of Clostridium beijerinckii (Clostridium butylicum)

Transcription

1 APPLID AND NVIRONMNTAL MICROBIOLOGY, Apr. 1987, p /87/ $02.00/0 Copyright X 1987, Americn Society for Microbiology Vol. 53, No. 4 Butnol-thnol Dehydrogense nd Butnol-thnol-Isopropnol Dehydrogense: Different Alcohol Dehydrogenses in Two Strins of Clostridium beijerinckii (Clostridium butylicum) STPHN F. HIU,t CHANG-XI ZHU,f RUN-TAO YAN, AND JIANN-SHIN CHN* Deprtment of Anerobic Microbiology, Virgini Polytechnic Institute nd Stte University, Blcksburg, Virgini Received 6 October 1986/Accepted 8 Jnury 1987 Alcohol-producing strins of Clostridium beijerinckii (Clostridium butylicum) produce, besides cetone, either n-butnol nd ethnol or n-butnol, ethnol, nd isopropnol s their chrcteristic products. Alcohol dehyrodgense hs been isolted from strin (NRRL B593) of C. beijerinckii producing isopropnol nd from strin (NRRL B592) not producing isopropnol. Butnol-ethnol dehydrogense ctivities were present in both strins, but isopropnol dehydrogense ctivity ws present only in the isopropnol-producing strin. The butnol-ethnol dehydrogense of strin NRRL B592 hd Mfr 66,000 nd. Km of 6,uM for butyrldehyde. In contrst, the butnol-ethnol-isopropnol dehydrogense of strin NRRL B593 hd Mr 100,000 nd Kms of 9.5 nd 1.0 mm for butyrldehyde nd cetone, respectively. In purifiction by four different types of seprtory methods (DA-cellulose, hydroxyptite, Sephcryl S-300, nd Mtrex Gel Red A), butnolethnol-isopropnol dehydrogense ctivities of strin NRRL B593 were purified up to 200-fold (10 to 30% yield), nd these ctivities were not seprted. Gel electrophoresis followed by ctivity stin lso reveled distinct mobilities for the butnol-ethnol dehydrogense of strin NRRL B592 nd the butnol-ethnolisopropnol dehydrogense of strin NRRL B593. In cell extrcts from both strins, higher lcohol dehydrogense ctivity ws mesured with NADP(H) thn with NAD(H). The 150- to 200-fold-purified lcohol dehydrogense from strin NRRL B593 did not show ny NAD(H)-linked ctivities. The Km for NADPH ws 31,uM (with butyrldehyde s cosubstrte) nd 18,uM (with cetone s cosubstrte) for the lcohol dehydrogense of strin NRRL B593. This study showed tht the lcohol dehydrogenses from two strins of C. beijerinckii differed significntly. Acetone, n-butnol, ethnol, nd isopropnol (solvents) re chrcteristic products of severl Clostridium species (6, 8, 14, 16, 28, 29). Production of these chemicls by fermenttion is n estblished industril prctice (28) nd my gin become commercilly importnt s n lterntive to the petroleum-bsed synthetic route (15, 26). thnol, isopropnol, nd n-butnol hve been suggested s feedstocks (with 2,3-butnediol) tht cn support mjor frction of the chemicl industry (27). They re lso useful s cosolvents nd octne number enhncers in blends of methnol nd gsoline s trnsporttion fuels. Clostridium cetobutylicum produces cetone, butnol, nd ethnol, wheres certin strins of Clostridium beijerinckii (some were previously known s Clostridium butylicum) produce isopropnol in ddition to cetone, butnol, nd ethnol (6, 8, 14). Cultures of isopropnolproducing C. beijerinckii ccumulte cetone only trnsiently (6). The solvent-producing properties of C. cetobutylicum nd C. beijerinckii re lrgely similr. However, the two species re distinct s shown by DNA homology nd other physiologicl chrcters (9, 14). C. beijerinckii ws chosen for this study becuse members of this species produce isopropnol. The butnol yield of C. beijerinckii is comprble to tht of C. cetobutylicum (both t 11 to 13 g/liter) (13, 25, 28). * Corresponding uthor. t Present ddress: IGN Biotechnology, Inc., Columbi, MD t Permnent ddress: Shnghi Institute of Plnt Physiology, Acdemi Sinic, Shnghi, Chin. Metbolic pthwys for solvent formtion hve been suggested (12, 17); however, the enzymology of these pthwys hs not been fully elucidted. In recent yers, enzyme ctivities specificlly relted to solvent formtion hve been mesured in cell extrcts (1, 13, 17, 29), but cetocette decrboxylse (ctlyzing the rection of cetocette to cetone plus C02) of C. cetobutylicum remins the only solvent-forming enzyme tht hs been purified nd whose moleculr properties hve been chrcterized (23). In butnol, ethnol, nd isopropnol formtion, the finl step is ctlyzed by lcohol dehydrogense. Although lcohol dehydrogenses show brod substrte specificity, they usully disply high selectivity towrd either primry lcohols nd ldehydes or secondry lcohols nd ketones (5, 19, 22, 24, 32). Becuse isopropnol is secondry lcohol, wheres butnol nd ethnol re primry lcohols, we wish to determine whether the isopropnol-producing strins of C. beijerinckii contin seprte secondry lcohol dehydrogense (for isopropnol formtion) in ddition to primry lcohol dehydrogense (for butnol nd ethnol formtion) nd whether similr butnol dehydrogense is present mong C. beijerinckii strins. Informtion concerning the type of lcohol dehydrogense present in solventproducing orgnisms will ber on the pproches tht cn be used for regulting the product pttern. In this pper, we report differences in the properties of the butnol-ethnol dehydrogense of C. beijerinckii NRRL B592 (not producing isopropnol) nd the butnol-ethnol- 697 beijerinckii NRRL B593 isopropnol dehydrogense of C. (producing isopropnol). The butnol-ethnol-isopropnol dehydrogense of strin NRRL B593 hs been purified up to

2 698 HIU T AL. 200-fold, nd butnol-, ethnol-, nd isopropnol-linked ctivities hve been copurified with no signs of seprtion. MATRIALS AND MTHODS Mterils. Tryptone nd yest extrct were obtined from Difco Lbortories; bovine serum lbumin, chloride determintion kit, dithiothreitol (DTT), DNse T, glutthione (reduced), immunoglobulin G (humn), p-iodonitrotetrzolium violet (INT), NAD, NADH, NADP, NADPH, phenzine methosulfte, RNse A, Sephcryl S-300 (Phrmci, Inc.), semicrbzide hydrochloride, N,N,N',N'- tetrmethyl ethylenedimine, nd thyroglobulin (bovine) were obtined from Sigm Chemicl Co.; Whtmn DAcellulose (D-52) ws obtined from Bodmn Chemicls; glycine, hydroxyptite (Bio-Gel HTP), dye-binding protein ssy kit, nd immunoglobulin G (bovine) were obtined from Bio-Rd Lbortories; Mtrex Gels Green A nd Red A nd ultrfiltrtion membrnes were obtined from Amicon Corp.; mmonium peroxydisulfte, bromophenol blue, - toluenesulfonyl fluoride, crylmide, nd N,N'- methylenebiscrylmide were obtined from stmn Kodk Co.; cetldehyde, cetone, butyrldehyde, butnol, nd isopropnol were obtined from Fisher Scientific Co.; Tris bse ws obtined from Clbiochem-Behring; nd methnol ws obtined from Burdick & Jckson Lbortories Inc Ȯrgnisms nd growth conditions. Stock cultures of C. beijerinckii NRRL B592 (VPI 13436) nd NRRL B593 (VPI 13437) were spores kept in liquid nitrogen (13). To strt growth, stock cultures were thwed, het treted, nd trnsferred to chopped-met-crbohydrte (CMC) medium (13, 18). After 24 h, cultures in CMC medium were trnsferred to tryptone-yest extrct-sucrose (TYS) medium; 5% (vol/vol) inoculum ws used (13). Actively growing cultures in TYS medium were trnsferred once (5%, vol/vol) into lrger cultures of TYS medium for generting cells for enzyme isoltion. All btch cultures in TYS medium were flushed with N2 nd incubted t 30 C. Cells in solventproducing phse were hrvested by centrifugtion, wshed once in 50 mm Tris chloride (ph 8.0), nd stored in liquid nitrogen or in freezer t -80 C. Preprtion of cell extrcts. Cell pste ws thwed under Ar in 50 mm Tris chloride (ph 8) (3 ml/g of cells) contining DNse I (0.1 mg/ml) nd o-toluenesulfonyl fluoride (0.3 mg/ml). All opertions were crried out under Ar or N2. Cells were disrupted by two pssges through French pressure cell t 18,000 lb/in2 nd then incubted t 25 C for 15 min. Cell debris were removed by centrifugtion t 37,000 x g for 30 min t 4 C. The superntnt (cell extrct) ws stored s frozen pellets in liquid nitrogen. Anlyticl procedures. Protein ws determined by the dye-binding ssy (4) with bovine immunoglobulin G s stndrd. Neutrl products in cultures were determined by gs-liquid chromtogrphy (14). The chloride concentrtion ws determined with the Sigm chloride determintion kit. nzyme ssys. Alcohol dehydrogense ctivities were routinely mesured in the direction of ldehyde or ketone reduction t 25 C under Ar. The ssy mixture (1 ml) contined 50 mm Tris chloride buffer (ph 7.5), 1 mm DTT, nd 0.2 mm NAD(P)H. The rection ws initited by the ddition of cetone (6.6 mm; cetone ws first diluted 10-fold with wter), butyrldehyde (5.4 mm for strin B592; 33 mm for strin B593; butyrldehyde ws first diluted 2- to 10-fold with methnol), or cetldehyde (5.1 mm; cetldehyde ws first diluted 10-fold with deoxygented wter kept APPL. NVIRON. MICROBIOL. on ice). Addition of ldehyde or ketone s the lst component of n ssy llowed the correction for ny diphorse ctivity in cell extrcts. Oxidtion of NAD(P)H ws monitored t 340 nm (-340 = 6.2 mm-1 cm-'). Alcohol oxidtion ws mesured in ssy mixtures contining 50 mm Tris chloride (ph 8), 6.2 mm semicrbzide, 3 mm NAD(P), nd lcohol (146 mm for butnol, 150 mm for isopropnol, nd 152 mm for ethnol). Production of NAD(P)H ws monitored t 340 nm. When tetrzolium (INT) ws included in the ssy, the rection mixture contined 50 mm Tris chloride (ph 8.2), 1 mm NADP, 0.1 mm phenzine methosulfte, 1 mm INT, nd lcohol t the bove concentrtions. An extinction coefficient of 14.3 mm-1 cm-' t 500 nm ws used for reduced INT (formzn). Polycrylmide gel electrophoresis nd ctivity stin. lectrophoresis ws performed in 7% polycrylmide gels (gel columns of 5 by 55 mm t 2.5 ma per gel) s described previously (11). Proteins were stined with niline blck. Alcohol dehydrogense ctivity bnds were locted by coupling lcohol oxidtion to INT reduction (13) in 0.1 M glycine-noh (ph 8.2). The lcohol concentrtion used ws 380 to 410 mm for butnol, ethnol, nd isopropnol. When glycine ws excluded from the procedure, Tris-borte (Tris, 3 g/liter; boric cid, 1.46 g/liter [ph 8.0]) ws the electrode buffer nd Tris chloride (0.1 M, ph 8.2) ws used in the ctivity stin. Purifiction of lcohol dehydrogense. Unless stted otherwise, 50 mm sodium phosphte buffer (ph 7) contining 0.1 M KCI nd 2 mm DTT ws used in column chromtogrphy. All opertions were performed under nerobic conditions nd t mbient temperture (bout 25 C). Active frctions were stored t 4 C overnight between purifiction steps. (i) DA-cellulose column. Cell extrcts (1.2 to 6.8 g of protein) were loded onto D-52 column (5 by 10 cm) which ws equilibrted with 50 mm Tris chloride buffer (ph 8). The column ws then wshed with 350 ml of the sme buffer nd with liner grdient of KCl (0 to 0.4 M in Tris buffer; totl volume, 0.8 to 1 liter). The flow rte ws 200 ml/h, nd 40-ml frctions were collected. In erlier trils, vrible mount of lcohol dehydrogense ctivity ws eluted t the beginning of the KCl grdient, but enzymtic properties of this erly frction were similr to those of the min frction. Incubtion of broken cells t room temperture, with DNse I present, for 15 min before centrifugtion essentilly eliminted the ppernce of this erly pek. (ii) Hydroxyptite column. Combined ctive frctions from the D-52 column were loded onto hydroxyptite column (Bio-Gel HTP; 5 by 14 cm) which ws equilibrted with sodium phosphte buffer. The column ws wshed with 300 ml of the sme buffer nd then with liner grdient of sodium phosphte buffer (0.1 to 0.5 M; totl volume, 1,400 ml). The flow rte ws 200 ml/h, nd 60-ml frctions were collected. Combined ctive frctions were concentrted through n Amicon Diflo membrne (PM-10). The concentrted smple ws diluted once with 50 mm sodium phosphte buffer nd reconcentrted. (iii) Sephcryl S-300 column. The concentrted smple from the hydroxyptite column ws pplied to Sephcryl S-300 column (2.6 by 42 cm) tht ws equilibrted nd eluted with sodium phosphte buffer. The flow rte ws 50 ml/h, nd 5-ml frctions were collected. Active frctions were combined. (iv) Mtrex Gel Red A column. Active frctions from the gel filtrtion step were pplied to Mtrex Gel Red A column (2.6 by 4.9 cm) which ws equilibrted with sodium

3 VOL. 53, 1987 ALCOHOL DHYDROGNASS OF CLOSTRIDIUM BIJRINCKII 699 phosphte buffer. The column ws wshed with 80 ml of the sme buffer nd then with liner grdient of KCl (0.1 to 1.0 M in phosphte buffer; totl volume, 100 ml). The flow rte ws 50 ml/h, nd 5-ml frctions were collected. Determintion of moleculr weight. Cell extrcts nd prtilly purified enzymes were pssed through Sephcryl S-300 column (see bove), nd the elution position of lcohol dehydrogenses ws determined. Moleculr weight stndrds used were thyroglobulin (Mr 669,000), humn immunoglobulin G (Mr 160,000), bovine serum lbumin (Mr 66,000), nd RNse A (Mr 13,700). Determintion of Michelis constnts. The Kms of the lcohol dehydrogenses were determined by using 50 mm sodiuum phosphte buffer (ph 7) contining 1 mm reduced glutthione. When used s the fixed substrte, the concentrtions of butyrldehyde, cetone, nd NADPH were 11, 132, nd 0.2 mm, respectively. Butyrldehyde ws diluted 10- to 100-fold with methnol, wheres cetone ws diluted 10- to 100-fold with wter. RSULTS Fctors ffecting ssys for lcohol dehydrogense. n- Butyrldehyde hs low solubility in wter (31), nd cler queous solution is not redily formed. This problem hmpered routine ssy for butnol dehydrogense in the physiologicl direction (reduction of butyrldehyde), but it ws circumvented by first dissolving butyrldehyde in methnol, which is not substrte for C. beijerinckii lcohol dehydrogenses. When ethnol dehydrogense ctivity ws mesured in the physiologicl direction, the substrte cetldehyde could contin substnce(s) tht oxidized NAD(P)H nonenzymticlly t rtes comprble to tht of the enzyme-ctlyzed rection (R.-T. Yn, C.-X. Zhu, nd J.-S. Chen, submitted for publiction). The interfering rection ws evident with cetldehyde bove 20 mm (up to 300 mm ws used by some workers in routine ssys). When cetldehyde ws below 5 mm, the rection might not be pprent, but the mesured enzyme ctivity could be severlfold lower with cetldehyde contining interfering substnce(s). Acetldehyde without prolonged contct with ir contined very little interfering substnce(s). Cell extrcts of C. beijerinckii hd high NADH-oxidizing (diphorse) ctivities in ir, which necessitted the use of nerobic conditions for ssying NADH-linked ctivities. However, diphorse ctivity ws completely seprted from lcohol dehydrogense by DA-cellulose column chromtogrphy, nd prtilly purified lcohol dehydrogense could be ssyed in ir. Cell extrcts of C. beijerinckii lso hd NADP-linked glycine-oxidizing ctivity, which complicted the use of glycine-contining buffers when the ctivity stin ws performed fter polycrylmide gel electrophoresis. Alcohol dehydrogense ctivities. Cell extrcts of C. beijerinckii B592 nd B593 were ssyed for both lcoholformtion nd lcohol-oxidtion ctivities. Substrtes tested were cetldehyde, cetone, butyrldehyde, ethnol, isopropnol, nd n-butnol. NADH, NADPH, NAD, nd NADP were tested s the electron donor or cceptor. A tetrzolium (INT) ws included in one series of ssys to fcilitte the mesurement of lcohol-oxidizing ctivities. Tble 1 lists ctivities found in the two strins. In both strins, higher ctivities were seen with NADPH thn with NADH. NADH-linked butnol dehydrogense ctivity ws 10 to 15% of NADPH-linked ctivity when NADH nd NADPH were used t sturting level (0.2 mm). TABL 1. Alcohol dehydrogense ctivities in cell extrcts of C. beijerinckii NRRL B592 nd NRRL B593 Sp ct (U/mg of protein) Assy NRRL NRRL B592 B593 Butnol dehydrogense NADH, butyrldehyde NADPH, butyrldehyde Butnol, NAD 0 0 Butnol, NADP Butnol, NADP, INT Isopropnol dehydrogense NADH, cetone 0 0 NADPH, cetone 0 89 Isopropnol, NAD 0 0 Isopropnol, NADP 0 78 Isopropnol, NADP, INT NDb 140 thnol dehydrogense NADH, cetldehyde NADPH, cetldehyde thnol, NAD thnol, NADP thnol, NADP, INT One unit (U) is the oxidtion of 1 nmol of NAD(P)H per min or the reduction of 1 nmol of NAD(P) or tetrzolium per min. The butyrldehyde concentrtion in the ssy ws 5.4 mm for strin NRRL B592 nd 33 mm for strin NRRL B593. b ND, Not determined. C. beijerinckii NRRL B592, which does not produce isopropnol, did not hve ny isopropnol dehydrogense ctivity. It contined similr level of ethnol nd butnol dehydrogense ctivities. C. beijerinckii NRRL B593, which produces butnol, isopropnol, nd ethnol, hd NADP(H)- linked butnol, isopropnol, nd ethnol dehydrogense ctivities. The isopropnol dehydrogense ctivity ws NADP(H) specific nd ws higher thn the butnol dehydrogense ctivity. nzyme stbility. The stbility of butnol nd isopropnol dehydrogense ctivities ws exmined to give clues on the possible multiplicity of lcohol dehydrogenses in this species. Under nerobic conditions (under Ar), butnol dehydrogense ctivity (both strins) nd isopropnol dehydrogense ctivity (strin NRRL B593) in cell extrcts showed similr stbility, since bout 80% of the strting ctivities remined fter 5 dys t 4 C. (Storge t -20 C, with or without 15% [vol/vol] glycerol, did not give higher stbility. nzymes from both strins were stble in liquid nitrogen.) Alcohol dehydrogense of both strins ws much less stble in ir thn under Ar t 4 C. A 10 to 30% loss in butnol dehydrogense ctivity ws seen when extrcts of strin NRRL B592 were kept in ir for 1 dy, nd 70 to 80% of the ctivity ws lost fter 7 dys. In comprison, loss of bout 70% of both butnol nd isopropnol dehydrogense ctivities occurred fter extrcts of strin NRRL B593 were kept in ir for 1 dy, nd 90 to 100% of both ctivities ws lost fter 2 dys. The study showed tht chnges in butnol nd isopropnol dehydrogense ctivities of strin NRRL B593 prlleled ech other under either erobic or nerobic conditions (dt not shown). Moleculr weight. Cell extrcts were nlyzed by gel filtrtion to show whether butnol dehydrogense of the two strins hd similr size nd whether butnol dehydrogense nd isopropnol dehydrogense ctivities of C.

4 700 HIU T AL. APPL. NVIRON. MICROBIOL. 0.40M4 in T 0b FRACTION NUMBR W FIG. 1 lution of butnol dehydrogense of C. beijerinckii NRRL rtwb592 50mlhxn from DA-cellulose rcin f10m column. A eecletd cell extrct (85 ml; e 50 mg of protein per ml) ws pplied to D-52 column (2.6 by 10 cm), nd the column ws developed by liner grdient of KCI(0 to 0.4 M in Tris chloride buffer [ph 8]; totl volume, 300 ml). The flow rte ws 50 ml/h, nd frctions of 10 ml were collected. See Mterils nd Methods for other conditions. beijerinckii NRRL B593 would seprte. Butnol dehydrogense ctivity of C. beierinckii NRRL B592 showed n pprent Mr of 66,000. In contrst, butnol nd isopropnol dehydrogense ctivities of C. beijerinckii NRRL B593 were eluted together in pek corresponding to Mr 100,000. Further mesurement with prtilly purified enzyme from both strins confirmed the difference in moleculr weight. lectrophoretic mobility. Following electrophoresis of cell extrcts on polycrylmide gels, lcohol dehydrogense ctivity on gels ws visulized with n ctivity stin. Butnol dehydrogense ctivity of strin NRRL B592 stined s doublet (Rf of 0.82 nd 0.87). The bnding pttern observed with NADP nd NAD ws comprble nd did not chnge fter the enzyme ws prtilly purified by DA-cellulose or Sephcryl S-300 column chromtogrphy. No ctivity bnd ws seen with isopropnol. NADP-linked butnol nd isopropnol dehydrogense ctivities of strin NRRL B593 ppered s single bnd with n Rf of c The bnd ppered much fster with isopropnol thn with butnol. This is consistent with the lower butnol-oxidizing thn isopropnol-oxidizing ctivity observed in spectrophotometric ssys (Tble 1). The electrophoretic mobility of butnol nd isopropnol dehydrogense ctivities of strin NRRL B593 remined the sme throughout the purifiction steps (see below). Chromtogrphy on DA-cellulose. To initite the purifiction nd chrcteriztion of lcohol dehydrogense from C. beijerinckii, extrcts from the two strins were chromtogrphed on D-52 columns. This ws n effective purifiction step (Fig. 1 nd 2). Agin, butnol nd isopropnol dehydrogense ctivities of strin B593 did not seprte (Fig. 2). The butnol dehydrogense ctivity of strin B592 ws eluted t higher chloride concentrtion (0.27 M versus 0.15 M) thn the butnol-isopropnol dehydrogense ctivities of strin B593, which indictes difference in chrge properties between the lcohol dehydrogenses of the two strins. Purifiction of lcohol dehydlrogense from C. beijerinckii NRRL B593. Alcohol dehydrogenses re usully specific for either primry or secondry lcohols, lthough some secondry lcohol dehydrogenses my rect with ethnol t 5 to 6% of the isopropnol-linked rte (5, 24). Since C. beijerinckii NRRL B593 contined comprble lev'els of butnol, ethnol, nd isopropnol dehydrogense ctivities (Tble 1) nd ll preliminry tests (gel filtrtion, electrophoresis, nd nion-exchnge chromtogrphy) did not seprte butnol dehydrogense ctivity from isoproponl dehydrogense ctivity, we decided to further purify the lcohol dehydrogense(s) from this strin. Both butnol nd isopropnol dehydrogense ctivities were monitored during ech step of purifiction. The four steps described here (Tble 2) gve n overll purifiction of 150- to 200-fold (results of three btches). These four steps, using DAcellulose, hydroxyptite, Sephcryl S-300, nd Mtrex Gel Red A, represent four seprtory methods bsed upon different moleculr properties of the proteins. Butnol dehydrogense nd isopropnol dehydrogense ctivities did not seprte throughout this purifiction. The rtio (I/B) of isopropnol-to-butnol-linked ctivities remined similr throughout the purifiction (Tble 2). A comprison of lcohol dehydrogense ctivities (in the physiologicl direction) in cell extrcts nd in highly purified smple (Tble 3) indictes tht the purified enzyme represented essentilly ll NADPH-linked lcohol dehydrogense species in strin NRRL B593. Results of gel electrophoresis followed by protein nd ctivity stins showed tht lcohol dehydrogense ws the mjor protein (with two dditionl fint bnds) in the 200-fold-purified enzyme preprtion (the best frction). Nevertheless, further work is needed to show more conclusively whether butnol-ethnolisopropnol dehydrogense ctivities of C. beijerinckii NRRL B593 reside in single enzyme or tightly ssocited enzyme complex. It is intriguing tht some NAD(H)-linked butnol nd ethnol dehydrogense ctivities were mesured in cell extrcts (Tble 1) but only NADP(H)-linked ctivities were mesured in the highly purified enzyme from strin NRRL B593. The possibility remins tht minor NAD(H)- linked primry lcohol dehydrogense exists in strin NRRL B593. ph nd enzyme ctivity. The effect of ph on lcohol dehydrogense ctivities ws studied in the direction of lcohol formtion. A significnt difference ws seen between butnol dehydrogense ctivities of strins NRRL B592 nd NRRL B593 (Fig. 3 nd 4). The butnol dehydrogense ctivity of strin NRRL B592 continued to increse with incresing ph between ph 6 nd 9; however, the butnol dehydrogense ctivity of strin NRRL B593 showed mximum ner ph 6.5. The isopropnol dehydrogense ctivity of strin NRRL B593 ppered to hve mximum ner ph 7.5. At ph 7.5, the lcohol dehydrogense ctivity ~ z. Wos I- ~~~~~~~~0w FRACTION NUMBR FIG. 2. lution of butnol dehydrogense (U) nd isopropnol dehydrogense (O) ctivities of C. beijerinckii NRRL B593 from DA-celluluose column. A cell extrct (85 ml; 16 mg of protein per ml) ws pplied to D-52 column (5 by 10 cm), nd the column ws developed by liner grdient of KCI (O to 0.4 M in Tris chloride buffer [ph 8]; totl volume, 1 liter). See Mterils nd Methods for other conditions.

5 VOL. 53, 1987 ALCOHOL DHYDROGNASS OF CLOSTRIDIUM BIJRINCKII 701 TABL 2. Purifiction of lcohol dehydrogense from C. beijerinckii NRRL B593' Isopropnol dehydrogenseb Butnol dehydrogenseb Step Amt of protein (mg) Totl U Sp ct Yield Purifiction B Totl U Sp ct Yield Purifiction IBC (U/mg) (%) (fold) (U/mg) ( (fold) Crude extrct 6, DA-cellulose 1, Hydroxyptite Ultrfiltrtion Sephcryl S Mtrex Gel Red A Alcohol dehydrogense ctivities were determined t mbient temperture (bout 25'C) in the direction of ldehyde or ketone reduction. The ssy mixture (1 ml) contined 50 mm Tris chloride buffer (ph 7.5), 1 mm DTT, nd 0.2 mm NADPH. The rection ws initited by the ddition of 6.6 mm cetone or 33 mm butyrldehyde (ll in finl concentrtions). The oxidtion of NADPH ws monitored t 340 nm. b U, unit (>Lmol of NADPH oxidized per min). c I/B, Rtio of isopropnol dehydrogense ctivity to butnol dehydrogense ctivity. ws much higher in Tris chloride buffer thn in sodium phosphte buffer. Michelis constnts. Alcohol dehydrogense ctivities in the direction of lcohol formtion were studied with cetldehyde, cetone, butyrldehyde, or NADPH s the vrying substrte. Apprent Km vlues re shown in Tble 4, nd significnt differences re evident between lcohol dehydrogenses of strins NRRL B592 nd NRRL B593. DISCUSSION The switch of metbolic pthwys during solvent fermenttion is useful for mechnistic studies of the control of gene expression in Clostridium spp. The environmentl signl(s) nd developmentl sequence tht cuse clostridi to switch from cid production to solvent production hve been the subject of ctive reserch in recent yers, nd in these studies, the ccumultion of solvents usully served s the indictor for trnsition of the cell into solvent production. The onset of solvent production is ccompnied by the ppernce of specific solvent-forming enzymes (1, 3, 13, 29; R.-T. Yn, C.-X. Zhu, nd J.-S. Chen, unpublished dt). Assys for these enzymes cn be more sensitive thn mesurements of solvents, nd these enzymtic ctivities cn thus serve s erlier indictors for the onset of solvent production. Identifiction of the very erly moment of onset should fcilitte the determintion of fctors tht re more TABL 3. Comprison of lcohol dehydrogense ctivities in cell extrcts nd prtilly purified enzyme from C. beijeirinckii NRRL B592 nd NRRL B593' - Sp ct (1tmol/min NRRL B592 mg of protein) for: NRRL B593 Assy Purified Purified Cell enzyme Cell enzyme extrcts (fold puri- extrcts (fold purifiction) fiction) Butnol dehydrogense NADPH, butyrldehyde (26) (147) Isopropnol dehydrogense NADPH, cetone None None (152) thnol dehydrogense NADPH, cetldehyde (24) b (153) The lcohol dehydrogense of strin NRRL B593 ws purified by the procedure described in Tble 2. The lcohol dehydrogense of strin NRRL B592 ws prtilly purified on Mtrex Gel Green A column (Yn nd Chen, unpublished dt). b At n cetldehyde concentrtion of 20 mm, the ctivity ws directly relted to triggering the switch into solvent production. To monitor these enzymtic ctivities, pproprite routine ssys re necessry. An ssy for cetocette decrboxylse ws previously estblished nd hs been used for this purpose (3). A stisfctory ssy for mesuring butnol dehydrogense in the direction of butnol formtion ws developed only in this study nd hs ided our efforts to mesure ll solvent-forming enzymes to define the progress of trnsition of cell into solvent production. The butnolforming ctivity in cell extrcts, s mesured under our ssy conditions, ws comprble to the butnol-producing ctivity of cells in btch cultures (dt not shown). Since the level of butyrldehyde in the in vitro ssy required the use of methnol to keep it in solution, it would be interesting to know the level nd stte of butyrldehyde in the cell. So fr, ll the properties of the extensively purified lcohol dehydrogense from strin NRRL B593 suggest tht single enzyme or tight enzyme complex is responsible for both isopropnol nd butnol dehydrogense ctivities in this orgnism. Alcohol dehydrogenses rective with both n- butnol (or n-butyrldehyde) nd isopropnol (or cetone) hve been found in Thermonerobium brockii (24), W 20 z 30 / ph FIG. 3. ffect of ph on butnol dehydrogense ctivity of C. beijerinckii NRRL B592. The rection mixture (1 ml) contined 50 mm buffer (sodium phosphte between ph 6 nd 7.5; Tris chloride between ph 7.5 nd 9), 0.2 mm NADPH, 0.54 mm butyrldehyde, 1 mm DTT, nd 0.23 mg of protein from cell extrct.

6 702 HIU T AL. APPL. NVIRON. MICROBIOL. z w FIG. 4. ffect of ph on butnol dehydrogense (O, *) nd isopropnol dehydrogense (0, 0) ctivities of C. beijerinckii NRRL B593. The conditions were s for the experiment in Fig. 3, except tht butyrldehyde ws t 33 mm for mesuring butnol dehydrogense ctivity, cetone ws t 6.6 mm for mesuring isopropnol dehydrogense ctivity, nd 0.12 mg of protein from cell extrct ws used. Thermonerobcter ethnolicus (5), Pseudomons fluorescens (20), nd Mycobcterium vcce (7), but, except for the enzyme from M. vcce, these lcohol dehydrogenses showed much lower ctivity with n-butnol thn with isopropnol. The lcohol dehydrogense of C. beijerinckii NRRL B593 lso showed much lower ctivity with butnol thn with isopropnol. However its ctivities (Tble 3) in the physiologicl direction were more comprble, s the rtio of ctivities with cetldehyde (20 mm), cetone (6.6 mm), nd butyrldehyde (33 mm) were bout 4:2: 1. Tht lcohol dehydrogenses of C. beijerinckii showed much lower ctivities in lcohol oxidtion my reflect their supposed physiologicl function for lcohol formtion. The rtio of NADPH-linked lcohol dehydrogense ctivities differed from the rtio of lcohols produced in btch cultures (with butnol higher thn isopropnol nd ethnol). It suggests tht mounts of lcohols formed re determined by the supply of enzymic substrtes. The rtio of solvents formed could be chnged by mnipulting the growth conditions (2, 10, 28, 34). These studies lso suggest tht metbolic pthwys providing substrtes for solvent-forming enzymes my be enhnced or tht competing pthwys my be inhibited to increse the solvent yield. The lcohol dehydrogense of C. beijerinckii NRRL B593 ctlyzes the finl rections for both the butnol nd the cetoneisopropnol brnches. It should be interesting to determine whether the two enzymtic ctivities cn be differentilly regulted nd to find the effects of such regultion on the rtio of lcohols produced. Alcohol dehydrogenses of C. beijerinckii showed higher ctivities with NADP(H) thn with NAD(H), nd specificity for NADP(H) ppered bsolute for the purified en- ph TABL 4. Michelis constnts of lcohol dehydrogenses of C. beijerinckii NRRL B592 nd NRRL B593 for vrious substrtes Km Substrte NRRL B592 NRRL B593 Acetldehyde <0.6 mm >15 mmb Acetone NDC 1.0 mm Butyrldehyde 6,uM 9.5 mm NADPH <1.9 p.md 18,uM, 31 p.me Activity remined constnt between 0.6 nd 5 mm. b Activity incresed linerly with cetldehyde between 1 nd 15 mm but decresed when cetldehyde ws bove 20 mm. c ND, No cetone-linked ctivity. d Activity did not chnge between 1.9 nd 85 F.M with butyrldehyde s cosubstrte, nd it ws techniclly difficult to mesure NADPH below 2 JIM. e With cetone s cosubstrte, Km ws 18 F.M; with butyrldehyde s cosubstrte, Km ws 31,uM. zyme from strin NRRL B593. NADP(H)-specific lcohol dehydrogense hs been isolted from other nerobes (21, 22, 24). The butnol dehydrogense of C. cetobutylicum ws reported to be more rective with NAD (for strin DSM 1732 [1]) or NADP (for strin NRRL B643 [29]). It is known tht nicotinmide nucleotide-linked enzymes for the sme physiologicl function my be NAD specific in one orgnism but NADP specific in nother for no pprent resons (30). However, electron trnsport processes vi NAD(H) nd NADP(H) hve different physiologicl implictions (33). Since butnol is produced in lrge mounts, butnol dehydrogenses differing in their specificity for nicotinmide nucleotides my involve different cellulr controls. This spect must be considered when ttempts re mde to mnipulte the product pttern of different solventproducing orgnisms. Judging from the retention of the ctivity rtio with different substrtes during the purifiction of lcohol dehydrogense from strins NRRL B593 (Tbles 2 nd 3) nd NRRL B592 (Tble 3; R.-T. Yn nd J.-S. Chen, unpublished dt), the lcohol dehydrogense we isolted from ech strin represent the mjor lcohol-forming enzymes in the two strins. The isolted lcohol dehydrogenses of C. beijerinckii NRRL B592 nd NRRL B593 were clerly different, s evidenced by their differences in stbility, moleculr weight, chrge properties, substrte rnge, Kms, nd effect of ph on ctivity. The mino cid sequences of these enzymes my give clues on how such differences rose in two strins of Clostridium species, nd the structure of their genes my suggest wys to generte new enzymic properties through genetic mnipultions. Further chrcteriztion of lcohol dehydrogenses by biochemicl nd genetic mens is thus being pursued. ACKNOWLDGMNTS This work ws supported by U.S. Deprtment of Agriculture grnt 83-CRSR , Deprtment of nergy grnt D-FG05-85R13368, nd project from the Commonwelth of Virgini. We thnk Christine Golemboski for excellent technicl ssistnce. LITRATUR CITD 1. Andersch, W., H. Bhl, nd G. Gottschlk Level of enzymes involved in cette, butyrte, cetone, nd butnol formtion by Clostridium cetobutylicum. ur. J. Appl. Microbiol. Biotechnol. 18:

7 VOL. 53, 1987 ALCOHOL DHYDROGNASS OF CLOSTRIDIUM BIJRINCKII Bhl, H., M. Gottwld, A Kuhn, V. Rle, W. Andersch, nd G. Gottschlk Nutritionl fctors ffecting the rtio of solvents produced by Clostridium cetobutylicum. Appl. nviron. Microbiol. 52: Bllongue, J., J. Amine,. Msion, H. Petitdemnge, nd R. Gy Induction of cetocette decrboxylse in Clostridium cetobutylicum. FMS Microbiol. Lett. 29: Brdford, M. M A rpid nd sensitive method for the quntittion of microgrm quntities of protein utilizing the principle of protein-dye binding. Anl Biochem. 72: Brynt, F., nd L. G. Ljungdhl Chrcteriztion of n lcohol dehydrogense from Thermonerobcter ethnolicus ctive with ethnol nd secondry lcohols. Biochem. Biophys. Res. Commun. 100: Chen, J.-S., nd S. F. Hiu Acetone-butnol-isopropnol production by Clostridium beijerinckii (synonym, Clostridium butylicum). Biotechnol. Lett. 8: Colemn, J. P., nd J. J. Perry Purifiction nd chrcteriztion of the secondry lcohol dehydrogense from propne-utilizing Mycobcterium vcce strin JOB-5. J. Gen. Microbiol. 131: Compere, A. L., nd W. L. Griffith vlution of substrtes for butnol production. Dev. Ind. Microbiol. 20: Cummins, C. S., nd J. L. Johnson Txonomy of the clostridi: wll composition nd DNA homologies in Clostridium butyricum nd other butyric cid-producing clostridi. J. Gen. Microbiol. 67: Dtt, R., nd J. G. Zeikus Modultion of cetonebutnol-ethnol fermenttion by crbon monoxide nd orgnic cids. Appl. nviron. Microbiol. 49: Dvis, B. J Disc electrophoresis. II. Method nd ppliction to humn serum proteins. Ann. N.Y. Acd. Sci. 121: Doelie, H. W Formtion of cetone, isopropnol, butnol, nd ethnol, p Bcteril metbolism, 2nd ed. Acdemic Press, Inc., New York. 13. George, H. A., nd J.-S. Chen Acidic conditions re not obligtory for onset of butnol formtion by Clostridium beijerinkii (synonym, Clostridium butylicum). Appl. nviron. Microbiol. 46: George, H. A., J. L. Johnson, W.. C. Moore, L. V. Holdemn, nd J.-S. Chen Acetone, isopropnol, nd butnol production by Clostridium beijerinkii (syn. Clostridium butylicum) nd Clostridum urntibutyricum. Appl. nviron. Microbiol. 45: Gibbs, D. F The rise nd fll (... nd rise?) of cetone/butnol fermenttions. Trends Biotechnol. 1: Gottwld, M., H. Hippe, nd G. Gottschlk Formtion of n-butnol from D-glucose by strins of the "Clostridium tetnomorphum" group. Appl. nviron. Microbiol. 48: Hrtmnis, M., T. Klson, nd S. Gtenbeck Uptke nd ctivtion of cette nd butyrte in Clostridium cetobutylicum. Appl. Microbiol. Biotechnol. 20: Holdemn, L. V.,. P. Cto, nd W.. C. Moore (ed.) Anerobe lbortory mnul, 4th ed. Virgini Polytechnic Institute nd Stte University, Blcksburg. 19. Hou, C. T., R. Ptel, N. Brnbe, nd I. Mrczk Stereospecificity nd other properties of novel secondrylcohol-specific lcohol dehydrogense. ur. J. Biochem. 119: Hou, C. T., R. N. Ptel, A. I. Lskin, I. Brist, nd N. Brnbe Thermostble NAD-linked secondry lcohol dehydrogense from propne-grown Pseudomons fluorescens NRRL B Appl. nviron. Microbiol. 46: Hsu, H. J., nd Z. J. Ordl Comprtive metbolism of vegettive nd sporulting cultures of Clostridium thermoscchrolyticum. J. Bcteriol. 102: Kleiner, D.., nd M. Johnston Purifiction nd properties of secondry lcohol dehydrogense from the prsitic protozon Tritrichomons foetus. J. Biol. Chem. 260: Kokesh, F. C., nd F. H. Westheimer A reporter group t the ctive site of cetocette decrboxylse. II. Ioniztion constnt of the mino group. J. Am. Chem. Soc. 93: Lmed, R. J., nd J. G. Zeikus Novel NADP-linked lcohol-ldehyde/ketone oxidoreductse in thermophilic ethnologenic bcteri. Biochem. J. 195: Monot, F., J.-R. Mrtin, H. Petitdemnge, nd R. Gy Acetone nd butnol production by Clostridium cetobutylicum in synthetic medium. Appl. nviron. Microbiol. 44: Moreir, A. R Acetone-butnol fermenttion, p In D. L. Wise (ed.), Orgnic chemicls from biomss. Benjmin/Cummings, Menlo Prk, Clif. 27. Plsson, B. O., S. Fthi-Afshr, D. F. Rudd, nd. N. Lightfoot Biomss s source of chemicl feedstocks: n economic evlution. Science 213: Prescott, S. C., nd C. G. Dunn The cetone-butnol fermenttion nd the butnol-isopropnol fermenttion, p Industril microbiology, 3rd ed. McGrw-Hill Book Co., New York. 29. Rogers, P Genetics nd biochemistry of Clostridium relevnt to development of fermenttion processes. Adv. Appl. Microbiol. 31: Smith,. L., R. L. Hill, I. R. Lehmn, R. J. Lefkowitz, P. Hndler, nd A. White Nicotinmide denine nucleotides, p Principles of biochemistry, generl spects, 7th ed. McGrw-Hill Book Co., New York. 31. Smith, T.., nd R. F. Bonner Acetldehyde, propionldehyde, nd n-butyrldehyde. Some physicl properties. Ind. ng. Chem. 43: Sund, H., nd H. Theorell Alcohol dehydrogenses, p In P. D. Boyer, H. Lrdy, nd K. Myrbck (ed.), The enzymes, 2nd ed., vol. 7. Acdemic Press, Inc., New York. 33. vn Diken, J. P., nd W. A. Scheffers Redox blnces in the metbolism of sugrs by yests. FMS Microbiol. Rev. 32: Yerushlmi, L., B. Volesky, nd T. Szcesny ffect of incresed hydrogen prtil pressure on the cetone-butnol fermenttion by Clostridium cetobutylicum. Appl. Microbiol. Biotechnol. 22: