Microbiology (1 994),140, 1687-1 695 Printed in Great Britain

~- -

Purification and characterization of an

extracellular serine protease from the
nematode-trapping fungus Arthrobotrys
oligospora
Anders Tunlid,’ Stefan Rosen,’ Bo Ek2and Lars Rask2

X u t h o r for correspondence: Xnders T d d . Tel: +46 46109614. Fa.;: +46 46104158.

e-ma 11 : A nde r s. T u n I1 d ((1 m IN oc k o I. I u .se.
1

1 Department of Microbial When grown in liquid cultures allowing the formation of nematode traps, the
Ecology, Lund University, fungus Arthrobotrys oligospora produced two extracellular proteases
Ecology Building,
Helgonavagen 5, 5-223 62 hydrolysing the chromogenic substrate Azocoll. The protease activity was
Lund, Sweden separated into two fractions (FI and FII) using anion-exchange
* Department of Cell chromatography. In bioassays, protease(s) present in FII immobilized the free-
Research, Swedish living nematode Panagrelhs redivivus indicating that the enzyme(s) might be
University of Ag r icu Itur al involved in the infection of nematodes. A protease designated PI1 was purified
Science, Box 7055, 5-75007
Uppsala, Sweden from FII to apparent homogeneity b y hydrophobic interaction and size-
exclusion chromatography, resulting in an approximately 15-fold increase in
specific activity. The purified enzyme was glycosylated, had a molecular mass
of approximately 35 kDa (gel filtration) and an isoelectric point of pH 4.6. PI1
immobilized P. redivivus in bioassays and hydrolysed proteins of the purified
cuticle. The enzyme hydrolysed several protein substrates including casein,
bovine serum albumin and gelatin, but not native collagen. Examination of
substrate specificity with synthetic peptides showed that PI1 readily
hydrolysed tripeptides with aromatic or basic amino acids includingN-benzoyl-
~-phenylalanyl-~-valyl-~-arginine-4-nitroanilide (Bz-Phe-Val-Arg-NA) and
succiny I-glycy I-glycy l-~-phen
y lalanine-4-nitroaniIide (Suc-GIy-GIy-Phe-NA).
Mono-peptides were hydrolysed a t considerably slower rates. PI1 had an
optimum activity between pH 7 and 9 and was susceptible to autodegradation.
PI1 was inhibited b y several serine protease inhibitors including
phenylmethylsulfonyl fluoride (PMSF), chymostatin and antipain. The protease
was N-terminally blocked, but the sequence of one internal peptide showed a
high homology with a region containing the active site histidine residue of the
subtilisin family of serine proteases.

Keywords : .4rthrobotg~.co/?jcgo.rpor~,extracellular serine protease, nematophagous fungus,

subtilisin, cuticle-degrading enzyme

INTRODUCTION cells (Jansson & Nordbring-Hertz, 1988). The molecular

mechanisms of this sequence are not well known, but, on
Nematophagous fungi infect their hosts through a se- the basis of studies of entomopathogens (St Leger, 1993),
quencc of events: attachment to the host surface, pen- it is likely that hydrolytic enzymes are involved in several
etration, followed by invasion and digestion of the host steps and processes during the infection : releasing nutri-
ents for pathogen growth, facilitating penetration by
. ... . . . . ... . ... . .. . ... , ... ..... . , . . . .. . . .. ... .. ... .. .. .. . ., , ... . ., , ., ., . . . ... . ..... . . . .. . .. . .. . .. . .. .... .. .. . ... ..
, , ,,
solubilizing the cuticle, inducing cytotoxic effects on the
Abbreviations: DCI, 3,4-dichloroisocoumarin; DIG, digoxigenin; E-64, nematode, digesting the host tissue, and inhibiting
~-trans-epoxy-succinyl-leucylamide-(4-guanidino)-butane; pCMB, p-
secondary invasion of micro-organisms.
hydroxymercuribenzoate; PU, proteolytic units; TFA, trifluoroacetic acid;
TLCK, tosyl lysyl chloromethyl ketone; TPCK, tosyl phenylalanyl chloro-
methyl ketone. Abbreviations for chromogenic peptides are defined in Among the hydrolytic enzymes proteases are of special
Methods. interest since the nematode cuticle is composed of
~~ -

0001-8912 0 1994SGM 1687

A. 'l'UN1,ID a n d O T H E R S
proteins, including collagens (Cox e t a/.,1981). Extra-
cellular proteases have also been detected a n d partly
characterized f r o m a few nematode-trapping fungi
(Schenk e t a/., 1980; Aswani & Jaffar, 1992), as well as
from endoparasites of cyst nematodes (Dackman e t a/.,
1989 ; Lopez-Llorca, 1990). However, the importance and
function of these proteases in the infection of nematodes
are n o t known.
B ' e recently demonstrated that the nematode-trapping
fungus A rthroDotr_vs oligovspora p rod u ce s ext r ace 11u 1a r se r ine
proteases when g r o w n in liquid medium allowing the
formation of nematode traps. Furthermore, incubating
the trap-bearing mycelium with inhibitors against serine
proteases significantly decreased the immobilization of
captured nematodes, indicating a n important function of
such proteases d u r i n g infection (Tunlid 8c Jansson, 1991).
I n this study, a n extracellular serine protease present in
the culture filtrates of A.oligospora has been purified a n d
characterized. The purified protease hydrolysed cuticle
proteins and immobilized free-living nematodes, indi-
cating that it may be a n important virulence factor for the
infection of nematodes by A.oligospora.
Cultures of A. oligospora and Panagrellus redivivus. A .
o/iCpo.pora Fres. (ATCC 24927) was maintained on cornmeal agar
(Difco) supplemented with 2 g I<H,HPO, 1-'. Liquid cultures
of A . o/z@spora were obtained by inoculating 4500 ml medium
with a water suspension of conidia prepared from a 2- to 3-
week-old culture grown on the cornmeal agar. Trap-containing
mycelium [T( +)] was grown in medium containing 0-01O/O soya
peptone (neutralized ; Oxoid) supplemented with 0.05 g phenyl-
alanine and 0.05 g valine 1-' (Friman e t a/., 1985). The cultures
were grown for 7 d at room temperature with constant aeration,
achieved by vigorous air bubbling from the bottom of the vessel
(Friman eta/., 1985). To compare proteases produced by hyphae
without traps [T(-)], the fungus was grown in the same
medium supplemented with a phosphate buffer to a final
concentration of 12 mM (pH 7.0).
The nematode P . redivivzds Id. (Goodey) was grown axenically in
a soya peptoneeliver extract medium (Nordbring-Hertz, 1977).
Purification of extracellular serine proteases. Typically,
mycelium from four vessels (4 x 4500 ml) was harvested by
filtering through a nylon mesh, and washed with 4 x 5 0 ml
10 mM Tris/HCl (pH 7.5).
(i) Binding to Q Sepharose. The pH of the culture filtrate was
adjusted to 7.5 by adding Tris/HCl to a final concentration of
10 mM. Approximately 20 ml of a suspension (80% in
0.01 M Tris/HCl pH 7.5) o f Q Sepharose (Pharmacia LKB
Biotechnology) was added per 4500 ml of culture filtrate and
placed on a magnetic stirrer at 4 "C for 15 min. After sedi-
menting for at least 3 h, the supernatant was discarded, and the
resin was transferred to a funnel with a Whatman glass
microfibre filter (GF/F). The Q Sepharose was washed with
5 x 10 ml 10 mM Tris/HCl (pH 7.5) and the proteases were
eluted with 50-100 ml 10 mM Tris/HCl (pH 7.5) and
0.5 hf NaCl (until no protease activity was detected). The extract
was concentrated by ultrafiltration (YM-3 membrane, cut-off
3 kDa; Amicon, W. R. Grace, Helsingborg, Sweden). Finally,
the extracts were desalted by passing through a Sephadex G-25
PD-10 column (Pharmacia) equilibrated with 10 mM Tris/HCl
(pH 7.5). This extract was designated crude protease extract.
1688
(ii) Anion-exchange chromatography. The crude protease
extract was applied to a Mono Q HR 5/5 column (Pharmacia)
connected to an HPLC system (Pharmacia LKB; pump 2248,
detector Variable Wavelength Monitor 2141, fraction collector
HeliFrac). Buffers used were: A, 10 m M Tris/HCl (pH 7.5); B,
10 mM Tris/HCl (pH 7.5) and 0.5 M NaCI. The gradient was
0.5 YOB for 5 min, 0.5 YOto 100 YOB in 35 min, and 100 YOB for
5 min with a flow of 1.0 ml min-'. Elution of proteins was
followed at 280 nm. Fractions of 1.0 ml were collected and
assayed for protease activity (Azocoll).
(iii) Hydrophobic interaction chromatography. Fractions con-
taining protease activity from the Mono Q column were pooled
and mixed with 3.4 M (NH,),SO, in a proportion of 3: 2 ( v / v )
sample :buffer. The sample was applied to a Phenyl Superose
HR 5/5 column (Pharmacia) connected to the Pharmacia HPLC
system. Buffers used were: A, 50 mM sodium phosphate buffer
(pH 7.0) and 1.7 M (NH,),SO,; B, 50 mM sodium phosphate
buffer (pH 7.0). The gradient was 0 'YO B for 5 min, 0 to 100 OO/ B
for 37 min, 100°/o B for 5 min, with a flow of 0-4 ml min-'.
Elution of proteins was followed at 280 nm. Fractions of0.4 ml
were collected and assayed for protease activity (Azocoll or
chromogenic peptides).
(iv) Sizeexclusion chromatography. Fractions containing
protease activity from the Phenpl Superose column were pooled,
concentrated by ultrafiltration (Filtron, cut-off 10 kDa, final
volume of sample < 0.35 ml), and applied to a Superdex
75 HR 10/30 size exclusion column (Pharmacia) connected to
the Pharmacia HPLC system. The buffer used was 0.100 M
NH,HCO, (pH 7.7) with a flow of 0.4 ml min-'. Elution was
followed at 280 nm. Fractions of 0.4 ml were collected and
assayed for protease activity (Azocoll or chromogenic peptides).
The enzyme purified from the Superdex column was designated
PII. Before biochemical characterizations, the ammonium
carbonate buffer was evaporated (Speedvac) and PI1 was
dissolved in the appropriate buffer.
Extraction of cell-bound proteases. The filtered mycelium of
A. oligospora was washed with 3 x 50 ml PBS (20 mM sodium
phosphate buffer pH 7.4 containing 0.15 M NaCl) and cell-
bound proteins were extracted using a homogenization
sonication procedure previously described (Tunlid e t a/., 1991).
The extract was concentrated by ultrafiltration (YM-3 mem-
brane), desalted (PD-10 column) and separated chromato-
graphically on the Mono Q column as described above.
Protease assays. Protease activity was measured in the extracts
and HPLC fractions using the chromogenic protein substrate
Azocoll (Sigma) (Chavira et al., 1984; Tunlid & Jansson, 1991).
Incubations were performed at 37 O C . The activities were
expressed as or as proteolytic units (PU) defined as the
increase of A,,,, ml-l min-l.
Proteolytic activity versus protein substrates and purified nema-
tode cuticle (see below) were assayed by mixing 10 pl PI1
(typically 0.01 pg pl-') with 100 pl 0.1 M Tris/HCl (pH 7.5)
containing 2.5 mg substrate ml-'. After appropriate periods of
incubation at 30 O C , the reaction was terminated by adding
200 pl5 O/O (w/v) trichloroacetic acid and the tubes were allowed
to stand for more than 1 h. Undigested protein was removed
by centrifugation and the released peptides were assayed by
measuring the absorbance at 280 nm. One unit is defined as the
increase in A,,,ml-' min-' (1 cm path length). The following
substrates were used : casein (BDH), bovine serum albumin
fraction V (Boehringer Mannheim), collagen (Sigma, type I
insoluble) and gelatin (Sigma). Fragments of cuticle were
prepared from the nematode P. redzviuzas by sonication and
treatment with 1 YO(w/v) SDS according to Cox e t al. (1981).
Before being used in the assays, the fragments were thoroughly
washed with 0.01 M Tris/HCl (pH 7.5) and lyophilized.

Hydrolysis of chromogenic peptide substrates was measured
using peptides conjugated to 4-nitroaniline. The following
peptides \'ere used : hr-benzoyl-~-arginine-4-nitroanilide.
(Bz-Arg-N , I ) , N-benzoyl-~~-lysine-4-nitroanilide.
Lys-NA), ,Y-benzoyl-~-tyrosine-4-nitroanilide
N -benzo!,l- L - phenylalanyl- L - valyl- L - arginine-4 -nitroanilide
( Bz- P h e - \' 21 1- A r g - N A ) , N-b e n z o y 1- p h e n y 1a 1an y 1- 1e u c y 1-
arginine-4-rii troanilide .acetate (Bz-Phe-Leu-Arg-Nh), suc-
cinyl-~-phenylalanine-4-nitroanilide (Suc-Phe-NA), succinyl-
~-alanyl-~-alanyl-~-phenylalanine-4-nitroanilide
Phe-NA), succinyl-~-alanyl-~-alanyl-~-alanine-4-nitroanilide
( Suc- h la- A I a - h la-N A), s u ccin y I-g1y c y 1-g1y cy 1- L - p hen y la la n i ne- column and conditions described above), and comparing the
4-nitroanilide (Suc-Gly-Gly-Phe-Nh). All were obtained from
Serva Fei n b iochemica, except Bz-Phe-Leu-A rg-N A, which
was from NovaBiochem. Stock solutions (10 mM) o f the
substrates \vere prepared in DMSO, and diluted with H 2 0 to
1.0 mM just before use. Ten microlitres of sample (typically
0.01 pg p1-l) was added to 70 pl 100 mM Tris/HCl buffer
(pH 7*5),and the reaction was started by adding 10 pl substrate
(final concentration 0.125 mM). After a suitable incubation time
(5-15 min) a t 25 O C , the reaction was stopped by adding 10 p1
50% ( v / v ) acetic acid, and the absorbance was measured at
405 nm. Specific activities are given in nmol 4-nitroaniline
liberated min-' (mg protein)-', using a molar absorption co-
efficient f o r 4-nitroaniline of 10500 M cm-' (Sarath e t al.,
1989).
The kinetic constants (K,,, k ~ , . ~were , ) determined from the initial
rate of hydrolysis of the substrate Bz-Phe-Val- Arg-N A at five
different concentrations by using Lineweaver-Burk plots.
Protease inhibitors and pH. The effects of inhibitors on the
protease actil-ity were examined by incubating 0.10 pg PI1 with
various inhihitors for 5 min at room temperature, before adding
the Tris bufter and the substrate Bz-Phe-Val-Arg-NA. The
proteolytic activity was measured as described above. The
following inhibitors were used : phenylmethylsulfonyl fluoride
(PMSF), tosyl lysyl chloromethyl ketone (TLCK), tosyl phenyl-
alanyl chloromethyl ketone (TPCK), A'-((S)-l-carboxy-iso-
pent y I) - c a r b:i m o y 1-a-(2- i min o h ex a h y d r o -4(J) - p y rim i d y I) - L - with Achromobacter iyticzls protease (Wako Pure Chemical
G 1y -Ph e-al) ( ch y mo st a t i n) , ((S)- 1-carbox y -2-p hen y let h y 1)-car-
bamoyl-ArS-Val-Arg-al (antipain), 3,4-dichloroisocoumarin
(DCI), p-hydroxymercuribenzoate (pCMB), L-tram-epoxy-suc-
cinyl-leucylaniide-(4-guanidino)-butane(E-64), pepstatin A,
1,10-phenanthroline, EDTA, and dithiothreitol (DTT). A11
inhibitors were obtained from Sigma, and they were prepared in
stock solutions and applied in concentrations within their
effective rang:", as given by Beynon & Salvesen (1989).
The effects o f pH on the proteolytic activity were investigated
by mixing enzyme extracts or purified PI1 with the Britton
Robinson universal buffer system at p H values between 3 and
12, followed h y protease assay.
Stability of PII. PI1 (10 pl, 0.02 pg PI-') was mixed with 70 pl
0.1 M Tris/HCl (pH 7.5) and incubated at 4, 20, 37 or 55 OC.
The protease activity was determined in the samples after 0, 1,
4 and 24 h using the peptide Bz-Phe-Val-Arg-NA.
PAGE, detection of glycoproteins and molecular mass.
Discontinuous SDS-PAGE was performed according to
Laemmli (1 970) using a Mini-Protean I1 electrophoresis unit
(Bio-Rad) and 12 YOrunning gels. Samples were diluted (1 : 1, by
vol.) with 50 mM Tris/HCl buffer (pH 6.8) containing 2 %
(w/v) SDS, 11.6 '/o (v/v) glycerol and 0.001 o/' (w/v) bromo-
phenol blue in the presence or absence of 5 % (w/v) D T T . The
samples were heated at 100 "C for 3 min. The molecular mass
was calculated using a Bio-Rad prestained SDS-PAGE standard
kit including phosphorylase b (106 kDa), bovine serum albumin
(80 kDa), ovalbumin (49.5 kDa), carbonic anhydrase
-.~
Serine protease from A . oligospora
(32.5 kDa), soybean trypsin inhibitor (27.5 kDa) and lysozyme
(18.3 kDa). Gels were stained with Coomassie Brilliant Blue.
HCI
Glycoproteins were detected on SDS-PAGE blots using a
acetate (Bz-
digoxigenin (DIG) glycan detection kit (Boehringer Mannheim)
(Bz-Tyr-NA),
according to the manufacturer's instructions. Approximately
2 pg PI1 was applied on the gels. Transferrin and creatinase
served as positive and negative controls for glycoproteins,
respectively .
(Suc-Ala-Ala- The apparent molecular mass of PI1 was determined by size-
exclusion chromatography (using the Superdex 75 HR 10/30
elution volume of the peak containing the protease activity with
those of bovine serum albumin (67 kDa), ovalbumin (45 kDa),
cx-chymotrypsinogen (25 kDa) and ribonuclease (13.7 kDa)
(Pharmacia low molecular weight standard kit).
lsoelectric focusing and chromatofocusing. Isoelectric fo-
cusing (Pharmacia Immobiline DryPlate, p H 4-7) was per-
formed in a Multiphore I1 system (Pharmacia) according to the
manufacturer's instructions with the temperature set at 10 "C.
The isoelectric point (PI) was estimated using a broad pI
calibration kit (pH 3-10, Pharmacia). The gel was stained with
Coomassie Brilliant Blue.
Chromatofocusing was performed with a Mono P HR 5/20
column (Pharmacia) connected to the Pharmacia HPLC system
(see above). The column was equilibrated with 0.025 M Bis-
Tris/HCl (pH 5.8) and the sample was eluted with Polybuffer 74
(Pharmacia) p H 3.5. The chromatography was performed at
21 "C with a flow rate of 1.0 ml min-l. Elution of proteins was
followed at 280 nm. Fractions of 1.0 ml were collected and
assayed for protease activity (using the peptide Bz-Phe-Val-
Arg-Nil).
Protein content. The protein content was determined by the
method of Bradford (1976) using bovine serum albumin as a
standard.
Protein cleavage. Purified PI1 (approx. 10 pg) was digested
Industries) in 2 M guanidine hydrochloride essentially as
described by Riviere e t al. (1991). The peptides were separated
by HPLC on a Brownlee C4 Aquapore column (2.1 x 30 mm).
Pumps were System Gold (Beckman) and the detector a photo
diode-array 990 (Waters). Buffers used for the reversed-phase
chromatography were: A, 0.1 % (v/v) trifluoroacetic acid
(TFA) in H,O; and B, 0.1 % TFA (v/v), 1 0 % H,O (v/v), 9 0 %
acetonitrile (v/v). The gradient was 2 to 62 % B in 60 min and
62 to 90 YOB in 3 min with a flow rate of 100 pl min-l. Elution
was followed at 214 nm and fractions were collected manually.
Peptide sequencing. Peptides were sequenced with a model
470A gas-phase sequenator (Applied Biosystems) equipped
with an on-line phenylthiohydantoin (PTH) amino acid analyser
(model 120, Applied Biosystems) according to the manu-
facturer's protocol.
Bioassay. P . redivivzls nematodes were washed thoroughly with
a 10 mM sodium phosphate buffer (pH 7.2) before being used in
the assays. Portions of the nematode suspension containing
20- 30 nematodes (100 pl) were transferred to microtitre wells
and 5-10 pl protease extracts were added. After incubating
(static) the wells at 20 "C for 20-22 h, the numbers of mobile
and immobile (i.e. with arrested movements) nematodes were
counted in a light microscope. The experiments were performed
with five parallels and repeated at least twice. Controls were
incubated without protease extracts. In one experiment, the
extract FII (from Mono Q) was boiled for 10 min before being
added to the nematodes. PMSF-treated FII was obtained by
1689
A. TUN1,ID a n d O T H E R S

The statistical significance of the differences in frequency of

0.7 mobile and immobile nematodes in treated versus control
samples was tested by ,yB analysis.
0.6

T
U

0
0-5
0.4
RESULTS
Production of serine proteases
qN0.3
When grown in dilute liquid medium allowing the
0.2 formation of trap cells [T(+)I, A.oligospora secreted very
0.1 low levels of proteins ( < 0.1 mg m1-l). Proteolytic
..I jo activity hydrolysing the chromogenic substrate Azocoll
5 10 15 20 25 30 35 40 45 was, however, detected in culture filtrates concentrated by
Time (min) ultrafiltration. Experiments showed that it was possible to
0.06 bind at least 9 5 % of this activity to the anion exchange
resin Q-Sepharose at pH 7.5.
0.05 1.5 n
Chromatography on Mono Q of the extracts eluted from
the Q-Sepharose separated the proteolytic activity into
T
W 0.04
0.03
1.0
W

d
two fractions, FI and FII (Fig. la). The proteolytic
activities in both FI and FII were briefly characterized.
Q 2 The activities in both fractions were completely inhibited
0.02 I" by the serine protease inhibitor PMSF, but not by the
0-5 metalloprotease inhibitor phenanthroline (FI, 107 % of
the activity in controls; FII, 101 YO),nor the aspartate
protease inhibitor pepstatin (FI, 118 '/o ; FII, 114 YO).The
proteolytic activities of FI and FII had their maxima at
5 10 15 20 25 30 35 40 45 50
Time (min) basic p H values (approx. 7.5-1 0.0). Further purification of
h

0.025 [ (c)
PII
i o.lo <s
W
qg 0.010 i . 0.05
0.005 1
10 20 30 40 50 60
Time (rnin)
Figrn1. Purification of the extracellular serine protease PI1 from
Arthrobotrys oligospora. (a) Anion-exchange chromatography
on Mono Q of a crude extracellular protease extract
concentrated from a culture filtrate of the fungus. The column
was equilibrated with Tris/HCI buffer (pH 7.5), and eluted with
a gradient of NaCl using a flow of 1 mi min-'. Fractions (1 ml)
were collected and assayed for protease activity using the
chromogenic substrate Azocoll. (b) Hydrophobic-interaction
chromatography on a Phenyl Superose column of fraction FII
recovered from the Mono Q column. The column was
equilibrated with 1.7 M (NH,),S0,/0.025 M sodium phosphate
buffer (pH 7.0), and eluted with a decreasing gradient of
1.7 M (NH,),SO, with- a flow of 0.4 ml min-'. Protease activity
was measured in 0.4 ml fractions using Azocoll. (c) Gel
chromatography (Superdex column) of the major protease
fraction (FII) from the Phenyl Superose column. The sample was
eluted with 0.100 M (NH,),HCO, buffer (pH 7-7) with a flow of
0.400 ml min-'. Fractions (0.4 ml) were collected and assayed for
protease activity using Azocoll.
adding PMSF to the extract (final concentration 1.0 mM),
incubating at room temperature for 30 min, and unbound
PMSF was removed by ultrafiltration (Filtron, cut-off 10 kDa).
T
FI and FII by size exclusion chromatography (Superdex
I
column) showed that the protease activity of FI eluted in
W
i one peak with an apparent molecular mass of 60 kDa and
0
FII in one peak of 35 kDa. Of the 4-nitroanilide peptide
substrates tested (see Methods), both FI and FII hydro-
.-
CL
lysed the tripeptide Bz-Phe-Val- Arg-NA best.
.->
CL
m FI and FII were also the major fractions containing
.-U proteolytic activity in extracts purified from fungal
s
-
0 cultures containing no traps [T(-)I. There was no
CL
L
significant differences in the ratio of the proteolytic
P activity of FI and FII between extracts recovered from
T( +) and T( -) cultures: the ratio of the maximum .iz,,,
of FI to maximum A,,, of FII in T ( +) was 0.334 (mean)
(SD, 0.288; n = 3) and in T( -) 0-202 (mean) (SD, 0.168;
n = 3).
Chromatography on Mono Q of cell-bound proteases
extracted from A. oligospora showed the presence of
protease activity in a fraction corresponding to FI of the
extracellular proteases, but no activity was detected
corresponding to FII. Chromatography of FI from the
cell extract on the Superdex column showed that the
protease activity eluted as a peak with an apparent
molecular mass of approximatrely 60 kDa.
Bioassays
Crude extracts from both T ( + ) and T(-) cultures
contained materials that immobilized the nematode P.
redivivzls in microtitre assays (Table 1). After chroma-
tography on Mono Q, the fraction FII immobilized the
nematodes, but FI failed to do so. N o significant effect
however, was observed after boiling or treating FII with
PMSF, indicating that the effect was due to the presence of

1690
 Serine protease from A.oligospora

Table 1. Immobilization of the nematode Panagrellus redivivus by protease extracts from A. oligospora
................................................................................................................................................................................................................................................................7

Extracts \rere incubated with nematodes in microtitre wells. After 20-22 h, the numbers of mobile and immobile nematodes were
counted in a light microscope.

Extract* No. of nematodes Statistics$

Mobile Immobile 2 P
~~

Crude T( + ) 1.36 4.4 254 87 (25.5)s 6.0 < 0.05

Crudc T( - ) 1-93 2.5 204 70 (25.5) 5.5 < 0.05
FI (hlono Q) 10.0 2.1 173 51 (22.8) 09 NS
FII (hlono Q) 1.2 10.4 131 175 (57.2) 50.7 < 0.001
FII, ~ ( J led
I 1.2 0-4 275 68 (19.8) 3.55 NS
FII, P3ISF 1.2 0.0 273 72 (20.9) 2.48 NS
PI I 1.0 17.5 70 232 (7643) 240 < 0.001
* Crude T( + ) and T ( -) extracts designate extracellular proteases concentrated from the medium using Q Sepharose. Mono Q FI and FII
are two pooled fractions containing protease activity purified by anion exchange chromatography; PI1 is the purified extracellular serine
protease (ci. Fig. 1).
j- PU, proteolytic units. For definition see Methods.
$ x 2 tests (c1.f. = 1) comparing the frequencies of mobilized and immobilized nematodes in treated versus control samples. NS, Not significant.
5 Percentagr (of the total numbers) of nematodes that were immobilized. Values in controls (without enzyme) varied between 17 and 22 YO.

Table 2. Purification of the major extracellular serine protease (PII) from A. oligospora
...............................................................................................................................................................................................................................

The extracts were recovered from trap-containing cultures [T(+)I. Crude extract was obtained by binding of proteases in culture filtrate
(4 x 4500 nil) to Q Sepharose, followed by elution in salt buffer and concentration by ultrafiltration. Crude extract was chromatographed
on Mono (2, followed by hydrophobic interaction and size exclusion chromatography as shown in Fig. l(a-c). T h e purification scheme
and calculations were repeated three times with similar results.

Purification step Total Total Specific Purification Yield

protein activity activity (fold) (%)
(mg) W)* (PU mg-I)

Crude extract 2.1 8 0-27 0.12 1 *00 100

Mono Q 0-33 0-22 0.67 5.47 82.4
Phenyl Superose 0.060 0.039 1.56 12.74 35.1
Superdex 0.01 3 0.03 1 1.88 15.32 9.29
~~

* PU, protcolytic units. Protease activity was assayed using the substrate Azocoll (see Methods).

serine proteases in FII. O n the basis of these data, we presence or absence of DTT, showed a major band at an
decided t o further purify the protease(s) present in FII. estimated molecular mass of approximately 40 kDa.
The amount of PI1 recovered from 4 x 4500 ml of culture
Purification of PI1 medium [(T+) or T( -)] varied and was relatively small,
A protease designated PI1 was purified from fraction FII typically between 3 and 30 pg. Growth in medium with a
by hydrophobic-interaction and size-exclusion chroma- higher concentration of soya peptone, in medium dialysed
tography (Fig. lb, c ; Table 2). The purification scheme to remove low molecular mass peptides and proteins (cut-
and calculations were repeated three times, giving a mean off 3 o r 12 kDa), or adding nematodes ( P . redivivus) to the
specific activity of 3-14 PU mg-' (SD, 1*68),a 15.5-fold mycelium did not considerably improve the amount of
increase in specific activity (SD, 2*0),and a yield of 15.5 % recovered PI1 (data not shown).
(SD, 16.7). Further purification of PI1 using chromato-
focusing (Mono P) or hydroxylapatite chromatography Molecular mass, isoelectric point and glycosylation
(Bio-Rad kITP cartridge) did not increase the specific Gel filtration of the purified PI1 showed that it corre-
activity of the protease. sponded to a protein with an apparent molecular mass of
PI1 was found to be almost homogeneous on SDS-PAGE approximately 35 kDa. The isoeiectric point of PI1 was
(Fig. 2). Electrophoresis of the purified protease, in the 4.6 on the Immobiline gel with the temperature set at

1691
X . T U N J A I Da n d O T H E R S

Table 3. Hydrolysis o f various protein substrates by the

serine protease PII, purified from A. oligospora

The maximum activity corresponding to 100 YOwas 2.79

(SD,0.21) (units x lo-')), for three replicates.

Substrate Relative
activity
(W
Casein (denatured)* 100
Bovine serum albumin 5.99
Gelatin 10.5
Collagen 0.42
Collagen (denatured)* 16.2
Nematode cuticlet 9.84

* Denatured by heating at 100 "C for 15 min.

t Fragments of cuticle prepared from the nematode P. rediiliiw.

Table 4. Substrate specificity o f the serine protease PI1

Fig. 2. SDS-PAGE of samples from the purification of PII. Lanes:
A, crude extract; B, Fraction FII from the Mono Q column (cf. Purified enzyme (0.10 pg) was incubated with the substrates
Fig. la); C, Fraction FII from the Phenyl Superose column (cf.
Fig. lb); D, PI1 from the Superdex column (cf. Fig. lc). Molecular
(1.0 mM) at 25 "C for 20 min. The maximum proteolytic activity
mass (kDa) of marker proteins is indicated a t the left of the corresponding to 100% was 1370 (SD, 85) nmol NA ml-' min-'
figure. (mg protein)-', for three replicates.

Peptide Relative
activity
10 "C. Using chromatofocusing at 21 OC, PI1 eluted as a
sharp peak at pH 4.4 in which the A,,,,and proteolytic (W
activitv coincided.
Bz- Arg-NA 1.4
PI1 was stained on blots using the D I G kit, indicating that Bz-L~s-NA 1.5
the protease was a glycoprotein. Bz-Tyr-N h 0.0
Suc-Phe-N A 1.6
Hydrolysis of protein substrates and nematode Bz-Phe-Val-Arg-N A 100.0
cuticle Rz-Phe-Leu- Arg-N A 28-9
Suc-Ala- Ala-Phe-N A 45.6
PI1 showed high hydrolytic activity against denatured Suc- Ala- Ala- Ala-N A 2.8
casein and moderate hydrolysis of BSA, gelatin, denatured Suc-Gly-Gly-Phe-N A 2.4
collagen and the preparation of cuticle from the nematode
P. redziiws (Table 3). The activity against native collagen
was very low.
The mean values ( n = 3) of K,, kcat, and catalytic
Hydrolysis of peptide substrates efficiency ( k C , J K m ) of PI1 for the hydrolysis of the
peptide Bz-Phe-Val-Arg-NA were 0.0962 mM (SD,
The substrate specificity of PI1 was investigated in more 0-0145),7.62 s-' (SD, 1*45),and 78900 M-' s-l (SD, 3200),
detail with a number of synthetic peptide substrates, respectively.
including those with one o r three amino acids. The
substrates were blocked at the N-terminus and bore the Inhibition and pH effects
chromogenic group at the C-terminus. The most com-
pletely cleaved substrate was the tripeptide Bz-Phe-Val- PI1 was completely inhibited by the serine protease
Arg-NA (Table 4). Substituting Val for Leu at position P, inhibitor PMSF (Table 5). The amino acid aldehydes
in this peptide (for notation see Schechter & Berger, chymostatin and antipain with a Phe and Arg residue,
1967) significantly decreased the rate of hydrolysis. PI1 respectively, were also inhibitory. Other serine protease
was also active against the tripeptide Suc-Ala-Ala-Phe- inhibitors, including the isocoumarin DCI, TLCK and
NA. Substitutions at position P, (-Ala-Ala-Ala-) or at P, TPCK were less inhibitory or completely ineffective. The
and P, (-Gly-Gly-Phe-) in this peptide significantly thiol reagent pCMB inhibited the activity of the enzyme.
decreased the rate of hydrolysis. The one-peptide sub- The cysteine protease inhibitor E-64, the aspartic protease
strates tested were not cleaved or were active at con- inhibitor pepstatin as well as DTT did not significantly
siderably lower rates. affect the activity of PII. Minor effects on the proteolytic

1692
 Serine protease from A.oligosporu

Table 5. Effects of various inhibitors on the protease 100 0

activity of PI1
.. .. ..... ., ., . .. ... . .... ., . .. .... . . .., . . .. .. . ... . .. .. . .. . . .. . . .. .. . .... . . .. . . .. . ., . .. , , .. . ... .. . . .. . . . .. . . .. . .. . .. . . . . ... . . .

T h e enzyme (0.10 pg) was incubated with the inhibitor at 25 "C

for 5 min prior to the addition of the substrate Uz-Phe-Val-Arg-
NA. The samples were incubated at 25 "C for 20 min.
Measurements were made in triplicate. T h e maximum proteolytic
activity in controls incubated without the inhibitors
(corresponding t o 100%) was 1590 (SD, 100) nmol N A ml-' min-'
(mg protein)- I .

Inhibitor Concn Enzyme activity as

(mM) % of control (SD)
~~

PhiSF 1.0 0.00 (0.00)

TLCK 0.0 10 86.2 (17.5)
TPCK 0.0 10 86.4 (10.7)
Chymosta tin 0.010 6.3 (3.9) incubation time (h)
A ntipai n 0.010 4.8 (4.6)
DCI 0.050 68.6 (4.4)
Fig. 3. Stability of Pi1 a t various temperatures. Approximately
pCMH 0.10 0.00 (0.00) 0.2 pg Pii was incubated in 0.100 M Tris/HCI buffer (pH 7.5) a t
E-64 0.010 92.8 (10.1) 4 "C (M), 20 "C (a),
37 "C (A)or 55 "C (0). The protease activity
Phenanthroline 1.0 98.9 (7.2) was assayed using the substrate Bz-Phe-Val-Arg-NA. The
EDTA 5.0 84.4 (7.5) maximum proteolytic activity in controls a t the start of the
incubation (corresponding t o 100%) was between 324 and
Pepstat in 0.010 92.2 (18.5)
480 nmol NA ml-' min-' (mg protein)-'. Bars indicate 1 SD
DTT 5.0 98.9 (7.2) (n = 3).

activity o f PI1 were observed after treating the enzyme G N G H'G T H C A G T I

with the metal chelator E D T A at 5 mM concentration. G N G H*G T H O A G T B A
PB E D L D
The proteolytic activity of PI1 had a broad p H optimum
at alkaline p H values (approx. 7-9) when using the PK S S R D
Britton-Robinson universal buffer system. Pri Q T D

Stability Fig. 4. Comparison of the amino acid sequence of a peptide

obtained by cleaving Pii with Achromobacter lyticus protease
The proteolytic activity of PI1 was sensitive to storage with those of the subtilisin-family enzyme CAI subtilisin
and the enzyme was subjected to rapid autolysis even at Carlsberg (from Nedkov et a/., 1985); PB, Protease B of
4 "C (Fig. 3). Saccharomyces cerevisiae (Moehle et a/., 1987); PK, Proteinase K
of Tritirachium album (Jany et a/., 1986); P r l , protease from
Metarhizium anisopliae (St Leger et a/., 1992). identical amino
Sequencing acids are boxed. The asterisk denotes the active-site His residue.

The N-terminal of PI1 was blocked. Cleavage with the ,4.

hticzts protease, however yielded a peptide with a sequence in these fractions was similar as examined by synthetic
showing high homology with the active site histidine
peptides, but the protease(s) present in FI had a higher
residue o f the subtilisin family of proteases (Fig. 4).
molecular mass than those in FII, and analyses of cell
extracts indicated that protease of FI, but not FII, was
DISCUSSION partly bound to the mycelium of the fungus. Furthermore,
in bioassays the protease present in FII immobilized the
In preliminary experiments extracellular proteases pro-
nematode P. redi~ivus,but that in FI did not.
duced from A. oligospora mycelium with traps [T( +)]
were comp;ired with those from vegetative mycelium The sensitivity of the enzyme PII, purified from FII, t o
[T( -)I. Analysis of proteases in culture filtrates using the inhibitors PMSF, antipain and chymostatin indicated
anion-exchange chromatography (Mono Q), peptide sub- that it was a serine protease. T h e relatively low molecular
strates (unpublished data), o r results from previous work mass (approx. 35 kDa) and isoelectric point (pH 4.6) of
using protease inhibitors and substrate gel electrophoresis PII, as well as the high p H optimum are common for
(Tunlid 8c Jansson, 1991) have so far not revealed any fungal serine proteases (North, 1982; Iiominami e t a/.,
differences in the production of extracellular set-ine 1981a; Ogrydziak & Scharf, 1982), including several
proteases between T( +) and T ( -) cultures. Both T( +) serine proteases purified from entomopathogens (Bi-
and T( - ) cultures contained two fractions of extracellular dochka & Khachatourians, 1987; St Leger ef d/., 1987a),
serine protease activity (FI and FII). T h e protease activity as well as from the nematophagous fungus 17erticillizmz

1693
\ . ’I’LIKJAIDa n d O T H E R S
.rztch/asporizutz (Lopez-Llorca, 1990). Several peptides have
been sequenced from PI1 (unpublished data). The re-
ported amino acid sequence from a peptide recovered
after A.hticas cleavage contained a sequence showing
high homology with the region of the active site histidine
residue of the subtilisin family of serine proteases (cf. Fig.
4). The total inactivation of PI1 by PMSF but not by the
trypsin and chymotrypsin inhibitors TLCK and TPCK
further supports the view that PI1 is a protease of the
subtilisin family (Kominami e t a/., 1981a; Ogrydziak &
Scharf, 1982; Ebeling e t a/., 1974; Ottesen & Svendsen,
1970).
The total loss of activity o f PI1 caused bypCMB indicates
that the protease requires a reactive sulfhydryl group. PI1
was not, however, inhibited by the cysteine protease
inhibitor E-64 nor the reducing agent DTT, which
suggests that -SH groups are not localized in the active
site of PII. The effects of pCMB may reflect the close
proximity of a cysteine residue to the active site of the
enzyme. In such cases it has been observed that binding o f
pCMB can interfere with the binding of the substrate to
the active site (Bai & Hayashi, 1979).
Studies of the various peptides showed that PI1 hydro-
lpsed both trypsin (Bz-Phe-Val- Arg-NA) and chymo-
trypsin (Suc-Xla-Ah-Phe-NA) substrates. Mixed trypsin/
chymotrypsin activities have previously been de-
scribed for at least one other fungal serine protease of the
subtilisin type, proteinase B from bakers’ yeast (Komi-
nami e t a/., 198lb). St Leger e t al. (1987b) demonstrated
the presence of acid serine proteases in several entomo-
pathogens with preference for the substrate Bz-Phe-Val-
Arg-NA but cleavage of chymotrypsin substrates was
not reported. The differences in the action o f PI1 on
tripeptides and monopeptides, and the effects of sub-
stitutions at position P, and P,, indicate that the active
site of PI1 extends over several amino acids and that
the reactivity of a particular bond (Pt-P;) is not only
dependent on P, and P; but also on the residues at
neighbouring subsites (cf. Schechter & Berger, 1967).
The comparative non-specificity of PI1 accounts for it
being a general protease with activity against a range of
protein substrates.
Previous experiments treating A.oligospora with various
protease inhibitors have indicated a role of fungal serine
protease for the immobilization of nematodes captured by
A.oligospora (Tunlid & Jansson, 1991). The most effective
inhibitors used in these experiments (PMSF, antipain and
chymostatin) were also the most potent inhibitors of PII.
The immobilization of free-living nematodes by PI1
further indicates that the purified serine protease can be
involved in the infection of nematodes by A.oligospora.
O n the basis of microscopic investigations it has been
proposed that the immobilization of nematodes by A.
oligospora occurs during the penetration of the cuticle
(Nordbring-Hertz e t a/.,1986). This is a possible mech-
anism for the immobilization effect of PI1 on the nematode
P. rediuiuiu, since the purified enzyme hydrolysed proteins
in fragments of cuticles prepared from this nematode.
Notably, PI1 did not degrade native collagen, which is
supposed to be the major class of protein in the cuticle o f
1694
nematodes (Cox e t a/., 1981). However, it has been shown
that the cuticle of Panagrelhs sihsiae contains proteins that
are susceptible to proteases such as papain (Martin r f a / , ,
1986), which is a rather nonspecific cysteine protease with
a preference for Arg or Lys at the subsite P, (Cleveland e t
a/., 1977). In another study it has been demonstrated by
scanning electron microscopy that adding papain to plant
parasitic nematodes can produce structural changes in the
cuticle (Miller & Sands, 1977). At this point, however, the
possibility cannot be excluded that the enzyme has a more
specific cytotoxic effect and that it can be transported
inside the nematode during pumping of the feeding
apparatus.
The relatively broad substrate specificity of PI1 and the
production of this enzyme by vegetative mycelium
indicates that PI1 has two independent functions, one
during saprophytic growth and one during the infection
of nematodes. Such a dual role has been suggested for the
well-characterized serine protease Prl purified from
Afetarhixizmz anisopliae (St Ixger, 1993). Notably, results
from the inhibitor experiments on the fungal mycelium
indicate that the active serine protease is present before
contact with the nematode (Tunlid e t a/., 1991). Other
proteases including collagenases might be synthesized
after contact with the cuticle. Induction of protease
activity in A.oligospora by peptone extract was demon-
strated by Aswani & Jaffar (1992), but the enzyme was
not purified and characterized. Collagenase(s) can be
induced in the nematophagous fungus Arthrobotys amero-
spora by growing the fungus in liquid medium containing
collagen as a substrate (Schenk e t a/., 1980). This enzyme
was not characterized in detail, but its sensitivity towards
inhibitors showed that it was not identical with the
purified PII.
There is an increasing demand to use nematophagous
fungi or their products as biological control agents for
plant and animal parasitic nematodes (Icerry, 1990). There
are some data indicating that hydrolytic enzymes in-
cluding proteases and collagenases can be added to soil to
control plant parasitic nematodes (Miller & Sands, 1977 ;
Galper e t a/., 1990). Isolation and characterization of
hydrolytic enzymes from nematophagous fungi involved
in the infection process can be used to develop more
effective preparations or strains of organisms to be used
for biological control of parasitic nematodes.
ACKNOWLEDGEMENTS
This study was supported by grants from the Swedish Council
for Forestry and Agricultural Research, the Banks of Sweden
Tercentary Foundation, and Carl Tryggers Stiftelse.
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Received 6 January 1994; revised 25 February 1994; accepted 10 March
1994.
1695