Core-binding domains of fungal cellulases from Trichoderma reesei were purified using a new and simple technique.
Cellulases were hydrolysed with papain and the binding domains were then separated from the digested
mixture by ultrafiltration. The enzymatic digestion process was monitored using capillary electrophoresis. This
methodology produced a yield of 85% of binding domains.Fundação para a Ciência e a Tecnologia (FCT) – PRAXIS XXI

Gama, F. M.

Lemos, M. A.

Mota, M.

Teixeira, J. A.

Biotechnology Letters

Universidade do Minho: RepositoriUM

Biotechnology Letters 22: 703–707, 2000.© 2000 Kluwer Academic Publishers. Printed in the Netherlands. 703A simple method to separate cellulose-binding domains of fungalcellulases after digestion by a proteaseM.A. Lemos∗, J.A. Teixeira, M. Mota & F.M. GamaCentro de Engenharia Biolo´gica-IBQF, Universidade do Minho, 4700-320 Braga, Portugal∗Author for correspondence (Fax: +351-253-678986; E-mail:adilia@deb.uminho.pt)Received 26 January 2000; Revisions requested 23 February 2000; Revisions received 6 March 2000; Accepted 7 March 2000Key words: capillary electrophoresis, core-binding domain, Trichoderma reesei, ultrafiltrationAbstractCore-binding domains of fungal cellulases from Trichoderma reesei were purified using a new and simple tech-nique. Cellulases were hydrolysed with papain and the binding domains were then separated from the digestedmixture by ultrafiltration. The enzymatic digestion process was monitored using capillary electrophoresis. Thismethodology produced a yield of 85% of binding domains.IntroductionThe purification and immobilisation of biologicallyactive proteins is an area of great importance in bothresearch and industry. Most protein immobilisationmethods require a chemical modification of the ma-trix, which at times can result in the introductionof toxic compounds. Moreover most of the matricesemployed are expensive materials. The choice of cel-lulose as an immobilisation matrix is attractive as itoffers an inexpensive and inert alternative. This ex-plains the recent attention on promoting the use ofcellulose-binding domains (CBDs), which have a nat-ural ability to bind to cellulose, as important ‘tools’in biotechnology. In relation to protein-based appli-cation areas, the CBD genes can be expressed aloneor in combination with other genes of interest to pro-duce recombinant or fusion proteins. These proteinscan be purified or immobilised on cellulose matri-ces. Many CBD fusion proteins have been produced,such as CBD-alkaline phosphatase (Greenwood et al.1989), CBD-β-glucosidase (Ong et al. 1991), CBD-proteinA (Ramirez et al. 1993) and more recentlyCBD-heparinase I (Shpigel et al. 1999). Furthermore,the use of CBDs in textile and paper industry can beadvantageous as CBDs may have potential applica-tions in the modification of polysaccharide fibres, forinstance in cotton, wood or paper (Din et al. 1991).The majority of the information concerning the roleof CBDs was gathered by the use of domain ex-change (Srisodsuk et al. 1997), domain removal (VanTilbeurgh et al. 1986), or site-directed mutagenesis(Reinikainen et al. 1992, 1995, Mattinen et al. 1997).The fungus Trichoderma reesei produces severalcellulases (Knowles et al. 1987), which show a similarstructural organisation, i.e., a catalytic domain (or coredomain), which is connected by a glycosylated linkerto a core-binding domain (CBD) located at either theN or C terminal of the protein (Van Tilbeurgh et al.1986, Abuja et al. 1988). All these CBDs consist of36–40 amino acids with very closed related sequences(Linder et al. 1995). Most of the CBDs investigatedin recent years were obtained by genetic engineering,however the separation of the two domains has alsobeen achieved using proteolysis (Van Tilbeurgh et al.1986, Tomme et al. 1988, Woodward et al. 1992,1994). In these cases the interest in proteolysis wasmore directed to the isolation of catalytic cores ofcellulases.In this work we have developed a simple methodto separate and isolate the binding domains fromcellulases (in this case Trichoderma reesei) usingproteolysis.704Materials and methodsEnzymesA crude cellulase preparation (Celluclast, NovoNordisk) was diluted (4×) in sodium acetate buffer(50 mM, pH 5), washed in an ultrafiltration cell(Amicon) with a 10-kDa nominal weight cut-off, poly-sulphone membrane to remove low molecular weightcompounds.Proteolysis was done with papain (papaya latex,Sigma). The protein concentration in the cellulasepreparation was determined using BCA Protein AssayKit (Pierce).Digestion conditionsDifferent cellulase to papain (w/w) ratios were taken,as well as duplicates for each experiment. The diges-tion was performed at room temperature (∼20 ◦C) fora maximum of 4 h. At intervals samples were checkedfor enzymatic activity and analysed using capillaryelectrophoresis (CE). Two digestions were performed(ratios 300:1 and 50:1) with the digested mixture ul-trafiltrated through both 10-kDa and 30-kDa nominalcut-off membranes to assure the separation of thecellulose-binding domains (CBDs) from the digestedmixture.Enzymatic measurementsCarboxymethylcellulose-CMC (1% w/v) was used assoluble substrate and filter paper (Whatman no. 1,3.9 mg) as insoluble substrate. The amount of sugarreleased was measured by the dinitrosalicylic acidmethod. Papain activity was assessed by QuantiCleaveProtease Assay Kit (Pierce).Capillary electrophoresisProtein separations were performed using a BioRad,BioFocus 2000 with an uncoated capillary (total length37 cm and 50 µm internal diameter). Electrophoreticinjection was applied to the sample at 10 kV for5 s, from negative to positive polarity. The appliedvoltage was 15 kV and the temperature was 20 ◦C.Protein fractions were separated by molecular weightand detected by monitoring the absorbance at 220 nm.For peptide analysis a coated capillary (total length24 cm and 25 µm internal diameter) was used. Injec-tion of the samples (filtrates) was done by pressure.Fig. 1. Effect of hydrolysis on the FPase activity after 4 h digestionfor different ratios cellulase:papain (w/w). Residual activities aregiven as percentages of the original.The run voltage was 10 kV and the absorbance wasmeasured at 200 nm.Adsorption assaysPurified CBDs were incubated at room temperature(∼20 ◦C) for 1 h with agitation. Samples were taken toevaluate the percentage of protein adsorbed into cellu-lose (Sigmacel, type 101–30 mg ml−1). The depletionof the protein was analysed by monitoring the decreasein absorbancy at 280 nm, after the centrifugation of thesuspensions.Results and discussionOptimisation of digestion conditionsThe effect of proteolysis on the enzyme activity andon the electrophoretic profiles is described below. Ini-tially the best enzyme:cellulase ratio was determined.Effect of proteolysis on the enzyme activityThe papain-digested solution was found to maintainits activity against carboxymethycellulose in all thepapain:cellulase ratios studied indicating that the cat-alytic activity was preserved.A decrease of activity was observed when an in-soluble substrate (filter paper) was used. The FPaseactivity decreased with incubation time when papainwas employed, with a greater decrease at higher pa-pain concentrations (Figure 1). These results suggestthat the protein cannot adsorb to the solid substratesurface and therefore the cellulases were split into705Fig. 2. Evolution of the digestion followed by capillary elec-trophoresis, using the uncoated capillary (20 ◦C), for the digestion300:1. The native cellulase, band A, is hydrolysed by papain givingrise to catalytic domains, band B, and the binding domain, band C.both the CBD and catalytic domain. This is in agree-ment with other published results (Van Tilbeurgh et al.1986, Tomme et al. 1988).Electrophoretic profiles of cellulases and digestedmixturesThe protein fractions were separated by molecularweight and an internal standard was used to correctmigration times. When the high ratio cellulase:papain(2000:1) was used the papain concentration was toolow to attack the cellulases, as the cellulase profilepattern did not change. However, for the lower ra-tios, it was evident that the cellulases were split inprotein fractions with smaller molecular weights, asa result of proteolysis. It is interesting to compare thedifferent electropherograms obtained with the diges-tion ratio of 300:1, where it is possible to follow thedigestion process with time (Figure 2). On the firstelectropherogram there is one main broad band (A).Part of the protein corresponding to this band is broken(because of the hydrolysis by the papain) leading theappearance of a second band (B) after 4 h of digestion.It can be seen that this ‘new’ protein has lower mole-cular weight (as the migration time is proportional tomolecular weight). After digestion for 24 h a furtherincrease in B with a concomitant decrease in A wasobserved and a third band (C) can also be seen in theelectropherogram. This is explained by the fact that thecellulases (A) were split in the catalytic domains (B)and binding domains (C). The latter fact is confirmedin the forthcoming section.Generally, if we look at the effect of the papainon the enzyme activity we can distinguish three maintypes of digestion: (I) corresponding at very light di-gestion (2000:1), (II) medium light digestion (300:1and 200:1), and (III) heavy digestion (50:1 and 10:1).In the very light digestion the effect of papain on theenzyme activity is not noticeable even after severaldays. The medium and heavy digestions seem to bethe optimal ratios in order to obtain the CBDs. In theseconditions the papain activity led to a decrease of theenzyme activity towards filter paper, meaning that thetwo cores were split. Considering this, and in orderto separate the CBDs from the digested mixture, twodigestions in medium and heavy conditions were per-formed and the CBD separation by ultrafiltration wasassayed.Separation of CBDs by ultrafiltrationThe digested mixtures were ultrafiltrated using mem-branes with nominal cut offs of 10 kDa (PM10)and 30 kDa (PM30). The same mixture was ultra-filtrated several times to obtain the maximum yieldof CBDs. Enzymatic measurements, electrophoresisanalysis and adsorption tests were performed to con-firm the presence of CBDs and to evaluate the extentof the proteolysis.Enzymatic activityCMCase and FPase activities were determined for thedigested mixture and filtrates. In the digested mixturethe CMCase activity was maintained, while a decreaseof FPase activity was observed (as seen previously).The filtrates obtained with the PM10 membrane didnot show any hydrolytic activity, however CMCase ac-tivity was detected in the filtrates obtained with PM30membrane showing that some catalytic cores managedto pass through the PM30 membrane.706Fig. 3. Adsorption of the different filtrates obtained sequentiallyfrom the 300:1 and 50:1 digestions (using the PM10 membrane)into cellulose. The % adsorbed was obtained by the difference inabsorbancy (280 nm) in the filtrate and the supernatant (obtainedby centrifugation after incubation of the filtrate with cellulose,30 mg ml−1, for 1 h at ∼20 ◦C).Electrophoretic profiles (filtrates)The purity of the filtrates was checked using CE. Theresults obtained displayed one peak corresponding toa protein with a molecular weight close to 9 kDa.The retention time obtained confirms that peak C inFigure 2 is likely to correspond to this peptide. Themolecular weight of the core-binding domain of fungalcellulases is close to 5 kDa, however the peptide re-leased can be heavily glycosylated thereby increasingthe peptide molecular weight to about 10 kDa (Tommeet al. 1988). Thus, it appears that we have obtained aglycosylated CBD.Adsorption testsThe affinity of the native cellulase and the proteolysedmixture for cellulose was evaluated. A significant re-duction of affinity towards cellulose occurred whenthe enzyme was submitted to proteolysis. Native en-zyme showed an adsorption of 71%, while when usingthe digested mixture at different ratios it was foundthat the lowest ratio (50:1) gave the lowest adsorption(41%, cf. 51%, for the ratio 300:1).The filtrates obtained showed an absorbance at280 nm, indicating that proteic material was present.In order to prove the presence of CBDs adsorptiontests were performed. Figure 3 shows the percentageof adsorption for the filtrates, obtained with a PM10membrane, from the digestion ratios 300:1 and 50:1.Adsorption studies of the filtrates obtained with thePM30 membrane were not performed since a residualcatalytic activity was found in these filtrates.Fig. 4. Comparison of electropherogram profiles of a filtrate (—)and supernatant (- - -), obtained by the peptide analysis with coatedcapillary. The supernatant was obtained by centrifugation after in-cubation of the filtrate with cellulose (30 mg ml−1) for 1 h at∼20 ◦C.The filtrates obtained from the 300:1 ratio ad-sorbed in a higher percentage than the ones from the50:1 digestion. This can be explained by the fact thatthe higher concentration of papain can lead to the de-struction and/or damage of the peptide chain givingrise to a loss of amino acids which, as proved byReinikainen et al. (1992) and Linder et al. (1995), areimportant in the binding of the CBDs to cellulose.To further ascertain the purity of the filtrate, an-other capillary electrophoresis method, in which themigration time depends on both the charge and mole-cular weight, was performed. A typical electrophero-gram is shown in Figure 4 displaying a dominant peak,thus the peptide appears reasonably pure. To confirmthat this peak represents the peptide corresponding toCBDs, microcrystalline cellulose was added to CBDssolutions. The supernatant was analysed by the samepeptide analysis method and, as a reduction in themain peak was observed (Figure 4), we suggest thatthis peak corresponds to the CBD peptide.ConclusionThis simple technique can be used to isolate theseCBDs, which can have further application in thepaper and textile industries. A ratio of 300:1 (cellu-lase:papain) was found to be the best ratio to performthe proteolysis. By considering that CBDs contributefor 10% of initial mass and calculating the concen-tration of the CBDs in the filtrates using the molarextinction coefficient 5545 cm−1 m−1 (Linder et al.7071995) we may conclude that these conditions giverise to yield 85% of CBDs. This methodology canbe extended to quickly obtain CBDs from differentmicrobial sources.AcknowledgementM. A. Lemos would like to thank Fundação paraa Ciência e Tecnologia (Programa Praxis XXI) forfinancial support.ReferencesAbuja PM, Pilz I, Claeyssens M, Tomme P (1988) Domain structureof cellobiohydrolase II as studied by small angle X-ray scatter-ing: close resemblance to cellobiohydrolase I. Biochem. Biophys.Res. Commun. 14: 180–185.Din N, Gilkes NR, Tekant B, Miller RC, Warren RAJ, KilburnDJ (1991) Non-hydrolytic disruption of cellulose fibres by thebinding domain of a bacterial cellulase. Bio/Technology 9: 1096–1099.Greenwood JM, Gilkes NR, Kilburn DG, Miller Jr RC, Warren RAJ(1989) Fusion to an endoglucanase allows alkaline phosphataseto bind cellulose. FEBS Lett. 1: 127–131.Knowles J, Lehtovaara P, Teeri T (1987) Cellulase families and theirgenes. 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A simple method to separate cellulose-binding domains of fungal cellulases after digestion by a protease

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A simple method to separate cellulose-binding domains of fungal cellulases after digestion by a protease

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