Degradation of cellulases in cultures of Cellulomonas fimi

ELSEVIER

Degradation

MICROBIOLOGY LETTERS FEMS Microbiology Letters 143 (1996) 7-12

of cellulases in cultures of Cellulomonas

fi

mi

L.E. Sandercock, A. Meinke *, N.R. Gilkes, D.G. Kilburn, R.A.J. Warren * Department of Microbiology and Immunology, University of British Columbia, 00-6174 University Blvd., Vancouver, B.C. V6T 123, Canada

Received 6 March 1996; revised 5 June 1996; accepted 9 June 1996

Abstract Endoglucanases CenA, CenB and CenD, cellobiohydrolases CbhA and CbhB, and the mixed function xylanaseexoglucanase Cex are degraded proteolytically in the supematants of cultures of Cellulomonasfimi growing with cellulose. All of these polypeptides are modular. The initial sites of proteolysis are within or adjacent to the linkers connecting the modules, leading to the appearance of discrete fragments of the enzymes which retain the functions of the component modules. Keywords: Cellulomonas; Cellulase; Degradation; Proteolysis

1. Introduction

The cellulase systems of bacteria are complex, and may comprise up to 20 or more genetically distinct components [l-3]. Many of the component enzymes are modular, with the linker sequences connecting the modules prone to proteolysis [4]. Some of the enzymes are glycoproteins [5]. The secreted enzyme profile of an organism frequently changes with the age of the culture and with the nature of the inducing substrate [6]. Analysis of the cellulase system of a particular organism may thus be confounded by the complexity of the system and by changes in some of its components as a consequence of proteolysis and deglycosylation. Cloning of the genes encoding the components of a cellulase system proves to be the * Corresponding author. Tel.: +1 (604) 822-2376; E-mail: [email protected] ’ Present address: Department of Microbiology and Genetics, University of Vienna, Dr. Bohr-Gasse 9, 1030 Vienna, Austria.

most direct approach to characterizing distinct components of that system 171. In many instances, however, the protein encoded by a cloned gene has not been identified in the organism from which the gene was obtained. Consequently, much is known about the individual components of many cellulase systems [l-3], but relatively less is known about the entire systems. The genus Cellulomonas is characterized by the ability to hydrolyze cellulose [8,9]. The cellulase systems of several strains have been examined in greater or lesser detail. The systems are complex [6,1&13], and multiple components have been purified from some of them [10,14-161. In some instances the complexity was demonstrated by PAGE, with or without probing with polyvalent antisera [6,1 l-131. However, these methods do not take into account immunological cross-reactivity between conserved regions within the different proteins or the fragmentation of proteins by proteolysis, and in none of the analyses were individual components identified unequivocally. Supematants from cultures of Cellulomonas jimi

0378-1097/96/$12.00 Copyright 0 1996 Published by Elsevier Science B.V. All rights reserved PIfSO378-

1097(96)00297-2

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L. E. Sundercock

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7-12

2. Materials and methods 200

116 ts7

CenACex A 4s 31 Fig. 1. Variation with age in the cellulose-binding polypeptides in the supematant of a culture of C. jimi growing with 0.1% Avicel. Samples were removed from the culture at intervals, the residual cellulose and the cells removed by centrifugation, and the Cbps in the supematant adsorbed to Avicel. The adsorbed polypeptides were analyzed by SDS-PAGE as described in Section 2. Lanes: 1, 26 h; 2, 51 h; 3. 74 h: 4. 95 h; 5. 121 h: 6. 142 h: 7, 171 h; 8, molecular mass standards (kDa). The intact enzymes were identified from their molecular masses and N-terminal amino acid sequences [17-191 and Table I.

ATCC 484 grown with carboxymethylcellulose as inducer contain five major cellulose-binding polypeptides (Cbps) with molecular masses of 120, 110, 95, 75 and 53 kDa [17] (Fig. 1). The N-terminal amino acid sequences of the Cbps identify them as CbhB, CenB, CbhA, CenD and CenA, respectively [17-191. CbhA and B are cellobiohydrolases; CenA, B and D are endoglucanases. C. fimi also produces a xylanasel exoglucanase, Cex, of molecular mass 49 kDa [20]. This paper describes the appearance and stabilities of these polypeptides in cultures of C. jimi ATCC 484 growing with cellulose.

2. I. Muterids Avicel PH 101 (a microcrystalline cellulose preparation derived from wood) was from FMC International, Little Island, Ireland. Carboxymethylcellulose (CM-cellulose; sodium salt; low viscosity grade) was from Sigma Chemical Co., St. Louis, MO. Bacterial microcrystalline cellulose (BMCC) was prepared from Acetobacter xylinum ATCC 23769 as described previously [21]. 2.2. Bucteriul strain The C. $mi strain used was ATCC 484.

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CbhB CenBl

200 116 96: 31

Fig. 2. Variation with age in the polypeptides adsorbed to the Avicel in a culture of C. Jimi growing with Avicel. The residual cellulose from the samples shown in Fig. 1 was washed then boiled with loading buffer. The polypeptides released were subjected to SDS-PAGE. Lanes as in Fig. 1.

Fig. 3. Polypeptides in the culture supernatant. A culture was grown for 48 h in the presence of Avicel. Cells and residual cellulose were removed by centrifugation. The polypeptides in the supernatant were concentrated by precipitation with ammonium sulfate and analyzed by SDS-PAGE. Left lane, molecular mass standards; right lane, concentrated culture supematant.

L. E. Sandercock et al. iFEMS

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Table 1 Putative identities of polypeptides Polypeptide”

N-terminal

amino acid

Putative identity

Amino acids Fraament”

1 2 3 4d 5d 6 7d 8d 9 10 11 12 13

1AVDGEYAQRFL 1APTYNY 1APVHVDNPYAG 1APTYN 1APVHV 1ATGDDWLXVEG IATGDD 1ATGDD 1APGCRV 1AXTLKEAADG 640DDGT”TTPDT 432GTTPVEDLAV 429GTVTDTTA

CbhB CenB CbhA CenB CbhA CenD CenD CenD CenA Cex CbhB CbhA CbhA

intact intact intact CD-Fn3-Fn3 CD-Fn3-Fn3-Fn3 intact CD-Fn3-Fn3 CD-Fn3 intact intact Fn3-Fn3-Fn3-CBD Fn3-Fn3-Fn3-CBD Fn3-Fn3-CBD

1037 1012 832 - 820 N 720 708 N 600 -500 418 443 398 401 304

Predicted molecular mass (kDa) 110 106 85 90 80 75 66 55 44 47 44 44 33

“The numbers refer to the polypeptides indicated on Fig. 3, except for 13, which is the polypeptide of molecular mass N 35 kDa in Fig. 1, lane 3. bThe number before the first amino acid shows its position in the sequence of the native enzyme; X indicates that the amino acid was not identified. ‘CD, catalytic domain; Fn3, fibronectin type III repeat; CBD, cellulose-binding domain. dPolypeptides which do not adsorb to cellulose.

2.3. Growth conditions Cultures were grown at 30°C in basal salts medium containing 0.1% cellulose [17].

mined as described previously [17]. The intact enzymes were identified from their molecular masses and N-terminal amino acid sequences [17-191. The pattern of bands is quite reproducible.

2.4. Analysis of proteins

3. Results

Cells and undegraded cellulose were removed from samples of culture by centrifugation. The cellulose was separated from the cells by repeated settling and resuspension in buffer, and then boiled in SDS-PAGE loading buffer to release any polypeptides adsorbed to the cellulose. The cellulose-binding polypeptides (Cbps) in the culture supernatants were recovered by adsorption to fresh Avicel as described previously [17]. Total polypeptides in the supernatants were concentrated by adding 1.2 ml of saturated (NH&S04 to 0.5 ml of supernatant; after leaving on ice for 30 min, the precipitates were recovered by centrifugation in a microfuge. The supernatant was removed, the tube allowed to drain in an inverted position, and each pellet dissolved by boiling in 2.5 ~1 SDS-PAGE loading buffer. All samples were analyzed by SDS-PAGE [22]. The N-terminal amino acid sequences of polypeptides were deter-

3.1. Degradation of cellulases in cultures of C. jimi Cellulases appeared within 27 h in the supematants of cultures of C. jimi grown with Avicel (Fig. 1, lane 2). CbhA and B, CenA, B and D, and Cex were present, with CbhA appearing to be the most abundant. CbhB, CenA and Cex increased during the next 26 h, but CenD virtually disappeared and the amount of CbhA decreased (Fig. 1, lane 3). Traces of a number of other polypeptides were also present after 52 h, together with a considerable amount of a polypeptide of molecular mass -33 kDa (Fig. 1, lane 3). Most of the polypeptides gradually disappeared during the next 119 h, but CbhB and Cex persisted longer than most (Fig. 1, lanes 48). Identical profiles were obtained over the same time for cultures growing with CM-cellulose instead of Avicel (not shown).

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L. E. Sandercock et al. IFEMS

Cex

10

CenA

9

CenB

2 4

CenD

Microbiology Letters 143 (1996) 7-12

,! :.:.. .\... ...... Fn3 1 CBQI ] 1 CBDlll 1 Fn3 1 Fn3 b,~,~,~,~,)~,.,~,~,,,~~,I.,,.,.,,.,. .. .,.,.. .., : CfQll 1 F,.,3 1 ‘y::::::: ::::..:::.:.I::~::!j:::~:: :j.:. :.:.:.:....A.. :.:.:.:.:.:.:.~:.:.:.:,:,:,:,:::::; :;:,..,.., .A.: .::.‘.::::::.:.:.::::::::::i.i:“.”:.,.:~,:~:~:~:~~~~,:~~~~...~ . ..~:‘::.~:::::::.~:::::::::::::,:,::::::::::::::::,:,:.:.~.~.. :,:i:::: .:::,.::: ,.:: ::7.: ,...I.: Fn3 .,.,,.,.,.,.............................. ::,,:::.:, ,.,. .:.c:;:;,::.:. .,.:.:::::: ..:.,.:).j:.,.::):;: :,::::j::::

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6 7 8

CbhA

3 5 12 13

CbhB

1 11

1 Fn3 I CB& Fn3 1 Fn3 1 Fn3 I CBD,

Fig. 4. Primary sites of proteolysis of the cellulases in C. fimi cultures. The numbering corresponds to that in Fig. 3. J, primary cleavage sites; Fn3, fibronectin type repeat; CBD~I and CBD ~1, type II and type III cellulose-binding domains, respectively (see [7]). Catalytic domains are stippled. Linkers are black.

The residual Avicel in the culture was screened for adsorbed polypeptides. All six enzymes were present on the cellulose after 27 h, but a greater proportion of each was in solution (Fig. 1, lane 2; Fig. 2, lane 2). CbhA was the most abundant enzyme both on the cellulose and in solution. CenB and CenD had disappeared from the cellulose after 52 h, and the

amount of CbhA was greatly reduced (Fig. 2, lane 3). Only Cex was present on the cellulose throughout the incubation (Fig. 2). After 48 h, the culture supernatant contained a number of polypeptides which did not adsorb to cellulose as well as some which did adsorb to cellulose but did not correspond to any of the cellulases (Fig.

L.E. Sandercock et al. IFEMS Microbiology Letters 143 (1996) 7-12

3). Such fragments would result from proteolysis separating the cellulose-binding domain (CBDs) from the other domains in the proteins. 3.2. IdentiJication of degradation products The identities of a number of the polypeptides present after 48 h incubation were deduced from their N-terminal amino acid sequences, their molecular masses as determined by SDS-PAGE, and their abilities to bind to cellulose (Fig. 3; Table 1). They were the intact enzymes and discrete fragments of them produced by proteolysis in or near sequences connecting domains. It was known that a crude protease preparation from C. jimi culture supernatants hydrolyses purified CenA and Cex initially in the proline-and threonine-rich linkers connecting the catalytic domains and the CBDs of the glycanases [20]. The initial hydrolysis of CenB, CenD, CbhA and CbhB in C. fimi cultures also separated their catalytic domains and their CBDs (Fig. 4).

4. Discussion The glycanases secreted by C. jimi during growth with cellulose do not persist in the culture as the substrate is degraded. Initially, the enzymes are cleaved into discrete fragments, with separation of the catalytic domains and the CBDs. Removal of their CBDs reduces the activities of CenA and Cex on cellulose but it does not affect their activities on soluble substrates [20]. Removal of the CBDs could enhance the hydrolysis of cellulose fragments accumulating in the culture. The molecular masses of the enzymes do not correspond to the values calculated from their amino acid sequences [ 17-201; except for CenA and Cex, the causes of the discrepancies are unknown. The discrepancies for CenA and Cex arise from glycosylation of the enzymes and from their linkers [6,23,24]. CenA and Cex are glycoproteins [6,23]. The glycans are O-linked to the proline-threonine linkers in the enzymes (Sandercock, unpublished observations). The glycans protect the purified enzymes from the C. jimi protease when the enzymes are adsorbed to cellulose but not when they are free [23]. In the cultures, CenA disappears after 122 h, but some Cex

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is still present after 171 h. CbhB also appears to be longer lived, but it is not glycosylated. The virtually complete degradation of the extracellular proteins produced by C. jmi is striking. Although individual enzymes appear to differ in their susceptibilities to proteolysis, the relative rates and periods of synthesis will also affect the profiles. The production of discrete fragments of the enzymes may be an integral stage in the process of cellulose degradation. Experiments are in progress to examine this possibility.

Acknowledgments This research was supported by the Natural Sciences and Engineering Research Council of Canada.

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[18]

L. E. Sandercock et al. IFEMS of a bacterial cellulase and its two isolated domains to crystalline cellulose. J. Biol. Chem. 267, 67436749. Kolios, G., Papadopoulos, G.K., Roussis I. and Dramas, C. (1991) Immunological versatility and carbon regulation of Cellulomonas fimi endo-l A-S-glucanases. Biotechnol. Appl. Biochem. 14, 365-371. Poulsen, O.M. and Petersen, L.W. (1989) Electrophoretic and enzymatic studies on the crude extracellular enzyme system of the cellulolytic bacterium Celiulomonas sp. ATCC 21399. Biotechnol. Bioeng. 34, 59-64. Sami, A.J. and Akhter, M.W. (1989) Multiplicity of the endo1,4-B-o-glucanase activity in CellulomonasJIavigena. Biochem. Sot. Trans. 17, 580-581. Poulsen, O.M. and Petersen, L.E. (1989) Purification of two immunologically distinct endoglucanases without affinity for microcrystalline cellulose from CeNulomonas sp. ATCC 2 139. Biotechnol. Bioeng. 34, 65-71. Sami, A.J. and M.W. Akhter. (1990) Purification and characterization of two native extracellular carboxymethylcellulases of Cellulomonas flavigena. Biochem. Sot. Trans. 18, 649650. Sami, A.J. and Akhter, M.W. (1990) Purification and characterization of three extracellular carboxymethylcellulases of Cellulomonas javigena. Biochem. Sot. Trans. 18, 65 1. Meinke, A., Gilkes, N.R., Kilburn, D.G., Miller, R.C. Jr. and Warren, R.A.J. (1993) Cellulose-binding polypeptides from CeNulomonas fmi: endoglucanase D (CenD), a family A B1,4-glucanase. J. Bacterial. 175, 191&1918. Meinke, A., Gilkes, N.R., E. Kwan, D.G. Kilburn, R.A.J. Warren and R.C. Miller, Jr. (1994) Cellobiohydrolase A

Microbiology Letters 143 (1996) 7-12

[19]

[20]

[21]

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[24]

(CbhA) from the cellulolytic bacterium Cellulomonas jmi is a B-1,4-exocellobiohydrolase analogous to Trichoderma reesei CBH II. Mol. Microbial. 12, 413422. Shen, H., Gilkes, N.R., Kilburn, D.G., Miller, R.C. Jr. and Warren, R.A.J. (1995) Cellobiohydrolase B, a second exo-cellobiohydrolase from the cellulolytic bacterium Cellulomonas fimi. Biochem. J. 311, 67-74. Gilkes, N.R., Warren, R.A.J., Miller, R.C. Jr. and Kilburn, D.G. (1988) Precise excision of the cellulose-binding domains from two Cellulomonasjimi cellulases by an homologous protease and the effect on catalysis. J. Biol. Chem. 263, 1040110407. Beguin, P. and Eisen, H. (1978) Purification and partial characterization of three extracellular cellulases from Cellulomonas sp. Eur. J. Biochem. 87, 525531. Laemmli, U.K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 277, 680685. Langsford, M.L., Gilkes, N.R., Singh, B., Moser, B., Miller, R.C. Jr., Warren, R.A.J. and Kilburn, D.G. (1987) Glycosylation of bacterial cellulases prevents proteolytic cleavage between functional domains. FEBS Lett. 225, 163-167. Shen, H., Schmuck. M., Pilz, I., Gilkes, N.R., Kilbum, D.G., Miller, R.C., Jr. and Warren, R.A.M. (1991) Deletion of the linker connecting the catalytic and cellulose-binding domains of endoglucanase A (CenA) of Cellulomonas fimi alters its conformation and catalytic activities. J. Biol. Chem. 266, 11335-11340.