Transglycosylation activity of cellobiohydrolase I from Trichoderma longibrachiatum on synthetic and natural substrates

8iochiraica el BiophysicaActm 1073 (1991)481-485

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© 1991 Else'ActSc/encePublishers$.V. 0304-4165791/$03.50 ADONIS 0304416591001"~23 BBA(3EN 23476

Transglycosylation activity of cellobiohydrolase I from Trichoderma longibrachiatum on synthetic and natural substrates A l e x a n d e r V+ G u s a k o v , O k s a n a V. Protas, V l a d i m i r M. C h e m o ~ a z o v , A r k a d y P. Sinitsyn, G a l i n a V+ K o v a i y s h e v a , O l g a V. S h p a n c h e n k o a n d O l g a V. Ermolova Del2~rtmentof Chemistry. M. V. Lom~o~v MoacQ,vStare Universiff. Moscow ( U S.S.R+)

{Received 1 ~epterabctD89) (l~e~isedman~scriptr~=~:ived25 Oclober 19~) Key wet&s: Exo-celloblohydrol:L~e;~Methylumbellife~'[fl-D-cellohioside;TraJ~gIy¢osylatioa: ~ long~brachiazum Using 4-methylumbelliferyl (MUF) ~-~-eellnbioside as a substrate, the abilRy of cdlobiohydrolase I Item Tr/choderma longibrachiatum to catalyze/ransgly~sy~ation has been demonstrated. At substrate ¢oncen~afio~ greater than 2 raM,

the formation of M1LIF.tetmsaccharide was detected using HIPLC. In the ¢otase of enzymatic reaction, a concentration of the transglycosylation product passed Ibxough a maximum, shace at later stages of the reaction the product was furlher hydmlyzed. At MUF-~-Zy-cellobioside ¢o¢~enlrations of 2-18 raM. *.hemaximum weight content of MUF-tetrasaeehedde amounted to I-4% of the total cement of s~¢chari4es, In the reactims system, containing 2.5 mM MUF-~-D-cellobinside and 10 mM MUF-~-D-81ucoside , MUF4risa~haride was formed as the Inain h'aasglycosylation prodltet. In hydrolysis of nahural sul~lxates (cellulose and celhilrinse) in the Imre~enee of MUF-~-D-glueaside a formation of MUF-Irisaccharide was also @bserved. Introduction

Cellulase enzyme system produced by various cdlulolyric microorganisms is known to consist of three major components differing in their specffzcity [1,2]: 1,4-/]-Dglucan glueanohydrolase (EC 3.2.1.4) which is often referred to as endoglueanase, 1,4-//-l>-glaean cellohiohydrolase (EC 3,2,1,91) and ~-glucosidase (EC 3+2.1,21)+ Each component may have multiple forms. One of the interesting properties inherent to some cellulolytic enzymes is transglycosylation, i.e., the transfer of carbo+ hydrate moiety to an acceptor, which cart be another carbohydrate moteeule, leading to elongation of the saccharide chain. Trausglyeosytation activity was reported for endoghicanases [3-6] and ~-ghicosidases [79]. However, for cdlobiohydrolases, isolated from various sources and tho~roughly investigated [10 15]. the

Abbreviations: MUF, 4-meth2/lumbelliferylor 4-rneth~/lumbelllfearone; HPLC, high-performanceliquid chromatographs,; CMC, carboxymechylcenulos¢. Correspondence: A_V. GusaIco¢, Department of Chemistry, M.V. LomonosovMo~w Stale University,Moscow117234, U.S.S.R.

phenomenon was not ob~rved experimentally, though its possibility was discussed [16|. In this paper, we report the transglycosylation a~.tivity for ceRobiohydro|ase t from Trichoderma Iongibr~chialum. The reaction was studied using MUF-~-D-cellobioside+ micr0crystalliae cellulose and cellotriose as subslrotcs, MUF-derivatives of glucose and cellobiose were used as accepters of cenobiosyl residue in transglycosylation. MUF-fl-D-celtootigosaccharides were intro.. duced as substrales for ceilobiohydsotase I by van Ti/beurgh et al. [17]. Due to the high extinction coeffi+ cient of MUF, it was possible to detect various products of the reaction with high sensitivity. Materials and Methods E n z y m e purification and prope*zies

Cellobiohydrolase ! was purified from a culture filtrate of T. tongibrachiatum 7-26. The culture conditions are described in detail elsewhere []8]. The endoglucanase (Cx) activity of the culture filtrate, determiiled by viscometrical method [19.20l using CMC as a substrate, was 1.8 units/ml (0.9 units/rag protein), filter paper activity [21] was 0.5 units/ml, activity Inwards MUF-~-D-cellobioside was 0.04 umts/ml (0.02

482 units/rag protein), and /3-glucosidasc (ccllobiase) activity [20] was negligibly low ( < 0.01 units/ml). In all cases one unit of activity corresponds to l #reel of hydrolyzed bonds per min. The procedure of purification included the following steps. To 500 ml of culture filtrate, containing approx. 1 g of protein, ammonium sulphate was added (80~ of saturation). The imxture was incubated for i2 h at 4°C~ the protein precipitate was centrifuged and dissolved in 50 ml of 0.1 M acetate buffer (pH 4.5). The solution was desalted on Bio-Gei P-2 (Bio-Rad). The united protein fraction was separated by ion-exchange chromatography on DEAE-Spheron C-1000 (25-40 /tm, Chemapo], Czechoslovakia). The column was equilibrated with 0.I M acetate buffer (pH 4.5), proteins were eluted using gradient of NaC1 (up to 1 M). Fractions, having low endoglucanase (viscometric) activity and relatively high activity towards MUF-fl-D-cellobioside. were subjected to chromatography and then rechromatography on Mono-Q eohimn, using Pharma¢ia FPLC System. As a result, 60 mg of protein was obtained (6% yield). The isolated enzyme showed homogeneity in polyacrylamide gel electrophoresis and analytical isoelectdcfocusing. According to the results of elcctrophoresis and gel chromatography, the molecular weight of cellobiohydrolas¢ was 65000 and 54000, respectively; the isoelectric point was 3.g. Activity of the enzyme towards various substrates was studied (Table I). When acting on CMC, the enzyme gave practically no decrease in viscosity of the substrate. Its specific activity determined by viscomctrical method [19,20] was less than 0.0005 units/me (0.5% CMC, ,10" C, pH 4.5, assay time 30 rain, enzyme concentration 0.02 mg/m]). However, a small amount of reducing sugars (0.1 mM in glucose equivalents) were formed from 1% CMC after 10 h of reaction (30°C, pH 4.5, enzyme concentration 0.08 mg/ml). This corresponds to < 1% of the glueoside linkages split and may be accounted for by ccllobiohydrolase action on non-substituted ends of CMC molecules. Thus, the endoglucanase activity was practically absent in the purified preparation, fl-Glucosidase (cellobiase) activity was also absent, since the enzyme (0.08 mg/ml) did not split 10 mM cdlobiose at all after 24 h of reaction at 3 0 ° C and pH 4.5. HPLC analysis has shown that the main product of the action of the enzyme on microcrystalline cell/rose was cellobiose ( > 90%), and trace amounts of glucose ( < 10%) were also formed. Using HPLC, the hydrolysis of ¢dlooligosaccharides and their MUF-derivatives was investigated. In the hydrolysis of eellotriose, glucose and celIobiose were formed at equimolar ratio. The only detectable product of eellotetraose hydrolysis was ce|lobiose. Onty cellobiose and MUF-fl-o-glucoside were formed in hydrolysis of MUF-fl-D-cellotrioside. This fact allowed us to conclude that the enzyme split ce]lobiosyl

residues from the non-reducing end of ohgosacchaddes and their MUF-derivatives. In all cases the enzyme was inhibited by the end product (eellobiose).

Substrates MUF-fl-D-eellobiostde, MUF-fl-D-ghicoside, ceilobiose, CMC (medium viscosity) flora Sigma, and .,nietocrysta/line cellulose for column chromatograpi~2t .~rom Chemapol (Czechoslovakia) were used in the experiments. The microcryslalline cellulose had mean particle size 23/zm and degree of polymerization of 170. MUFfl-l~-cellotrioside and MUF-fl-D-cellotetraoside were synthesized and presented by Y.V. Vozny (Institute of Biochemistry, Armenian Academy of Sciences, Yerevan, U.S.S.R.). Cellotriose and eeilotetraose were obtained by HCI hydrolysis of cellulose powder followed by separation on charcoal-celite (1 : 1) using gradient elution with ethanol-water [22].

HPLC analysis of products The products of enzymatic reaction were analyzed by HPLC on a Bio-Rad Series 700 Chromatographic System (including Model 1350 Soft-Start Pump, Model 1706 UV/Visible Monitor, Differentia/ Refraetometer, and IBM XT compatible computer for data collection and analysis), using Silasorb NH z column (4.6 × 250 ram) and acalonitrile/water mixture of various composition as a mobile phase. The acetonitrite/water ratio 90:10 was used for the quantitative analysis of MUF, MUF-~-D-glucoside and MUF-,//-v-cellobioside; under these conditions, however, the pe~ks of MUF-trisaccharide and MUF-tetrasaceharide, formed as transglycosylation products, had a long time and were widened. Thus, for the analysis of the transglycosylation products the acetonitrile/water ratio 80:20 was used. Under these conditions, the peaks of MUF and IVfUF-fl-D-glucoside were unresolved. In order to minimize the retention time of MUF-tetrasaeehatO.de, a gradient of flow rate of 1.5-3 m l / m i n was used. The MUF and MUFoligosaccharides were detected at the wavelength of 318 nm. Glucose and cdlooligosaccharides were detected with differential refractometer.

Reaction conditions The experiments were carried out in thermostatistically controlled cells at 3 0 " C and pH 4.5 (0.l M sodium acetate buffer). Prior to analysis, samples of the reaction system were diluted 2.5-times with acetonitrile and filtered. Results and Discussion

The enzymatic hydrolysis of MUF-,8-D-eellobioside catalyzed by cellobiohydrolase 1 from T. Iongibrachialure was carried out with substrate concentrations of 0.5-10 raM. At all MUF-/l-D-cellobiuside concentra-

483 2o I'

i

2

l

w

'

K T,rre [rr,,n:

Fig. I. H P L C analysis of M U F an~ MUF-dLerlvalivca tern,el in the reaetit~n system eontaie~tlg l 0 mM of MOF-~-D~ellobio~ide as a sub,gtrate and 0.08 me/rot o1" cellobioh'~drola~:, Reaction time 16 h. p H 4,5, 30" C_ Gradient of flow talc 1.5-J ral/min was used. l, M U F and MUF-~-o.gluc0side (unte-solvedl; 2, MUF./~.a-¢¢ltobioside; and 3, M U F-to trasaccha r;d .',

course or enzymatic reaction, the concentration of MUF-tetrasaccharide reached a maximum and then decreased due to its further hydrolysi~ (Fig. 2). The maximum was reached at the approx. 40-50% degree of substrate conversion. Under the experimental conditions used, the maximum weight content of MUF-tetrasacchafide amounted to 1-4~% of the total content of sacchatides and MUF-defivatives in the system. It shoald be noted that in control experiments, where MUF-fl-D-ce!lobioside ,:,,as incubated under the same conditions but in the absence of cellobiohydrotase, no reactions were observed. The elongation of carboh3~drate chain in the enzymatic reaction due to transglycosylation was reported earlier for c,dogtncanase~ [3-61 and /~-glucosidases [7~'], The simplest kinetic scheme allowing for hydrolysis and transglycosylation in the reaction system s~adied in this paper may he presented as'. E+G:MUF ~

tides, eellobiose and MUF were formed as major prodacts. The specific activity of cellohiohydtolase, calculated as the ratio of maximum rate of the reaction to protein concentration, was 80 n m o l / m i n per mg protein. The ~race amounts of MUF-/~-o-glucosidc (not more than 4 - 3 5 ) were also formed under the action of enzyme, the percentage of tiffs product was not dependent on the substrate cot~entration. At 0.5-2 mM MUF-~v-cdlobioside concentrations, no other produets were detected. However, at higher substrate concontrations, the formation of an tmkrtown MUF.dedvatire was observed (Fig. 1). Using MUF-,8-D-cellotfioside and MUF-B-D-cellotetraoside as standards, this compound was identified as a MUF-tetrasaccharide. The formation of MUF-tetrasacchadde increased with increasing MUF-.O-~-cellobioside concentration. In the

10C

O!

10

2.1

~

Q 7.2

Tirr~ 1~.1

Fig. 2. Kinetic profiles of M U F and MUF-dcdvatives formed in the re.action s3~stem containing l0 mM of MUF-fl-l:-cellobiosid¢ as a subsltat¢ and 0~18 m g / e ~ l Of ~¢llobiohydtdas~. p H 4.5. 3 0 ° C . MUFB-t:~cltob~oside (o); M U F (~,); MUF-fl-a-glucaslde (O); and MUFlelrasa~haride (U).

k.i

EGzMU F

h xlH~Ol k~[O:MUF] EG 2

~ E + O4MUF

MUF I

[

~d:,}

(i)

' E+G,A

Dunng the f i r s t s t e p o f the reaction, the enzyme-substrate complex, EG2MUF. is formed. In the second step. a M U F molecule cleave~ off from a molecule of substrate with the formation of cellobiosyl-e.nzym¢, EG 2. During the third step, the cellobiosyl residue in the celloiffosyl-en~me is attacked by a nucleophile, i.e.. a water or MUF4/-o-cellobioside molecule, to form mol~gules of cellobiose, G 2, or the transglycosylation product G~MUF, respectively. An acceptor of a carbohydrate residue in transglycosylation may be not only another substratc molecule but any other carbohydrate present in the reaction system. On the kinetic scheme, a 8eneralized accepter is denoted as [A]. Thus, for example. if MUF-~l-~-glucoside were present in excess in the re~ction syste~a, the preferential formation of MOF-tr~saccharide as transglycosylation product could be expected. In order to cheek this idea, lhe hydrolysis of MUF-flI>cellobioside (2.5 raM) was performed in the presence of an excessive amount of M U F - ~ g l u c o s i d e (10 raM). In tbfis case, as expected, the formation of MUF-trisaccharide was observed (Fig. 3). Its maximum concentration was about 0.15 raM. Thus, this experiment confirraed an ability of ceHobiohydro!ase ! to catalyze trmrsgiycosylatiom It should be noted that the peak of MUF-trisaccharide was not uniform (Fig. 3). This can be explained by formation of MUF4risacchatides with different types of glycoside linkages. It is known ,.hat in

484 I

E

upon the rado botwocn the rates of formation and consumption of this product. In our case the hydrolysis of generalized donor of cetlobiosyl residue ((3 2R), which can be MUF-,8-o-cealobioside, insoluble cellulose or eellotrinse, in the presence of ao~eptor (MUF-B-D-glueoside) may be represented as k E+G2R ~

~. EG2R~

k_~ c

p

,

,

,

,

,

~.me ~tn,n~ Fig. 3. HPLC analysis of M U F and MUff-derivatives ton'ned in the reaction system containing 2.5 mM of MUF-,a-D-cellobioside, 10 mM of MUF-#-o-glucosid¢ ~nd 0.06 mg/rnl of cellobiohydrola~e. Reaction time 8 h, pH 4.5, 3 0 ° C . n o w rate 1.5 ml/min. L M U F and MUF-~-D-g]ucoside iunresolved); 2, MOF-~-o-eellobio~id~; and 3. M UF.trisaccharide.

the ir~nsglycosylation process ~lycoside linkages other than /3-1,4 can be formed. For example, in t ramglycosylation catalyzed by/Lglucosldase the preferential synthesis of saccharides having B-1,6-1inkage has been observed [9]. Since MUF-fl-D-eeUobioside is a synthetic substrate and may affect the enzyme specificity, a question may arise - does the enzyme retain an ability for transglyco~ylation when acting on natural substrates? Unfortunately~ the sensitivity of differential refraetometer does not allow the detection of transglycosylation products when unmodified cellooligosacoharides are used as accepters. This is a possible reason why transgiycosylation was not observed in t_He hydrolysis of cellooligosaccharides. Therefore, the hydrolysis of natural substrates (microcrystalline cellulose and cellotriose) in the presence of chromophoric MUF-/~-D-glucoside as accepter was studied. In the reaction system, contaimng 20 mM of eel]otciose and 10 mM of MUF-/~-D-glucoside, an intensive formation of MUF-trisacchadde was observed. Its maximum concentration achieved 0.3-0.4 raM. In the hydrolysis of microcrystalline cellulose (20 m g / m l ) in the presence of 10 mM MUF-/~-D-glucoside, the rate of soluble product (cellobiose) formation was low and decreased significantly after 1-2 h of the reaction. However, the formation of trace amounts of MUF-trisaceharide (about 0.01 mM) was also observed at 0.5-2 h. Thus, the transglycosylation product was detected only at the initial period of the reaetion, when the rate of hydrolysis was higher. The observed regularities of MUF-trisaccharide formation in hydrolysis of synthetic and natural substrates can be explained in terms of kinetic theory. According to the theory, the maximum concentration of intermediate product (e,g., MUF-trisaceharide) depends

I

k~lH20] ~

E+G2 (2)

E¢~~ k,IGMON R

' E+G~MUF

According to this scheme, at the same aeeeptor concentration (10 raM) the rate of MUF-trisaccharide formation (and the relevant ratio between the rales of formation and consumption of this intermediate produc0 is proportional to the rate of hydrolysis of cellobiosyl donor. The latter rate increases in the following order: insoluble cellulose < MUF-~-D-cellobioside < cellotriose (Table I). Thus, the maximum concentration of transglycosylation product should also increase in the same order. Namely such results can be seen from the experiments (see above). Thus, transglycosylation activity of cellobiohydrolase was observed during the enzyme action on both natura~ and synthetic substrates. In discussion of a mechanism of catalysis by carbohydrases, the researchers often connect an ability for transglycosylation with a stereoehemical course of enzsTac action. These two characteristics are also widely used for classification of eelhilase& It is known that endoglucanases and ~8-ghicosidases, which catalyze hydrolysis of //-1,4-glucans with a retention of glycone anomerie configuration, are able to catalyze transglycosylation [16,23]. On the other hanti, coUobiohydrolases, which usually reverse #ycone anomcric configuration in hydrolysis, are considered to be unable to catalyze transglycosytation [16,23]. However, in a recent report Knowles et al. [24] have shown that stereochemical courses of the action of cellobiohydrolases I and I1 from T. reese~ are different. When acting on 8-celTABLE |

Speci./i¢activities of cellobiah£drolazeI towards varioussubst~tes (30°C pH 4.5) Sttbstrat+

Specific activity ( n m o l / m i n per nag prolein)

CMC D CMC ~ Midocrystalline cellulose C¢llobiosc Cellotriase Cdlotetraose M U F-.~-D-¢cll obi oside M UF-~-D-cellot fiosirte

< 0.5 2 40 no activity 310 1040 80 420

a Vise.ometrle acti'nty (40 o CL b ,, Imtlal rate of re,duemg sugar formation.

485 lobiosyl fluoride as a substrate, eellobiohydrolase l c a t a l j z e d hydrolysis with a retention of anomeric configuration and g a v e fl-cellobiopyranose as the first product, whereas cellobiohydrolase 1I gave a-ceI!obiopyranose, In our case, the ability of cetlobiohydrolase I f r o m T. longibrachiatum to catalyze transglycosylation w a s found. T h o u g h the enzy.,~te wa~ isolated Crom a n o t h e r source, its moleenlar characteristics (molecular weight 5 4 0 3 0 - 6 5 0 0 0 , p l 3.8) are very simitar to those of cellohlohydrolase I f r o m T, reesei (molecular weight 60 0 0 0 - 6 5 0()0 [12,15,16}, p l 3.6-4.2 [12,13,15,16]}, Thus, although in o u r case the stereochemical m o d e of celIobiohydrolas¢ action w a s nol investigated, it m a y b e similar m that o f cellobiohydrolase I I~rom 7"_ reesel. In such case, the results obtained in the present p a p e r together with the results o f Knowles et al. [24] m a y support the idea that retention of glycone a n o m e r i c configuration and transglycosylation are j e i n t p h e n o m ella. I n conclusion, the abifity o f cellobiohydrolase to catalyze transslycosylation shown here allows study o f this enzyme, a n d classification o f cellulases in general, from another point of view a n d gives a n e w information about the catalytic properties o f cellobiohydrolases.

Admowledsment T h e authors t h a n k D r . Y N , Vozny (Institute of Biochemistry, A r m e n i a n A c a d e m y o f Sciences, Yerevan, U.S.S.R.) for p r o v i d i n g MUF-/]-n-cellotrioside and M U F-B-D-cellote traoside. References I Ryu. D.D.Y, and Mandcts, M. [i980) Et~ymc Mk~:ob. Tec_~not. 2, ~|-102.

2 Ladisch. MR., I.in. K.W.. Voloeh, M. and Tsao, G.T. (1993} Enzyme Microb. Technol, 5, 82-102. 30ka.da. Q. and Niskawa. K, 0975) J. Bioehem. 7g, 297 306. 4 Maksiruov. V.1. and Churilova, I.¥. (1'985) Pdkl. Biokhim. Mi~robiol. 21, d56-460. 5 Fukum~ri. F.. Kudo, T. and Horikcshi. K- (198.5) J. Gan. Microbiol. 131, 3339-3345. 6 Kr~.~va,N.E, Rablnow/tch, M.L., Kiyosov, A.A and Bere-fin.I.V. (].986} Dokl. AN SSSR 290, 484~186. 7 Hash. J.H. and King, K.W. (1958) J. Biol. Chem. 232, 381-402. 8 Kanfer, I.N. and Raghavea. S,S. (1975) Bix~:him. Biophys. Aeta 391, 129 140. 9 Gu~tkov, A,V,, Sinitsym A.P., Goldstems~ G.hL and Klyosov~ A.A. [19841 En,~ym¢Mierob, T~haoL 6. 275-282, t0 Wood. T.M., MeCtan. S.I. and Maefarlane. C.C. (1980) Biochem. J. 189, 51 65. ii Fagetstarn, L~ and PC~IC~'~On,G, (19'~0) FEB$ LeU. 119. 97-100. 12 Nummi. M., Niku-Paavola. M.-L, Lappalaiaerk A,, F.nail. T,-M. and Raunlo, V. (t983) Biochem. J. 215, 677-693. 13 Van Tilbeorgh. H,, Bhikhabhai, IL, Pettets~oa, G. and ClaeysSens, M, (19~4) FEBS l..¢tLL69. 21~-218, 14 Patil. RN. and Sadaae.. J.C. (t984-) Can, J. "uiochen~ Cell Biol. 62. 920-926. 15 Beldman, G , Searte-Van Lee)~wen, M.F., Rambouts, F.M. and Voragen, G.J. (1985) Ear. J. Bioehem. 146, 301-30S. 16 Rabirtowitch, M.L., Chemoglazov, V.M and Klyosov. A.A. (1988) Classifical~on of Cdldases, Their Spreading, Multiple Forms and Mechanism of Action (in Russian), VIN1TY Press, Mo~.ow. 17 Van Tilbeurgh. H. Clae3t~m~. M. aed De B~yla~ C,K. ('1982) FEBS Lett, 149.152-156. 18 Chemoglazov, V.M., Elmolov~. 0.'4., Perer~ H., Shpanchenko. O.V. and Get'net,/~V, (1~9~')Mik~bicdogiya. in pre..s~ 19 Klyt~.~v,A.A. mad Rabinowirr_h, M.L (19g0) m Enzyme Engineering - Future Di~ctions (Wingard ctal,,eds,), pp. 83-165, Plenum Press. New York. 20 Kiyo~ov+A.A,, Rabinowitch, M_L~ Sh'tlts~a, A.P,, Churilova, IN, and Grigora~h. $.Yo- (lq&0) Bi~rg. Khim. 6, 122~-1241. 21 Maadels. M. Atldl~#tti, R. and Roche., C. ([976) Biot~heol, Bioen8. Symp. 6, 21-23. 22 Jermytl, M.A, (1957) Au.~I. J. Chmm. 10, S$-59. 23 Khcrrfin.A.Y. (1974) in Etru~um and Funetlonx of Active Sites of Enzymes lin Russian), pp. 39-69, "Naulm,Moseaw. 24 K~owles, J.K.C., Lentov~axa, P., Mgrtay, M. and Sinaott. M.L (1988) t. Chem. Soe., Chum. Cow~'~'.~. 2!. !~J11-1402.