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31 FEMS MicrobiologyLetters 120 (1994) 31-36 © 1994 Federation of European Microbiological Societies 0378-1097/94/$07.00 Published by Elsevier FEMSLE 06025 The 92-kDa chitinase from Streptomyces olivaceoviridis contains a lysine-C endoproteinase at its N-terminus H . H . R a d w a n .,a, H . J . P l a t t n e r a, U . M e n g e b a n d H . D i e k m a n n a a Institut fiir Mikrobiologie, Universitiit Hannover, Schneiderberg 50, 30167 Hannover, FRG, and b Gesellschaft fiir Biotechnologische Forschung, Mascheroder Weg 1, 38124 Braunschweig, FRG (Received 26 February 1994; revision received 14 April 1994; accepted 21 April 1994) Abstract: Serine proteinases of 42, 22 and 14 kDa were purified from the culture fluid of Streptomyces olivaceoviridis by FPLC. The first 14 amino acids at their N-termini were identical and coincide with the N-terminal amino acid sequence of 92-kDa chitinase, which was found to hydrolyse casein. The four proteins hydrolyse synthetic substrates at the carboxyl group of lysine and (more slowly) arginine. The 14-kDa endoproteinase releases only two fragments of 42 and 43 kDa from/3-galactosidase. When the pure 92-kDa chitinase was incubated at 37°C in Tris.HCl buffer, it was cleaved into a 70-kDa chitinase and a 22-kDa proteinase which in its part is rapidly degraded to a 14-kDa proteinase. Key words: Streptomyces olivaceoviridis; Chitinase; Serine proteinase; Autocatalytic degradation Introduction A f t e r the a s s u m p t i o n h a d b e e n m a d e that the multiplicity of chitinases from Bacillus circulans is d u e to proteolytic m o d i f i c a t i o n [1], we h a d shown previously [2] that four chitinases in the c u l t u r e fluid of Streptomyces olivaceoviridis [3] o r i g i n a t e from a c o m m o n 92-kDa p r e c u r s o r protein. Low c o n c e n t r a t i o n s a n d the t r a n s i e n t n a t u r e of the 92-kDa chitinase h a d p r e v e n t e d a d e t a i l e d * Corresponding author. SSDI 0378-1097(94)00171-M study of this largest of k n o w n chitinases from o u r Streptomyces strain. W e have now c h a r a c t e r i z e d several proteases from the same c u l t u r e fluid a n d can p r e s e n t u n a m b i g u o u s results that 92-kDa chitinase is a self-processing protein. Materials and Methods Microorganisms Streptomyces olivaceoviridis A T C C 11238 was m a i n t a i n e d o n yeast extract agar slants a n d grown in a 10-1 b i o r e a c t o r (Biostat V; B r a u n Diessel Biotech, M e l s u n g e n , F R G ) . 32 Chemicals Chromozym PL, Chromozym TH, fl-galactosidase from Escherichia coli and endoproteinase Lys-C from Lysobacter enzymogenes were from Boehringer Mannheim (Mannheim, FRG). Arylamide 2 × , ammoniumpersulfate, Coomassie brilliant blue G250, N,N'-methylenebisacrylamide 2 × , N,N,N'N'-tetramethylethylendiamine, and Tris(hydroxymethyl)-aminomethane were from Serva Feinbiochemica (Heidelberg, FRG). Casein and Pefabloc SC were from Merck (Darmstadt, FRG). Trypsin from bovine pancreas was from Sigma Chemie (Deisenhofen, FRG). Purification of 92-kDa chitinase and proteinases The protein concentrate from the culture fluid of Streptomyces olivaceoviridis ATCC 11238 was prepared and the enzymes purified as described [2,3] with the following modifications: (i) fermentation was terminated after 4 days, and after ultrafiltration 24 mg Pefabloc SC per 1 concentrate were added. (ii) 92-kDa chitinase was purified from enriched preparations by HIC on PhenyI-Superose and subsequent chromatography on MonoQ with a descending pH gradient. 100 nmol Pefabloc SC m1-1 was added to the fractions immediately after separation. (iii) For the preparation of 22- and 14-kDa proteinases, 200 ml of the ultraconcentrate (240 mg protein) were applied to a Q-Sepharose 16/10 column and proteins were eluted by KC1 gradient in 20 mM Tris" HC1 buffer (pH 9.0). Fractions with high proteinase activity were pooled and concentrated on PM 10 membranes (Amicon, Witten, FRG). After adjusting the pH to 7.0 and adding ammonium sulfate to 0.9 M, the proteins were separated on a Phenyl Sepharose HiLoad 16/10 column with a descending ammonium sulfate gradient. Active fractions were pooled, diafiltrated and rechromatographed on Phenyl-Superose by the descending ammonium sulfate gradient (1 M to zero). 40/xl of 10 mM Pefabloc SC solution were added immediately to the active fractions. Proteolysis of [3-galactosidase 3-Galactosidase was denatured with 0.1% SDS in Glycin/NaOH buffer (pH 9.0) and hydrolysed with 50 /xl each of 14-kDa proteinase (0.9 mg ml-1), endoproteinase Lys-C (5 /~g m1-1) and trypsin (5/xg ml-1). The reaction was stopped by addition of sample buffer and boiling for 5 min at 100°C. Products were analysed by SDS-PAGE. Autoproteolysis of 92-kDa chitinase Pefabloc SC was removed from protein samples by diafiltration in Microcon-10 concentrators (Amicon, Witten, FRG) at 4°C. Samples (500/xl) were first concentrated to 50/xl by centrifugation in a Sigma 202M cone (Sigma, Osterode, FRG) at 10000 rpm for 30 min; then they were diluted with 500 /zl Tris. HCI buffer (pH 9.0) and concentrated again. This step was repeated two more times. The dialysed samples (400/xg protein ml- ~) were stored in an ice bath. 40/zg enzyme protein were incubated in 100 /xl of 50 mM Tris. HC1 buffer (pH 9.0) at 37°C. Aliquots of 15 /xl were withdrawn at different times, and immediately 5 ~I Pefabloc SC (400/xM) were added. The samples were heated in 7.5 /xl sample buffer for 5 min at 100°C for SDS-PAGE. Analytical methods Protease activity with casein as a substrate was determined according to Nakayama et al. [4]. One protease unit was defined as the amount of enzyme which yields an E540 of 1.0 per hour. Activity against chromogenic substrates was tested in 25 mM glycine/sodium hydroxide buffer (pH 9.0) containing 0.5 mM substrate at 37°C. One unit was defined as the amount of enzyme which releases 1 /xmol 4-nitroaniline (e405 = 9.62 c m 2 txmo1-1) per min. SDS-gel electrophoresis was performed according to Laemmli [5] using a slab gel electrophoresis system from Biometra (G6ttingen, FRG). Staining was done using the silver stain method of Blum [6]. Calibration proteins were from Pharmacia Biosystems (Freiburg, FRG). Purified proteins were blotted from SDS-PAGE onto Immobilon P membranes (Millipore, Eschborn, FRG) by semi-dry method with transfer buffer CAPS (pH 11.0), using a Fastblot apparatus (Biometra). Aminoterminal amino acid sequences were determined by a 470A sequencer and on-line analysis of phenylthiohydantoin-derived amino acids 33 Table 1 N-terminal amino acid sequences of three proteinases and 92-kDa chitinase [2] from S. olivaceoviridis Size Position (kDa) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 42 22 14 92 Ala Ala Ala Ala Gly Gly Gly Gly Gln Gin Gin Gin Glu Glu Glu Glu Ser Ser Ser Ser Xa Ala Ala Ala X X X Arg Pro Pro Pro Pro Asp Asp Asp Asp Gly Gly Gly Gly Leu Leu Leu Leu Tyr Tyr Tyr Tyr X X X Arg Thr Thr Thr Thr Pro X Pro Gly Gly Gly X, not identified. with 120A HPLC equipment, both from Applied Biosystems (Weiterstadt, FRG). Results A 42-kDa proteinase had been purified from the ultraconcentrate of S. olivaceoviridis before [7] and more recently a serine proteinase of 22 kDa (Breves, unpublished) was characterized in our laboratory. We isolated 42-, 22- and 14-kDa proteinases by FPLC from the protein concentrate in the presence of Pefabloc SC. The proteins were blotted and the N-terminal amino acid sequence determined. As can be seen from Table 1, the amino acid sequences of the three proteinases are identical. For comparison the Nterminal sequence of 92-kDa chitinase [2] is included in Table 1. Initially we had assumed that the instability of chitinases from S. olivaceoviridis was due to contamination with proteases. Later it was shown that despite extensive purification some residual proteolytic activity was retained in the 92-kDa chitinase while the 70-kDa chitinase was inactive against casein and chromogenic Lys-C substrate. The specific activities of the proteinases and the 92-kDa chitinase against casein are very similar when the different molecular masses are considered (Table 2) but were much lower than with trypsin (6600 mU nmol-t). The same is true for the four S. olivaceoviridis enzymes when the chromogenic substrates Tosyl-Gly-Pro-Lys-4-nitranilide-acetate (Chromozym PL; 2.04-2.350 mU nmo1-1) or Tosyl-Gly-Pro-Arg-4-nitranilide-acetate (Chromozym TH; 0.02-0.19 mU nmo1-1) were used (compared to 369 mU nmo1-1 for trypsin). The specific activity of the Lys-C endoproteinase against Chromozym PL is 1000-fold higher than that of the 14-kDa enzyme. When /3-galactosidase was used as a substrate for 14-kDa endoproteinase only two major degradation products of 42 and 43 kDa were observed in SDSPAGE. In order to detect the suspected autocatalytic degradation of 92-kDa chitinase the protein was freed from Pefabloc SC by diafiltration in Microcon concentrators at 4°C. Thereafter the Pefabloc-free 92-kDa chitinase in Tris. HCI buffer was incubated at 37°C. A Pefabloc-containing sample served as a control. Aliquots of 15 /zl withdrawn at 0, 15, 30, 60 and 120 min were supplied immediately with Pefabloc SC and heated in sample buffer. Then the samples were analysed in SDS-PAGE. Whilst the 92-kDa chitinase was unchanged in the presence of Pefabloc SC, the dialysed samples showed a rapid degradation of the 92-kDa protein and concomitant appearance of bands at 70, 30, 26 and 14 kDa (Fig. 1). Activity staining with Chromozym PL revealed that, at the onset of incubation, protease activity Table 2 Proteolysis of casein by proteinases and 92-kDa chitinase from S. olivaceoviridis Protein 42-kDa 22-kDa 14-kDa 92-kDa Enzyme activity a proteinase proteinase proteinase chitinase a Casein units. mU (mg protein)- 1 mU (nmol protein)- 1 8 330 14400 22 200 2 950 350 318 311 271 34 Fig. 1. Autoproteolysis of 92-kDa chitinase. Lanes 1, 8, standard molecular mass proteins; lane 2, 92-kDa chitinase+ Pefabloc SC; lanes 3-7, samples after diafiltration and 0, 15, 30, 60 and 120 min incubation at 37°C. was only detectable at the 92-kDa protein band. During the incubation period, increasing protease activity became visible at the 42-, 22- and 14-kDa protein bands, but not at the 70- and 30-kDa protein bands. Discussion By the addition of the serine proteinase inhibitor Pefabloc SC during the purification procedure it was possible to prepare pure proteinases and the 92-kDa chitinase (single bands in SDSPAGE). It could be shown that the three proteinases have the same amino acid sequence at the N-terminus which is also identical with the N-terminus of the 92-kDa chitinase. The proteolytic activity of 92-kDa chitinase and other proteinases from S. olivaceoviridis against casein is low (5%) compared to trypsin, and their activity against the Chromozym substrates is low compared to the activity of S. aureus V8 protease against its chromogenic substrate carbobenzoxyPhe-Leu-Glu-4-nitranilide (data not shown) which might be explained by a very high specificity. By comparison of the /3-galactosidase degradation products with the known fragments of endoproteinase Lys-C degradation [8] it can be deduced that the 14-kDa endoproteinase hydrolyses only Lys-Ala bonds. This assumption is substantiated by the fact that the first amino acid at the Nterminus of 70-kDa and 30-kDa chitinase is Ala [2,9]. This unique specificty has so far only been reported for a protease from A c h r o m o b a c t e r lyticus M497-1 [10]. As soon as the protease inhibitor was removed by diafiltration, degradation of the 92-kDa chitinase occurred. It was concluded that the proteolytic activity must be connected with the 22-kDa degradation product which was temporarily detected by S D S - P A G E in the autoproteolysis experiment. These results point unequivocally to the fact that the 22-kDa terminus of the 92-kDa chitinase has proteolytic activity. It is split off in an autocatalytical process to release the 70-kDa chitinase. Other fragments are 42 kDa and 14 k D a proteinases. All three proteinases form identical peptides from 70-kDa chitinase (data not shown). Acknowledgement We would like to thank Dr. H e m b e r g e r from E. Merck, Darmstadt, for the first information that the 42-kDa proteinase is lysine-C-specific. References 1 Watanabe, T., Oyanagi, W., Suzuki, K. and Tanaka, H. (1990) Chitinase system of Bacillus circulans WL-12 and importance of chitinase A1 in chitin degradation. J. Bacteriol. 172, 4017-4022. 2 Romaguera, A., Menge, U., Breves, R. and Diekmann, H. (1992) Chitinases of Streptomyces olivaceoviridis and significance of processing for multiplicity. J. Bacteriol. 174, 3450-3454. 3 Romaguera, A., Tschech, A., Bender, S., Plattner, H.J. and Diekmaun, H. (1993) Protoplast formation by a mycolase from Streptomyces olivaceoviridis and purification of chitinases. Enz. Microb. Technol. 15, 412-417. 4 Nakayama, M., Tomita, Y., Suzuki, H. and Nisizawa, K. (1976) Partial proteolysis of some cellulase components from Trichoderma viride and the substrate specificity of the modified products. J. Biochem. (Tokyo) 79, 955-966. 5 Laemmli, U.K. (1970) Cleavage of the structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. 35 6 Blum, H., Beirer, H. and Gross, H.J. (1987) Improved silver staining of proteins, DNA and RNA in polyacrylamide gels. Electrophoresis 8, 93-99. 7 Romaguera, A. (1991) Proteinchemische Untersuchungen der Enzyme des chitinolytischen Systems und proteolytische Spaltung der Chitinasen aus Streptomyces olivaceoviridis. Dissertation Universit~it Hannover. 8 Henrissat, B. (1990) Weak sequence homologies among chitinases detected by clustering analysis. Protein Seq. Data Anal. 3, 523-526 and HUSAR, EMBL, Heidelberg. 9 Breves, R. (1993) Multiplizit~it der Chitinasen aus Streptomyces olivaceoviridis: Substratspezifit~it und proteinchemische Charakterisierung. Dissertation Universit~it Hannover. 10 Masaki, T., Fujihashi, T., Nakamura, K. and Soejima, M. (1981) Studies on a new proteolytic enzyme from Achromobacter lyticus M497-1. II. Specificity and inhibition studies of Achromobacter protease I. Biochim. Biophys. Acta 660, 51-55.