Purification and characterization of two low-molecular weight endoglucanases of Cellulomonas flavigena

Purification and characterization of two low-molecular weight endoglucanases of Cellulomonas flavigena Amtul Jamil Sami and M. Waheed Akhtar Section o f Biochemistry, Institute o f Chemistry, University o f Punjab, Lahore, Pakistan

Two isoforms of endoglucanases ofCellulomonas llavigena were purified to homogeneity after a series of purification steps including precipitation with 80% acetone, gel filtration, ion-exchange chromatography, and nondenaturing gradient 5-20% preparative polyacrylamide gel electrophoresis. Each of the endoglucanases, CM-Cellulase I and CM-Cellulase 2, appeared as a single band on SDS-PAGE and had an apparent molecular weight of 20,400. Both of the endoglucanases were comparable to the substrate-bound endoglucanases and the recombinant DNA-derived endoglucanases of C. flavigena expressed in Escherichia coli. The two isoforms of endoglucanase, CM-Cellulase 1 and CM-Cellulase 2, were different in their mobilities on native nondenaturing 5-20% gradient PAGE. The purified endoglucanases displayed significantly similar characteristics, including pH optima (6.5 and 7) and temperature optima (50°C). The purified CM-Celhdases were unable to hydrolyze Avicel, xylan, and filter paper and were only active against CMC. The purified proteins had a K,,, value of O.78 g I t Heavy metal ions like Ag 2+ and Fe 2" inactivated the enzymes. NaCI and CaCI2 had no effect on enzyme activities. [3-Mercaptoethanol also inhibited enzyme activity, possibly due to the involvement of sulfhydryl groups.

Keywords: Endoglucanase; Celhdomonasflavigena; carboxymethylcellulase; carboxymethylcellulose: polyacrylamide gel electrophoresis;CM-Cellulase

Introduction Cellulose is the main structural constituent of plant cell walls and consists of unbranched polymers of glucose. This polymer can be hydrolyzed into glucose units by the actions of ceilulases. Due to the complex nature of the cellulose molecule, a group of enzymes acting synergistically with different binding sites is required. Cellulase enzymes are produced as a system by a number of microbes, including yeast, fungi, and bacteria. Among the bacterial strains, Cellulomonas is considered one of the potent strains for the production of an efficient system of cellulases for the hydrolysis of cellulose into glucose. Workers have reported the production of cellulases by different strains of Celhdom o n a s on natural and synthetic carbon sources.l-4

Address reprint requests to Dr. Samiat the Departmentof Biochemistry and Chemistry, Royal Free Hospital, Schoolof Medicine, University of London, Hampstead, London NW3 2PF, UK Received 29 July 1992; revised 4 November 1992

586

Enzyme Microb. Technol., 1993, vol. 15, July

Cellulomonas, like other microbes, produces multiple forms of cellulases when grown on cellulosic substrates. 4-6 This multiplicity is primarily genetic in origin and secondarily physiological. 4-~ These enzymes could be the different isoforms of the cellulase activity. Langsford et al. s have reported the production of at least I0 different endoglucanases (EC 3.2.1.4) in the culture supernatant of C. fimi. Previously, we have reported the appearance of at least seven active fractions in the culture supernatants of C. flavigena at different fermentation periods.4 Thus, identification of the native forms of the enzymes is required before purification and characterization, which could be helpful in elucidating the mode of action of cellulases on the cellulose. There are reports on the purification of more than one endoglucanase from Cellulomonas sp.12-14 The enzymes purified and characterized are of high molecular weight and are not considered as isoforms. These endoglucanases had great affinity for insoluble cellulosic substrates and Sephadex. Here we report the purification and characterization of two isoforms of endoglucanases of C. flavigena and

~ 1993 Butterworth-Heinemann

Endoglucanases of Cellulomonas flavigena: A. J. Sami and M. W. Akhtar their expression in Escherichia coli which had great affinity for crystalline cellulose and Sephadex.

Materials and methods

Microorganism and culture methods Cellulomonas flavigena was isolated locally and was identified by the National Collection of Industrial and Marine Bacteria Ltd., Aberdeen, Scotland, as described earlier. 4 The bacterium was cultured according to the method described previously: For the production of extracellular cellulases, C. flavigena was grown in i-1 Erlenmeyer flasks containing 0.5% crystalline cellulose (Avicel) as a carbon source, 0.2% yeast extract, and 0.1% Tween-80 in a salt medium at 30°C for 72 h. 4 After specific hours of fermentation, the culture broth was centrifuged at 10,000g for 30 rain at 4°C in a Beckman Model J2-21 to remove the bacterial cells and residual crystalline cellulose. The supernatant obtained was used as an enzyme source. For the isolation of the substrate-bound cellulases of C. flavigena after specific hours of fermentation, the residual Avicel was removed by centrifugation at 1000g for 5 rain at 4°C. The pellet was suspended in a volume of distilled water that was double the volume of the original culture broth. Avicel was then subjected to sonication at amplitude 20 in an MSE sonicator three times, 20 s each. The supernatant was removed after centrifugation at 6000g for 10 min at 4°C. Avicel was washed three times similarly. The supernatant obtained was used as a source for the substrate-bound enzymes. An Escherichia coli clone carrying plasmid pMW1149 for the expression of DNA-derived recombinant endoglucanase of C. flavigena was grown in 100 mi of culture containing 1 g Trypton (Sigma), 1 g yeast extract, 0.5 g NaCI, 20 tzg ampicillin ml-~, and isopropyl-/3-thio-galactosidase 20/zg ml-I in a 500-ml conical flask. The microbe was grown for 22 h at 37°C. The bacterial pellet was obtained by centrifugation at 10,000g in a J2-21 Beckman centrifuge for 15 min at 4°C and was suspended in 5 ml 20 mM Tris-HCi buffer, pH 8.3. The bacterial cell wall was broken down by sonication at amplitude 20 in an MSE sonicator three times, 20 s each. The supernatant was removed after centrifugation (3,000g for 5 rain), and the cells were washed twice. This supernatant was used as a source of the recombinant DNA-derived endoglucanases.

Protein estimation Proteins were estimated by the dye-binding method with a slight modification of the original method, reported by Bradford. 15 The method was standardized for estimating diluted protein solutions ranging from 1 to 15 p.g of protein in 1 ml of sample. Reagent was prepared as follows: 450 mg of Coomassie brilliant blue G-250 (Shandon) was dissolved in 50 ml of absolute ethanol and 100 ml of orthophosphoric acid (BDH). The volume was made up to 1 1 with distilled water. The reagent was filtered twice using Whatman filter paper No. 3 to remove any insoluble dye. For estimat-

ing the proteins, I ml reagent was mixed with 1 ml protein solution prepared in 20 mM Tris-HC! buffer pH 8.0. After 5 min, absorbance was read at 595 nm against the reagent blank containing I ml appropriate buffer. Bovine serum albumin (Sigma) was used as a standard protein for plotting a standard curve. After ion-exchange chromatography, proteins were also determined by taking absorbance at 280 nm on a spectrophotometer.

Enzyme assays Endoglucanases (CM-Cellulase) and exoglucanase assays were carried out according to the method described previously. 4

Purification procedures The supernatant broth obtained as above (see microorganism and culture methods), after 72 h of fermentation, was concentrated with 80% acetone as follows: 200 ml culture supernatant was mixed with 800 ml chilled acetone (kept in the freezer at -20°C for 2 h) and incubated overnight at -20°C. The pellet was obtained after centrifugation at 10,000g for 30 min at 4°C. The pellet was resuspended in 20 ml double distilled water and lyophilized. Thus the powder obtained was stored at -20°C until further use. Two hundred milligrams of this crude enzyme preparation was dissolved in 20 ml 20 mM Tris-HCI buffer pH 8.3 and loaded onto a Sephacryl Superfine-300 (Pharmacia) column (1.6 × 44 cm). The column was previously equilibrated with the same buffer. Proteins were eluted at a flow rate of 0.5 ml min -~ at 4°C. A total of 100 fractions was collected, 3 ml each. Endoglucanase enzyme activity and protein concentrations were analyzed in all the fractions. The fractions showing enzyme activity were pooled together and loaded onto an ion-exchange column (1.6 x 20 cm) packed with DEAE-Sephadex in a 20 mM Tris-HCI buffer pH 8.3. Proteins were eluted at 4°C with a linear salt gradient ranging from 0.05 to 0.750 M NaCI. Before introducing, the salt gradient column was washed with 2 column volumes of buffer to elute any loosely bound proteins. Elution was carried out at a flow rate of 20 ml h-J. A total of 150 fractions was collected; fraction size was 3 ml each. All the fractions were tested for protein concentration by taking the absorbance at 280 nm. Fractions were also analyzed for endoglucanase activity. The fractions showing enzyme activity were further purified on preparative nondenaturing gradient polyacrylamide gel electrophoresis (PAGE 5-20%) at 4°C. Gradient gel was prepared by gradually mixing two solutions containing 5 and 20% acrylamide in a gradient mixer (Pharmacia Fine Chemicals, Uppsala, Sweden). The gel cassette was filled with the solution gradually from bottom to top. The composition of the 5% acrylamide solution was 4.7 g acrylamide (Sigma), 0.3 g bisacrylamide (Sigma), 50 p.1 N,N,N',N'-tetramethylene diamine (Sigma), 15 mg ammonium persulfate, and 0.9 g sucrose (Sigma) in 100 ml 0.05 M Tris-borate-

Enzyme Microb. Technol., 1993, vol. 15, July

587

Papers EDTA buffer, pH 8.5. The composition of the 20% acrylamide solution was the same, except that it contained 18.6 g acrylamide, 1.3 g bisacrylamide, and 3.6 g sucrose per 100 ml. A 3-mm-thick gel was used for preparative PAGE according to the method described previously/ After the electrophoretic run, proteins were eluted from the gel as follows: The gel was sliced into 0.3-mm slices with a sharp razor. Each slice was then crushed by passing through a 5-ml disposable syringe with 3 ml of distilled water under pressure. The gel matrix was further broken down by freezing it at -20°C, and then the proteins were allowed to elute via diffusion overnight at 4°C. Each fraction was analyzed for protein concentration and enzyme activity. Each purification step was monitored by running the samples on a non-denaturing 5-20% gradient PAGE and then locating the endoglucanase activity on a carboxymethyl cellulose (CMC)-agar plate using the zymogram technique as described previously. 4

Molecular weight determination The molecular weights of the purified proteins were determined according to the method of Laemmli. 16The molecular weight markers used were supplied by Pharmacia Fine Chemicals, Uppsala, Sweden.

Paper chromatography The hydrolytic products of the enzymes were identified by paper chromatography on Whatman filter paper No. I as follows: 100 ml of the enzyme solution along with 1 ml of substrate buffer (1.5% CMC 100 mM citrate buffer, pH 6.5) was incubated at 50°C for 24 h. Ten microliters of the hydrolysate was applied on the chromatographic paper. One microgram of reference sugars (glucose and cellobiose) in a volume of 10 tzl was also applied. The chromatogram was run for 20 h at room temperature. The solvent system was composed of Nbutanol : pyridine : water (6 : 4 : 3 v/v). Products were identified by dipping the chromatogram into AgNO3 prepared as follows: 0. I ml of saturated aqueous solution was diluted to 20 ml with acetone, and water was added dropwise until AgNO 3 redissolved. The chromatogram was dried and sprayed with 0.05 M NaOH in 50% ethanol. Carbohydrates appeared immediately as black spots against a light brown background.

Results Two hundred milligrams of powdered crude preparation was obtained after precipitating the 72-h culture broth with 80% acetone, as described in Materials and methods. This preparation was passed through a Sephacryl S-300 column. The cellulase system of C. flavigena was eluted in one peak (Figure 1). Proteins were further purified by ion-exchange chromatography using DEAE-Sephadex on a salt gradient, as described in Materials and methods. The results are shown in Figures 2 and 3. The chromatogram shows the presence of three main peaks for endoglucanase activity: DI, D2, and D3. Fraction D I (7% of the eluted activity)

588

Enzyme Microb. Technol., 1993, vol. 15, July

0.6

EO.4 o

< 0.2

O

20

40

Fraction

60

NO.

Figure 1 Gel filtration of acetone-precipitated crude preparation of broth soluble extracellular cellulases on Sephacryl S-300 (1.6 x 40 cm). Proteins were eluted at a flow rate of 0.5 ml rain -1 in a 20 mM Tris-HCI buffer pH 8.3. (11) Endoglucanase; (-I) exoglucanase; (A) A~0

was eluted with 0.05 M NaCI; D2 (37% of the eluted activity) was eluted with salt concentration of 0.075-0.25 M NaCI; D3 (57% of the eluted activity) was eluted with a higher salt concentration, 0.5-0.7 M NaCI. Analysis o f D l , D2, and D3 on 5-20% nondenaturing gradient PAGE shows that all the fractions were composed of more than one band (Figure 2, lanes c-eL DI was composed of two bands. D2 showed four active bands active against CMC on a zymogram. Fraction D3 was composed of two bands which move very close to each other on 5-20% gradient PAGE. These two bands also coincided with the two fast-moving fractions of the substrate-bound enzymes of C.flavigena at early stages of fermentation (Figure 4, lane a). Three hundred micrograms of fraction D3 was further purified on 5-20% gradient PAGE. After the electrophoretic run, proteins were eluted from the gel as described in Materials and methods. The results are shown in Figure 5. Two peaks were obtained for endoglucanase activity. Peak 1 contained 30% of the total eluted activity, while peak 2 formed 70% of the total eluted activity. The results showed that there were two endoglucanases and they appeared as single bands on the zymogram (Figure 4, lanes d and e). After all the purification steps, the purification factor of carboxymethyl (CM)Cellulase I increased fivefold, and that of CM-Cellulase 2 increased sixfold (for summary, see Table 1). The purified enzymes were compared with the recombinant DNA-derived endoglucanases of C. flavigena expressed in E. coli. As shown in Figure 4, lane f plasmid pMW expressed four different endoglucanases; two of them were comparable to the CM-Cellulase 1 and CMCellulase 2. SDS-PAGE (10% gel) of the purified CMCellulases showed that each protein was a single polypeptide with apparent molecular weight of 20,400

Endoglucanases of C e l l u l o m o n a s f l a v i g e n a : A. J. Sami and M. W. Akhtar

f e

d

c

b

a

e

d

c

b

a

Figure 4 Location of endoglucanase activity after 5-20% nonde-

Figure 2 Analysis of CM-Cellulase activity on 5-20% gradient PAGE, After electrophoretic run, gradient gel was incubated for 20 min on an agar plate at 50°C and then stained with 0,2% (w/v) Congo red, Excess dye was washed with 1 M NaCI. (a) Acetoneprecipitated sample; (b) sample after gel filtration; (c,d,e) fractions after ion-exchange chromatography: (c) D1 fraction; (d) D2 fraction; (e) D3 fraction

naturing gradient PAGE for 17 h at 4°C, on 0.05% CMC (w/v) and 2% agar. The zymogram was incubated for 40 min at 50°C. Remaining text as for Figure 2. (a) Substrate-bound endoglucanase activity after 24 h of fermentation; (b) free endoglucanase activity after 72 h of fermentation; (c) D3 fraction after ionexchange chromatography; (d) CM-Cellulase 1; (e) CM-Cellulase 2; (f) recombinant DNA-derived endoglucanases. Lanes d and e after purification of preparative gradient PAGE

0-8

--0.4

E

D2

\

°,

¢o

A

0.1.

%.'"'"" i

03

30 ~ --~

0.2

20<

0-4 z

00.2

=E

,, I

0

40 Fraction

80

% ~ 0.1

10

120

No.

10

20

Fraction

Figure 3 Ion-exchange chromatography on a DEAE-Sephadex

A-50 column (1.6 x 30) of endoglucanase activity of C.flavigena, obtained after gel filtration. Proteins were eluted at a flow rate of 0.2 ml rain -1 at 4°C with a linear salt gradient in 25 mM TrisHCI buffer pH 8.3. (11) Endoglucanase; (---) i NaCI; (©) A=B0

30

~0

50

No-

Figure 5 Purification of D3 fraction on nondenaturing preparative gradient 5-20% PAGE. Proteins were electrophoresed for 17 h at 4°C with a current supply of 2 mA cm -1 of the gel. ( t ) Endoglucanase activity; (--) PAGE gradient; (r-I) Aza0

E n z y m e M i c r o b . T e c h n o l . , 1993, v o l . 15, J u l y

589

Papers Table 1 Steps involved in the purification of CM-Cellulase 1 and CM-Cellulase 2 of C. flavigena Purification step

U endoglucanase

1. 72 h broth 2. Precipitation with 80% acetone 3. Gel filtration on Sephacryl column 4. Ion-exchange chromatography on DEAE-Sephadex A-50 D3 fraction 5. Preparative gradient PAGE 5-20% CM-Cellulase 1 CM-Cellulase 2

Protein (rag)

Purification factor

10,000 9,000

100 75

-1.2

7,200

30

2.4

1,600

4

4.0

0.6 1.2

5.0 6.0

300 720

(Figure 6). Enzyme activity was not reconstituted after SDS-PAGE. The characteristics of the purified CM-Cellulase I and CM-Cellulase 2 were studied and compared. Both the enzymes were only active against CMC and were unable to hydrolyze Avicel, xylan, and filter paper. The hydrolytic products of both the enzymes were glucose, along with cellobiose and some oligosaccharides. CMCellulase 1 was most active at pH 6.5, while CM-CelIu-

lase 2 had a pH optimum of 7 (Figure 7). Each of the enzymes had optimum activity at a temperature of 50°C (Figure 8). Enzymes were inactivated by/3-mercaptoethanol (Figure 9). Heavy metal ions, such as Ag 2+ and Fe 2÷, also inhibited the enzyme activities (Figures 10 and 11). NaCI and CaCI z (Figure 12) had no effect on the enzyme activities. The kinetics of both the endoglucanases were studied, and it was found that the

5

96000

z,

E3

66OOO

2 1 I

0

=

Z~ 5

,

i

i

6 pH

7

8

Figure 7 Effect of pH on purified CM-Cellulase 1 and CM-Cellulase 2 of C. flavigena. (&) CM-Cellulase 1; (O) CM-Cellulase 2

2O4OO

14000

I

I

I

t

i

Z0 3O ~0 5O °6O a

b

c

Figure 6 Molecular weight determination of purified CM-Cellulase 1 and CM-Cellulase 2 on 10% SDS-PAGE. (a) Molecular weight markers; (b) CM-Cellulase 1; (c) CM-Cellulase 2

590

E n z y m e M i c r o b . T e c h n o l . , 1993, v o l . 15, J u l y

Temperature C

Figure 8 Effect of temperature on purified CM-Cellulase 1 and

CM-Cellulase 2 of C. flavigena. (&) CM-Cellulase 1; (©) CM-Cellulase 2

Endoglucanases of C e l l u l o m o n a s

f l a v i g e n a : A. J. S a m i and M. W. Akhtar

=Z

=2

1

1

3

6

0

9

7

lZ,

21

/JM fl.Mercopfoethonol

mM AgN0 3 Effect of AgNO3 on the activity of purified C M-Cellulase 1 and CM-Cellulase 2 of C. f/avigena. (&) CM-Celtulase 1; (©) CM-Cellulase 2 Figure 9

Figure 12 Effect of/3-mercaptoethanol on the activity of purified CM-Cellulase 1 and CM-Cellulase 2 of C. f/avigena. (&) CM-Cellulase 1; (©) CM-Cellulase 2

3

/4

8

12

mM FeSOt, Figure 10 Effect of FeS04 on the activity of purified CM-Cellulase 1 and CM-Cellulase 2 of C. flavigena. (&) CM-Cellulase 1; (©) CM-Cellulase 2

1

./ - 1.5

I

1-5

I

I

3.0

4.5

I

6-0

!

7"5

1IS Figure 13 Determination of kinetic constants of CM-Cellulase 1 and CM-Cellulase 2 of C. flavigena with CM-Cellulose (Sigma, medium viscosity) as substrate. (&) CM-Cellulase 1; (A) CMCellulase 2

3 =2 1

0

I

I

3

6

I

9

mH CaCI 2 Figure 11 Effect of CaCI2 on the activity of purified CM-Cellulase 1 and CM-Cellulase 2 of C. f/avigena. (&) CM-Cellulase 1; (©)

CM-Celtulase 2

Lineweaver plot of CM-Cellulase 1 was superimposable on the CM-Cellulase 2 plot. Both had a K m value of 0.78 g 1-1 (Figure 13). Discussion

Microbial cellulases are generally difficult to purify due to their very nature: They are reported to exist in com-

plex form, tend to aggregate with other proteins, and have some binding capacity for the gel matrices used in column chromatography. 4'9'17-~9 The two endoglucanases of C. flavigena reported here had some affinity for Sephadex, as well as for the crystalline cellulose, which was used as a carbon source in the fermentation (Figures 3 and 4, lane a). The two endoglucanases, CM-Ceilulase 1 and CM-Cellulase 2 of C. flavigena, displayed similar characteristics, e.g., molecular weight (20,400), optimum temperature (50°C), and K m value (0.78 g 1-I). The only considerable difference was mobility on 5-20% gradient PAGE (Figure 4, lanes a-f). Two forms of the CM-Cellulase activity were not the aggregated forms of the same enzyme, as in the case of Erwinia chrysanthemi CM-Cellulases and Rumin©coccus albus. 2°'21 The two purified Sephadex-binding endoglucanases of C. fimi reported by Moser E n z y m e M i c r o b . T e c h n o l . , 1993, v o l . 15, J u l y

591

Papers et al.t3 were higher-molecular-weight endoglucanases (120,000 and 130,000) and were closely related in their amino acid sequence at the C-terminal. ~2 In the case of C. fermentans endoglucanases, they had molecular weights of 40,000 and 57,000 and were considered as two different proteins.14 It is possible that CM-Cellulase 1 and CM-Cellulase 2 of C. flavigena are isoforms of the same endoglucanase activity. When two isoforms were expressed in E. coli, they appeared as two distinct bands on the zymogram (Figure 4, lane f) and were comparable to the wild-type endoglucanases of C. flavigena (broth-free or substrate-bound; Figure 4, lanes a,b,f). The endoglucanase activity produced in E. coli was intracellular, whereas in C.flavigena it was extracellular. Thus, postsecretional modification could be ruled out. It is possible that these two isoforms are the result of the expression of two very closely related genes, or perhaps a result of posttranslational modifications. Characterization of the endoglucanase genes of C. flavigena will be helpful to answer this question. Acknowledgements We thank the Pakistan Atomic Energy Commission for providing a fellowship to A.J.S., and the International Foundation for Science, Stockholm, for financial assistance. References 1

592

Rajoka, M. I. and Malik, K. A. Biotechnol. Lett. 1984, 6, 597-600

Enzyme Microb. Technol., 1993, vol. 15, July

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Nakamura, K. and Kitamura, K. J. Ferment. Technol. 1982, 60, 343-348 Rapp, P. et al. Biotechnol. Bioeng. 1984, 26, 1167-1175 Sami, A. J., Akhtar, M. W., Malik, N. N. and Naz, B. A. Enzyme Microb. Technol. 1988, 10, 626-631 Langsford, M. L., Gilkes, N. R., Wakarchuk, W. W., Kilburn, D. G., Miller, R. C., Jr., and Warren, R. A. J. J. Gen. Microbiol. 1984, 130, 1367-1376 Gilkes, N. R., Warren, R. A. J., Miller, R. C., Jr., and Kilburn, D. G. J. Biochem. 1988, 263, 10401-10407 Wong, R. W. K., Gerhard, B., Guo, Z.-M., Kilburn, D. G,, Warren, R. A. J. and Miller, R. C., Jr. Gene 1986, 44, 315-324 Gilkes, N. R., Langsford, M. L., Kilburn, D. G., Miller, R. C., Jr., and Warren, R. A. J. J. Biol. Chem. 1984, 259, 10455-10459 Akhtar, M. W., Duffy, M., Dowds, B. C. A., Sheehan, M. C. and McConnell, D. J. Gene 1988, 74, 549-554 Sami, A. J. and Akhter, M. W. Biochem. Soc. Trans. 1989, 17, 580-581 Langsford, M. L., Gilkes, N. R., Singh, B., Moser, R. C., Miller, R. C., Jr., Warren, R. A. J. and Kilburn, D. G. FEBS Lett. 1987, 225, 163-167 Beguin, P. and Eisen, H. Eur. J. Biochem. 1978, 87, 525-531 Moser, B., Gilkes, N. R., Kilburn, D. G., Warren, R. A. J. and Miller, R. C., Jr. Appl. Environ. Microbiol. 1989, 55, 2480-2487 Bagnara, C., Gaudin, C. and Belaich, J.-P. Biochem. Biophys. Res. Commun. 1986, 140, 219-229 Bradford, M. M. Anal. Biochem. 1976, 72, 248-254 Laemmli, U. K. Nature 1970, 277, 680-685 Beguin, P. H., Eisen, H. and Roupas, A. J. Gen. Microbiol. 1977, 101, 191-196 Ait, N., Creuzet, N. and Forget, P. J. Gen. Microbiol. 1979, 113, 399-402 Calza, R. E., Irwin, D. C. and Wilson, D. B. Biochemist~. 1985, 24, 7797-7804 Boyer, M. H., Chambost, J. P., Magan. M. and Cattaneo, J. J. Biotechno/. 1984, 1, 241-252 Wood, T. M., Wilson, C. A. and Stewart, C. S. Biochem. J. 1982, 205, 129-137