Vol. 54, No. 10

Cloning of the Thermomonospora fusca Endoglucanase E2 Gene inStreptomyces lividans: Affinity Purification and Functional

Domains of the Cloned Gene ProductGURDEV S. GHANGAS* AND DAVID B. WILSON

Section ofBiochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853

Received 29 March 1988/Accepted 22 July 1988

Thermomonosporafusca YX grown in the presence of cellulose produces a number of 0-1-4-endoglucanases,some of which bind to microcrystalline cellulose. By using a multicopy plasmid, pUJ702, a gene coding for one

of these enzymes (E2) was cloned into Streptomyces lividans and then mobilized into both Escherichia coli andStreptomyces albus. The gene was localized to a 1.6-kilobase PvuII-ClaI segment of the originally cloned3.0-kilobase SstI fragment of Thermomonospora DNA. The culture supernatants of Streptomyces transformantscontain a major endoglucanase that cross-reacts with antibody against Thermomonospora cellulase E2 and hasthe same molecular weight (43,000) as T. fusca E2. This protein binds quickly and tightly to Avicel, from whichit can be eluted with guanidine hydrochloride but not with water. It also binds to filter paper but at a slowerrate than to Avicel. Several large proteolytic degradation products of this enzyme generated in vivo lose theability to bind to Avicel and have higher activity on carboxymethyl cellulose than the native enzyme. Othersmaller products bind to Avicel but lack activity. A weak cellobiose-binding site not observed in the nativeenzyme was present in one of the degradation products. In E. coli, the cloned gene produced a cellulase thatalso binds tightly to Avicel but appeared to be slightly larger than T. fusca E2. The activity of intact E2 fromall organisms can be inactivated by Hg2+ ions. Dithiothreitol protected against Hg2+ inactivation andreactivated both unbound and Avicel-bound Hg2+-inhibited E2, but at different rates.

The hydrolysis of cellulosic substrates such as Avicel,filter paper, or Solka Floc by cellulolytic microorganismsoccurs extracellularly, producing primarily cellobiose andglucose. These products are then taken up by the organismsand metabolized further. Although alternate modes of cello-biose utilization exist, many of these organisms utilize an

intracellular P-glucosidase for the hydrolysis of cellobiose.In principle, therefore, three activities-,(1-4)-endoglu-canase, exoglucanase (cellobiohydrolase), and cellobiase-are all that are required for growth on cellulose. However,cellulolytic microorganisms display enormous diversity intheir cellulolytic enzymes, and some produce as many as 20new proteins when grown on cellulose (2).Thermomonosporafusca YX is a thermophilic filamentous

bacterium, originally isolated from decaying wood (3). Thisorganism grows in minimal medium supplemented withcellulose. Culture supernatants of T. fusca can contain as

many as 15 different peptides that hydrolyze carboxymethylcellulose (CMC). Five of these peptides have been purifiedand, on the basis of numerous criteria, appear to be distinctgene products (5, 14, 24). To further understand the role ofthese and other proteins in cellulose utilization, the genescoding for T. fusca cellulases are being cloned. In a previousstudy, we demonstrated that Streptomyces lividans is a goodhost for cloning and expressing T. fusca cellulases. Thispaper describes the isolation, by cloning in S. lividans, of a

3.0-kilobase fragment of T. fusca DNA that codes forcellulase E2. The gene was also introduced into Escherichiacoli for expression and subcloning. The binding of E2 tomicrocrystalline cellulose, as well as the effects of proteoly-sis on binding and activity, was analyzed with E2 producedin S. lividans, E. coli, and Streptomyces albus. The effects ofHg2+ on activity and Avicel binding were also determined. A

* Corresponding author.

simple purification scheme based on the differential bindingof E2 and its proteolytic products to Avicel was developed topurify milligram quantities of E2.

MATERIALS AND METHODS

Enzymes and chemicals. Restriction endonucleases and T4DNA ligase were purchased from Bethesda Research Labo-ratories, Inc., New England BioLabs, Inc., and AmershamCorp. Tryptone soya broth was from Difco Laboratories.Thiostrepton was a generous gift from the Squibb Institute,Princeton, N.J. Polyclonal antibodies to T. fusca cellulaseswere produced in rabbits (5). Goat anti-rabbit immunoglob-ulin G-alkaline phosphatase conjugate used for Westernblotting (immunoblotting) was from Bio-Rad Laboratories.Avicel type PH-102 microcrystalline cellulose was fromFMC Corporation, Philadelphia, Pa. Cellodextrins were a

gift from Richard V. Greene, Northern Regional ResearchLaboratory, Peoria, Ill. Other reagents were obtained fromeither Sigma Chemical Co. or Fisher Scientific Co.

Bacterial strains and plasmids. S. lividans TK24 (str-6) (6),plasmid pIJ702 (16), and S. albus J1074 (ilv), which is a

restrictionless mutant of S. albus G (7), were provided byD. A. Hopwood, John Innes Institute, Norwich, England. E.coli HB101 (F- hsdS20 [rB- mB-] recA13 ara-14 proA2 lacYgalK rpsL strA xyl mtl supE A-) was used as a host forsubcloning. Plasmid pBR327 in E. coli RRI was provided byX. Soberon, Universidad National Autonoma de Mexico,Mexico City, Mexico (18). T. fusca YX36, previously clas-sified as a Thermoactinomyces sp., was provided by W. D.Bellamy, formerly at Cornell University (3).Media and culture conditions. Media for the sporulation of

S. lividans and preparation and regeneration of protoplastshave been described previously (13). Unless indicated oth-erwise, the S. lividans cultures were grown on tryptone soyabroth at 30°C in baffled flasks or in flasks with stainless steel

2521

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1988, p. 2521-25260099-2240/88/102521-06$02.00/0Copyright © 1988, American Society for Microbiology

2522 GHANGAS AND WILSON

springs. E. coli was grown in Luria broth and plated on Luriaagar plates. Thiostrepton was used at a concentration of 10,ug/ml in liquid media and at 50 ,ug/ml in plates. The ampi-cillin concentration was 50 ,ug/ml, both for liquid culturesand for plates. The tetracycline concentration in plates was10 ,ug/ml.Recombinant DNA techniques. Preparation of DNA from

S. lividans, E. coli, and T. fusca, construction of recombi-nant plasmids, protoplast transformation, and plasmid sub-cloning techniques were described earlier (8, 12, 13). Trans-formants were screened for cellulase activity as described inreference 13 except that the overlay agar was tempered to40°C before pouring and the CMC-overlaid plates wereincubated at 30°C.Enzyme assays. Cellulase assays on Streptomyces cultures

were carried out at pH 6.5 and 55°C as previously described(13). Unless otherwise specified, CMC was used as thesubstrate at 0.25% final concentration. The zymogram tech-nique of Beguin (1) was used to locate P-1-4-endoglucanasebands in sodium dodecyl sulfate (SDS) gels.Enzyme preparations. Culture supernatants were sepa-

rated from S. lividans mycelial mass by filtration throughglass wool. Cellulase present in culture supernatants wasprecipitated at 4°C with 40% ammonium sulfate, dissolved in1/100 volume of 10 mM potassium phosphate buffer (pH 6.7)(buffer I), and centrifuged at 10,000 x g for 10 min. Thesepreparations could be stored at -20°C without appreciableloss of enzyme activity and served as partially purifiedenzyme.

Avicel affinity purification of E2 cellulase from S. lividans.Avicel PH-102 was suspended in buffer I, about one-third ofthe fines were removed, and the remaining Avicel waswashed with cold buffer I. The binding of cellulase to Avicelwas carried out at 0 to 4°C by mixing the washed Avicel withthe partially purified enzyme. Typically, 5 g of washedAvicel was suspended in 75 ml of buffer I containing 2 Mguanidine hydrochloride and mixed with 25 ml of partiallypurified cellulase containing 1,000 IU. The Avicel wasrecovered by centrifugation and quickly washed with 100 mlof 2 M guanidine hydrochloride, 500 ml of water, and 100 mlof 2 M guanidine hydrochloride. The tightly bound enzymewas eluted at room temperature with 100 ml of 6 M guanidinehydrochloride, cooled to 4°C, concentrated, and desalted inan Amicon cell with a PM10 membrane.

Protein blotting. Proteins were separated on SDS-acryl-amide gels, electrophoretically blotted (22) onto 0.45-,um-pore-size nitrocellulose (Schleicher & Schuell, Inc.), andreacted with the appropriate serum. Proteins that boundantibody were detected by using an alkaline phosphatase kitfrom Bio-Rad Laboratories.

Other procedures. Protein was determined by the Bradfordprocedure (4) or by the method of Lowry et al. (17).Electrophoresis of proteins on polyacrylamide gels or onSDS-polyacrylamide gels was performed by standard proce-dures (9, 12). Cellulase bands were isolated from SDS gelsby electroelution (15). Protein glycosylation was estimatedby the method of Dubois et al. (10). Proteins were chemicallydeglycosylated with trifluoromethanesulfonic acid as de-scribed by Stewart et al. (19). For N deglycosylations, theN-glycanase and protocol were obtained from Genzyme,Inc., Boston, Mass.

RESULTS

Isolation and subcloning of the gene for cellulase E2.Cellulase E2 is a 43,000-molecular-weight extracellular 3-1-

Sst

Cia

Sph

PvuIPvuXho

Sst

pIJ702 T. fusca DNA

Sst Portiol

1. Ligate2.Transform S. livida3.Isolate cmc'ase positives

p0085 ApaI pBR327

.9a:° .BamH I *Sal I digest/ . 2. Purify large fragmentw

1. Xho .BglIIdgest2. Purify 2.6 kb piec

1. Mix)Iigate,and transform E.coli HB1012. Isolate ompt tets and cellulose positive clones

,6_ X //. gi11Sstl~~~~~Ss

EcoRI~~/4jk~~~utI SphI1

2. XPvu IPvu 1IXho I

FIG. 1. Cloning strategy and construction of plasmids pGG85and pGG86 that replicate in S. lividans and E. coli, respectively, andcarry T. fusca DNA coding for cellulase E2. , pBR327 se-quence; =zJ, pIJ702 sequence; _, Thermomonospora sequencescarrying the cellulase E2 gene. tsr, Thiostrepton resistance genewhich is expressed in S. lividans; amp, ampicillin resistance geneexpressed in E. coli; Ori, origin; cmc'ase, CMCase.

4-endoglucanase which has the highest specific activity oncrystalline cellulose of any purified T. fusca cellulase (5, 24).The enzyme is also active on CMC, and therefore transfor-mants expressing the E2 gene can be identified by incubationof CMC overlays followed by staining of overlays withCongo red (13, 20).

SstI partial digests of T. fusca DNA ligated into Strepto-myces plasmid pIJ702 were used to transform S. lividansprotoplasts. Cloning into the SstI site of pIJ702 inactivatesthe tyrosinase gene, and therefore recombinant plasmidsproduce colonies lacking melanin (Mel- phenotype). Onesuch clone produced cellulase E2 (G. S. Ghangas and D. B.Wilson, FEMS Symp. 1987, abstr. no. P3-05, p. 69) andcarried a pIJ702 derivative, pGG85, which carries a 3.0-kilobase-pair insert in the SstI site. The cellulase producedby pGG85 resembles its T. fusca counterpart in its mobilityon native and SDS-polyacrylamide gels, its heat stability,and its product profiles. The E2 gene is also expressed in E.coli, and the gene was localized to a 1.6-kilobase PvuIl-ClaIfragment of the original plasmid insert by subcloning in E.coli with plasmid pBR327. The strategies for cloning andsubcloning the E2 gene are given in Fig. 1, and restrictionmaps of the plasmids that were constructed are given in Fig.2.

Plasmid pGG86 (Fig. 1) was constructed by replacing thesmaller BamHI-SalI fragment of pBR327 (18) with a BglII-

APPL. ENVIRON. MICROBIOL.

CLONING OF T. FUSCA E2 GENE 2523

Plosmid No.Sstl

pGG85 '-

pGG86

pGG87

pGG88pGG89

pGG90

pGG91

Xholp4P4\I

EcoRI

IL

CellulosePhenotype

IsnSL 1 +

Soil -c

0m

-g(-)C

4,a-LI

¢ +

PvuIll lXhol Coll +

FIG. 2. Restriction maps of plasmid subclone inserts with theircellulase expression phenotypes.

XhoI fragment of pGG85. The BglII-SstI segment of theinsert in pGG86 comes from pIJ702, and the SstI-XhoIsegment comes from T. fusca DNA. Plasmid pGG87 is aderivative of pGG86 from which the smaller EcoRI segmenthas been eliminated. Plasmid pGG88 is a derivative ofpGG86 in which the orientation of the smaller EcoRI frag-ment has been reversed (only the relevant portion of theinsert is shown in Fig. 2). Plasmid pGG89 is a derivative ofpGG86 from which the small Sail fragment has been elimi-nated. Plasmid pGG90 is a derivative of pGG86 in which the0.25-kilobase-pair PvuII fragment has been deleted, whilepGG91 was derived from pGG90 by removing the small ClaIfragment.

Expression of the E2 gene in S. lividans, S. albus, and E.coli. Untransformed S. lividans and S. albus grown intryptone soya broth produced about 0.02 and 0.06 U ofcarboxymethyl cellulase (CMCase), respectively, per ml.The low level of host activity did not interfere with the CMCoverlay selection assay of cellulase-producing clones. Thecellulase activity of S. lividans and S. albus transformedwith plasmid pGG85 was much higher than that of strainslacking the E2 gene. The transformant colonies of S. lividansand S. albus selected from plates and grown in tryptone soyabroth produced about 3 to 4 and 8 to 10 U of CMCaseactivity, respectively, per ml. After cultivation in liquidmedium for several generations, these values drop to about1.5 U/ml for S. lividans and about 5 U/ml for S. albus.However, there was no obvious change in the yield and sizeof the isolated plasmids from these cultures. The activityfound in extracts of stationary-phase cultures of E. colitransformants, where the activity was greatest, was about 1/300 that of Streptomyces transformant cultures. The culturesupernatants of Streptomyces transformant cultures containE2 as the major protein. These cultures also contain variousamounts of different E2 degradation products (described inthe next sections).

Binding to cellulose of S. lividans E2. The purified E2 fromT. fusca cultures has significant filter paper- and Avicel-degrading activity (5). Nevertheless, S. lividans transfor-mants producing E2 do not grow in minimal media contain-ing filter paper as the sole carbon source. When filter paperis added to culture media containing another carbon source,the enzyme content in a 2-day culture supernatant is about30% that of a culture lacking cellulose, while the remaining70% of the activity is bound to filter paper. Binding of E2 toAvicel is rapid and independent of temperature between 0and 22°C, but the extent of binding to filter paper is markedlyinfluenced by temperature and incubation time (Fig. 3 and 4).

Properties of Avicel-binding and nonbinding forms of E2-cellulase. Experiments using filter paper indicated that at

Percent Filter Paper

V .4 hr.,room temp.g75- .!81 hr.,room temp.

_54 ~~~~~~~~~~~~~1 hr.,4°C

-~50-

~0. 0.5 1.0 1.5 2.0 2.5Percent Avicel

FIG. 3. Binding of cellulase activity, as assayed on CMC, fromS. lividans culture supernatant to filter paper (A) and Avicel PH-102(B). Supernatant (5 ml containing 1 to 1.5 IU of CMCase per ml) ofa pGG85 transformant culture grown in tryptone soya broth wasmixed in plastic vials with various amounts of filter paper discs orAvicel and continuously mixed on a rotary shaker. At the indicatedtimes, 10 to 20 ,ul of supematant, obtained by centrifugation, wasassayed for CMCase activity. The enzyme activity lost from thesupernatant was assumed to be bound.

least three classes of E2-related proteins were present in theS. lividans cultures: unbound, loosely bound, and tightlybound. The loosely bound activity could be recovered bywashing with buffer or water, while the tightly bound en-zyme could be eluted with 6 M guanidine hydrochloride. Tofurther study the properties of E2 and its derivatives, Avicelwas added to partially purified S. lividans enzyme to give afinal concentration of 10%. Two major protein bands wereadsorbed (Fig. 5). The 43,000-molecular-weight band (E23)corresponds to the purified E2 enzyme from T. fusca andbinds tightly to Avicel, while the 30,000-molecular-weightband (E'0) binds to Avicel at 0°C but not at 55°C (data notshown). The binding of E"0 to Avicel at 0°C is prevented bycellobiose but not by glucose, while neither sugar inhibitsE43 binding (Fig. 6). The binding of E30 to Avicel at 0°C isalso prevented by higher oligosaccharides up to cellohe-xaose (data not shown).

Various amounts of another E2 product, E25, can beobserved in the CMC overlays (Fig. 7). E35 also binds lesstightly to Avicel than E23, and on CMC overlays E25 and E30appear to be more active in CMC hydrolysis than is E2.

VOL. 54, 1988

Sgh I rSal I cial SstiI

it 11

I

II IL-J

11

L--i=j

2524 GHANGAS AND WILSON

cIm 75p

it

0

<L 0 5 0 15 2

Time,minFIG. 4. Time course of binding of E2 activity at O0C to micro-

crystalline cellulose. Avicel PH-102 (50 mg) was mixed with 5 ml ofS. lividans culture supernatant containing 1 to 1.5 IU of E2 per ml,and binding was measured as described in the legend to Fig. 3.

These results suggest that Avicel binding of E2 at 550Crequires the fragment or fragments that are removed fromE41 to generate E15.

2 25

In vivo generation of proteolytic products of E2 inS. lividansand E. coli. To confirm the precursor-product relationship ofE2 and its degradation products and to analyze further theproteolytic processing of E2, the proteins were fractionatedon SDS gels and then blotted onto nitrocellulose filters. Thefractionated proteins were probed with antibody againstpurified T. fusca E2. The results shown in Fig. 8 clearly

44ognrt 3 s

2~~

2~~~~~~

FIG. 5. Gel analysis of the binding of E2 gene products toAvicel. Partially puified S. lividans supenatant (10 IU of CMCasein 1 ml) was mixed on ice with 100 mg of Avicel and centrfugedwithin 5 min. Supernatant (20 ILI) was loaded onto an SDS-poly-acrylamide gel, and proteins were localized by staining with Coo-massie brilliant blue dye. Lane 1, Total proteins; lane 2, supernatantproteins after Avicel adsorption. The proteins removed by Avicel(molecular weights of approximately 43,000 and 30,000) are indi-cated by arrows.

FIG. 6. Inhibition by cellobiose of binding to Avicel of the30,000-molecular-weight E2 product In a final volume of 1 ml of 10mM potassium phosphate (pH 6.5) buffer, 10 IU of CMCase activity,and 100 mg of Avicel were mixed on ice for 5 mm. The mixtureswere then centrifuged and the supernatants were analyzed on anSDS-10% polyacrylamide gel. Additions: lane 1, none; lane 2, 1%glucose; lane 3, 2% glucose; lane 4, 5% glucose; lane 5, 10% glucose;lane 6, 1% cellobiose; lane 7, 2% cellobiose; lane 8, 5% c'ellobiose;lane 9, 7.5% cellobiose.

show that a 43,000-molecular-weight protein is the majorform of E2 present in S lividans cultures. Cultures of S.lividans that do not contain the E2 gene lack proteins thatreact with E2 antibodies A 14 000- molecular-weight peptidethat binds to Avicel is produced by both S. lividans and E.coli (Fig. 8). The smallest peptide that is present in Strepto-myces cultures and binds tightly to cellulose has an apparentmolecular weight of 3 p000 (data not shown).

Hgm2 ions preferentially inhibit the catalytic activity of E2.A number of metal ions, includingHig2f , are known to

Coomassie Activity1 2 1 2

A BFIG. 7. Activation of the CMCase activity and loss of Avicel

binding upon proteolytic digestion of the E2 protein in S. lividanscultures. In a final volume of 1 ml of buffer, 10 U of CMCase (50%ammonium sulfate cut) was mixed with 10% Avicel and electropho-resed on SDS-17% polyacrylamide gels in duplicate. (A) Proteinsstained with Coomassie blue. (B) Zymogram replica of gel in panelA showing CMCase activity. Lanes: 1, controls without Avicel; 2,supernatants after Avicel adsorption.

APPL. ENVIRON. MICROBIOL.

%%.I% --P=Mm=Mmft

CLONING OF T. FUSCA E2 GENE 2525

1 2 3 4 5 6 7 8 9

=.I _*f mm lw 's+4Xt:.,,,̂tai-qp-

m- ---W iu*o ,,, -~544* *446

TABLE 2. Purification of endoglucanase E2 fromS. lividans pGG85

Step Enzyme fraction Vol Protein Total %(ml) (mg) units Sp act Recovery

1 Supernatanta 1,600 64.0 1,600 25 1002 (NH4)2SO4, 40% 20 30.0 1,410 47 883 Avicel purified 6 2.4 500 208 30a Two 1,000-mI cultures grown for 2 days were filtered through Pyrex

filtering fiber (Coming Glass Works) to obtain the starting supernatant.

FIG. 8. Production of E2 cellulase and time course of proteolyticdigestion as measured by immunoblotting. Lanes 1 to 8 represent S.lividans cultures, and lane 9 represents E2 products in E. coli thatbind to Avicel. For S. lividans, the culture was centrifuged, washedthree times with tryptone soya broth, and taken up in 10 volumes oftryptone soya broth-thiostrepton medium. Samples in lanes 1through 8 were withdrawn at 10 h, 22 h, 30 h, 48 h, 3 days, 4 days,5 days, and 6 days, respectively, and centrifuged, and supernatantswere frozen at -20°C. Each lane represents 20 p.l of supernatant.For E. coli E2, the HB101(pGG91) culture grown overnight wascentrifuged and 0.05 U of E2 was adsorbed to 5 mg of Avicel andeluted with 1% SDS. The proteins were separated on an SDS-12%polyacrylamide gel and probed with E2 antibody after electroblot-ting as described in the text. The arrows indicate the major andminor bands detected by immunostaining having molecular weightsfrom 43,000 (highest) to 14,000 (lowest).

inhibit T. fusca E2 (5). The E2 produced by S. lividans andS. albus transformants is quickly and completely inhibited inthe presence of micromolar amounts of HgCl2. Theseamounts of HgCl2 do not affect the binding of E2 to Avicelwhen the enzyme is incubated at 0, 22, 37, and 55°C prior toincubation with Avicel and assays are carried out at 55 or37°C. Dithiothreitol (DTT) protects the enzyme against Hg2+inactivation (Table 1). Furthermore, the inactivated enzymein solution, or bound to Avicel, can be regenerated by DTT.However, the rate and extent of activation of the bound E2were lower than those of the soluble form (data not shown).

Purification of E2 and its products from S. lividans cultures.To purify E2 and its products from S. lividans cultures, theprocedure described in Materials and Methods was followedthe results of a typical purification are given in Table 2. Mostof the degradation products of the E2 gene product are

TABLE 1. Protection of cellulase E2 against Hg2+inactivation by DTT'

Enzyme and addition % Activity

None ........................................... 1001mMDTT ........................................... 11075 ,uM HgCl2........................................... 475 p.M HgCl2, then 1 mM DTT................................... 1101 mM DTT, then 75 p.M HgCl2................................... 107

present in the ammonium sulfate supernatant, while most ofthe E"3 is present in the pellet (Fig. 9).

Effects of deglycosylation treatments on S. lividans E2. T.fusca E2 was found to be glycosylated (5), but E2 from S.lividans contained less than 1% carbohydrate. E2 from T.fusca and S. lividans displayed identical enzymatic proper-ties and electrophoretic mobilities on SDS gels. Chemicaland enzymatic deglycosylation of T. fusca E2 with N-glycanase also did not change its mobility on SDS gels. Thefollowing experiments indicate that glycosylation of E2 isnot required for binding to cellulose or catalysis. First,partially purified E2 was subjected to periodate oxidationfollowed by reduction, and it retained both catalytic andbinding activities. Second, E2 produced in E. coli whichdoes not appear to glycosylate proteins was catalyticallyactive and bound to Avicel (Fig. 8).

DISCUSSION

The cloned E2 gene is stably maintained under selectivepressure in S. lividans, S. albus, and E. coli. As comparedwith E. coli cultures, the S. lividans and S. albus culturesproduce over 300 times more enzyme, and the bulk of theenzyme in S. lividans and S. albus is excreted. The mecha-nism for the decline to a lower level of CMCase activity ingrowing Streptomyces cultures appears to be a complexadaptation phenomenon and is, at present, poorly under-stood.The enzyme purified from T. fusca was active on CMC

and other forms of cellulose (5). Hagerdal et al. (14) reported

1 2 3 4

FIG. 9. Purification of E2. Lane 1 contains supernatant concen-trated 15-fold by freeze-drying; 15 ,ul of this fraction was loaded.Lane 2 contains the 40% ammonium sulfate fraction. Lane 3contains the proteins in the 40 to 80% ammonium sulfate fraction.Lane 4 contains the Avicel-affinity-purified fraction. Equal amountsof cellulase activity were loaded in each lane.

a 0.025 U of enzyme in 300 p. of 50 mM buffer I was mixed with HgCl2 orDTT and allowed to stay on ice for 5 min, followed by addition of DTT orHgCl2 as indicated. The mixture was left on ice for 5 min, mixed with 100 pulof 1% CMC, and incubated at 55°C. Appropriate controls without enzyme,substrate, DTT, etc., were treated similarly.

VOL. 54, 1988

2526 GHANGAS AND WILSON

that up to 50% of T. fusca cellulases were adsorbed toresidual cellulose in growing cultures and that the activitycould be desorbed under mild conditions. The results re-ported in this paper show that E2 excreted by S. lividanstransformants binds tightly to insoluble substrates such asfilter paper and Avicel and that the enzyme eluted withguanidine hydrochloride from these substrates retains fullactivity. This observation is consistent with the fact that, ofthe mixture of enzymes in the T. fusca supernatant, E2 bindsmost rapidly to Avicel (unpublished data).The results of in vivo proteolysis experiments demon-

strate that the E2 cellulose binding site is different from thecatalytic site. This conclusion is consistent with the fact thatHg2+ ions preferentially inhibit the catalytic activity of E2without affecting its ability to bind to Avicel. A bipartitedomain structure for two cellobiohydrolases from Tricho-derma reesei has recently been reported (21, 23). Unlike ourobservation, however, these workers did not report en-hanced activity towards CMC of any Trichoderma cellulasedegradation products. Similar domains appear to exist insome cellulases from other organisms (2). These observa-tions are based largely on DNA sequences which indicatethat regions of conserved amino acid sequences are commonto these proteins.

In addition to the cellulose-binding site, a cellobiose-binding site was identified on a 30,000-molecular-weightpeptide which also carries the catalytic site. The cellobiose-binding site may be involved in the regulation of cellulosehydrolysis of cellulase E2, as cellobiose has been observedto inhibit the activity of some cellulases, including E2 (5).

Inorganic mercuric salts reversibly inactivate the catalyticactivity of E2 without impairing its ability to bind to Avicel.The fact that DTT protects against Hg2+ inactivation andregenerates the inactivated enzyme suggests that thiolgroups may play a role in cellulose hydrolysis by E2.The reasons for the higher activity on CMC of smaller

forms of E2 observed on gels are not clear. A similarobservation showing activation of endoglucanases by prote-ases was made in Sporotrichum pulverulentum by Erikssonand Pettersson (11). In that study, however, Eriksson andPettersson did not compare the cellulose-binding ability ofthe proteolytic products and the precursor proteins. TheAvicel purification scheme developed here is simple andyields milligram quantities of E2 free from major contami-nants.

ACKNOWLEDGMENTS

This work was supported by National Science Foundation grantPCM-9319432 and by grant DEFG02-84-ER13233 from the Depart-ment of Energy.We thank S. J. Lucania for the continuous supply of thiostrepton.

LITERATURE CITED1. Beguin, P. 1983. Detection of cellulase activity in polyacryl-

amide gels using Congo Red-stained agar replicas. Anal. Bio-chem. 131:333-336.

2. Beguin, P., N. R. Gilkes, D. G. Kilburn, R. C. Miller, Jr., G. P.O'Neill, and R. A. J. Warren. 1987. Cloning of cellulase genes.Crit. Rev. Biotechnol. 6:129-162.

3. Bellamy, W. D. 1977. Cellulose and lignocellulose digestion bythermophilic actinomycetes for single cell protein production.Dev. Ind. Microbiol. 18:249-254.

4. Bradford, M. 1976. A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding. Anal. Biochem. 92:248-254.

5. Calza, R. E., D. C. Irwin, and D. B. Wilson. 1985. Purificationand characterization of two ,-1-4-endoglucanases from Thermo-monospora fusca. Biochemistry 24:7797-7804.

6. Chater, K. F., D. A. Hopwood, T. Kieser, and C. J. Thompson.1982. Gene cloning in Streptomyces. Curr. Top. Microbiol.Immunol. 96:69-95.

7. Chater, K. F., and L. C. Wilde. 1976. Restriction of a bacterio-phage of Streptomyces albus G involving endonuclease Sall. J.Bacteriol. 128:644-650.

8. Collmer, A., and D. B. Wilson. 1983. Cloning and expression ofa Thermomonospora YX endocellulase gene in E. coli. Bio/Technology 1:594-601.

9. Davis, B. J. 1964. Disc electrophoresis. II. Method and applica-tion to human serum proteins. Ann. N.Y. Acad. Sci. 121:404-427.

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