Effect of Tween 80 on Hydrolytic Activity and Substrate Accessibility of Carbohydrolase I (CBH I) from Trichoderma viride

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1 684 Effet of Tween 80 on Hydrolyti Ativity nd Sustrte Aessiility of Crohydrolse I (CBH I) from Trihoderm viride Wnje Kim, Yuko Gmo, Yhy Mohmmed Sni, Yimiti Wusimn, Storu Ogw 1 Shuihi Krit nd Mskzu Goto* Fulty of Bioresoures, Mie University, 1507 Kurimmhiy-ho, Tsu , Jpn 1 Shool of Mediine, Mie University, 1507 Kurimmhiy-ho, Tsu , Jpn ABSTRACT : The present study exmined the effets of Tween 80 on the tthment nd hydrolyti tivity of ellulse enzyme ginst ll-milled ellulose (BMC), using the whole omponent (ntive CBH I) nd the tlysis module (ore CBH I) of rohydrolse I purified from Trihoderm viride (Meielse, Meiji Seik, Tokyo, Jpn). The effets were evluted s protein onentrtions in the superntnt fter mixing enzyme nd sustrte with Tween 80 t room temperture. Tween 80 deresed the dsorption of ntive CBH I nd ore CBH I onto BMC (p<0.001) nd inresed the mount of reduing sugrs relesed from BMC y ntive CBH I (p<0.001). However, Tween 80 did not enhne the hydrolyti tivity of ore CBH I. Oservtions using SEM reveled tht Tween 80 used ellulose filter pper to swell nd enhned surfe rks nd filments used y ntive CBH I ut not y ore CBH I. These results suggested tht Tween 80 dereses enzyme dsorption to its sustrte ut enhnes enzymti tivity. (Asin- Aust. J. Anim. Si Vol 19, No. 5 : ) Key Words : Enzymti Ativity, Celloiohydrolse, Tween 80 INTRODUCTION Surftnts enhne the enzymti hydrolysis of ellulolyti mteril for industril (Fendler nd Fendler, 1975; Cstnon nd Wilke, 1981; Kim et l., 1982; Ooshim et l., 1986; Helle et l., 1993; Eriksson et l., 2002; Alksrwi et l., 2003) nd griulturl pplitions (Aksenov et l., 1994; Goto et l., 2003, 2003; Wng et l., 2003). Surftnts lso inrese ruminl ellulse tivity (Kmde et l., 2000; Goto et l., 2003; Lee nd H, 2003), totl numers of teri in vitro (Goto et l., 2003; Lee et l., 2003) nd improve the effiieny of eef prodution (Wng et l., 2003). A totl mixed rtion of diry ttle treted with firolyti enzyme nd Tween 80 produes less methne without ffeting VFA prodution in vitro (Kim et l., 2005). However, the improved enzymti hydrolysis of forge rops depends on the onentrtion nd type of surftnt. Thus, little is understood out the mehnism wherey Tween 80 enhnes ellulse tivity in the rumen. Sine ellulses re omposed of severl funtionl modules inluding tlyti nd ellulose inding domins (Byer et l., 1998), the mehnism through whih Tween 80 enhnes tivity my e lrified y studying its influene on these funtionl regions. Severl ellulolyti enzymes of Trihoderm sp. re extensively used in the degrdtion of rystlline ellulose (Vn Tileurgh et l., 1986; Tomme et l., 1988; Lemos et * Corresponding Author: M. Goto. Tel: , Fx: , E-mil: goto@io.mie-u..jp Reeived June 1, 2005; Aepted Deemer 14, 2005 l., 2003). The tivity of the ommeril enzyme omplex, Meielse (P-1, Meiji Seik, Tokyo, Jpn) is ttriutle not only to severl endoglunses nd elloiohydrolses (Srisodsuk, 1994; Sloheimo et l., 1997), ut lso to ellulose inding module linked through glyosylte to the tlyti module. We exmined the reltionships mong surftnt, ellulolyti enzyme nd firolyti sustrte e determining the extent to whih whole CBH I of Meielse nd its tlyti module dsor onto ll-milled ellulose (BMC). Furthermore, the extent to whih Tween 80 enhned the enzymti hydrolysis of BMC ws ssessed y mesuring the relese of reduing sugrs. Snning eletron mirosopy (SEM) determined whether Tween 80 used morphologil hnges in BMC. MATERIALS AND METHODS Purifition of CBH I nd experimentl tretments Either whole CBH I (ntive CBH I) or its tlyti module (ore CBH I) were mixed with the following onentrtions of Tween 80: ntive CBH I lone (C0), nd with 1% (C1) or 2% (C2) Tween 80; ore CBH I lone (P0), nd with 1% (P1) or 2% (P2) Tween 80. All rtios of Tween 80 were mixed on w/w (sustrte) sis nd the onentrtions of ntive nd ore CBH I were djusted to 1% of the weight of BMC dissolved in 0.1 M potssium phosphte uffer (ph 7.0). Ntive CBH I ws purified from Meielse P-1 ( ellulse derived from Trihoderm viride; Meiji Seik, Tokyo, Jpn) y nion exhnge hromtogrphy with HiTrp Q olumn (1 ml, Amershm Phrmi Bioteh AB,

2 EFFECT OF TWEEN 80 ON ENZYMATIC CELLULOLYSIS 685 Tle 1. Effet of Tween 80 on onentrtion of enzyme proteins in superntnt Time (h) Tween 80 (%, w/w) Signifint effet Enzyme (En) En Tw En Tw 10 min (µg/ml) p<0.001 p<0.001 p<0.141 C 13.83±5.42 e 52.58± ±2.92 P 40.04±0.83 d 70.08± ± h (µg/ml) p<0.001 p<0.001 p<0.002 C 14.20±1.05 e 51.54± ±0.87 P 40.26±1.06 d 70.33± ± h (µg/ml) p<0.001 p<0.001 p<0.015 C 14.12±0.57 e 51.69± ±1.24 P 41.63±3.65 d 71.75± ± h (µg/ml) p<0.001 p<0.001 p<0.007 C 14.24±1.01 e 51.33± ±2.26 P 39.20±2.31 d 69.86± ± h (µg/ml) p<0.001 p<0.001 p<0.216 C 15.89±1.31 e 50.03± ±1.16 P 42.12±3.00 d 70.80± ± h (µg/ml) p<0.001 p<0.001 p<0.198 C 14.90±0.81 e 50.05± ±2.56 P 39.16±2.09 d 70.31± ±1.83,,, d Signifint differenes y Tukey s multiple test. Vlues re mens of 3 replites. Sweden, Uppsl) using liner grdient (Bhikhhi et l., 1984). The purifition ws monitored y sodium dodeyl sulfte-polyrylmide gel eletrophoresis (SDS-PAGE), nd enzymti tivity on roxymethyl ellulse ws mesured y photometry. The tlyti module of CBH I ws otined from the protese, ppin (Wko Pure Chemils, Tokyo, Jpn), whih hydrolyzes the linkge of the tlyti nd elluloseinding modules (Vn Tileurgh et l., 1986). Core CBH I ws isolted y nion exhnge hromtogrphy. Proteolysis ws onfirmed y 12% SDS-PAGE, using moleulr mss mrkers (LMW Clirtion, Amershm Biosienes, New Jersey, USA) s stndrds. Cellulose (BMC, W-300G, Nippon pper Chemils Co. Ltd., Tokyo, Jpn) ws prepred y grinding in ermi ll mill (Tkno et l., 1992). The surftnt Tween 80 (polyoxyethylene soritn monoolete) ws purhsed from Nli Tesque In. (Kyoto, Jpn). Mesurements of dsored CBH I onto sustrte nd enzymti tivity The dsorption of ntive nd ore CBH I onto llmilled ellulose (BMC) ws estimted s the protein onentrtion in the superntnt fter mixing for 10 min nd 1, 2, 3, 4 nd 5 h with vrious rtios of Tween 80, euse preliminry investigtion showed tht ellulolyti tivity delined fter 4 h t room temperture nd entrifugtion t 7,000 rpm for 2 min. The protein onentrtion in the superntnt ws determined y the Folin-Lowry retion using BSA s the stndrd (Lowry et l., 1951). Cellulse tivity ws ssyed fter 3% (w/v) BMC dissolved in the uffer desried ove, ws inuted t 38 C for 1, 2, 3, 4 nd 5 h without shking. Smples were entrifuged t 7,000 rpm for 5 min, nd reduing sugrs in the superntnt were determined using dinitrosliyli id (Miller, 1959) SEM oservtion of filter pper treted with ntive nd ore CBH I Smples of filter pper (No.1, Whtmn Interntionl Ltd, Midstone, UK) were soked in C0, C1, P0 nd P1 t 38 C for 10 min, fixed in 2.5% (v/v) glutrldehyde for 2 h nd then wshed with 0.1 M sodium phosphte uffer (ph 7.2) for 30 min three times t 30 min intervls. The filters were dehydrted through grded ethnol series, dried y ritil-point drying with liquid CO 2, nd sputtered with gold (Bosworth et l., 2001). Smples were oserved using SEM (Hithi S-4000, Tokyo, Jpn). Sttistil nlysis Dt were nlyzed nd men differenes were determined using Tukey s multiple rnge test ording to the SAS (Sttistil Anlysis Systems Institute In. 1989) proedure. RESULTS AND DISCUSSION The moleulr mss of the purified ntive nd ore CBH I proteins were 65 nd 56 kd, respetively (Figure 1). The mounts of ntive nd ore CBH I in the superntnt

3 686 KIM ET AL. 94 kd 67 kd 56 kd 45 kd M 1 2 M Figure 1. Confirmtion of limited proteolysis of CBH I y ppin. M, Mrker; 1, Core CBH I (56 kd); 2, ntive CBH I (65 kd). inresed (p<0.001) with inresing onentrtions of Tween 80 (Tle 1). This finding indited tht Tween 80 deresed the mounts of the ntive nd ore CBH I tht dsored onto BMC, findings tht were in greement with the results of studies of non-ioni surftnts (Prk et l., 3,000 2,500 C0 P0 C1 P1 C2 P2 1991; Helle et l., 1993; Eriksson et l., 2003). The onentrtions of ntive CBH I t 3 onentrtions of Tween 80 were lso lower thn the orresponding onentrtions of ore CBH I. The dsorption of ntive or ore CBH I onto BMC ws not ffeted y the inution period, suggesting tht the enzymti retion ws generlly mintined. The mount of reduing sugrs relesed from BMC y ntive CBH I ws inresed (p<0.001) y Tween 80 (Figure 2). Espeilly t 1 h of inution, the mounts onsiderly differed mong Tween 80 onentrtions, lthough the differenes were redued with inresing inution time. In ontrst, the mount of reduing sugrs relesed y ore CHB I remined low, suggesting little response to Tween 80 for ny length of time. The surfe of untreted filter pper ws smooth nd sleek (Figure 3), wheres tht fter exposure to Tween 80 ws swollen nd flky (Figure 3). The surfe exposed to ntive CBH I ws remrkly disordered nd peeled (Figure 3), wheres ore CBH I used some rks nd filments (Figure 3d). After immersion in ntive CBH I nd Tween 80, the filter pper surfe ws oviously disordered nd eroded (Figure 3e). In ontrst, ore CBH I nd Tween 80 used limited morphologil hnges with some rks, flkes nd filments (Figure 3f). The surftnt Tween 80 onsiderly improved Meielse hydrolysis, whih ws in greement with the findings of others (Fendler nd Fendler, 1975; Cstnon nd Wilke, 1981; Kim et l., 1982; Ooshim et l., 1986; Helle µg/ml 2,000 1,500 1,000 d d Hour Figure 2. Conentrtion of reduing sugrs in superntnt fter retion with ll-milled ellulose (BMC). C0, ntive CBH I; C1, ntive CBH I nd 1% Tween 80; C2, ntive CBH I nd 2% Tween 80; P0, ore CBH I; P1, ore CBH I nd 1% Tween 80; P2, ore CBH I nd 2% Tween 80. All rtios (%) re w/w sustrte.,,, d Mens with supersripts signifintly differ (p<0.05).

4 EFFECT OF TWEEN 80 ON ENZYMATIC CELLULOLYSIS 687 d e f Figure 3. SEM oservtion of surfe stte of filter pper. ) No-Tween 80, ) Tween 80, ) ntive CBH I, d) ore CBH I, e) ntive CBH I nd Tween 80, f) ore CBH I nd Tween 80. t l., 1993; Eriksson et l., 2002; Goto et l., 2003, 2003). We lso found tht Tween 80 inresed the ility of CBH I with, ut not without its ellulose-inding module to enzymtilly hydrolyze ellulose. This finding suggests tht Tween 80 is losely ssoited with intertion etween the ellulose-inding module nd sustrte, s indited y the onsistently higher onentrtions of ore CBH I proteins in the superntnt ompred with those of ntive CBH I. These findings were lso onsistent with those of Tomme et l. (1988), who showed tht the mount of reduing sugrs relesed y ore CBH I isolted from Trihoderm reesei ws deresed to one-seventh of tht relesed y ntive CBH I. However, within the enzymti tivity of ntive CBH I, the onentrtion of the reduing sugrs ws higher in the presene, thn in the sene of Tween 80, suggesting tht the responses of ntive nd ore CBH I re ntgonisti. Therefore, the enzyme-sustrte intertion improved y surftnt Tween 80 would e ssoited with redued intivtion of the enzyme ound to sustrte nd inresed moiliztion of the enzyme mong sustrte retion sites (Cstnon nd Wilke, 1981; Helle et l., 1993; Kr nd Holzpple, 1998; Eriksson et l., 2002). In ddition, redued therml denturtion or denturtion y sher fores inrese enzymti stility nd llow the enzyme to remin tive for longer period (Kim et l., 1982; Kr nd Holzpple, 1998). The response of ntive CBH I to the surftnt onentrtion ws inonsistent with the response of enzyme

5 688 KIM ET AL. inding to BMC t vrious Tween 80 onentrtions. This finding ws in greement with those of Goto et l. (2003), who showed tht the degrdility of dry mtter from lef ldes of young orhrd grss ws inresed t Tween 80 onentrtions over 0.01%, ut tht the enzyme inding pity ws inresed only t 0.2% Tween 80. In ddition, our SEM oservtions of filter pper soked in Tween 80 showed hnges in the morphologil struture of the surfe. Thus, surftnts n initilly lter ellulose ultrstrutures to inrese vulnerility to enzymti ttk (Helle et l., 1993). Enzyme proteins might ind to hydrophoi regions of ellulose fter surftnt hs inresed its wettility (Goto et l., 2003). In onlusion, the results of the present study suggest tht the improvement of enzymti hydrolysis y surftnts depends upon synergism etween enzyme stility nd sustrte essiility. However, further reserh is needed to eluidte the effet of surftnts on the rte nd extent of enzyme turnover. REFERENCES Aksenov, L. A., M. V. Dunev, E. A. Zk, Y. F. 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