Precise Excision of the Cellulose Binding Domains from Two ...

Vol. 263, No. 21, Issue of July 25, pp. 10401-10407,1388 Printed in U.S. A.

THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology. Inc.

Precise Excision of the Cellulose Binding Domains fromTwo Cellulomonas fimi Cellulases by a HomologousProtease and the Effect on Catalysis* (Received for publication, March 24, 1988)

Neil R. GilkesS, R. AntonyJ. Warren, Robert C.Miller, Jr., and Douglas G. Kilburn From the Department of Microbiology, University of British Columbia, Vancouuer, British Columbia, Canada V6T 1 W5

An endo-8-1,4-glucanase (CenA) and an exo-B-1,4- mechanistic differences, the primary structures of CenA and glucanase (Cex) were prepared from Escherichia coli Cex derived from DNA sequence analyses (4,5) reveal striking expressing recombinant DNA of the cellulolytic bac- similarities in overall protein architecture (11).A 112-amino terium Cellulomonas fimi. Purification was facilitated acid amino-terminal region of CenA and a 108-amino acid by the high affinities of these enzymes for cellulose. carboxyl-terminal region of Cex show 50% sequence homolAn extracellularC. fimi protease cleaved both enzymesogy. In both enzymes this conserved region is separated from i n vivo in a highly specific manner. Theaffinity of the a larger, nonconserved region by a conspicuous sequence of parent enzyme for cellulose was contained independ- 20-23 amino acids consisting of alternating prolyl and threonyl residues termed the Pro-Thr box (11).We have ently in an amino-terminal fragment (p20) ofCenA and a carboxyl-terminal fragment(p8) of Cex. These proposed that the nonconserved regions comprise catalytic domains (11,12). Both containsequences which resemble the fragments contained homologous amino acid sequences which were proposed to comprisecellulosebinding sequence at the active site of hen egg white lysozyme (11). domains.Correspondingfragments, p30 fromCenA When the catalytic domains of Cex and CenA are fused and p35 from Cex, which were unable to bind to cel- genetically, a hybrid protein with catalytic activities characlulose, contained catalytic domains. In both enzymes, teristic of both enzymes is produced (13). the two functional domains were joined by a hinge Like many bacterial and fungal cellulases, Cex and CenA bind strongly to cellulose (6, 8, 12). We have exploited the region consistingsolely of prolyl and threonyl residues. The binding domain was excised from CenA by proteo- unusual specificity of an extracellular C. fimi serine protease to investigate the structuralbasis for this phenomenon. Prolytic cleavage immediatelyadjacenttothecarboxyl terminus of this hinge. Cex was cleaved at an exactly teolytic cleavage of CenA and Cex generates fragmentswhich lose the ability to bind to cellulose analogous site. p30 and p35 retained several of the retain catalytic activity but catalytic functions of their parent enzymes. However,(12). From these preliminary studies, we proposed that the p30 was less active than intact CenA against micro- conserved regions comprise cellulose binding domains. Our crystalline cellulose implying a critical role for the present investigation provides direct evidence for this probinding domain of CenA in thehydrolysisof crystalline posal. C. fimi proteasecleaves both Cex and CenA in a precise substrate. and analogous manner. The products comprise catalytic and cellulose binding domains which function independently. However, the substrate specificities of intact CenA and its isolated catalytic domain show significant differences. Cellulose degradation by bacteria and fungi is effected by EXPERIMENTALPROCEDURES extracellular multienzyme systems whose underlying mechaBacterial Strains and Plasmids-E. coli C600 (pUC12-1.1 (PTIS)) nisms, despite intensive efforts, remain poorly understood. JMlOl (pUC18-1.6 cenA) were grown to late logarithmic phase Our studies of the process in the bacterium Cellulomonasfimi and on LB medium containing 100 pg of sodium ampicillin/ml and 2 mM have implicated the involvement of three /3-1,4-glucanase isopropylthiogalactopyranoside,a t 30 "C (14). Plasmid construction genes (1-6). Of these, cenA and cenB encode endoglucanases has been described (14, 15). C. firni (ATCC 484) was grown on 3% ( 2 , 3 , 6 ) ,while cex encodes an exoglucanase (3).cenA, cex, and (w/v) Avicel or 0.1% glycerol, in basal salts medium, at 30 "C, as (3, 12). their encoded products, CenA and Cex,' respectively, have described Enzyme Substrates and Assays-Cm-cellulose was the low viscosity been examined by us in some detail (1-5,7-11). CenA hydro- grade from Sigma (No. C8758, nominal degree of substitution: 0.7; lyzes Cm-cellulose randomly while Cex shows preference for nominal degree of polymerization: 400). Avicel PHlOl (FMC Interterminal linkages (3). CenA-catalyzed hydrolysis proceeds national, Ireland) is a microcrystalline cellulose preparation; crystalwith retention of anomeric carbon configuration, Cex with linity index = 88.1 (16). Phosphoric acid-treated cellulose was prepared as described by Ooshima et al. (17) and is amorphous. Cellulose inversion (10). Although these results indicate fundamental azure is a dyed cellulose preparation from Calbiochem (No. 219481) * This work was supported by grants from the National Sciences and Engineering Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisernent" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed. To conform with accepted practice, the endoglucanase encoded by cenAhas been renamed CenA. Similarly, the exoglucanase encoded by cex is referred to as Cex. CenA = EngA (12-14) = EA (6) = Eng (6, 7 , 9) = endoglucanase (11) = CB1 (3, 10). Cex = Exg (4-7, 9, 1215) = exoglucanase (11)= CB2 (3, 10).

and is also amorphous (18).MN300 cellulose (Macherey, Nagel and Co., West Germany) is a microcrystalline preparation; crystallinity index = 80.3 (16). Hide powder azure (HPA)' was from Sigma (No. H4631). The abbreviations used are: HPA, hide powder azure; pNPC, p nitrophenyl-P-D-cellobioside; gCenA and gCex, the glycosylatedforms of CenA and Cex from C. fimi; ngCenA and ngCex, the nonglycosylated forms of CenA and Cex from recombinant E. coli; RPC, reversephase chromatography; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; a-Pro/Thr, rabbit antiserum directed againstsynthetic Cex Pro-Thr box; PMSF, phenylmethylsulfonyl fluoride.

10401

10402

Excision of Binding Domains from Bacterial Cellulases

Cm-cellulase activity was assayed at 30 “C using hydroxybenzoic acid hydrazide reagent to detect the appearance of reducing sugars, as described (12). The substrate concentration in the assay was 4.0 mg/ml (= 24Kmfor ngCenA, Ref. 12). Resultsare expressed as glucose equivalents. pNPCase was determined as described previously (12) with 6.3 mM substrate in the assay (= 9 K,,, for ngCex, Ref. 12). Activities againstphosphoric acid-treated cellulose, Avicel, and MN300 cellulose were determined by incubation with 15 mgof substrate suspended in 1.5 ml of 50 mM sodium citrate, pH 7.0,0.2% bovine serum albumin, 0.02% NaN3 (citratebuffer), at 37 “C. Reducing sugar release into the reaction supernatant was determined with hydroxzybenzoic acid hydrazide reagent after 4 h (phosphoric acidtreated cellulose) or 18 h (Avicel, MN300 cellulose) of incubation. Activity against cellulose azure was determined by incubation with 40 mg of substrate suspended in 2.0 ml of citrate buffer, for 18 h a t 37 “C. Release of solubilized substrate into thereaction supernatant was quantitated from the absorbance a t 585 nm. One unit of cellulose azureactivity gives an increase in A m ofl.O/h. C. fimiprotease activity was monitored by incubation with 10 mg of HPA suspended in 1.5 ml of phosphate buffer, for 1 h a t 37 ‘C. Release of soluble substrate into the reaction supernatant was determined from the absorbance at 585 nm. One unit of HPAse activity gives a change in A S of l.O/h. Protein determination hasbeen described (3). Preparation of Crude C. fimi Protease-The culture supernatant from C. fimi grown on 0.1% glycerol medium to early stationary phase at 37 “C was obtained by centrifugation, followed byfiltration through a 0.2-pm pore polysulfone membrane (Acrodisc, Gelman Sciences) and dialysis against 50 mM potassium phosphate, pH 7.0,0.02% NaN3 (phosphate buffer), or 50 mM sodium citrate, pH 7.0,0.02% NaN3, to equilibrium. Preparations were concentrated, if necessary, by ultrafiltration through a YM-2 membrane (Amicon). Purification of Glycosylated and Nonglycosylated Cellulases-Purification of gCex and gCenA from C. fimi by guanidine hydrochloride elution, lectin affinity chromatography, and anion-exchange chromatography were as described previously (3, 12) except that chromatography of gCex on Mono Q resin (Pharmacia LKB Biotechnology Inc.) employed a 20-600 mM ammonium formate,pH 8.0, gradient to permit subsequent lyophilization of the purified product. Similarly, chromatography of gCenA used a 20-700 mM ammonium acetate, pH 9.8, gradient. ngCex and ngCenA were purified from cell extracts of E. coli C600 (pUClZ-l.l(PTIS)) and JMlOl (pUC18-1.7 cenA), respectively. Cells were disrupted in phosphatebuffer and treatedwith PMSF as described (6). The clarified extracts were made 1.5% in streptomycin sulfate and the precipitate removed by centrifugation. Preliminary purification was by cellulose affinity chromatography using the procedure described by Owolabi et ai. (6). Enzymes were eluted withH20 (6) or with 8 M guanidine hydrochloride in phosphate buffer. In the lattercase, the eluate was concentrated, desalted, and the buffer exchanged with 20 mM ammonium acetate, pH 9.8 (ngCenA), or 20 mM ammonium formate, pH 8.0 (ngCex), by ultrafiltration through a PM-10 membrane (Amicon). In both instances, final purification was achieved by anion-exchangechromatographyon Mono Q resin, employing the gradient systems described above for gCex and gCenA. The purified enzymes were then lyophilized. Purification of ngCenA and ngCex Proteolytic Fragments-p3O was prepared by proteolytic digestion of ngCenA followedby anionexchange chromatographyin ammonium acetate,pH 9.8, as described above. p35, p33, and p8 were purified by proteolytic digestion of ngCex followed by RPC asdescribed in the legend to Fig. 5, or anionexchange chromatography in ammoniumformate, pH 8.0, as described above. Electrophoresis-Routine SDS-PAGE was performed as described by Laemmli (19). Gels were fixed in 3% sulfosalicylic acid, 10% trichloroacetic acid, and stained with 0.25% Coomassie Blue where appropriate. Calibration standards were myosin, rabbit muscle (212 kDa); 8-galactosidase, E. coli (130 kDa);phosphorylase B, rabbit muscle (97.4 kDa); albumin, bovine (68 kDa); glutamate dehydrogenase, bovine liver (53 kDa); ovalbumin (45 kDa); glyceraldehyde-3phosphate dehydrogenase, rabbit muscle (36 kDa);and carbonic anhydrase, bovine erythrocyte (29 kDa). p8 was analyzed using the electrophoreticsystem described by Swank and Munkres (20) for small polypeptides. Gel composition was:12.5% acrylamide, 1.25% bis-acrylamide, 0.1% SDS, and 8 M urea, in 165 mM Tris phosphate, pH 6.8. Fixation and staining were as described above. Calibration standards were: carbonic anhydrase, bovine erythrocyte (29.0 kDa); trypsininhibitor, soybean (21.1 kDa); myoglobin and its cleavage fragments,equine heart (17.0,14.4,8.2,6.2, and 2.5 kDa); and cytochrome c, equine heart (12.4 kDa).

Other Procedures-HPAse was inhibited with PMSF, added as a 0.1 M solution in anhydrous acetone by rapid mixing, a t a level of 25 pmollunit HPAse. Avicel was pretreated by autoclaving and buffer washing as described previously (6). Other experimental procedures were as described or referenced in figure legends.

RESULTS

Purification of ngCenA and ngCex-ngCenA and ngCex were expressed in E. coli from recombinant plasmids(pUC181.6 cenA) and (pUClZ-l.l(PTIS)), respectively. The enzymes were purified to apparenthomogeneity (Fig. 1, lanes 1 and 3) by cellulose affinitychromatographyand anion-exchange chromatography. Details of ngCenA purification are given in Table I. Similar resultswere obtained for ngCex. Unlike their native counterparts purified from C. fimi (Fig. 1, lanes 2 and 4, the recombinant enzymes are not glycosylated. The resultant shifts in their apparent molecular masses are evident in Fig. 1 and documented in Table11. C. fimi Protease-Crude protease was preparedby dialysis C. fimi of the culture supernatant from early stationary phase cells grown on 0.1% glycerol medium. Suchpreparations kDa

-212 - 130 -97 -68

- 53 -45 -36 -29

FIG.1. Analysis of purified cellulases by SDS-PAGE. ngCex ( h e I), gcex ( l a n e 2), ngCenA ( l a n e 3), and gCenA ( l a n e 4 ) were purified as described under “Experimental Procedures” and analyzed by SDS-PAGE (8% acrylamide). Each lane was loaded with approximately 2 pg of protein. TABLE I Summary of ngCenA purification The enzyme was purified from a crude extract of E. coli JMlOl (pUC18-1.6 cenA) by cellulose affinity chromatography using guanidine hydrochloride elution and anion-exchange chromatography as described under “ExDerimental Procedures.” Total Specific Total activity ?6 Fraction protein Cm-cel- of Cm- recovery ‘ulase” cellulase ~

mg

units

mg

2.7 100 833.7 308.0 Cell extract 80 667.5 28.3 23.6 Guanidine HCI eluate from affinity column 67 37.6 14.8 555.9 Desalted eluate 46.2 61 11.0 508.0 Anion-exchange column pool ,. Cm-cellulase activity isexpressed as micromoles of glucose equivalents released per min a t 30 “C.

Excision of BindingDomain1s from Bacterial Cellulases TABLE I1

1

2

3

10403 4

5

kDa -212

Size determinations for ngCenA, ngCex, and their glycosylated counterparts NA = not applicable because glycosyl substitution has not been defined.

-130 -97 L

Enzyme

Total amino acid residues”

Apparentb molecular mass

Predicted‘ molecular mass

ngCenA-

kDa 48.7 43.8 ngCenA 421/418 418 53.0 NA gCenA 443 47.3 47.1 ngCex 443 49.3 NA gCex a Determined for the mature formsfrom deduced amino acid compositions (4, 5).Two numbers for ngCenA reflects the occurrence of two amino-terminal variants (see “Discussion”). Determined by SDS-PAGE. Determined for the mature formsby summation of the molecular masses of component anhydro amino acids from the deduced amino acid compositions (4.5).

.

1 2 3 4 5 6 7

1 2 3 4 5 6 7 k a a ‘ -212

0

A

-130 -97

-68 ngcex

-

P35. P”

p33

’

-!x3 ’-45

-36 -29

FIG. 2. T i m e course of proteolysis of ngCenA and ngCex. 300 p g of ngCenA ( A ) or ngCex ( B )dissolved in 725 pl of phosphate buffer containing 1.5 units of crude C. fimi protease was incubated a t 37 “C. Reactionswere sampled at 0,6,24,48,96, and 144 h (lanes I6, respectively), treated with PMSF and analyzed by SDS-PAGE (8% acrylamide). Control samples were incubated in the absence of protease, for 144 h (lane 7).All lanes were loaded with sample equivalent to 2.8 pg of initial protein.

typically contained 3-7 HPAse units/ml. Approximately 92% of this initial activity remained following incubation for 144 h in the absence of substrate, under the conditionsdescribed in the legend to Fig. 2. HPAse was previously shown to be inhibited by PMSF (8, 12). Proteolytic Cleavage of ngCenA and ngCex-C. fimi protease effected rapid cleavage of ngCenA and ngCex (Fig. 2). Under the conditionsdescribed, limited proteolysis wasevident even in samples to which PMSF was added prior to incubation, indicating incomplete inhibition (Fig. 2, A and B, lane I). Cleavage was not evident in the absence of protease (Fig. 2, A and B , lane 7). Cleavage of ngCenA was complete within 24 h (Fig. 2 A ) . The major product was a 30.4-kDa polypeptide (p30). Further cleavage of this product was not apparent. A product of 20.1 kDa (p20) was also evident in 6- and 24-h samples, but it disappeared after prolonged incubation. A further transient 36.5-kDa product (p36)was seen only after 6 h of incubation. Rapidly migrating components were visible at the dye front in 24-120-h samples. These various products were examined further by SDS-PAGE, either withor without pre-adsorption by cellulose (Fig. 3). Residual intact ngCenA, p36, p30, and p20 were evident in the complete reaction mixture (Fig. 3, lane I). Adsorption to cellulose, following proteolysis, removed p20 and residual ngCenA but left p36 and p30 (Fig. 3, lane 2).

PS-

~ 3 0 -w

-68 -53

-

-45 -36 -79

p20-

FIG. 3. Analyses of ngCenA proteolysis products. 100 pg of ngCenA in 200 pl of phosphate buffer containing 0.6 units of crude C. fimi protease was incubated a t 37 “C for 6 h, followed by addition of PMSF (see “Experimental Procedures”). One-half of the reaction mixture was then left untreated; the other half was incubated with 20 mg of pre-washed Avicel in suspension a t 0 “C for 1 h, after which the soluble fraction was recovered by centrifugation. The untreated sample (lane I ) and the soluble fraction following Avicel adsorption (lane 2) were analyzed by SDS-PAGE (8% acrylamide) and stained with Coomassie Blue. Duplicate lanes ( 3 and 4, respectively) were analyzed on a zymogram (6) to detect components with Cm-cellulase activity. Furtherduplicates (lanes 5 and 6) were analyzed on a Western blot probed with antiserum directed against synthetic Cex Pro-Thr box (0-ProlThr). Each lane was loaded with sample volumes equivalent to9 pg of initial protein.

Duplicate lanes were analyzed on a zymogram. Cm-cellulase activity was associated with ngCenA, p36, and p30 but not p20 (Fig. 3, lanes 3 and 4 ) . Western blot analysis of a further set of duplicate lanes(Fig. 3, lanes 5 and 6)revealed that only p20 was recognized by antiserum directed againsta synthetic peptide corresponding to the Pro-Thr box of Cex (a-Pro/Thr, Ref. 12). No immunoreactive products remained after cellulose adsorption. Proteolysis of ngCex (Fig. 2B) was faster than that of ngCenA. No intact enzyme remained after 6 h of incubation. The major product was a 35.4-kDa polypeptide (p35). Other minor 34.5- and 32.8-kDa products (p34 and p33, respectively) were evident inearlysamples, andtheir levels increased progressively during incubation.By120 h, a concomitant decrease in the level of p35 was apparent. Rapidly migrating products were evident at the front after incubation for 6-120 h. An analysis of these various ngCex proteolysis products, to cellulose, is shown in Fig. with and without pre-adsorption 4. This analysis used a 12% acrylamide gel to obtain better resolution of smaller polypeptides. p35, p34, p33, and a small component, later designated as p8, were visible in the complete reaction mixture (Fig. 4, lane I). p8 disappearedfollowing cellulose absorption (Fig. 4, lane 2). A Western blot of the complete reaction mixture revealed that p35, p34, and p33 were all recognized by a-Pro/Thr; p8was not recognized (Fig. 4, lane 3). ngCex proteolysis products were also analyzed byRPC (Fig. 5, upper panel).A minor UV-absorbing peak elutedwith 32% 2-propanol.Amajorpeak, containingpNPCase activity, eluted with 38%2-propanol. A shoulder was apparent on the trailing edge of the major peak. Adsorption of the reaction mixture with cellulose, prior to RPC, resulted in the disappearance of the minor peak; the major peak, itsshoulder, and the associated pNPCase activity remained unchanged (Fig. 5, lowerpanel).These resultsindicated that themajor and minor RPC peaks represented p35 and p8, respectively. This was confirmed by routine SDS-PAGE (notshown). Low levels of

Excision of Binding Domains from BacterialCellulases

10404 1

2

kDa

-212

-130 -97

.-68

-53 -45 P35 \ p 3 4 i u P33

P8

6 3 " -

-29

-

FIG.4. Analysis of ngCex proteolysis products. 100 pg of ngCex in 120 pl of phosphate buffer containing 0.6 units of C. fimi protease were incubated a t 37 "C for 90 min, followed by addition of PMSF (see "Experimental Procedures"). One-half of the reaction mixture was then left untreated. The other half was incubated with Avicel as described in the legend to Fig. 3. The untreated sample (lane I ) , and thesoluble fraction following Avicel adsorption (lane2), were analyzed by SDS-PAGE (12% acrylamide)andstained with Coomassie Blue. Lanes were loaded with sample volumes equivalent to 7.8 pg of initialprotein.Untreatedsample (2.6 pg) was also analyzed on a Western blotwith a-Pro/Thr asdescribed in the legend to Fig. 3.

I 0

10

x)

30

40

50

60

Fraction

FIG.5. Chromatographic separation and characterization of ngCex proteolysis products. Upper panel, 70 pg of ngCex in

p34 and p33 were alsoapparent,particularlyinfractions corresponding to the shoulder. RPC fractions were also ana- 200 p l of phosphate huffer containing 0.36 units of crude C. fimi lyzed by SDS-PAGE in urea buffer, using a highly cross- protease was incubated a t 37 "C for 18 h and then treated with PMSF (see "Experimental Procedures"). The entire reaction mixture was linked gel designed to resolve small polypeptides (Fig. 6). p8 applied to a proRPC 5/10 reverse-phase column (Pharmacia LKB (apparent molecular mass = 8.3 kDa) comprised >95% of the Biotechnology Inc.) equilibratedwith 5% (v/v) 2-propanol, 0.1% protein present in the minor peak(Fig. 6, lane I). A low level trifluoroacetic acid, and components were resolved with a 5-65% (v/ of a22-kDa component was also visible in this fraction. v) 2-propanol gradient in 0.1% trifluoroacetic acid, a t a flow rate of 0.3 ml/min. 0.33-ml fractions were collected, and aliquots were asFractions corresponding to the major peak contained the sayed for pNPCase. Enzyme activity is expressed as micromoles of larger ngCex products. released per min/ml fraction, a t 37 "C. Selected fractions Amino-terminal Amino Acid Sequences and Amino Acid product were analyzed by SDS-PAGE (see Fig. 6). Lower panel, an identical Compositions of Proteolysis Products-The amino-terminal reaction mixture was preincubated with 15 mg of pre-washed Avicel amino acid sequences determined for ngCenA, p30, ngCex, a t 0 "Cfor 1 h, following proteolysis and PMSF treatment. The p35, p33, and p8 are given inTable 111. The previously soluble fractionwas recovered by centrifugation and analyzed as determined sequences forthe amino termini of mature gCenA described above. and gCex purified from C. fimi (4,5) areincluded for comparison. The previously reported occurrence of two amino-ter- itself has a molar activity of 9.0 katals/mol (calculated from minal variants of ngCenA (14) was confirmed. Analysis of data in Ref. 12). A K,,, of 0.65 mM was determined for p33 p30revealed theaminoterminus:Val-Thr-Pro-Gln-Pro-. with pNPC, using conditions described previously (12). The Thismatchesamino acids 135-139 of the deduced CenA corresponding K,,, for ngCex is 0.70 mM (12). Aplot of change sequence. The partial aminoacid composition determined for in specific fluidity against reducing sugar production during p30 and the theoretical partial composition of the carboxyl- Cm-cellulase hydrolysis describesthe relative rates of internal terminal CenA fragment Val'3s-Trp4'Rare given in Table IV. versus terminal cleavage of @-1,4-glucosidicbonds. Cex and Analysis of ngCex revealed the same amino terminus as that CenA give characteristic plots (3, 13). A plot determined for previously determined for gCex. ngCex proteolysis products, p33 (not shown) was indistinguishable from that given by p35 and p33, also have thisaminoterminus.p8hadthe ngCex. termino terminus Ser-Gly-Pro-Ala-Gly-. This corresponds to Qualitative analysis (Fig. 3, lanes 3 and 4 ) had alsoindicated amino acids 336-340 of the deduced Cex sequence. The partial that Cm-cellulose activity was retained by p30. Again, a plot amino acid composition determined for p8 is given in Table of specific fluidity versus reducing sugar production for p30 IV, together with the theoretical partial composition of the (not shown)was the sameas that previously reported for the carboxyl-terminal Cex fragment Ser336-Gly443. intact enzyme (3). In a further experiment, themolar activiCatalytic Activities of ngCex, ngCenA, and Their Productsties of p30 were compared with intact ngCenA using a range It is evident from the data in Fig. 5 that p35 from ngCex of cellulosic substrates (Table V). ngCenA, before and after retains pNPCase activity. In separate experiments, p35 and incubation with protease, provided equimolar quantities of p33 were purified for amino-terminal sequencing. The molar intact enzyme and its truncated product, respectively. AppropNPCase activities of these p35 and p33 preparations were priate controls were included. The expected compositions of approximately 7.2 and 8.9 katals/mol, respectively. ngCex the sampleswere confirmed by SDS-PAGE (notshown). The

Excision of Binding Domains

2 3 4 5 6 7

(simcslmm

from Bacterial Cellulases

TABLE IV Partial amino acid composition ojp30 and p 8 Amino acids were quantitated on a Beckman 119CL analyzer after hydrolysis of p3O or p8with 6 N HCI, 0.1%phenol, 0.1% thiodiglycolic acid at 110 “C for 24 h in uacuo. Figures in parentheses indicate the theoretical partial compositions of the CenA fragment Val’”-Trp“* and the Cex fragment Ser336-Gly443, respectively, deduced from appropriate DNA sequences.

kDa -29.0 -21.1 -1 7.0 -14.4 -12.4 -8.2 -6.2

% composition

Amino acid P8

P30

A

R

-2.5

B Z G H I

FIG 6. Electrophoretic analysis of ngCex proteolysis products separated by RPC. Proteolysis products were separated by RPC as shown in Fig. 5 and selected fractions (see Fig. 5) analyzed by SDS-PAGE in urea, as described under“ExperimentalProcedures”. Lane I , fractions 28 and 29; lane 2, fraction 32; lane 3, fraction 33; lane 4, fraction 34; lane 5, fraction 35; lane 6, fraction 36; lane 7, fraction 37.

Amino-terminal sequences of ngCenA, ngCer, and their proteolysis products Proteins were purified as described under “Experimental Procedures.” Sequencing was by automatedEdmandegradationonan Applied Biosystems 470A gas-phase sequenator. The previously determined amino termini of s e x and gCenA, and the amino termini deduced from DNA analyses of the appropriate structural genes, are included for comparison. Polypeptide nsex

p35

P33 (4670)

A (1380) T (1230) T (1260) L (1180) K (1300)

A (4012) T (2614) T (2780) L (2038) K (3406)

(1500) (1180Y

1 2 3 L4 5 6 A7 A8

A (800)d T (300) T (300) (840) K (670) E (500)

$exb

p8 (500)

S (6) AA G (12) T P (11) T A (10) L L G (8) K E A A

Polypeptide

1 2 3 4 5 6 7

Predicted amino terminus‘

T T K E

Pre-

ngCenA (4000)

p30 (3200)

A (4060) Q. P (1020,2190) A, G (1960,1930) A (2870) P, R (1380,530)

V (3400) T (1130) P (1830) Q (690) P (1220)

L K F P S

T Y V

(1.1)

12.5 (13.8) 10.8 (10.9) 4.0 (5.2) 0.8 (1.0) 10.8 (10.8) 10.9 (9.9) 9.7 (9.7) 7.6 (6.9) 13.5 (13.4) (14.9) 15.8 1.1 0.4 (1.0) 2.6 (3.4) 0.7 (1.0) 7.4 (7.5) 2.9 (3.0) 4.1 (4.5) 0.7 (1.0) 1.8 (1.9) 5.7 (5.9) 6.2 (4.9) 5.7 (6.9) 6.1 (6.3) (11.9) 11.6 5.8 (5.6) 15.0 (15.8) 5.6 (4.1)