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Purification and Properties of Collagenase from

Cytophaga sp. L43-1 Strain

Yoshikiyo Sasagawa, Yoshiyuki Kamio, Yuko Matsubara, Yoshiko Matsubara,

Koki Suzuki, Hisao Kojima & Kazuo Izaki

To cite this article: Yoshikiyo Sasagawa, Yoshiyuki Kamio, Yuko Matsubara, Yoshiko Matsubara,
Koki Suzuki, Hisao Kojima & Kazuo Izaki (1993) Purification and Properties of Collagenase from
Cytophaga sp. L43-1 Strain, Bioscience, Biotechnology, and Biochemistry, 57:11, 1894-1898, DOI:

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Biosci. Biotech. Biochem., 57 (11), 1894-1898, 1993

Purification and Properties of Collagenase from Cytophaga sp. L43-1 Strain

Yoshikiyo SASAGAW A, Yoshiyuki KAMIO, Yuko MATSUBARA, * Yoshiko MATSUBARA, * Koki SUZUKI, *
Hisao KOJIMA, * and Kazuo IZAKI
Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, Sendai 981, Japan
* Research Institute of Nippi,
Incorporated, Adachi-ku, Tokyo 120, Japan
Received May 12, 1993

A collagenolytic bacterial strain was isolated from soil and was identified as Cytophaga sp. It produced
several kinds of collagenase and protease. From the supernatant of a culture, a collagenase was purified
as a single protein band upon SD8-PAGE and its molecular mass was estimated to be 120 kDa. Collagen
and gelatin were good substrates for this enzyme. {3-Casein was cleaved by this enzyme at several sites.

Collagenases are used in molecular biology experiments Polyacrylamide gel electrophoresis (PAGE). PAGE was done by the
or medical and chemical fields. For example, the enzyme method of Davis. 12 ) The molecular mass of the enzyme was estimated by
the method of Hedrick and Smith,13) using bovine serum alqumin (BSA)
disperses mammalian cells by hydrolyzing collagen, and the
as the standard. Protein was stained with Coomassie brilliant blue R-2S0.
enzyme from Clostridium histolyticum has been used as a
reagent to cleave the fusion protein linked to {3-galactosidase Sodium dodecyl sulfate-PAGE (SDS-PAGE). SDS-PAGE was done by
via the collagenase recognition sites. 1) Collagenases have the method of Laemmli.14) Molecular mass standards used were myosin
been obtained from a variety of microorganism such as (200 kDa), f3-galactosidase (116.2S kDa), phosphorylase (97.4 kDa), bovine
serum albumin (66.2kDa), ovalbumin (45 kDa), carbonic anhydrase
Clostridium histolyticum,2) Vibrio arginolyticus,3) Vibrio (31 kDa), soybean trypsin inhibitor (21 kDa), and lysozyme (14.4kDa).
B-30,4) Pseudomonas marinoglutinosa,5) and Streptomyces To detect enzyme activity in the cultural supernatant, 0.1 % gelatin was
Sp.6) The collagenase from Clostridium histolyticum has been added to the resolving gel 15 ) and electrophoresis was done at 4C. After
studied most extensively.2,7) However, only a few reports electrophoresis, the gel was washed with 2.S% Triton X-lOO at room
on the cloning and sequencing of collagenase genes of temperature and incubated at 30C for 3h in 10mM Tris-HCI, pH 7.5,
containing 4 mM CaCI 2 The gel was stained with amide black. The bands
microorganisms have been published. 8 ,9) of protein having proteolytic or collagenolytic activity were observed as
In this paper, we describe the purification and the clear zones.
properties of the collagenase from Cytophaga sp. strain
L43-1. Isoelectric focusing. Isoelectric focusing was done at 4C by using
Ampholine pH 4-6 in a 2.S x lOOmm rod gel. 16) Electrophoresis was done
at 200V for 14h and at 400V for 1 h. After isoelectric focusing, the gel
Materials and Methods was electrophoresed by SDS-PAGE, which was done with a gel containing
Organism. Strain L43-l was isolated from soil in Sakura City, Chiba 0.1 % gelatin to detect enzyme activity.
Prefecture, Japan and was identified taxonomically as Cytophaga sp. in
the Enzyme Research Laboratory, Research Institute of Nippi, Inc. 10 ) Assay for enzyme activity.
Hydrolysis of native collagen by a collagenolytic enzyme preparation from i) Insoluble collagen as substrate. 7) The reaction mixture contained 10 mg
Cytophaga sp. L43-l was investigated (Y. Matsubara et al. in preparation). of insoluble collagen, 0.8ml of 50mM Tris-HCl containing 4mM CaCI 2,
For the isolation of the strain, a soil sample was suspended in sterile pH 7.S, and 0.2 ml of enzyme solution. The reaction mixture was incubated
water, and the filtrate was spread on agar plates with a medium containing at 30C with shaking, and the reaction was stopped at various incubation
insoluble collagen and mineral salts. The plates were incubated at 25C times by the addition of 1.0 ml of 0.1 N acetic' acid. The initial rate of
and colonies producing halos were selected. increase in free a-amino groups was measured by the ninhydrin method. 17)
The contents of the tubes were centrifuged at 10,000 x g for 15 min and
Culture media. The bacterium was aerobically grown in the medium 0.1 ml of the supernatant was taken and 1.4ml ofO.2M citrate-sodium
containing O.S% Polypeptone, 0.1 % yeast extract, 0.2% gelatin, 0.0588% citrate buffer, pH 5.5, and 1.0ml of 2% ninhydrin in methylcellosolve
CaCl 2 '2H 2 0, 0.05% KH 2 P0 4, O.OS% K 2HP0 4, 0.02% MgS0 4 '7H 20, containing 0.05% SnCl 2 H 2 0 were added ana heated at 100C for IS min.
0.001 % MnS0 4 4H 2 0, 0.001 % NaCl, and 0.001 % FeS04 ' 7H 20, pH 7.0, After this was cooled with ice water, the absorbance at 570 nm was
at 26C for 15 h. measured. The specific activity' was expressed as j.tmol of leucine equiv-
alent per min per mg protein.
Chemicals. Collagens (insoluble type I from bovine achielles'tendon ii) Acid soluble collagen or gelatin as substrate. 18) Acid soluble collagen
and acid soluble type III from calf skin) and f3-casein were purchased from was dissolved in 0.01 M acetic acid. Gelatin was dissolved in water by
Sigma Chemical Co., S1. Louis MO. Casein was obtained from Merck, boiling. Concentrations of both substrates were 0.2% (w/v). The reaction
Rahway, NJ and gelatin was given to us by Nippi, Incorporated, Tokyo. mixture contained 0.3 ml of substrate solution, 0.2 ml of 150 mM Tris-HCI,
Synthetic oligopeptides (pz-PLGPR, Z-GPGGPA, and Z-GPLGP) were pH 7.5, containing 12mM CaCI 2, and 0.1 ml of enzyme solution. The
purchased from BACH EM Feinchemikalien AG. Other chemicals were reaction mixture was incubated at 30C and the reaction was stopped at
of analytical grade. various incubation times by addition of 0.6ml of 0.1 N HCI. The initial
rate of increase in free a~amino groups was measured by the ninhydrin
Protein measurement. Protein was measured Lowry's method 11) using method, as described above.
bovine serum albumin as the standard. iii) Casein as substrate. 18) The enzyme solution (0.05-0.1 ml) was
incubated with l.Oml of 0.6% casein solution in SOmM Tris-HCI, pH 7.5,

Abbreviations: Pz-PLGPR,p-phenylazobenzyloxycarbonyl-L-prolyl-L-leucyl-glycyl-L-prolyl-D-arginine; Z-GPGGPA, carbobenzoxy-glycyl-L-prolyl-

glycyl-glycyl-L-prolyl-L-alanine; Z-GPLGP, carbobenzoxy-glycyl-L-prolyl-L-Ieucyl-glycyl-L-proline; PAGE, polyacrylamide gel electrophoresis; SDS,
sodium dodecyl sulfate; CAPS, 3-cyclohexyl-aminopropanesulfonic acid; DFP, diisopropyl fluorophosphate; EDTA, ethylenediaminetetraacetic acid;
PVDF, polyvinylidene difluoride.

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Purification of Collagenase of Cytophaga sp. L43-1 Strain 1895

containing 4 mM CaCl 2 at 30C. The reaction was stopped at various Results

incubation times by adding 0.5 ml of 10% trichloroacetic acid. The amount Production of collagenolytic enzyme
of acid-soluble peptide formed was estimated by measuring the absorbance Production of collagenolytic enzyme increased in
at 273 nm. The specific activity was expressed as p.mol of tyrosine
equivalent per min per mg protein.
proportion to the concentration of gelatin (within 0.2%)
iv) Synthetic oligopeptides as substrates. Hydrolysis of Z-GPGGPA and added to the culture medium. When the enzyme activity
Z-GPLGP by collagenase was assayed as described by Yoshida and was measured using insoluble collagen as substrate, the
Noda. 18 ) The reaction mixture contained 0.1 ml of 5 mM substrate solution, enzyme activity was maximal at the end of exponential
0.3 ml of 50mM Tris-HCI, pH 7.5, containing 4mM CaCI 2 , and 0.1 ml of growth phase (Fig. I). The collagenolytic activity in the
enzyme solution (15 p.g/ml). The reaction mixture was incubated at 30C
for 0-60 min and the reaction was stopped by the addition of 0.5 ml of culture supernatant was from the production of several
0.1 N HCI. The initial rate of increase in free a-amino groups was measured kinds of collagenase and protease (Fig. 2).
by the ninhydrin colorimetric method as described above.
Hydrolysis of Pz-PLGPR by collagenase was assayed as described by Purification of a collagenase
E. Wunsch and H. G. Heidrich. 19 ) The reaction mixture contains O.4ml
A collagenase was purified as described in Materials and
of 0.1 % substrate dissolved in 50mM Tris-HCI, pH 7.5, containing 4mM
CaCl 2 and 0.1 ml of enzyme solution (15 p.g/ml). The reaction mixture was Methods. The steps in the collagenase purification proce-
incubated at 30C for 0-60 min and the reaction was stopped by addition dure and the yield from each step are shown in Table I.
of 1 ml of 0.5% citric acid. pz-PL that was produced by the enzymatic In the first DEAE-Sepharose column chromatography,
reaction was extracted by 5 ml of ethyl acetate. The extract was dehydrated collagenolytic activity was detected in both adsorbed and
by 0.3 g of Na 2 S0 4 , and the absorbance of the extract was measured at
non-adsorbed fractions. The enzyme in the non-adsorbed
fraction had both collagenolytic and caseinolytic activities.
Purification ojcollagenase. The bacteria was aerobically grown in 10 liters The caseinolytic activity recovered in this fraction was about
of the medium described above at 26C for 15 h. The cells were removed 80% of the amount put on. Initially, the collagenolytic
by centrifugation and the supernatant was used as a source of the emzyme. activity was eluted as a sharp peak (fraction No. 6-15), and
The supernatant was concentrated by ultrafiltration using a Minitan
Ultrafiltration System (MILLIPORE). The concentrated supernatant was
then a big broad peak of the collagenolytic activity was
dialyzed against lOmM Tris-HCI, pH 7.5, containing 4mM CaCl 2 and the eluted. Most caseinolytic activity of the adsorbed fraction
precipitates that formed were removed by centrifugation at lO,OOO x g for
30 min. The concentrate was put on a DEAE-Sepharose CL-6B column
(1.5 x 28cm) equilibrated with lOmM Tris-HCI, pH 7.5, containing 4mM
CaCI 2 The column was washed with lOmM Tris-HCI, pH 7.5, containing
4 mM CaCI 2 Elution was done with NaCI (0-3 M). Rechromatography was ~ 0,1
done with a DEAE-Sepharose column (1.55 x 13.8 cm). Elution was !i!
performed with NaCI (0-1.25, 1.25-2 M). Insoluble collagen was used to
measure collagenolytic activity in each step. j
j 0,01 L - -_ _ _ _ _ _ _ _ _- - '

Effects oj pH on enzyme activity and stability. The effects of pH on the 3 6 9 12 15 1821 2427 30
Time (hours)
collagenolytic activity were measured with acid-soluble collagen as the
substrate by the method described above. Fig. 1. Production of Collagenolytic Enzyme during Bacterial Growth.
To measure the pH stability, the enzyme solution was incubated at Cells were grown in the medium described in Materials and Methods. Collagenolytic
various pHs with 4 mM CaCl 2 at 30C for 30 min, cooled on ice, and the activity was measured by using insoluble collagen. Symbols: 0, absorbance at 660 nm
remaining activity was measured at pH 7.5 by with acid-soluble collagen as cell density; ,A., collagenolytic activity
as the substrate.
The buffers used in this experiment were acetate (pH 3.5-5.5),
tris-maleate (pH 5.5-8.5), borate (pH 8.5-10.0), and CAPS (3-cyclohexyl- kDa
aminopropanesulfonic acid}-NaOH (pH lO.0-11.0).
Effects oj temperature on enzyme activity and stability. The effects of
temperature on enzyme activity were measured at various temperatures
from IS-55C using gelatin as the substrate. In this experiment, native _116.25
collagen was not used as substrate, since it was denatured above 40C. - 97.4
The stability of enzyme activity at various elevated temperatures was
measured with acid-soluble collagen as the substrate at pH 7.5. Enzyme _ 66.2
dissolved in 10 mM Tris-HCI, pH 7.5, containing 4 mM CaCl 2 was incubated
at various temperatures from IS-55C for 30 min and the remaining activity
was measured with acid-soluble collagen as substrate.
- 45
Effects oJ various inhibitors on enzyme activity. The enzyme solution was
incubated at 30C for 30 min with various protease inhibitors indicated in
Table III in 10mM Tris-HCI, pH 7.5. The remaining activity was measured
with acid-soluble collagen as the substrate.

Analysis oj peptide Jragment Jrom f3-casein. The peptide fragments from

f3-casein by the enzyme were separated by SDS-PAGE. After' elec-
trophoresis, electroblotting of bands of protein in the gel onto PVDF
membrane was done by the method of Matsudaira. 20 ) The N-terminal
sequence of the protein electro blotted onto a polyvinylidene difluoride
(PVDF) membrane was analyzed by Edman degradation with a ABI 473A
protein-sequencer. Fig. 2. Identification of Gelatinolytic Enzymes (Collagenases and
Proteases) in SDS-PAGE Containing 0.1 % Gelatin.
The details are described in Materials and Methods. A culture supernatant was
electrophoresed. Protein molecular mass markers (kOa) are indicated on the right
Clear zones showing proteolysis are indicated by arrows in the left.

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1896 Y. SASAGAWA et al.

Table I. Summary of Purification of Collagenolytic Enzyme from Cytophaga sp. U3-1 Strain

Total Total Specific Caseinolytic

Volume Yield
Purification step protein activity activity activity
(ml) (%)
(mg) (units) (units/mg) (units)

Culture supernatant 10,870 2650 1130 0.426 (1) 100 565

Ultrafiltration 480 576 538 0.934 (2.19) 47.6 58.6
DEAE-Sepharose 1st 128 71.2 87.4 1.22 (2.86) 7.73 0.660
DEAE-Sepharose 2nd 220 3.32 23.2 7.02 (16.5) 2.06 "'n.d.

"'n.d., activity was not detectable.


1.0 ....I 0.02 .~
Q) :::l
>-(.) \.../
.... c:
.- ~ 1. 0
~ c:
..... 0
(.) rJl
> o
'" I~ ....
~ 0.5 (.)\.../

0.01 ('(l 1.0~c:
>- ....>- Q)
Q: u

o 8 o
o ~
10 40 80
Fraction No.

Fig. 3. Elution Pattern of the Second DEAE-SepharoseCL6B Column Chromatography.

Fraction No. 16-33 in the first DEAE-Sepharose CL6B column chromatography was put on the colmn. Elution was done with NaCI (0-1.25, I.25-2M). Symbols: . ,
collagenolytic activity; 0, caseinolytic activity; ----, concentration of protein; ---, concentration of NaCl.

was eluted together with the sharp peak of collagenolytic

kDa activity (fraction No. 6-15). The eluted fractions were
separated and divided into three parts which were fraction
200 No. 6-15 (fraction A), 16-33 (fraction B), and 34-99
(fraction C). Fraction B, which contained the major enzyme
activity, was rechromatographed on a DEAE-Sepharose
116. 25 column. The collagenolytic activity in the adsorbed fraction
was separated into two peaks, one sharp peak eluted with
97.4 a low concentration of NaCI (O.1-O.2M), named peak I, and
a broad peak eluted with a high concentration of NaCI
66.2 (about 0.5 M), named peak II (shown in Fig. 3). Peak I
contained many proteins and several kinds of caseinolytic
and collagenolytic activities, but the peak II had only
45 collagenolytic activity. The collagenase in peak II was
purified about 17-fold. SDS-PAGE of the purified enzyme
preparation showed a single band of protein (Fig. 4).

31 Properties of the collagenase

The properties of the collagenase are shown in Table II.
21. 5 The molecular weight of the native enzyme was 96,200 by
the method of Hedrick and Smith with different
14.4 concentrations of acrylamide on PAGE. The molecular
weight of the denatured enzyme was 120,000 by SDS-PAGE
Fig. 4. SDS--PAGE Pattern of Purified Collagenase Preparation. (Fig. 4). The results indicate that the native enzyme is a
Stained with Coomassie brilliant blue R-250. Protein molecular mass markers (kDa)
are indicated in the right.
The isoelectric point of the enzyme was 4.8 by isoelectric

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Purification of Collagenase of Cytophaga sp. L43-1 Strain 1897

Table II. The Properties of the Purified Enzyme Preparation Time Chou rs)
Molecular weight 120,000 (SDS-PAGE)
96,200 (Hedrick-Smith) o O. 5 1 2 3 4 5 6 8 12 24
pI 4.8
Optimal pH 7.S
Optimal temperature 30C
pH stability 6.0--8.0 p -casein-
Thermal stability <40C (100%), 4SoC (SO%)

Table III. Effects of Various Protease Inhibitors on Hydrolysis of

Acid-soluble Collagen by the Purified Enzyme

concentration Remaining
(mM) activity (%)

None 100
EDTA 0.1 o Fig. 5. Cleavage of tJ-Casein by the Purified Enzyme.
1 o SOS-PAGE analysis of the digested products from fJ-casein by the purified enzyme
DFP 0.1 30.1 was done. {J-casein and enzyme (the ratio of weight of (J-casein to enzyme was 50: l)
were incubated at 30C for several hours and the reaction mixtures were put on
1 o SOS-PAGE with a 15-20% gradient acrylamide gel containing 3 M urea. The
Iodoacetamide 1 94 enzymatic products were numbered from I to 8 in order of molecular mass. The
Iodoacetic acid I 87.S positions of each products from fJ-casein are shown at right and the position of
Soybean trypsin inhibitor 0.1 mg/ml 102 fJ-casein is shown at left.

Table V. Edman Degradation of the Enzymatic Products from tJ-Casein

Table IV. Substrate Specificity of the Purified Enzyme
Reaction cycle Corres-
Activity No. of
Substrate ponding
(units/mg) product
10 sequence of
2 3 4 S 6 7 8 9
Milk casein o
Insoluble collagen 7.02 1-10
1-4 and 7 Arg Glu Leu Glu Glu Leu Asn Val Pro Gly
Acid-soluble collagen 3.93
6 Gly Pro lIe Pro Asn Ser Leu Pro GIn Asn 64-73
Gelatin 6.19 103-112
8 Ala Pro Lys His Lys Glu Met Pro Phe Pro
Cleavage of f3-casein by the purified enzyme
Cleavage of f3-casein by collagenases from Achromobacter
iophagus and Clostridium histolyticum has been reported by
The optimal pH of the enzyme activity was 7.5 when the Gilles and Kei1. 21 ) Although caseinolytic activity of the
activity was measured from pH 3.5 to 8.5 using acid-soluble purified enzyme could not be detected by the method
collagen as substrate. At pH 6.0 and 8.5, the activity described in Materials and Methods, cleavage of f3-casein
decreased by 50 % The optimal temperature was estimated by the purified enzyme was demonstrated by SDS-PAGE
to be 30C when the enzyme activity was examined at analysis (Fig. 5). Eight fragments from 1 to 8 in order of
various temperature from 15C to 55C using gelatin as a molecular weight were observed as digested products from
substrate. The purified enzyme was stable from pH 6.0 to f3-casein and their molecular weight were estimated to be
8.0 and at temperatures below 40C. 21.2, 20, 17.2, 13, 11.2, 9.1, 7.7, and 7.4kDa, respectively.
N-terminal sequences of eight fragments were analyzed
Effects of inhibitors on activity (shown in Table V). The N-terminal sequences of 1-4 and
The effects of several protease inhibitors on enzyme 7 corresponded to that of f3-casein. The N-terminal ends of
activity are shown in Table III. EDTA inhibited the activity 6 and 8 corresponded to 64 and 103 amino acid residue of
completely at the concentration of 0.1 mM. DFP inhibited f3-casein, respectively. Not enough of the product of No.5
the activity partially (70%) at 0.1 mM and completely at could be obtained to analyze the N-terminal sequence.
1 mM. Iodoacetamide (l mM), Iodoacetic acid (l mM), or
soybean trypsin inhibitor (0.1 mg/ml) had no inhibitory Discussion
activity. As described above, several kinds of collagenase and
protease were found in the culture supernatant in which
Substrate specificity Cytophaga sp. L43-1 strain was grown (Fig. 1). These
The specificity of the purified enzyme was tested using enzymes were separated into three peaks by DEAE-
various substrates. The purified enzyme digested insoluble Sepharose chromatography. The non-adsorbed fraction
collagen, acid-soluble collagen, and gelatin as shown in contained an enzyme that hydrolyzed casein, while the
Table IV. The enzyme did not hydrolyze casein or three adsorbed fraction had only a little caseinolytic activity. A
synthetic oligopeptides, Pz-PLGPR, Z-GPGGPA, and sharp peak eluted with a low NaCI concentration contained
Z-GPLGP. several kinds of collagenases and other proteins. On the

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1898 Y. SASAGAWA et al.

1 58
Arg-Glu-Leu-Glu-Glu-Leu ----Ser-Leu-Val-Tyr-Pro-Phe-Pro---------

'" 64

ttl 125
Me~fAla-prO-LYS-HiS-LYS-GIU se~t;eU-Thr-Leu-Thr-Asp-val----

A c
199 203
----Leu-Gly-Pro-Va I-Arg-Gly-Pro-Phe-Pro-Ile-II e-Val

Fig. 6. Cleavage Sites of p-Casein by the Collagenases.
Arrows indicate the cleavage sites by the collagenases from Achromobacter iophagus (A), Clostridium histolyticum (C), and Cytophaga sp. L43-1 strain (Cyt) purified in this report.

other hand, a broad peak eluted with high NaCI concen- protein from that of Achromobacter because N-terminal
tration contained an almost pure single enzyme having sequences of both proteins are different 9) (unpublished
collagenolytic activity and this enzyme was purified by results).
re-chromatography on DEAE-Sepharose. The purified en- When acid-sol.uble collagen, gelatin, and p-casein were
zyme had only collagenolytic activity but no caseinolytic used as substrates, similar pH and heat stabilities of the
activity. The molecular mass was estimated to be 120kDa. purified enzyme and also similar effects of inhibitors on the
Generally, bacterial collagenases are reported to be metal activity were observed (data not shown). Therefore, the
proteases with an optimal pH at neutral or slightly alkaline enzymatic reaction with collagen, gelatin, and p-casein
conditions that are inhibited by chelators such as EDTA. seems to be catalyzed by the same reaction site in the purified
The purified collagenase in this report was similar to enzyme.
previously reported collagenases but it was inhibited
partially by 0.1 mM and completely by 1 mM DFP. Bacterial References
collagenase inhibited by DFP has been reported,1) but this 1) M. Tajima, T. Iida, T. Kaminuma, M. Yanagi, and S. Fukushima, J.
enzyme was not inhibited by EDTA. Ferment. Bioeng., 72, 362-367 (1991).
The purified enzyme had no activity toward three 2) S. Sefter and E. Harper, The Enzymes, 3, 649-697 (1971).
synthetic oligopeptides, Pz-PLGPR, Z-GPGGPA, and 3) G. C. Reid, D. R. Woods, and F. T. Robb, J. Bacteriol., 142,447--454
Z-GPLGP which are hydrolyzed by the usual collagenases
4) J. R. Merkel and J. H. Dreishback, Biochemistry, 17, 2857-2863
at the bond ofX-G in sequences such as X-G-P. Commonly, (1978).
bacterialcollagenases recognize X-G-P in the collagen and 5) K. Hanada, T. Mizutani, M. Yamagishi, H. Tsuji, T. Misaki, and
cleave the X-G bond. It is assumed that the sequence in J. Sawada, Agric. Bioi. Chem., 37, 1771-1781 (1973).
collagen recognized by the purified enzyme from Cytophaga 6) A. Endo, S. Murakawa, H. Shimizu, and Y. Shiraishi, J. Biochem.,
102, 163-170 (1987).
is not X-G-P like Pz-PLGPR, Z-GPGGPA, and Z-GPLGP. 7) B. Peterkofsky, Methods Enzymol., 82,453--471 (1982).
Probably a longer sequence containing X-G-P is required 8) J. Fukushima, H. Takeuchi, E. Tanaka, K. Hamajima, Y. Sato, S.
to be recognized by this enzyme. Kawamoto, K. Morihara, B. Keil, and K. Okuda, Microbiol.
From the analysis of enzymatic products from p-casein Immunol., 34, 977-984 (1990).
by the purified enzyme from Cytophaga, two cleavage sites 9) H. Takeuchi, Y. Shibano, K. Morihara, J. Fukushima, S. Inami, B.
Keil, A. M. Gilles, S. Kawamoto, and K. Okuda, Biochem. J., 281,
(Pr063-Gly64 and Metl02-AlaI03) were demonstrated in 703-708 (1992).
p-casein (Fig. 6). These two cleavage sites are the same sites 10) H. Kojima, Japan Kokai Tokkyo Koho, 3-91478 (Apr. 17, 1991).
cleaved by the collagenase from Achromobacter iophagus. 21 ) 11) O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J.
The other cleavage sites have not been identified. However, Bioi. Chem., 193, 265-275 (1951).
from N-terminal sequence analysis of eight products from 12) B. Davis, Ann. N. Y. A cad. Sci., 121,404--427 (1964).
13) J. L. Hedrick and A. J. Smith, Arch. Biochem. Biophys., 126,155-164
p-casein and estimation of the molecular weights of these (1968).
products, it is assumed that the purified enzyme from 14) U. K. Laemmli, Nature, 227, 680-685 (1970).
Cytophaga cleaved p-casein at least five sites. Identification 15) C. Heussen and E. B. Dowdle, Anal. Biochem., 102,196-202 (1980).
of other cleavage sites is now under way by analysis of both 16) D. E. Garfin, Methods Enzymol., 182,459--477 (1990).
N-terminal and C-terminal sequences of the products. 17) W. H. Stein and S. Moore, J. Bioi. Chern., 176, 337 (1948).
18) E. Yoshida and H. Noda, Biochim. Biophys. Acta, 105, 562-574
Although the collagenase from Cytophaga is similar to (1965).
that from Achromobacter in properties including cleavage 19) E. Wunsch and H. G. Heidrich, Z. Physiol. Chem., 333, 149-151
sites in p-casein, the former did not hydrolyze small peptides (1963).
(Pz-PLGPR, Z-GPGGPA, and Z-GPLGP) unlike the latter 20) P. Matsudaira, J. Bioi. Chem., 262, 10035-10038 (1987).
21) A. M. Gilles and B. Keil, FEBS Lett., 65, 369-372 (1976).
enzyme. Also, the collagenase of Cytophaga is a different

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