Download The 92-kDa chitinase from Streptomyces olivaceoviridis contains a

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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 ) .
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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
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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
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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
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35
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