted i ,cellobiohydrolas (CBHs)are the most abundant enzymes,comprising roughly 60%of the cellulas produced by this fungus.The are key enzymes f
or the degradation of crystalline cellulo and the abnce of CBHs reduces the rate of cellulo hydrolysis by as much as 70%(Lynd et al.,2002).In general,the isoforms CBHI and CBHII act on the reducing and nonreducing ends of the cellulo chain,respectively,cleaving the internal glycoside bonds to relea the disaccharide cellobio (Hui et al.,2002).The enzymes are known to be glycosylated proteins with acidic pIs (Medve et al.,1998).Structurally,CBHI is a two-domain enzyme with a tertiary structure arrangement that is characteristic of glycosyl hydrolas (GHs)of family 7(Coutinho &Henrissat,1999).CBHs consist of a carbohydrate-binding module (CBM)and a cata-lytic core domain (CCD)parated by a heavily glycosylated peptide linker that is rich in prolines,rines and threonines (Gruno et al.,2004).Cellulo-binding modules (CBMs)are found in most cellu-las (Linder &Teeri,1996)and play an important role in the interaction of GHs with the insoluble cellulo (Palonen et al.,1999),which is crucial for the initiation and processivity of exoglucanas (Teeri et al.,1998).The activity of the enzyme towards insoluble polymeric substrate in the abnce of the CBM is frequently con-siderably lower than that of the full-length protein (Mattinen et al.,1997).Structural studies have demonstrated that the active site of the CBHI i is centrally located and is formed mainly by
a
山东省外事翻译学院#2010International Union of Crystallography All rights rervedto mother
large antiparallel -sandwich followed by a 50A
˚long cellulo-binding tunnel compod of four surface loops.As the cellulo chain pass through the tunnel the hydrolytic product cellobio is formed (Divne et al.,1994).
T.harzianum is a filamentous fungus that crets a cellulolytic complex that is able to efficiently hydrolyze a range of substrates to their reducing sugars (Saddler et al.,1985).With respect to the three major groups of cellulas,T.harzianum displays well balanced -glucosida,endoglucana a
nd exoglucana activities,efficiently hydrolyzing cellulolytic substrates into monomeric gluco (de Castro et al.,2010).Sequencing of the gene for T.harzianum exoglucana type I (CBHI)has shown that the enzyme is cloly related i CBHI (81%quence identity;Guilfoile et al.,1999).To date,little is known about the three-dimensional structure of CBHI from T.harzianum .In order to structurally characterize orthologues i CBHI and to better understand possible differences in the mechanisms of action of cellobiohydrolas,we have purified and crystallized the catalytic domain of CBHI from T.harzianum for X-ray diffraction analysis.
2.Experimental methods
2.1.Protein production
The enzymes of the cellulolytic complex were produced by sub-merged fermentation of T.harzianum in a 7.5l BioFlo 110bioreactor (New Brunswick Scientific)with an operating volume of 5l.The culture was maintained under 50%oxygen saturation by aeration and agitation at a temperature of 298K for 120h.The fermentation medium was prepared as described previously (Mandels et al.,1976),with microcrystalline cellulo (Avicel)as the carbon source to induce the expression of cellulas.
2.2.Protein isolation and purification
Full-length CBHI (amino acids 1–505,with a theoretical molecular weight of 53kDa)was purified from
a 5l culture of T.harzianum .On the fifth day the culture was harvested for 20min at 8000g and 277K and the clarified extract was submitted to filtration and concentration using hollow-fibre technology.The extract was then applied onto a Q-Sepharo (GE Healthcare)column equilibrated with 50m M Tris buffer pH 7.0and eluted with a linear gradient of 0–1M sodium chloride in the same buffer.The CBHI-containing fractions were concentrated with a Vivaspin concentrator and further purified by hydrophobic interaction chromatography on a Phenyl Sepharo column (GE Healthcare)equilibrated with 50m M Tris buffer pH 7.0and eluted with a linear gradient of 0–1M ammonium sulfate in the same buffer.The protein fractions were monitored for exoglucana activity using 1%(w /v )Avicel in 50m M sodium citrate pH 5.0as a substrate (Gho,1987)and the 3,5-dinitrosalicylic acid (DNS)method for quantification of the produced reducing sugars (Tomme et al.,1988).Finally,the fractions containing CBHI activity were pooled and concentrated.The Avicela activity of pure CBHI was deter-mined according to the procedures of the IUPAC Commission on Biotechnology (Gho,1987)using cellobio as a standard curve.As the next step,the CBHI was submitted to photolytic cleavage by the addition of papain at a protein:enzyme ratio of 50:1(w :w ).The reaction was carried out in 40m M potassium phosphate buffer pH 6.0at room temperature for 30min.Size-exclusion chromatography was applied for further purification of the catalytic core domain using a Superdex 7510/30column (GE Healthcare)equilibrated with 50m M Tris–HCl pH 7.0a
nd 300m M sodium chloride.The eluted protein was dialyzed overnight against 50m M Tris–HCl pH 7.0and con-
centrated prior to crystallization.Protein quantification was carried out by the method of Bradford (1976)using bovine rum albumin (BSA)as a standard.
2.3.Crystallization
The purified catalytic core domain of CBHI (46kDa;amino acids 18–449)at a concentration of 8mg ml À1was submitted to crystal-lization screening using the sitting-drop vapour-diffusion technique.Drops of 2m l final volume (1:1ratio of protein and screen solutions)were t up automatically with a Honeybee 931crystallization robot (Genomic Solutions Inc.)using a variety of commercially available screens (Qiagen)and maintained at a temperature of 291K.Needles grew using 35%PEG 4000as a precipitant.Crystal-growth optimi-zation trials were carried out in both hanging-drop and sitting-drop plates (24-well Linbro plates).
2.4.Data collection and processing
The cluster of needle-like CBHI CCD crystals was manipulated using an acupuncture needle and tra
nsferred to a cryosolution con-taining a mixture of 35%PEG 4000and 20%PEG 400.One single needle-like protein crystal was mounted in a cryoloop and directly flash-frozen in a nitrogen stream prior to X-ray data analysis.The diffraction data were collected on beamline MX2at the Synchrotron Light Source Laboratory (LNLS),Campinas,Brazil using a MAR
charge-coupled device detector (Guimara
˜es et al.,2009).Data covering 180
were collected using the oscillation method at a
headlineswavelength of 1.46A
˚.Data integration was carried out using the program XDS (Kabsch,2010)and data were scaled with the program SCALA (Collaborative Computational Project,Number 4,1994).
3.Results and discussion
3.1.Enzyme purification and crystallization
As expected,CBHI comprid the majority of the protein that was produced in the extracellular extract of T.harzianum (Fig.1,lane 1).Highly pure full-length CBHI could be obtained after the cond purification step via hydrophobic interaction chromatography (Fig.1,lane 3).Its enzymatic activity was determined to be 0.3U mg À1on Avicel substrate at pH 5.0and 323K.This value is at the upper limit or just slightly above the reported Avicela activity values obtained i CBHI,which range from 0.014to 0.26U mg À1depending
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CBHI catalytic domain Acta Cryst.(2010).F 66,
1041–1044
Figure 1
SDS–PAGE gel.Lane 1,supernatant of T.harzianum culture broth;lane 2,full-length CBH1eluted from the anion-exchange column;lane 3,full-length CBH1eluted from the hydrophobic interaction column;lane 4,CBHI CCD after cleavage with papain and size-exclusion chromatography purification.
Lane M contains protein markers (labelled in kDa).
on the initial substrate concentration and the experimental conditions (Tomme et al.,1988;Elgogary et al.,1989;Vehmaanpera et al.,2009).Following protein cleavage with papain,the CCD of CBHI was successfully parated from the CBM by size-exclusion chromato-graphy (Fig.1,lane 4).According to the size-exclusion chromato-graphy elution volume and SDS–PAGE,the CBHI CCD exhibits a molecular weight of about 50kDa,which is clo to the quence-bad theoretical molecular weight of the CBHI CCD (46kDa).The CBHI CCD was crystallized using a crystallization robot and veral commercial crystallizations kits covering more than 650different conditions.Crystals only grew in only one condition,which contained 35%PEG 4000as a precipitant.Small needles appeared very rapidly using this crystallization condition,so that the needle cluster was completely formed after 10h of incubation time (Fig.2).Various attempts were carried out to attempt to reduce the number of nuclei being formed and to favour single-crystal growth.Variations in the pH and the protein and precipitant concentrations and tests of different additives were ineffective,resulting exclusively in a shower of microcrystals or clusters of needles.Needles grown in clusters were tested for X-ray diffraction quality.Surprisingly,a small single needle-like crystal of approximate dimensions 10Â5Â5m m showed
a diffraction pattern that extended to a resolution of about 2.9A
˚(Fig.3).A complete native data t was collected on the dedicated
wiggler beamline MX2(LNLS,Brazil;Guimara
˜es et al.,2009).The data t was reduced in the orthorhombic space group P 222,with
unit-cell parameters a =56.9,b =69.3,c =90.7A
˚.Analysis of the systematic abnces revealed the prence of twofold screw symmetry along all three unit-cell axes.According to the Matthews
coefficient of 1.77A
˚3Da À1(Matthews,1968),there is only one molecule in the asymmetric unit and the solvent content of the crystal is 31%.Data-collection and processing statistics are shown in Table 1.
4.Conclusions
The cellula CBHI was successfully purified from the soluble extracellular fraction of T.harzianum c
ultured in a bioreactor.The catalytic core domain of the enzyme was submitted to crystallization and diffraction-quality crystals were obtained.An X-ray diffraction data t from a single needle-like CBHI catalytic core domain crystal was collected using a synchrotron source.
Acta Cryst.(2010).F 66,1041–1044Colussi et al.
CBHI catalytic domain语言学习
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Figure 2
Crystals of CBHI CCD grew as clusters in hanging drops using 35%(w /v )PEG 4000as precipitant.The black bar corresponds to 10m
m.
Figure 3
(a )Diffraction pattern collected from one single needle-shaped crystal extending tobureauveritas
a resolution of 2.9A
˚.(b )Clo-up view of the diffraction image with enhanced contrast.
Table 1
Data-collection and processing statistics.
Values in parenthes are for the highest resolution shell.Beamline
MX2,LNLS Wavelength (A
˚) 1.46Space group
P 212121
Unit-cell parameters (A ˚)a =56.9,b =69.3,c =90.7Resolution range (A ˚)
55.1–2.9(3.0–2.9)No.of unique reflections 8493(1176)Mosaicity ( )0.5
Redundancy
7.0(6.7)Completeness (%)99.6(99.6)R merge †(%)11.9(29.5)h I / (I )i
6.5(2.8)
†R merge =P hkl P i j I i ðhkl ÞÀh I ðhkl Þij =P hkl Pidealistic
i I i ðhkl Þ,where I i (hkl )is the obrved intensity of an individual reflection and h I (hkl )i is the average intensity of that reflection.
We are grateful to the MX2staff members for beamline support and to the Brazilian Synchrotron Light Source(LNLS).This work was supported by Fundac¸a˜o de Amparo a`Pesquisa do Estado de Sa˜o Paulo(FAPESP)via rearch grant Nos.08/56255-9,2007/08706-9 and2009/05349-6,Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior(Capes)and Conlho Nacional de Denvolvimento Cientı´fico e Tecnolo´gico(CNPq)via INCT do Bioetanol and grant No.471834/2009-2.
References
Bhat,M.K.(2000).Biotechnol.Adv.18,355–383.
Bradford,M.M.(1976).Anal.Biochem.72,248–254.
Castro,A.M.de,Pedro,K.C.,Cruz,J.C.,Ferreira,M.C.,Leite,S.G.& Pereira,N.Jr(2010).Appl.Biochem.Biotechnol.doi:10.1007/s12010-010-8986-0.
Collaborative Computational Project,Number4(1994).Acta Cryst.D50, 760–763.
Coutinho,P.M.&Henrissat,B.(1999).Recent Advances in Carbohydrate Bioengineering,edited by H.J.Gilbert,G.J.Davies,B.Henrissat&B. Svensson,pp.3–12.Cambridge:Royal Society of Chemistry.
Divne, C.,Stahlberg,J.,Reinikainen,T.,Ruohonen,L.,Pettersson,G., Knowles,J.K.C.,Teeri,T.T.&Jones,T.A.(1994).Science,265,524–528. Elgogary,S.,Leite,A.,Crivellaro,O.,Eveleigh,D.E.&Eldorry,H.(1989). Proc.Natl Acad.Sci.USA,86,6138–6141.morocco
Gho,T.K.(1987).Pure Appl.Chem.59,257–268.
Gruno,M.,Valjamae,P.,Pettersson,G.&Johansson,G.(2004).Biotechnol. Bioeng.86,503–511.
Guilfoile,P.,Burns,R.G.,Zu-Yi,Amundson,M.&Chang,F.-H.(1999).J. Minn.Acad.Sci.64,18–22.Guimara˜es,B.G.,Sanfelici,L.,Neuenschwander,R.T.,Rodrigues,F.,Grizolli, W.C.,Raulik,M.A.,Piton,J.R.,Meyer,B.C.,Nascimento,A.S.& Polikarpov,I.(2009).J.Synchrotron Rad.16,69–75.
Hui,J.P.M.,White,T.C.&Thibault,P.(2002).Glycobiology,12,837–849. Kabsch,W.(2010).Acta Cryst.D66,125–132.
Kubicek,C.P.,Messner,R.,Gruber,F.,Mach,R.L.&Kubicekpranz,E.M. (1993).Enzyme Microb.Technol.15,90–99.
Linder,M.&Teeri,T.T.(1996).Proc.Natl Acad.Sci.USA,93,12251–12255.
Lynd,L.R.,Weimer,P.J.,van Zyl,W.H.&Pretorius,I.S.(2002).Microbiol. Mol.Biol.Rev.66,506–577.
Mandels,M.,Andreotti,R.&Roche,C.(1976).Biotechnol.Bioeng.Symp.6, 21–33.
Matthews,B.W.(1968).J.Mol.Biol.33,491–497.
Mattinen,M.L.,Kontteli,M.,Kerovuo,J.,Linder,M.,Annila,A.,Lindeberg, G.,Reinikainen,T.&Drakenberg,T.(1997).Protein Sci.6,294–303. Medve,J.,Lee, D.&Tjerneld, F.(1998).J.Chromatogr.A,808,153–165.
Palonen,H.,Tenkanen,M.&Linder,M.(1999).Appl.Environ.Microbiol.65, 5229–5233.
Saddler,J.N.,Hogan,C.M.&Louisize,G.(1985).Appl.Environ.Microbiol. 22,139–145.
Teeri,T.T.,Koivula,A.,Linder,M.,Wohlfahrt,G.,Divne,C.&Jones,T.A. (1998).Biochem.Soc.Trans.26,173–178.
Tomme,P.,Vantilbeurgh,H.,Pettersson,G.,Vandamme,J.,Vandekerckhove, J.,Knowles,J.,Teeri,T.&Claeysns,M.(1988).Eur.J.Biochem.170, 575–581.mcjd
Vehmaanpera,J.,Alapuranen,M.,Puranen,T.,Siika-aho,M.,Kallio,J., Hooman,S.,Voutilainen,S.,Halonen,T.&Viikari,L.(2009).US Patent 20090042266.
Zhou,J.,Wang,Y.-H.,Chu,J.,Zhuang,Y.-P.,Zhang,S.-L.&Yin,P.(2008). Bioresour.Technol.99,6826–6833.
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