Cellulases of Pseudomonas fluorescens var. cellulosa

200

CELLULOSE

[21]

Sections are placed on Formvar-coated copper grids and coated with a thin layer of nickel in a vacuum evaporator. Grids are attached to a carbon post using spectroscopically pure carbon paint and analyzed with an energy dispersive spectrometer in conjunction with a scanning electron microscope and transmitted electron detector (STEM). The distance of the X-ray detector, accelerating voltage, condenser aperature, and electron beam spot size are all held constant. Bromine distribution profiles along a scan line, corresponding to lignin concentration, are presented for sound wood and wood decayed by Phellinus pini, a white rot fungus that preferentially removed lignin. Although the image quality of micrographs from electrons transmitted through thick sections is not excellent, it illustrates the exact location of the scan line (Figs. 9-11). The bromine (L-series) X-ray distribution profile provides the relative concentration of bromine among the different morphological regions. Lignin is most concentrated in the middle lamella region and secondary wall layers have a reduced concentration. Phellinus pini removes the lignin from the secondary wall layer from the lumen toward the middle lamella region. In advanced stages of decay, lignin is removed from throughout the secondary wall and middle lamella. SEM-EDXA can also provide X-ray distribution mapping and point analysis in addition to the line scan analysis.

[2 1] Cellulases of Pseudomonas fluorescens var. cellulosa B y K U N I O Y A M A N E a n d HIROSHI SUZUKI

Cellulose is an insoluble and highly ordered polymer of glucose. Microorganisms growing on cellulose as a sole carbon source should secrete cellulases to the outside of the cells. The cellulases hydrolyze the highly polymerized structure of cellulose to yield soluble cellooligosaccharides.l.2 Most studies on cellulases have been performed using extracellular cellulases. However, considerable amounts of cellulases have also been found inside c e l l s . 3'4 Cellulolytic bacteria are suitable for studies not only on the nature of the cellulolytic enzyme system but also on the regulation mechanism of E. 2 K. 3 K. 4 B.

T. Reese, R. G. H. Siu, and H. S. Levinson, J. Bacteriol. 59, 485 (1950). Selby, Adv. Chem. Ser. 95, 34 (1969). W. King, J. Dairy Sci. 42, 1848 (1959). Norkrans, Adv. Appl. Microbiol. 9, 97 (1967).

METHODS IN ENZYMOLOGY, VOL. 160

Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

[21]

CELLULASESOF P. fluorescens

201

cellulase formation in microorganisms, because the growth rate of bacterial cells is higher and experimental conditions are easier to control than in fungal cells. Pseudomonas fluorescens var. cellulosa produces two extracellular cellulases (A and B), and one cell-bound cellulase (C). Cellulase C differs from cellulases A and B in enzymatic properties and also in physiological responses to culture c o n d i t i o n s ) :

Assay Method Principle. The activity is measured by following the increase in reducing sugars due to enzymatic hydrolysis of cellulose. The enzyme activities for a soluble cellulose derivative, carboxymethylcellulose (CMC), and for an insoluble crystalline cellulose, Avicel, are called CMCase and Avicelase, respectively. Reagents Mcllvaine's buffer (0.2 M Na2HPO4 • 2H20-0.1 M citric acid), pH 7.0 CMC solution, l g CMC (degree of substitution = 0.6, Daiichi Kogyo Seiyaku Co., Kyoto, Japan) in 100 ml of H20 Avicel suspension, 1 g Avicel (American Viscose Co. Ltd.) in 100 ml of H20 Somogyi's copper reagent 8 and Nelson's arsenomolybdate reagent 9 for determination of reducing sugars ~° Procedure. The reaction mixture consisting of CMC solution or Avicel suspension, McIlvaine's buffer, and enzyme solution (1:2: 1, v/v/v) is incubated at 30 °. After an appropriate period (1 hr for CMCase and 24 hr for Avicelase activity), 1.0 ml of the mixture is withdrawn and the reducing power increase is determined by the method of Somogyi and Nelson. J0 Color development is measured by the absorbance at 660 nm with a spectrophotometer. Definition of Unit. One unit of CMCase activity is defined as the amount of enzyme which produces 1.0/zmol of reducing sugars as glucose/min/ml of enzyme solution and 1 unit of Avicelase activity is defined as for CMCase except that the rate is in/zmol/hr. 5 K. Yamane, H. Suzuki, M. Hirotani, H. Ozawa, and K. Nisizawa, J. Biochem. (Tokyo) 67, 9 (1970). 6 K. Yamane, H. Suzuki, and K. Nisizawa, J. Biochem. (Tokyo) 67, 19 (1970). 7 H. Suzuki, K. Yamane, and K. Nisizawa, Adv. Chem. Set. 95, 60 (1969). 8 M. Somogyi, J. Biol. Chem. 195, 19 (1952). 9 N. Nelson, J. Biol. Chem. 153, 375 (1944). ~0R. G. Spiro, this series, Vol. 8, p. 7.

202

CELLULOSE

[21]

Origin and Culture Medium of Organism

Pseudomonas fluorescens var. cellulosa (IAM 12622, the culture collection of Institute of Applied Microbiology, The University of Tokyo, Bunkyo-ku, Tokyo, 113 Japan) isolated in 1948 by Ueda et al. ~l from soil was provided by Dr. Ueda. This cellulolytic bacterium is a gram-negative straight rod (0.5-0.7 × 1.2-3.0/zm) with a polar flagellum. Its taxonomic position is being recharacterized. Bacteria are maintained on Dubos minimal medium ~2consisting of 0.1 g of K2HPO4, 0.05 g of MgSO4 • 7H20, 0.1 g of NaNO3, 0.05 g of NaCI, and a trace of FeCI3 in 100 ml of tap water (pH 7.0-7.2). A strip of filter paper is the sole carbon source. NaCI is generally omitted and 0.02-0.05% FeC13 • 6H20 provides optimal growth. Purification of Pseudomonas Cellulases

Pseudomonas fluorescens produces two extracellular components (cellulase A which moves rapidly to the cathode and cellulase B which moves slowly), and one cell-bound component (cellulase C), upon zone electrophoresis on cellulose acetate film (Fig. 1). Cellulases A and B are obtained from the supernatants of cultures grown on cellulose as the carbon source. In contrast, cellobiose (G2), one of the major products of cellulolysis, stimulates only the formation of ceUulase C, which seems to be located at the wall or periplasmic space of the c e l l s Y Purification of cellulases A, B, and C is summarized in Table 1.6,7 Preparation of Cellulases A and B The organism is grown on Dubos minimal medium containing 0.5% (w/v) Avicel in 20-liter jar fermentors for 4 days at 37°. After centrifugation at 10,000 g, the supernatant (approximately 120 liters in all from 6 batches of the culture) is concentrated to about 1/10 volume using a flash evaporator under reduced pressure at 30-35 °. Cellulases were precipitated therefrom by ammonium sulfate (65 g/100 ml) at pH 7, collected by filtration through Celite 535, and extracted with 3 liters of distilled water. The dissolved material was dialyzed against tap water and then distilled water. The dialyzed solution was concentrated under reduced pressure and lyophilized (yield, about 12 g). All following procedures are carried out at 2-5 ° unless otherwise stated using Na2HPO4-KH2PO4 buffers. Chromatography on Sephadex G-IO0 (Separation of Cellulase A from B). Half a gram of the lyophilized preparation was dissolved in 50 ml of 20 11 K. Ueda, S. Ishikawa, T. Itami, and T. Asai, J. Agric. Chem. Soc. Jpn. 26, 35 (1952). 12 R. J. Dubos, J. Bacteriol. 15, 223 (1928).

[21]

CELLULASESOF P. fluorescens

203

1.0

% ¢,D

._> <

0.5

0 0.50

b

/L

t

i i ¢D r,J

0.25

8 Fraction

®

9

10

Number

G

FIG. 1. Zone electrophoretic patterns of an extracellular cellulase preparation from a 0.5% cellulose culture (a) and a cell-bound cellulase preparation from a 0.5% cellobiose culture (b) on cellulose acetate films (2 x 11 cm). Arrows indicate the origins. A, B, and C represent cellulases. Electrophoresis was conducted at about 2° for 2 hr with a constant current of 0.6 mA/cm in Veronal buffer, pH 8.6. After the run, the films were cut crosswise into l-cm segments and the cellulase (CMCase) activity in each was assayed.

m M buffer at pH 7.0. After dialysis against the same buffer overnight, the cellulase solution was passed through a Sephadex G-100 column (42 × 650 mm), and eluted with the same buffer; fractions of 100 ml were collected. Most of the cellulase activity was found in peak fractions 3-6, but some activity tailed into fractions 7-15 or 17. The former and latter combined fraction (AI and BI) consisted mainly of cellulase A and cellulase B, respectively. AI and BI from 22 chromatographic runs were concentrated. Chromatography on DEAE-Sephadex A-50. AI and BI were dialyzed against 20 m M buffer, pH 6.0. The solution was divided into five portions and each was applied to a DEAE-Sephadex A-50 column (42 × 650 mm) equilibrated with the same buffer. Upon stepwise treatment with 1.5 liter each of the buffers of 20 mM, pH 6.0, 50 m M pH 7.0, 100 mM, pH 8.0, and 100 mM, pH 9.0 with 0.5 M NaCI, cellulase AI elutes as a major peak with 100 m M buffer at pH 8.0; cellulase BI elutes as a major peak with 50 m M buffer, pH 7.0. The fractions in each major peak were combined, concentrated, and dialyzed against 20 m M buffer, pH 7.0. The combined

204

CELLULOSE

[2 1 ]

TABLE I PURIFICATION OF EXTRACELLULARAND CELL-BOUND CELLULASESAND THEIR RECOVERIES Recovery Total activity a Purification step

Cellulase A Crude cellulase preparation Sephadex G-100 (cellulase AI) DEAE-Sephadex A-50 (cellulase AII) Sephadex G-100 (cellulase AIII) DEAE-Sephadex A-50 (cellulase A) Cellulase B Crude cellulase preparation Sephadex G-100 (cellulase BI) DEAE-Sephadex A-50 (cellulase BII) Starch zone electrophoresis (cellulase B) Cellulase C Cells EDTA-lysozyme-suerose treatment Sephadex G-150 (cellulase CI) DEAE-Sephadex A-50 (ceUulase CII) Sephadex G-150 (cellulase C)

(× 10-2)

Specific activity b

1030 760 -344

80 (390) (640) (1260) 1150

1030 96

80 (10)

--

(lOO)

--

12 380 --

210 -59

90 5 (30)

(70) (1520) 1860

a CMCase activity units. b Units per Lowry protein as bovine serum albumin. Figures in parentheses are calculated from the protein measured by the absorbance at 280 nm, providing that a 0.1% solution of protein gives an absorbance of 1.0.

preparations, named cellulase AII and BII, were almost free of amylase and/3-glucosidase activities. Gel Filtration of Cellulase AH with Sephadex G-IO0. All (40 ml) was applied to a Sephadex G-100 column (42 x 700 mm) and eluted with 20 mM buffer, pH 7.0. A peak of cellulase activity, fractions 7-11 (250 ml), was concentrated (AIII). Rechromatography of AIII on DEAE Sephadex A-50 Column. AIII was subjected to DEAE-Sephadex A-50 column chromatography under the same conditions as above. Eluates with 100 mM buffer, pH 8.0, were combined, concentrated, dialyzed against 2 mM buffer, pH 7.0, and lyophilized. The preparation was called cellulase A. Starch Zone Electrophoresis of Cellulase BII. BII was divided into four portions of I0 ml each and each portion was subjected to starch zone electrophoresis in a plastic tray (35 x 10 x 1.5 cm) for 40 hr at 0-2 ° with a

[21]

CELLULASES

/

OF P. fluorescens

'--' ,__,,

205

/

100

1.5

'~

1.0

50

e-

0.5

¢.~ 0

0 i0

20

30

40

50

~

o

Fraction Number

F]o. 2. Gel filtration pattern of a crude extracellular cellulase preparation obtained from a 0.5% Avicel culture on a BioGel P-150 column (4 x 70 cm). Eluates were collected every 10 ml. (0) CMCase activity; (O) Avicelase activity; dotted line, absorbance at 280 nm.

constant current, 2.3 mA/cm, in Veronal buffer of pH 8.6 (0.05/x). After the run, the starch block was cut crosswise into 1 cm widths, each block is extracted with 10 ml of 50 m M buffer, pH 7.0, and measured for CMCase activity. The major peak, which behaved as component B on cellulose acetate film electrophoresis, was combined, concentrated, dialyzed against 2 m M buffer, pH 7.0, and lyophilized. This was called cellulase B. Alternative Procedure for Fractionation and Purification of Extracellular Cellulases A and B. Figure 2 shows the gel filtration pattern on a BioGel P-150 of CMCase and Avicelase activities of the crude extracellular cellulase preparation obtained from the 0.5% Avicel culture. Five peaks (I-V) were detected, whose molecular weights are estimated to be >10 × 104, 6.5 × 10 4, 6 x 10 4, 5 X 10 4, and 4 x 104, respectively. Peak II showed the highest Avicelase activity. Peak I mainly consists of cellulase B and peak V contains only cellulase A, whereas peaks II-IV are composed of both cellulase A and B. Cellulase A in peak V was purified by DEAE-Sephadex A-50 column chromatography. Cellulase B in peak I can be further purified by gel filtration on BioGel P-300 column and DEAESephadex A-50 column chromatography. 13 13 T. Yoshikawa, H. Suzuki, and K. Nisizawa, Sci. Rep. Tokyo Kyoiku Daigaku 16, 87 (1975).

206

CELLULOSE

[21]

Preparation of Cellulase C Bacteria are grown at 37° for 8 hr by shaking in 150 500-ml flasks each containing 200 ml Dubos minimal medium with 0.5% (w/v) G2. Cells, harvested by centrifugation at 10,000 g, were suspended in 4 liters of 0.32 M sucrose in 25 m M Tris-HC1 buffer, pH 8.0, containing 2 g of lysozyme (Seikagaku Kogyo Co. Ltd., Tokyo, Japan) and 8 g of EDTA, and incubated at 30° for 30 min under gentle stirring. After removing the resultant spheroplasts by centrifugation at I0,000 g for 1 hr, cellulase in the supernatant was precipitated by ammonium sulfate (65 g/100 ml), cellected by centrifugation, and dissolved in 20 m M buffer at pH 7.0. Gel Filtration on Sephadex G-150. After desalting by passing through a Sephadex G-25 column the crude preparation of cell-bound cellulase was divided into 20 portions of 25 ml each and each portion was subjected to gel filtration on Sephadex G-150 columns (42 x 650 mm) equilibrated with 20 m M buffer, pH 7.0. The eluate with the same buffer was collected every 50 ml. The peak of ceUulase activity, fractions 13-17, was pooled and concentrated (CI). Chromatography on DEAE-Sephadex A-50. CI was dialyzed against 20 m M buffer, pH 6.0, and divided into four portions. Each was applied to a DEAE Sephadex A-50 column (42 × 650 mm) under conditions similar to those described for extracellular cellulases. The cellulase activity was recovered in the eluate with 20 m M buffer, pH 6.0, and was completely separated from amylase and fl-glucosidase activity. The peak fractions were pooled and concentrated (CII). Second Filtration on Sephadex G-150. CII was dialyzed against 20 m M buffer, pH 7.0, and divided into six portions of 4.5 ml each. Each was applied to a Sephadex G-150 column (24 x 1,000 mm) and eluted with the same buffer. The peak of cellulase activity, fractions 18-21 (60 ml), were combined, concentrated, dialyzed against 2 m M buffer, pH 7.0, and lyophylized. This was designated cellulase C. Purity of Cellulases A, B, and C The three preparations showed a single cellulase peak in zone electrophoreses on starch and on cellulose acetate film, and in analytical ultracentrifugation, although a small amount of heavier component was detected in the ultracentrifugal pattern of cellulase C.

Properties of Cellulases A, B, and C Chemical Analyses. Cellulases A, B, and C showed no significant differences in their amino acid composition. 6 All three contain carbohy-

[21]

CELLULASESOF P. fluorescens

207

drate, amounting to about 13, 36, and 33%, respectively. 6 The main constituent is galactose in cellulase A and glucose in cellulases B and C. p H Optima and Heat Stability. The optimal pH of CMCase activity is 8.0 for cellulase A and B and 7.0 for cellulase C. All three are most stable at pH 7.0-8.0, and completely inactivated by heating at 60° for 10 min, although cellulase B is slightly activated by treatment at 40 ° for I0 min. Activities toward Cellooligosaccharides, Celluloses, and Their Derivatives. Cellulases A, B, and C are similarly incapable of attacking Gz and p-nitrophenyl-fl-glucoside, but they hydrolyze various substrates such as cellooligosaccharides, amorphous celluloses, and Avicel. The activities of each cellulase toward these substrates are markedly different from one another. Cellulase B generally shows much lower activity toward soluble substrates including CMC than cellulase A and C (approximately 1/10 to 1/20), whereas their activity toward highly polymerized substrates such as insoluble swollen cellulose, Avicel, and DEAE-cellulose is not so different. The most conspicuous difference is found in the activity toward cellotriose (G3) and cellotriosylsorbitol (G4H). Only cellulase C hydrolyzes them easily. This indicates that at least three consecutive glucosyl residues seem to be necessary for the hydrolysis of cellooligosaccharides by cellulase C. In contrast, four consecutive glucosyl residues are necessary for hydrolysis by cellulases A and B, since they hydrolyze cellotetraose (G4) and cellotetraosylsorbitol (GsH) but not G4H. Table II is a summary of relative hydrolysis rates of each bond of nonreduced and reduced cellooligosaccharides by the three cellulases. Cellulase C mostly binds the G2 residue of the nonreducing end to cleave it when it forms an enzymesubstrate complex. Cellulases A and B similarly bind a G2 residue at the nonreducing end of lower cellooligosaccharides, but they more easily bind the G3 residue of the nonreducing end of higher cellooligosaccharides. There is no significant difference in the degree of randomness in CMC hydrolysis by the three cellulases, as shown by the relationship between the decrease in viscosity and the increase in reducing power during incubation. Therefore, these cellulases hydrolyze CMC in a similarly random mechanism. Effect of Culture Conditions on Cellulase Formation Bacteria can grow on various carbohydrates, amino acids, and organic acids. 14However, cellulase production is remarkably affected by the kind 14K. Yamane, H. Suzuki, K. Yamaguchi, M. Tsukada, and K. Nisizawa, J. Ferment. Technol. 43, 721 (1965).

208

CELLULOSE

[21]

E~

<

E~

~ ~ A~ ~_ A A _ A~ A A ~ ~ A m AA A

A A A

Z

E)

Q. o

O

E~ e.~

o ~--

A Z <

S

~

~

A

A A ~ A A A A ~ ~ ~ ~

A

A

A

~ A .,~. ~" A ~ All A

z

<

<

~'~

O

A ,-1

~

~

A

A

A

A

A

A

A

A ~

II

~

A .~- A ~ All A

0

~A~

m

V Ot

..t e~

O Q O 0

v

m >

~A~

O O O O O 0

< m

~1~1~1~1

O

O O Q Q Q O Q

- QI -QI -OI -OI -OI -QI -QI O O O O O O O

dfl~t~z

[21]

CELLULASESOF P. fluorescens

209

of carbohydrates (0.5% w/v) in batch cultures at 37°. Glucose supports excellent growth, but does not stimulate cellulase formation. In contrast, cellulose and sophorose (2-O-fl-glucosyl-o-glucose) increase the production of cellulases, especially the extracellular form. Sophorose is an inducer of cellulase in the cellulolytic fungus, Trichoderma viride. ~5,j6 G2 stimulates the formation of a cell-bound cellulase but not the extracellular enzyme. Growth rates (increase in turbidity at 610 nm/hr) in the logarithmic phase of growth on glucose, G2, sophorose, and cellulose were 0.183, 0.174, 0.183, and 0.03, respectively. Sophorose supported excellent growth (as did glucose) and hyperproduction of the extracellular cellulase (as did cellulose). Effects of starch, lactose, galactose, and xylose on cell growth and cellulase formation were similar to that of glucose, while those of cellooligosaccharides (G3, G4, Gs, G6, and G25) were similar to that of G2.5 In the cellulose culture supporting the enhanced cellulase production, the concentration of soluble sugars in the medium should not be high. Slow feeding of cultures was then attempted with glucose, G2, and sophorose. The growth rate in these cultures was suppressed to about one-third of that found in the batch cultures. Cellulase formation, particularly extracellular cellulase formation, in all the slowly fed cultures was stimulated to the level comparable to the cellulose batch culture. The extracellular cellulase was also composed of cellulase A and B. 5,17 The level of the cell-bound cellulase in these cultures was almost equal to the levels in batch cultures on G2 and cellooligosaccharides. The cell-bound cellulase C can be solubilized during spheroplasting with the treatment of lysozyme and EDTA in an isotonic solution. Therefore, this component seems to be located at the wall or periplasmic space of the cells. ~7 Sophorose highly stimulates the production of extracellular cellulase not only in the batch culture but also in the slowly fed culture. To investigate the sophorose effect further, the effects of various concentrations of carbohydrates on cellulase and amylase formation were analyzed using washed cell suspensions. The results suggest that sophorose does not act as an actual inducer for cellulase formation in this bacterium, and that the observed stimulation of cellulase formation was rather closely related to release from so-called catabolite repression, although we have not yet clarified why sophorose supports such excellent growth and hyperproduct5 M. Mandels, F. W. Parrish, and E. T. Reese, J. Bacteriol. 83, 400 (1%2). ~6T. Nisizawa, H. Suzuki, M. Nakayama, and K. Nisizawa, J. Biochem. (Tokyo) 70, 375 (1971). 17 K. Yamane, T. Yoshikawa, H. Suzuki, and K. Nisizawa, J. Biochem. (Tokyo) 69, 771 (1971).

210

CELLULOSE

[21]

tion of cellulase. Since the formation of cell-bound cellulase is only slightly influenced by culture conditions, it seems to be less sensitive to catabolite repression than that of extracellular cellulases. 18 In Vitro Conversion of Cellulase B to Cellulase A

Cellulases A and B are secreted into the culture medium and are similar in their substrate specificity; but they differ in their specific activity, electrophoretic mobility, and carbohydrate content. Characteristics of cellulase B such as high adsorbability on Sephadex and slow electrophoretic mobility, possibly due to a high adherence to cellulose acetate film, may be due to its high glucose content. The proportion of cellulase B to A decreases with the culture period not only in the Avicel culture but also in the culture slowly fed with g l u c o s e . 17A9 An attempt was made to convert cellulase B to A in vitro using Trichoderma viride fl-glucosidase as a modifying agent. The B-glucosidase preparation, free from protease activity for casein as well as from CMCase and other glycanases, was obtained from a commercial cellulase preparation of Trichoderma viride, cellulase Onozuka (Yakult Co. Ltd., Tokyo Japan) by consecutive column chromatography on Amberlite CG-50 and DEAE-Sephadex A-50. 2° When a purified cellulase B preparation from peak I (Fig. 2) was incubated with the/3-glucosidase at 30° for 72 hr at pH 7.0, cellulase B was completely lost and an electrophoretically rapid-moving cellulase A-like component was detected. Similar changes in cellulase B by fl-glucosidase treatment were observed in peaks II-IV (Fig. 2).13 Although we have not yet obtained evidence other than the electrophoretic behavior for identification of the conversion product with cellulase A, the results strongly indicate the possibility of an enzyme-catalyzed conversion of cellulase B to A. In contrast, no changes in CMCase activity and electrophoretic mobility occurred upon the fl-glucosidase treatment of cellulase A and C, and peak V. These results suggest that cellulase B is an aggregated form of cellulase A, which differs from cellulase C.

18 H. Suzuki, in "Symposium on Enzymatic Hydrolysis of Cellulose" (M. Bailey, T. M. Enari, and M. Linko, eds.), p. 155. Technical Research Centre of Finland, Helsinki, Finland, 1975. ~9T. Yoshikawa, H. Suzuki, and K. Nisizawa, J. Biochem. (Tokyo) 75, 531 (1974). 20 G. Okada, K. Nisizawa, and H. Suzuki, J. Biochem. (Tokyo) 63, 591 (1968).