Characterization of a Thermomonospora curvata endoglucanase expressed in Escherichia coli

307

Journal of Biotechnology, 29 (1993) 307-320

© 1993 ElsevierScience PublishersB.V. All rights reserved 0168-1656/93/$06.00

BIOTEC 00880

Characterization of a Thermomonospora curvata endoglucanase expressed in Escherichia coli David G. Presutti, T h o m a s A. H u g h e s and Fred J. Stutzenberger Department of Microbiology, Clemson University, SC, USA (Received 8 November1991;revisionaccepted3 October 1992)

Summary A Thermomonospora curvata endoglucanase gene was subcloned into Escherichia coli-Bacillus subtilis shuttle vector pLP1202. E. coli strain DH1 harboring

the recombinant cellulase plasmid pTC15 produced an endoglucanase with pH and temperature optima of 8.0-8.5 and 70°C, respectively. This cellulase was produced during exponential growth with > 80% secretion ( ~ 60% in periplasmic space and ~ 25% in culture fluid). The activity of the enzyme was inhibited by antiserum produced against crude T. curvata extracellular proteins. Expression of endoglucanase was not detected in B. subtilis transformants harboring plasmids containing a functional T. curvata endoglucanase gene. Bacillus subtilis; Cellulase; momonospora curvata

Cloned endoglucanase; Escherichia

coli;

Ther-

Introduction Microbial degradation of native cellulose to glucose requires the participation of at least three enzyme activities: endo-l,4-/3-glucanase (EC 3.2.1.4), which cleaves the polymer at random internal sites to produce free chain ends; exo-l,4-/3glucanase (EC 3.2.1.91), which cleaves cellobiose units from the non-reducing chain ends; and /3-glucosidase (EC 3.2.1.21), which converts cellobiose to glucose (Eriksson, 1979). Correspondence to: F.J. Stutzenberger, Clemson University, Clemson, SC 29634-1909, USA. Fax

803-656-1127.

308 Cellulase genes have been cloned and expressed in organisms other than E. coli such as B. subtilis (Koide et al., 1986; Robson and Chambliss, 1986), Streptomyces lividans (Ghangas and Wilson, 1987), Z y m o m o n a s mobilis (Misawa et al., 1988), Saccharomyces cereviseae (Sacco et al., 1984; Skipper et al., 1985), Lactobacillus plantarum (Scheirlinck et al., 1989), and L. acidophilus (Baik and Pack, 1991) Thermomonospora curvata, a facultative, thermophilic actinomycete, secretes a thermostable, multicomponent cellulase complex that degrades crystalline cellulose (Fennington et al., 1982; Stutzenberger, 1972). A variety of ether extracellular enzymes are also produced by T. curvata including amylases (Glymph and Stutzenberger, 1977), proteases and xylanases (Bernier et al., 1988; McCarthy and Cross, 1984) and polygalacturonate lyase (Stutzenberger, 1987). Presutti et al. (1991) reported the cloning of three endoglucanase genes from T. curvata. Here we describe the characterization of the recombinant cellulase produced by one of these genes in E. coli and the feasibility of employing B. subtilis as an alternate host.

Material and Methods Bacterial strains and plasmids

Bacterial strains and plasmids used in this study are listed in Table 1. Materials

Restriction endonucleases, T 4 D N A ligase, RNase A, lysozyme, calf alkaline phosphatase, and isopropyl-/3-D-thiogalactopyranoside (IPTG) were purchased from B R L (Gaithersburg, MD), Sigma Chemical Co. (St. Louis, MQ) or USB (Cleveland, OH). Carboxymethylcellulose (CMC; types 7L and 7H) was donated by Hercules, Inc. (Wilmington, DE).

TABLE 1 Bacterial strains and plasmids Strains/plasmids E. coli DH1 B. subtilis PSL1 T. curvata

Plasmids pLP1202 pTC520 pTC12 pTC13 pTC15

Genotype or phenotype

Source

Reference

F-, recA1, endA1, gyrA96, thi-1, SupE44, recA1 stp, leuA8, arg-15, thrA, recE4, hsr R, hsr M

E.L. Kline

Hanahan, 1983

G.H. Chambliss

/3-1,4-glucanase

This laboratory

Ostroff and Pene, 1984 Stutzenberger, 1971

Ap r, Tcr, Cmr Ap r,/3-1,4-glucanase Ap ~, Cm~,/3-1,4-glucanase Ap r, Cmr,/3-1,4-glucanase Ap r, Cmr,/3-1,4-glucanase

T.A. Hughes

Hanahan, 1983 this study this study this study this study

309

Culture media Thermomonospora curvata was grown in minimal medium (Stutzenberger, 1972). For cellulase production the carbon source was ground surgical grade cotton (Johnson and Johnson Co.) or native cotton (8 g 1-1). E. coli strains were grown in Luria broth (LB) containing Tryptone [Difco], 10 g 1-1; yeast extract [Difco)], 5 g 1-1; NaCI, 10 g 1-1; pH 7.5, or M9 salts (Maniatis et aI., 1982) containing 0.2% glycerol as a carbon source. Ampicillin (50 /zg ml-1), tetracycline (25/~g ml-1), 0.1%, CMC type 7L, and 1.5% agar were added where necessary. Plasmids were isolated from E. coli strains by the methods of Birnboim and Doly (1979) or Holmes and Quigley (1981). Recombinant plasmids were isolated from Bacillus subtilis strains by a modification of the method of Birnboim and Doly (1979): 3 ml of an overnight culture were grown in LB containing 5 ~g ml-a chloramphenicol, then incubated with their solution 1 for 45 min at room temperature. Plasmid DNA was suspended in 20/zl TE (10 mM Tris buffer, pH 7.5 with 1 mM EDTA). Plasmid preparations were also further purified by cesium chloride-ethidium bromide density gradient centrifugation. Preparation of anticellulase antiserum Cell-free culture fluid was obtained by centrifugation of a 500 ml culture of T. curvata shaken in mineral salts-cotton medium at 53°C for 48 h. Extracellular proteins were concentrated under N 2 (10 PSI) at 4°C on YM10 ultrafiltration membranes (Amicon) to approx. 1 mg ml ~ 1 and filter sterilized. The sterilized concentrate was aseptically mixed 1 : 1 with Freund's complete adjuvant (Difco). New Zealand white rabbits were subcutaneously injected at 6 weekly intervals with 0.25 ml of the mixture in each shoulder and 0.25 ml intramuscularly in each hindquarter. After the second week Freund's incomplete adjuvant (Difco) was used to avoid necrosis at injection sites. Rabbits were bled from the marginal ear vein one week after final injection. Transformation E. coli DH1 cells were transformed by the method of Hanahan (1983) except hexamine cobalt (III) chloride and dithiothreitol were omitted from the competency buffer. Transformation of B. subtilis was facilitated by electroporation with a Bio-Rad Gene Pulser Transfection Apparatus. B. subtilis PSL1 was grown in LB broth to an OD600 of about 0.4. Harvesting and resuspension in sucrose electroporation buffer was done as described for electroporation of E. coli in the Instructions and Applications Guide No. 87-0365 0588. Each of three samples containing 780/zl of the PSL1 cell suspension were placed in Gene Pulser cuvettes and mixed with 20/xl of pTC15 (containing approx. 1 /zg DNA). After incubation on ice for 10 rain the samples were pulsed at 1.0 kV. The transformation mixtures were allowed to express antibiotic (chloramphenicol) resistance for 1 h at 37°C. The mixtures were then plated out on LB plates containing 5/xg ml-1 chloramphenicol. A total of 600/~1 of each transformation mixture were plated. Transformants were selected for on I,B plates containing 5/zg ml-1 chloramphenicol.

310

Screening of clones for CMCase (endo-l,4-[3-glucanase) activity Ap r, Tc r transductants were screened for CMCase activity by the technique of Teather and Wood (1982) modified as previously described (Presutti et al., 1991).

Subcloning of cellulase gene contained in pTC520 The cellulase gene contained on pTC520 was subcloned to a 6 kb EcoRI-SphI fragment which encoded cellulase activity (pTC15). The subcloning protocol is shown in Fig. 1.

D

0

pLPI202

| Digested~Hl,

i0 kb b a n d p u r i f i e d f r o m gel.

i

i

|

Digeated~mHI, a l k a l i n e p h o s p h a t a s e treated.

i

i pl.pl202

Q

E

a

g e s t e d Eu~RI religated. z

(partial), and

sz

Digested ~hI (partial) a n d religated.

Q

Fig. 1. Protocol for subcloning of endoglucanase gene into E. coli-B, subtilis shuttle vector pLP1202. E = EcoRl,

B = BamHI,

S = SphI.

311

Assays Endoglucanase (EG) activity from recombinant organisms was determined viscometrically with a Canon-Fenske type (200) viscometer. Standard assay mixtures contained 4.75 ml 1% CMC (Hercules type 7H) in 0.1 M 4[2-hydroxethyl]-lpiperazine-ethanesulfonate, sodium salt (HEPES) buffer, pH 8.0, and 250 /zl of the enzyme sample (preheated before mixing to 65°C). The viscometric assays were performed and enzyme units calculated as previously described (Presutti et al., 1991). Protein concentrations were measured by the method of Bradford (1976) using bovine serum albumin as a standard.

SDS-PAGE electrophoresis SDS-PAGE was performed by the method of Laemmli (1970) using a Bio-Rad Protean 16CM vertical electrophoresis unit. Enzyme samples Were run on 10% resolving gels with 3% stacking gels.

Zymograms Endoglucanase zymograms were prepared from SDS-polyacrylamide gels by the method of Beguin (1983), using 50 mM HEPES buffer, (pH 8.0) in the overlay. Electrophoresis grade agarose was substituted for agar. Microscope slides (0.8 mm) were used as spacers. Polyacrylamide gel-CMC agarose overlay sandwiches were incubated at 60°C for 1-4 h to detect hydrolysis zones.

Preparation of EG from E. coli DH1 (pTC15) was grown to late exponential phase (OD600 of about 1.0), centrifuged at 5000 g at 4°C for 10 min, washed twice with 10 mM potassium phosphate buffer (pH 7.0) and resuspended in a small volume (approx. 1 ml per 100 ml of culture) of buffer. Cells were sonicated and debris was removed by centrifugation. The supernatant was heated to 65°C for 15 min and centrifuged to remove native host proteins.

Localization of EG activity in E. coli cells E. coli cells harboring recombinant cellulase plasmids were grown at 37°C with shaking to late exponential phase in M9 medium containing 0.2% glycerol as a carbon source, with 1 mM thiamine, 50/~g ml-1 ampicillin and 1 mM IPTG. The cultures were fractionated into cytoplasmic, periplasmic and extraeellular fractions by the method of Cornelis et al. (1982). Periplasmic and cytoplasmic fractions were concentrated 30-100 fold by ultrafiltration with Amicon YM10 membranes. The cytoplasmic fraction was obtained by resuspending the final pellet in 1 ml of 10 mM Tris, pH 7.5, in an ice bath and sonicating for three 1-min bursts at 60% of maximum with a Fisher Sonic Dismembrator 300. Cell debris was removed by centrifugation for 5 min in an Eppendorf Centrifuge 5414. The EG activity in these fractions was determined as described above. /3-Galactosidase, employed as a cytoplasmic marker, was assayed by the method of Pardee et al. (1959). /3-Lactamase was used as a periplasmic marker and was assayed by the microiodometric method described by Ross and O'Callaghan (1975).

312

Time course of cellulase production in E. coli E. coli DH1 (pTC15) was grown using 1% inoculum in l-liter volumes of M9 medium supplemented as previously described. The culture was shaken at 37°C to stationary phase. Forty ml samples were also removed at intervals for assay of total EG activity. The E. coli cells were harvested by centrifugation at 5000 g at 4°C for 10 min. The pellet was suspended in a small volume of supernatant and sonicated as described above. Cell debris was removed by centrifugation. Extracellular fluids were concentrated in stirred ceils on YM10 membranes (Amicon) and added to the cell-free extracts for determination of total EG activity. Determination of optimum p H and temperature The optimum temperature of the endoglucanase produced by E. coli DH1 (pTC15) was determined by performing the standard viscometry assay at temperatures ranging from 40°C to 80°C. The optimum pH was also determined using the standard assay. One percent CMC was dissolved in one of the following buffers: 0.1 M acetate (pH 4.0-6.0), 0.1 M 2(N-morpholino) ethanesulfonic acid (MES, pH 5.5-6.5), 0.1 M N-(2-hydroxy-ethyl) piperazine-N'-(2-ethanesulfonic acid) (HEPES, pH 6.5-8.0), 0.1 M N-(2-hydroxyl,l-bis-(hydroxymethyl)ethyl amino-l-propanesulfonic acid (TAPS, pH 8.0-9.0), or 0.1 M 2-N-cyclohexylamino]ethane-sulfonic acid (CHES, pH 8.0, 9.5). Antibody inhibition of enzyme activity Anti-serum diluted 1 : 10 was used to inhibit enzyme activity of DH1 (pTC15) endoglucanase and extracts from B. subtilis transformants. Diluted antiserum was mixed with enzyme samples at ratios of 1:12. Identical enzyme samples were added at same ratios to pre-immune rabbit serum. After incubation at 37°C for 1 h, viscosity reduction assays were performed on the serum-enzyme mixtures.

Results

Restriction mapping of pTC15 Restriction mapping of pTC15 was performed with the following enzymes: BamHI, Bcll, BglII, EcoRI, EcoRV, HindIII, KpnI, PstI, PvuII, SphI, XbaI, and XhoI (Fig. 2). No sites were found in pTC15 for the enzymes BclI, BglII, XbaI, and HindIII (the 378 bp fragment of pLP1202, contained between the EcoRI and BamHI sites was removed during the subcloning process). Time course and location of EG production The time course of EG production during growth of E. coli DH1 (pTC15) is shown in Fig. 3. The location and activity of endoglucanase activity contained in intracellular, periplasmic and extracellular extracts of exponential phase E. coli DH1 (pTC15) are shown in Table 2. Most of the EG was secreted into the periplasmic space; about 1 / 4 of the total activity was found in culture fluid.

313

0

1

2

3

4

5

6

7

8

9

I0

II

12

13

i

I

i

I

i

i

i

I

i

I

I

i

I

i

Kilobases Fig. 2. Restriction map of pTC15. T. curvata DNA (

; pHC79 DNA ( ~ ) .

5O

10

40

o 0-

' 40

.01 0

10

20

Time

30

2O

Q"

10

g (5 U.l

0 50

(h)

Fig. 3. Production of endoglucanase (e) by E. coli DH1 (pTC15) during growth (©; OD550).

Determination o f optimum p H and temperature T h e effects of t e m p e r a t u r e o n the activity of E G o b t a i n e d from E. coil D H 1 (pTC15) a n d t h a t of c r u d e extracellular T. curvata e n d o g l u c a n a s e were c o m p a r e d . M a x i m a l activities u n d e r s t a n d a r d i n c u b a t i o n c o n d i t i o n s were observed at 70°C a n d 65°C, respectively (Fig. 4). A n o p t i m u m p H for E. coli D H 1 (pTC15) E G

TABLE 2 Localization of endoglucanase activity in E. coli DH1 (pTC15) Location Culture supernatant Shock fluid Cell extract

Percent of total activity fl-Gal ~

fl-Lac b

EG

1.5 0.2 98.3

8.6 89.8 1.6

24.5 59.4 16.1

a fl-Gal = fl-Galactosidase (cytoplasmic enzyme marker). = fl-Lactamase (periplasmic enzyme marker).

b ~-Lac

314

100 >

80

(3

-~ E

60 40 20 ,

30

~

40

•

J

50

,

J

,

60

J

,

70

Temperature

.

.

80

90

(°C)

Fig. 4. Optimum temperature for activity of E. coli DH1 (pTC15) endoglucanase (o) compared to T. curuata crude extracellular endoglucanase (e).

activity was determined to be pH 8.0-8.5 (Fig. 5). This optimum differed significantly from the optimum (pH 6.0-6.2) of T. curvata crude endoglucanase. SDS-PAGE and zymograms E. coli DH1 (pTC15) periplasmic extracts and T. curvata extracellular proteins were electrophoresed on 10% SDS-PAGE gels. After zymogram overlay, three major activity bands with mobilities corresponding to M r values of approx 82 K, 50 K and 36 kDa plus at least three minor bands, were obtained from periplasmic extracts (Fig. 6). Transformation of pTC15 in B. subtilis PSL1 The 46 Cm r transformants were scored on LB agar plates supplemented with 5 Izg ml - t chloramphenicol. Since B. subtilis PSL1 possessed some natural endoglucanase activity, colonies with increased z o n e s were recorded. However, none of the transformants produced zones larger than B. subtilis PSL1 controls. In order to determine if PSL1 transformants harbored recombinant plasmids containing functional cellulase genes, plasmids were extracted from ten random 120 >.

100

>_

80

~ 6o ~

40 20 4

5

6 pH

7

8

9

10

Fig. 5. The effect of pH on the activity for activity Of T. curvata cellulase and E. coli DH1 (pTC15) endoglucanase. T. curvata cellulase in buffers (0.1 M): acetate (+), and MES (A); and E. coli endoglucanase in buffers (0.1 M): acetate (o), MES ([]), HEPES (e), TAPS (,x), and CHES (I1).

315

1

2

97400 66000

1 ®iii

45000

29000

Fig. 6. Cellulase zymogram performed at pH 8.0. Lane 1 = T. curvata extracellular fluid. Lane 2 = E. coli DH1 (pTC15) concentrated shock fluid. Molecular weights are shown to the left for comparison. Arrows at right indicate the major active bands.

transformants. Plasmid D N A from these strains were electrophoresed on 0.7% agarose gels. Deletions occurred in most of the transformants (Fig. 7). Three of the larger plasmids (from Bacillus transformants 13, 17, and 110) were used to transform competent E. coli D H 1 cells. Ten transformants from each transformation (as well as 10 colonies of D H l [ p T C 1 5 ] from a fresh streak plate) were picked and scored on L B - A m p plates containing 0.1% CMC. After incubation at 37°C overnight and further incubation overnight at 60°C, the colonies were screened by the Congo R e d plate assay. Results obtained by isolating plasmid D N A from the B. subtilis transformants and transforming E. coli with the same plasmids indicated that some of the Bacillus transformants contained the functional T. curvata cellulase gene (Table 3). Antibody inhibition of endoglucanase activity was determined as a means to detect E G activity of products from a T. curvata cellulase gene in PSL1 transformants (Table 4). While T. curvata and E. coli D H 1 (pTC15) E G activities were d e a r l y inhibited by antiserum against T. curvata cellulase, no specific reduction in E G activity from B. subtilis P S l l ( p B T l l 0 ) was observed.

316

1

3

6

9

12

Fig. 7. Gel electrophoresis of plasmids (undigested) isolated from B. subtilis PSLI transformants. L, ne 1 = Isolate No. 110 (pBTll0). Lane 2 = Isolate No. 19. Lane 3 = Isolate No. 18. Lane 4 = Isolate No. 17. Lane 5 = Isolate No. 16. Lane 6 = Isolate No. 15. Lane 7 = Isolate No. 14. Lane 8 = Isolate No. 13. Lane 9 = Isolate No. 12. Lane 10 = Isolate No. 11. Lane 11 = pTC15. Lane 12 = pLP1202.

TABLE 3 Detection of functional endoglucanase genes from plasmids isolated from 13. subtilis PSL1 isolates Source of plasmid D N A

Percent of E. coli transformants showing CMC + phenotype

PSL1 isolate 13 PSL1 isolate 17 PSL1 isolate 110 E. coil DH1 (pTC15)

0 90 100 100

TABLE 4 Inhibition of EG activity by antiserum prepared against crude extracellular T. curvata EG EG activity (% of unihibited control)

T. curuata DH1 (pTC15) PSI1 (pBT110)

Pre-immune serum

Anti-EG serum

118 106 56

10 13 63

317 Discussion

The levels of expression of T. curvata cellulase genes were similar to those achieved in E. coli with cellulase genes-from other actinomycetes (Collmer and Wilson, 1983; Hu and Wilson, 1988; Yablonsky et aI., 1988). The ceUulase plasmid pTC15, a subclone of pTC520, on E. coli-B, subtilis shuttle plasmid pLP1202, was able to transform B. subtilis PSL1 by electroporation. Although most PSL1 transformants suffered deletions, some of the transformants retained the intact cellulase gene, as determined by retransforming E. coli DH1. However, no activity was detected above that already present in PSL1. Concentrated PSL1 (pBTll0) extracellular cellulase was not specifically inhibited by antiserum as was T. curvata cellulase and E. coli (pTC15) cellulase (although even normal rabbit serum inhibited the B. subtilis cellulase). It is not known if the apparent lack of expression in B. subtilis is due to transcriptional effects, translational effects or instability of the proteins in B. subtilis, but similar results were obtained by Ghangas and Wilson (1987) when the T. fusca E 5 cellulase gene was cloned into B. subtilis. A high background of deletions among the Bacillus transformants was also observed, and in clones containing intact genes, no expression could be detected. This evidence suggests that B. subtilis PSL1 is not a suitable host for the cloning of Thermomonospora genes. The expression of the cellulase gene on pTC15 was studied in more detail in E. coli. This gene did not hybridize to the pD318 (E 5) cellulase gene of T. fusca (Presutti et al., 1991) but its restriction map appeared strikingly similar to the restriction map of the E 2 cellulase gene of T. fusca (Ghangas and Wilson, 1988; Lao et al., 1991). We have not yet determined if these two genes show homology to each other by hybridization. The recombinant cellulase did react specifically with antiserum prepared against crude T. curvata cellulasel as shown by inhibition of enzyme activity. T. curvata cellulase and E. coli cellulase having equivalent activities were inhibited to an equal extent by the same antiserum. Almost 85% Of the EG was secreted by E. coli. Most of the activity was found in the periplasmic space. These results are similar to those of Collmer and Wilson (1983) for T. fusca EG genes, particularly that of recombinant strain D334 which contained 52% of the activity in the periplasm and 20% extracellularly. It is common for heterologous proteins containing signal sequences to be transported to the periplasm in E. coli (Emr et al., 1980). Thus the results here were consistent with those from other cellulase genes cloned in E. coil from Fibrobacter succinogenes cel-3 gene (McGavin et al., 1989), and from alkalophilic Bacillus species (Fukumori et al., 1986; Sashihara et al., 1984). Cloned cellulases are rarely secreted largely into the culture medium by E. coli except when encoded by genes from Bacillus species (Sharma et al., 1987; Ix) et al., 1988). In spite of the similarity of the restriction maps of pTC15 and the E 2 gene of T. fusca, the pH optima, temperature optima, and apparent molecular weights of the enzymes are quite different. The temperature optimum of 70°C for EG from E. coli (pTC15) was similar to that of purified E 1 from T. fusca (Calza et al., 1985). However, a rather large difference was observed between pH optima (pH 8.0-8.5 in this study

318

versus 6.1 for purified E 1 and E 2 from T. fusca and the crude EG from T. curvata culture fluid) This may be due to differences in post-translational processing in the host bacterium (however, it is interesting that xylanases, proteases and pectinases from T. curvata have similar high pH optima). It is possible that these apparent differences may be generated, at least in part, by multiple cellulase genes present on the 6 kb segment of T. curvata DNA in pTC15. Alternatively, since there is much similarity between the banding patterns formed by T. curvata cellulase and E. coli (pTC15) cellulase, it is possible that the pTC15 cellulase is proteolytically processed in E. coli as in T. curvata and that the digestion products retain at least a portion of their activity. Calza et al. (1985) found that almost one-third of the T. fusca E1 endoglucanase (M r = 94000) could be proteolytically removed with no subsequent reduction of activity, while Ghangas and Wilson (1988) reported that the E 2 cellulase of T. fusca was proteolytically processed into smaller fragments with altered kinetic characteristics.

References Balk, B.-H. and Pack, M.Y. (1991) Expression of Bacillus subtilis endoglucanase gene in Lactobacillus acidophilus. Biotechnol. Lett. 12, 919-924. Beguin, P. (1983) Detection of cellulase activity in polyacrylamide gels using congo red-stained agar replicas. Anal. Biochem. 131, 333-336. Beguin, P., Millet, J. and Aubert, J.P. (1987) The cloned cel (cellulose degradation) genes of Clostridium thermocellum and their products. Microbiol. Sci. 4, 277-280. Bernier, R., Kopp, M., Trakas, B. and Stutzenberger, F. (1988) Production of extracellular enzymes by Thermomonospora curvata during growth on protein-extracted lucerne fibres. J. Appi. Bacteriol. 65, 411-418. Birnboim, H.C. and Doly, J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acids Res. 7, 1513-1523. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. Calza, R.E., Irwin, D.C. and Wilson, D.B. (1985) Purification and characterization of two/3-1,4-endoglucanases from Thermomonospora fusca. Biochemistry 24, 7797-7804. Clegg, S. and Allen, B.L. (1985) Molecular cloning and expression of an extracellular nuclease of Serratia marcescens in Escherichia coli. FEMS Microbiol. Lett. 27, 257-262. Collmer, A. and Wilson, D.B. (1983) Expression of a Thermomonospora Y X endocellulase gene in E. coli. Bio/Technology 1, 594-601. Cornelis, P., Digneffe, C. and Willemot, K. (1982) Cloning and expression of a Bacillus coagulans amylase gene in Escherichia coli. Mol. Gen. Genet. 186, 507-511. Emr, S.D., Hall, M.N. and Silhavy, T.J. (1980) A mechanism of protein localization: the signal hypothesis and bacteria. J. Cell Biol. 86, 701-711. Eriksson, K.E. (1979) Biosynthesis of polysaccharases. In: R.C.W. Berkeley, G.W. Gooday and D.C. Ellwood (Eds.), Microbial Polysaccharides and Polysaccharases. Academic Press, London, pp. 285-296. Fennington, G., Lupo, D. and Stutzenberger, F. (1982) Enhanced cellulase production in mutants of Thermomonospora curvata. Biotechnol. Bioeng. 24, 2487-2497. Fukumori, F., Kudo, T., Narahashi, Y. and Horikoshi, K. (1986) Molecular cloning and nucleotide sequence of the alkaline cellulase gene from the alkalophilic Bacillus sp. Strain 1139. J. Gen. Microbiol. 132, 2329-2335.

319 Ghangas, G.S. and Wilson, D.B. (1987) Expression of a Thermomonospora fusca ceUulase gene Streptomyces lividans and Bacillus subtilis. Appl. Environ. Microbiol. 53, 1470-1475. Ghangas, G.S. and Wilson, D.B. (1988) Cloning of the Thermomonospora fusca endoglucanase E 2 gene in Streptomyces lividans: affinity purification and functional domains of the cloned gene product. Appl. Environ. Microbiol. 54, 2521-2526. Glymph, J.L. and Stutzenberger, F.J. (1977) Production, purification and characterization of a-amylase from Thermomonospora curvata. Appl. Environ. Microbiol. 34, 391-397. Hanahan, D. (1983) Studies on transformation in Escherichia coli with plasmids. J. Moi. Biol. 166, 557-580. Holmes, D.S. and Quigley, M. (1981) A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 14, 193-197. Hu, Y.-J and Wilson, D.B. (1988) Cloning of Thermonomospora fusca genes coding for beta 1-4 endoglucanases El, E 2 and E 5. Gene 71, 331-337. Kohchi, C. and Toh-e, A. (1985) Nucleotide sequence of Candida pelliculosa/3-glucosidase gene. Nucl. Acids Res. 13, 6273-6282. Koide, Y., Nakamura, A., Uozumi, T. and Beppu, T. (1986) Molecular cloning of a cellulase gene from Bacillus subtilis and its expression in Escherichia coli. Agric. Biol. Chem. 50, 233-237. Lao, G., Ghangas, G.S., Jung, E.D. and Wilson, D.B. (1991) DNA sequences of three /3-1,4-endoglucanase genes from Thermomonospora fusca. J. Bacteriol. 173, 3397-3407. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Lo, A.C., MacKay, R.M., Seligy, V.L. and Willick, G.E. (1988) Bacillus subtilis fl-l,4-endoglucanase products from intact and truncated genes are secreted into the extracellular medium by Escherichia coli. Appl. Environ. Microbiol. 54, 2287-2292. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY. Margaritis, A. and Merchant, R.F.J. (1986) Thermostable cellulases from thermophilic microorganisms. CRC Crit. Rev. Biotechnol. 4, 327-367. McCarthy, A.J. and Cross, T. (1984) A taxonomic study of Thermomonospora and other monosporic Actinomycetes. J. Gen. Microbiol. 130, 5-25. McGavin, M.J., Forsberg, C.W., Crosby, B., Bell, A.W., Dignard, D. and Thomas, D.Y. (1989) Structure of the cel-3 gene from Fibrobacter succinogenes $85 and characteristics of the encoded gene product, endoglucanase 3. J. Bacteriol. 171, 5587-5595. Misawa, N., Tomoyuki, O. and Nakamura, K. (1988) Expression of a cellulase gene in Zymomonas mobilis. J. Biotechnol. 7, 167-178. Moser, B., Gilkes, N.R., Kilburn, D.G., Warren, R.A.J. and Miller R.C., Jr. (1989) Purification and characterization of endoglucanase C of Cellulomonas fimi, cloning of the gene and analysis of in vivo transcripts of the gene. Appl. Environ. Microbiol. 55, 2480-2487. Ostroff, G.R. and Pene, J.J. (1984) Molecular cloning with bifunctional plasmid vectors in Bacillus subtilis. II. Transfer of sequences propagated in Escherichia coli to B. subtilis. Mol. Gen. Genet 193, 306-311. Pardee, A.B., Jacob, F. and Monod, J. (1959) The genetic control and cytoplasmic expression of 'inducibility' in the synthesis of ~-galactosidase by E. coli. J. Mol. Biol. 1, 165-178. Presutti, D.G., Hughes, T.A. and Stutzenberger, F.J. (1991) Cloning of three endoglucanase genes from Thermomonospora curvata into Escherichia coli. J. Biotechnol. 17, 177-188. Robson L.M. and Chambliss, G.H. (1986) Cloning of the Bacillus subtilis DLG/3-1,4-glucanase gene and its expression in Escherichia coli and B. subtilis. J. Bacteriol. 165, 612-619. Ross, G.W. and O'Callaghan, C.H. (1975) fl-Lactamase assays. Methods Enzymol. 43, 69-85. Sacco, M., Millet, J. and Aubert, J.P. (1984) Cloning and expression in Saccharornyces cerevisiae of a cellulase gene from Clostridium thermocellum. Ann. Microbiol. 135A, 485-488. Sashihara, N., Toshiaki, IC and Horikoshi, K. (1984) Molecular cloning and expression of cellulase genes of aikalophilic Bacillus sp. Strain N-4 in Escherichia coli. J. Bacteriol. 158, 503-506. Scheirlinck, T., Mahillon, J., Joos, H., Dhaese, P. and Michiels, F. (1989) Integration and expression of a-amylase and endoglucanase genes in the Lactobacillus plantarum chromosome. Appl. Environ. Microbiol. 55, 2130- 2137.

320 Sharma, P., Gupta, J.K., Vadehra, D.V. and Dube, D.K. (1987) Molecular cloning and expression in Escherichia coli of a thermophilic Bacillus sp. PDV endo-/3-1,4-glucanase gene. Enzyme Microb. Technol. 9, 602-606. Skipper, N., Sutherland, M., Davies, R.W., Kilburn, D., Miller, R.C., Jr., Warren, A. and Wong, R. (1985) Secretion of a bacterial cellulase by yeast. Science 230, 958-961. Stutzenberger, F. (1971) Cellulase production by Thermomonospora curvata isolated from municipal solid waste compost. Appl. Microbiol. 22, 147-152. Stutzenberger, F.J. (1972) Cellulolytic activity of Thermomonospora curvata: Optimal assay conditions, partial purification, and product of the cellulase. Appl. Microbiol. 24, 83-90. Stutzenberger, F. (1987) Inducible thermoalkalophilic polygalacturonate lyase from Thermomonospora fusca. J Bacteriol. 169, 2774-2780. Stutzenberger, F.J. and Carnell, R. (1977) Amylase production by Thermomonospora curvata. Appl. Environ. Microbiol. 34, 234-236. Stutzenberger, F.J., Kaufman, A.J. and Lossin, R.D. (1970) Celluiolytic activity in municipal solid waste composting. Can. J. Microbiol. 16, 553-560. Teather, R.M. and Wood, P.J. (1982) Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl. Environ. Microbiol. 43, 777-780. Whittle, D.J., Kilburn, D.G., Warren, R.A.J. and Miller, R.C., Jr. (1982) Molecular cloning of a Cellulomonas cellulase gene in Escherichia coli. Gene 17, 139-145. Wilkes, C.R., Yang, R.D., Sciamanna, A.F. and Freitas, R.P. (1981) Raw materials evaluation and process development studies for conversion of biomass to sugars and ethanol. Biotechnol. Bioeng. 23, 163-183. Yablonsky, M.D., Bartley, T., Elliston, K.O., Kahrs, S.K., Shalita, Z.P. and Eveleigh, D.E. (1988) Characterization and cloning of the cellulase complex of Microbispora bispora. In: J.P. Aubert, P. Beguin and J. Millet (Eds.), Biochemistry and Genetics of Cellulose Degradation, Academic Press, New York.