Purification and characterization of a digestive alkaline protease from the larvae of Spilosoma obliqua.

Archives of Insect Biochemistry and Physiology 51:1–12 (2002)
Purification and Characterization of a Digestive
Alkaline Protease From the Larvae of Spilosoma obliqua
Adil Anwar1* and M. Saleemuddin2
A digestive protease from Spilosoma obliqua (Lepidoptera: Arctiidae) fifth instar larval guts was purified and characterized.
The protease was purified using ammonium sulfate fractionation, ion-exchange chromatography, and hemoglobin-sepharose
affinity chromatography. The purification procedure resulted in a 37-fold increase in the specific activity of the protease.
Protease thus obtained was found to be electrophoretically pure under native and denaturing conditions. The purified protease
had a molecular mass of 90 kDa as determined by gel filtration, and a pH optimum of 11.0. The purified protease optimally
hydrolyzed casein at 50°C. A Km of 2 ´ 10–6 M was obtained using BApNA as a substrate for the purified alkaline protease.
The ability of S. obliqua protease and bovine trypsin to hydrolyze various synthetic substrates (BApNA, BAEE, and BAME), and
the inhibition patterns of S. obliqua and bovine trypsin with “classical” trypsin inhibitors are also reported. Arch. Insect
Biochem. Physiol. 51:1–12, 2002. © 2002 Wiley-Liss, Inc.
KEYWORDS: Spilosoma obliqua; larval gut protease; purification; synthetic substrate hydrolysis; protease inhibitors
INTRODUCTION
Spilosoma obliqua, commonly known, as “Bihar
hairy caterpillar” is a polyphagous pest that causes
damage by defoliating many economically important crops, including grain legumes. S. obliqua is a
pest in many Asian countries including India, particularly in the North and Northwest regions of
India. As the larvae of S. obliqua enter the third
instar stage of their development, it is difficult to
control them, due to the presence of long hairy
tufts all over their body, which in turn make them
resistant to many of insecticides. As with many
other species of insect pest, S. obliqua tends to develop resistance to a wide range of pesticides. It is
well established that proteolytic enzymes in insect
guts are primarily responsible for the breakdown
of leaf proteins. Proteins are digested in insects by
enzymes that are active in slightly alkaline to
slightly acidic pH (Applebaum, 1985).
Lepidopteran larvae have been extensively studied because of their impact on economically important plants. The digestive enzymes of these
larvae are of interest both as a target for insect control and also because of their unusual ability to
function in alkaline microenvironment in the lepidopteran guts pH 10.0–12.0 (Christeller et al.,
1992). This observation substantiates that proteases
from the lepidopteran guts are finely designed to
work optimally in alkaline conditions of the guts
(Applebaum, 1985). It is well documented that
protein digestion in lepidopteran larvae is medi-
1
Department of Pharmaceutical Sciences, Program in Molecular Toxicology, School of Pharmacy, University of Colorado Health Sciences Center, Denver, Colorado
2
Department of Biochemistry, Faculty of Life Sciences and Institute of Biotechnology, Aligarh. M. University, Aligarh, India
Abbreviations used: BApNA = N-a-benzoyl-DL-arginine p-nitroanilide; BAME = N-benzoyl-L-a-arginine methyl ester; BAEE = N-benzoyl-L-a- ethyl ester;
DEAE-cellulose = Di ethyl amino ethyl cellulose; TLCK = N-a-p-tosyl-L-lysine choloromethyl ketone; TPCK = N-tosyl-L-phenyl alanine choloro methyl ketone;
SBTI = soybean trypsin inhibitor; Hb = hemoglobin; SDS = sodium dodecyl sulfate; PAGE = polyacrylamide gel electrophoresis.
*Correspondence to: Dr. Adil Anwar, Department of Pharmaceutical Sciences, Box C-238, School of Pharmacy, UCHSC, 4200 E.9 Th Ave., Denver, CO 80262.
E-mail: adil.anwar@uchsc.edu
Received 17 January 2002; Accepted 6 April 2002
© 2002 Wiley-Liss, Inc.
DOI 10.1002/arch.10046 Published online in Wiley InterScience (www.interscience.wiley.com)
2
Anwar and Saleemuddin
ated by the concerted action of digestive enzymes
in the larval guts (Terra, 1990), particularly trypsin
(EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1),
which are the primary digestive proteases in most
insect groups (for a review see Terra et al., 1996).
Since proteolysis is an essential part of food digestion in insects, studies on insect proteases are
important. Disruption of protein digestion by protease inhibitors represents an alternative approach
to pest control (for review see Reek et al., 1997).
However this requires characterization of proteases
that are present in the larval guts and the way they
interact with various protease inhibitors (Ortego
et al., 2000).
In earlier studies, we have reported the potential of gut hydrolytic enzymes from S. obliqua, in
various bio-formulations (Anwar and Saleemuddin,
1997, 2000). Enzymes that are active in extremes
of alkaline pH are industrially important for many
applications including detergent, dairy, and leather
industries (for a review see Anwar and Saleemuddin, 1998). More recently, we have reported
some biochemical properties from the gut homogenate from S. obliqua larvae in relation to various
developmental stages, food rationing, and starvation (Anwar and Saleemuddin, 2001).
To date the larval protease of S. obliqua gut has
not been purified and characterized. We report the
results obtained for the purification of a protease
isolated from the fifth instar larvae of S. obliqua.
The biochemical and kinetic parameters of the purified alkaline protease from S. obliqua were investigated. Comparision between the purified alkaline
protease from S. obliqua and bovine trypsin, in hydrolyzing various synthetic substrates like BApNA,
BAEE, and BAME and the inhibition patterns of
bovine trypsin and the larval protease with “classical” trypsin inhibitors are also reported.
MATERIALS AND METHODS
Chemicals
Vitamin-free casein was purchased from ICN.
Bovine serum albumin, hemoglobin, sodium lauryl sulfate, Coomassie Brilliant Blue (G-250 and
R-250), Tris hydroxy amino methane, and all the
synthetic substrates were purchased from Sigma
Chemical Co (St. Louis, MO). The Ion-exchanger
DE-52 (DEAE) was a product of Whatman (Madistone, Kent, UK). Enzyme inhibitors were purchased from E. Merck (Germany). All other
chemicals used were of the highest purity available commercially. Glass distilled water was used
in all the experiments described in the study.
Larval Rearing
S. obliqua larvae were bred in rearing jars in a
climate-controlled incubator maintained at 28 ±
1°C and 60% relative humidity. The larvae were
fed on fresh castor bean leaves. Fifth instar larvae
were used unless mentioned otherwise.
Linking Hb to sepharose 4B. For affinity chromatography on Hb-sepharose matrix, sepharose 4B was
activated as described by Porath et al. (1967).
Briefly, 10 g sepharose was washed thoroughly with
distilled water in a sintered glass funnel. The gel
was sucked dry and suspended in 10 ml of distilled water and 10 ml of 2.0 M Na2CO3, was added
and mixed thoroughly by placing on a magnetic
stirrer. Two grams of CNBr dissolved in 2.0 ml of
acetonitrile was added to the sepharose. and was
mixed thoroughly for 10 min. The gel was then
transferred to a glass sintered funnel and was
washed again with 0.1 M bicarbonate buffer, pH
8.5, distilled water, and followed by the buffer
again. The activated sepharose was dried and resuspended in 10 ml of 0.1 M bicarbonate buffer
pH 8.5. Hb (5–25 mg/g gel) was dissolved in buffer
and stirred with CNBr activated sepharose for 24
h at 4°C. The sepharose matrix with bound Hb
was separated from the unbound by centrifugation
and the protein in the supernatant was quantitated
in order to determine the bound Hb. The Hb
bound matrix was thoroughly washed in 0.1 M bicarbonate buffer pH 8.5 containing 1.0 M NaCl,
and the washed suspension was treated with 0.1
ml of 98% ethanolamine for 2 h at 4°C. The Hb
matrix was washed with bicarbonate buffer, pH 8.5,
containing 1.0 M NaCl, distilled water, and 0.1M
sodium acetate buffer, pH 4.5. The Hb bound
Archives of Insect Biochemistry and Physiology
Purification of Protease From S. obliqua Larvae
sepharose was then re-suspended in an equillibrating buffer (0.1 M Tris-HCl, pH 8.0). Affinity
chromatography was carried out as described by
Smith and Turk (1974).
3
Active fractions were pooled, concentrated, and
were used for further studies described in this
manuscript.
Gel Electrophoresis
Purification of the Protease
Crude enzyme extract preparation. Fifth instar larvae were dissected and their guts were removed using forceps. Guts were then collected in ice-cold
normal saline, and were squeezed out by gently
tapping the intestines. The gut contents were then
centrifuged at 4°C at 4,000 rpm for 20 min and
the supernatant was used as an enzyme source for
further purification.
Ammonium sulfate fractionation. Crude gut extract
that was obtained after the centrifugation was subjected to ammonium sulfate fractionation (0–
60%). Saturation was carried out in 0.1 M Tris-HCl
buffer, pH 8.0, at 4°C for 8 h to ensure complete
precipitation. The resulting precipitate was dissolved in 0.1 M Tris-HCl buffer, pH 8.0, and dialyzed extensively against the same buffer.
DEAE-cellulose chromatography. The dialyzed enzyme solution obtained from the ammonium sulfate fractionation was applied on a DEAE-cellulose
column (2.6 ´ 30 cm), equilibrated in 0.1 M TrisHCl buffer, pH 8.0. The column was thoroughly
washed to remove the unbound protein. The
bound protein was eluted with a gradient of 0.0–
1.0 M NaCl in the same buffer, and 4-ml fractions
were collected at a flow rate of 40 ml/h.
Hb-sepharose affinity chromatography. The dialyzed
active and pooled fractions of the S. obliqua protease obtained after DEAE-cellulose chromatography were applied onto Hb-sepharose matrix and
allowed to bind for 4 h at 4°C. The matrix was
packed in a 1.5 ´ 10 cm column, and was equilibrated with 0.1 M Tris-HCl buffer, pH 8.0, and the
flow rate was adjusted to 20 ml/h. The column
was washed thoroughly to get rid of the unbound
protein, the bound protein was eluted using 1.0
M NaCl in 0.1 M Tris-HCl buffer, pH 8.0. Fractions (2 ml) were collected and monitored for protein content according to Bradford (1976) and
assayed for the caseinolytic activity (Kunitz, 1947).
September 2002
Polyacrylamide gel electrophoresis (PAGE and
SDS-PAGE) was performed according to the
method described by Laemmli (1970) using trisglyine buffer pH 8.3. Routinely a 7.5% acrylamide
gel was used. Stock solutions of 30% acrylamide
containing 0.8% bisacrylamide, 1.5 M Tris (pH 8.8
and 6.8) in absence of SDS for native gel or in
10% SDS for denaturing gels were prepared. Bromophenol blue and 10% glycerol in absence of
SDS and b-mercaptoethanol was used as tracking
dye for native gels, while the denaturing gels had
SDS and b-mercaptoethanol in the tracking dye.
Enzyme Assays
Determination of protease activity. Proteolytic activity was measured according to the method of
Kunitz (1947) with the following modifications
using 2% casein as a substrate. For this purpose,
0.5 ml of casein was prepared in 0.1 M phosphate
buffer of appropriate pH and was incubated with
equal volumes of suitably diluted enzyme. After
incubation at 37°C for 15 min, the reaction was
terminated by the addition of 0.5 ml of 20% TCA
and the acid soluble peptides were quantified using Folin-phenol reagent (Lowry et. al., 1951). Appropriate blanks were used in all the experiments.
One unit of protease is referred as the amount that
caused the formation of 1 mg of TCA soluble Lowry
positive material per minute under the experimental conditions.
Determination of trypsin like activity. Peptidase activity was measured by the method as described
by Hayakawa et al. (1980). The substrate solution
was prepared by dissolving 43.5 mg of BApNA hydrochloride in 1 ml of DMSO and diluting the solution in 0.1 M Tris-HCl buffer, pH 8.0, to 100 ml
(care was taken to ensure that all the BApNA was
dissolved prior to the addition of buffer). To 0.2
ml of appropriately diluted enzyme, 0.8 ml of 0.1
4
Anwar and Saleemuddin
M Tris-HCl buffer pH 8.0 was added, followed by
2.0 ml of the substrate solution. After 30 min of
incubation at 40°C the reaction terminated by the
addition of 1 ml of 30% (v/v) acetic acid. The
quantity of the liberated p-nitroanilide was followed spectrophotometrically at 410 nm. The
amount of the substrate hydrolyzed by the enzyme
was calculated using the molar extinction co-efficient of 8,800 M–1 cm–1 for p-nitroanilide at 410
nm. Under the experimental conditions, one enzyme unit was equivalent to change in O.D. at 410
nm. One unit of peptidase activity is the amount
that caused the hydrolysis of 1 mmol of BApNA
per minute.
Determination of esterase activity. The esterase activity was determined using BAEE and BAME as a
substrate. The reaction mixture in a total volume
of 3.0 ml contained 50 mg of BAEE/BAME in 0.1
M Tris-HCl, pH 8.0, containing 0.05 M CaCl2 and
appropriately diluted enzyme. Alterations in the
absorbency at 253 nm were followed at 30°C on a
Beckman DU spectrophotometer. The amount of
substrate hydrolyzed by the enzyme was calculated
using the molar extinction coefficient of 808 M–1
cm–1 at 253 nm.
One enzyme unit is equivalent to the amount
that causes hydrolysis of 1 mmol substrate per
minute.
Protein estimation. Protein analysis of the gut contents was carried out according to Bradford (1976).
This method was selected in view of the presence
of high concentration of plant phenols in the
preparation and the tolerance of this method towards the plant polyphenols (Saleemuddin et al.,
1980). Suitably diluted gut extract/purified enzyme
containing 10–100 mg protein in a volume of 100
ml was pipetted out in a test tube, and was made
up to 1.0 ml with distilled water. Five milliliters of
the reagent (0.1% w/v Coomassie Brilliant blue G250, 4.7% absolute alcohol and 75% w/v orthophosphoric acid) was added to the test tubes and
the contents were mixed by vortexing; absorbency
was measured at 595 nm. A calibration curve was
prepared using BSA as a standard.
RESULTS
Isolation and Purification of S. obliqua Protease
The crude extract of the larval gut contents of
S. obliqua contained dark brown pigments presumably derived from the castor leaves on which the
larvae feed. In order to remove these pigments, the
gut homogenate was subjected to ammonium sulfate fractionation (0–60% saturation) in 0.1 M TrisHCl buffer, pH 8.0. The precipitate was dissolved
in the same buffer and extensively dialyzed. Ammonium sulfate fractionation resulted in a threefold purification and a 76% yield (Table 1). The
enzyme’s solution obtained after the ammonium
sulfate fractionation was purified by DEAE-cellulose chromatography, and fractions (4 ml) were
collected. Suitable aliquots were removed and
monitored for caseinolytic activity and protein content. At this stage, almost all the pigments were
removed and this purification step resulted in a 7fold purification and a yield of 35%. Active fractions were pooled from the first peak (shown by
arrows, Fig. 1), concentrated and dialyzed against
0.1M Tris-HCl, pH 8.0, at 4°C. The pooled and
dialyzed protease activity obtained after the DEAEcellulose chromatography was further purified by
affinity chromatography on Hb-sepharose. Frac-
TABLE 1. Purification of S. obliqua Protease
Purification stepa
Crude gut contents
0–60% (NH4)2So4
DEAE-chromatography
Affinity-chromatography
Total volume
(ml)
Total activity
(units/ml)b
Total protein
(mg/ml)c
Specific activity
Purification fold
Yield (%)
150
36
19
26
79,200
60,480
19,500
12,116
562
113
20.9
2.34
140.8
533
933
5177
1.0
3.0
7.0
37.0
100
76
35
15
a
All steps were carried out at 4°C.
One unit of protease is equivalent to the amount that resulted in the formation of 1 mg TCA soluble Lowry positive material.
c
Determined according to Bradford (1976).
b
Archives of Insect Biochemistry and Physiology
Purification of Protease From S. obliqua Larvae
5
Fig. 1. DEAE-Cellulose column chromatography. Ammonium sulfate precipitated gut protein obtained from 150 ml of gut contents, were
dissolved in 36 ml of 0.1 M Tris-HCl buffer
pH 8.0. Sample was dialyzed extensively against
the same buffer at 4°C. Sample was then loaded
on a 2.6 ´ 30 cm column. After allowing the
protein to bind on the ion-exchanger for 60
min, the column was washed extensively with
0.1 M Tris-HCl buffer, pH 8.0. The bound protein was subsequently eluted with a linear gradient of 0–1.0 M NaCl in 0.1 M Tris-HCl buffer,
pH 8.0. Fractions were collected and were assayed for protease activity and protein content.
Arrows indicate the pooled fractions used for
further purification.
tions (2 ml) were collected and the elution profile obtained is shown in Figure 2. The proteolytic
activity was eluted as a single sharp peak with a
37-fold purification and a yield of 15%. The purification procedure was repeated six times and com-
parable values were obtained each time. The only
exception was that of an additional small peak,
which was proteolytically active, and was obtained
twice during the DEAE-cellulose chromatography.
However, the fractions from the second peak were
Fig. 2. Hb-sepharose affinity chromatography.
Fractions containing the protease activity obtained
after the DEAE-cellulose chromatography were
pooled, concentrated, and dialyzed against 100
mM Tris-HCl buffer, pH 8.0, and applied on a 1.5
´ 10 cm Hb-sepharose column. The column was
washed thoroughly to remove weakly adsorbed
proteins. Protease bound on the column was
eluted in 0.1M Tris-HCl buffer, pH 8.0, containing 1.0 M NaCl. The column was eluted at 20 ml/
h and fractions (2 ml) were collected. Fractions
were assayed for activity and protein content as
described in Materials and Methods.
September 2002
6
Anwar and Saleemuddin
not used in the experiments described in this
present study. The purification process was also
monitored by polyacrylamide gel electrophoresis
(Laemmli, 1970) under native and denaturing conditions. The purified protease was found to be homogenous under both the conditions migrating as
a single band (Fig. 3A,B). The gels were also subjected to silver staining to check the purity of the
protease and no additional bands could be detected in the gels (data not shown).
Molecular Weight Determination:
The molecular weight of the purified alkaline
protease was determined by SDS-PAGE (Fig. 3B).
The purified alkaline protease migrated as a single
band when subjected to electrophoresis in the presence of SDS under reducing conditions. The purified protein migrated between myosin (220 kDa)
and b-galactosidase (116 kDa) markers. The mo-
Fig. 3. Native PAGE (A) and SDS-PAGE (B) of the purified alkaline protease. Electrophoresis of the purified alkaline protease was carried out using a 7. 5% gel as
described in Materials and Methods according to Laemmli
(1970). A: Lane A: Crude gut extract. Lane B: 0–60%
lecular weight of the purified alkaline protease was
calculated from the mobilities of the marker proteins by the procedure reported by Weber and
Osborn (1969). The mobilities were plotted against
the logarithm of molecular weight. The least square
analysis of the data indicated a linear relationship
between logM and relative mobility (Rm) and the
molecular weight of the purified protease was estimated to be 120 kDa as determined by SDS-PAGE.
Molecular weight of the purified alkaline protease was also determined by gel chromatography
using sephadex G-100 (Andrews, 1964). For gel filtration studies, a column (2.1 ´ 50 cm) was equilibrated with 0.05 M Tris-HCl pH 8.0. The column
was calibrated with b-galactosidase, hemoglobin,
ovalbumin, soybean trypsin inhibitor, cytochromec, and insulin. About 1.5 mg of the purified enzyme was chromatographed on the same column
and the elution was performed with the equilibrated buffer. Fractions (2 ml) were collected at a
(NH4)2So4 fraction. Lane C: DEAE-cellulose fractions.
Lane D: Affinity chromatography fractions. B: Lane Mw:
Molecular weight markers (kDa). Lane A: Purified alkaline protease.
Archives of Insect Biochemistry and Physiology
Purification of Protease From S. obliqua Larvae
7
speed of 15 ml/h and were analyzed for protein.
Measuring the BApNA lytic activity monitored the
elution of the alkaline protease. The elution volume of the marker proteins was noted and the plot
of ve/vo vs. logarithm of molecular weight, according to the procedure of Andrews (1964) (Fig. 4).
The molecular weight of the alkaline protease was
found to be about 90 kDa.
Enzymatic Properties
Influence of pH. The effect of pH on the purified
protease from S. obliqua is shown in Figure 5. The
proteolytic activity increased almost linearly with
the pH between pH 7.0–11.0 when casein was used
as a substrate for determining the pH optima of
the purified protease from the larval guts. This was
followed by a rapid decrease in activity at pH 12.0.
Like most of the proteases from other lepidopterans (Ahmad et al., 1976, 1980; Christeller et al.,
1992), the alkaline protease from S. obliqua ap-
Fig. 4. Determination of molecular weight of the purified alkaline protease by gel filtration. The void volume
(Vo) of the column was determined using blue dextran.
The ratio of elution volume (Ve) to (Vo) was plotted against
the logarithm of molecular weights of the marker proteins:
(1) b-galactosidase 116,00, (2) hemoglobin 66,000, (3)
ovalbumin 45,000, (4) soybean trypsin inhibitor 20,100,
(5) cytochrome-c 12,400, (6) insulin 6,000. Arrow indicates the position of the alkaline protease from S. obliqua.
September 2002
Fig. 5. Effect of pH on the proteolytic activity of the purified S. obliqua alkaline protease. The reaction mixture in
a total volume of 1.0 ml containing 5 mg of protein in
100 mM phosphate buffer of the indicated pH and 10 mg
of casein was dissolved in buffer of the desired pH. Incubation of the reaction mixture was carried out for 15 min
at 37°C. Reaction was terminated by the addition of 0.5
ml of 20% TCA. Proteolytic activity was determined as
described in Materials and Methods. Buffers used were
KH2PO4-K2HPO4 (pH 7.0–8.0), KH2PO4-NaOH (pH 9.0)
and K2HPO4-NaOH (10.0–12.0).
pears to have a pH optimum of 11.0. In a separate
study, Peterson et al. (1995) suggested that the
presence of the large number of arginine residues
in Manduca chymotrypsin sequence may be responsible for the stabilization of the enzymes at highly
alkaline pH. The arginines apparently contribute
to the stability by remaining protonated even at
highly alkaline pH and this may also be the case
in the purified protease from S. obliqua.
Effect of temperature. Figure 6 shows that the
maximal caseinolytic activity was exhibited by the
alkaline protease at 50°C. While the enzyme retained about 20% of the activity at 70°C. Moderately high temperature optimums of alkaline
proteases have been reported earlier from various
insect sources (Thomas and Nation, 1984; Teo and
Woodring, 1988).
8
Anwar and Saleemuddin
Fig. 6. Effect of temperature on the proteolytic activity
of S. obliqua. The assay conditions were the same as described in Figure 5 except that the reaction was carried
out at the indicated temperatures for 15 min. The reaction was terminated by the addition of 0.5 ml 20% TCA.
Protease activity was determined as described in Materials and Methods.
Lineweaver Burk plot. A linear double reciprocal
plot was obtained with BApNA as a substrate. The
Km was computed from the intercept and the slope
and was found to be 2 ´ 10–6 M (Fig. 7).
Substrate specificity. The ability of the purified alkaline protease from S. obliqua larval guts to hydrolyze synthetic substrates was investigated by
using BApNA, BAME, and BAEE. It is interesting
to note that all the substrates tested were hydrolyzed by the larval protease (Table 2) exhibiting
different degrees of hydrolytic activity. A comparison was also made between the larval protease and
bovine trypsin. Bovine trypsin hydrolyzed the synthetic substrates in the order of BAME>BApNA>
BAEE, whereas the larval protease hydrolyzed the
substrates in the following order BAEE>BAME>
BApNA. Thus S. obliqua protease can be classified
as a serine protease like trypsin because these substrates are specifically hydrolyzed by trypsin and
trypsin like enzymes. The hydrolysis of BApNA by
the alkaline protease was similar with that of bovine trypsin. However, BAEE was a better substrate
for the larval protease.
Fig. 7. Lineweaver Burk plot of S. obliqua protease with
BApNA as a substrate. The assay mixture in a total volume of 3.0 ml contained 10 mg purified enzyme and 0.75
´ 10–5 mM to 19 ´ 10–5 mM BApNA. The reaction was initiated by the addition of substrate and was carried out at
37°C for 30 min. Reaction was stopped by the addition of
0.5 ml of 30% acetic acid. Formation of p-nitroanilide
was measured at 410 nm as described in Materials and
Methods. The inverse rate of reaction was plotted against
inverse substrate concentration.
Effect of various protease inhibitors. In order to further elucidate the nature of the purified alkaline
protease from the S. obliqua larval guts, the effect
of various inhibitors on the BApNA lytic activity
of the protease were investigated. Table 3 shows
that the inhibitors that were highly effective against
bovine trypsin were also predominantly inhibitory
towards the larval protease except ovomucoid,
TABLE 2. Activity Action of Alkaline Protease on Various Synthetic
Substrates*
Activity (units)
Substrate
BApNA
BAEE
BAME
Trypsin
Alkaline protease
0.33 ± 0.1
0.26 ± 0.1
3.70 ± 0.5
0.39 ± 0.1
4.10 ± 0.3
1.75 ± 0.5
*The reaction mixuture in a total volume of 3.0 ml contained suitably diluted
enzyme and substrates. Reaction was carried out in 0.1 M Tris-HCl, pH 8.0,
buffer and the activity of each substrate was determined and units were calculated as described in Materials and Methods. Details of the individual assays are
given in Materials and Methods. Each value represents the mean ± SD of four
determinations.
Archives of Insect Biochemistry and Physiology
Purification of Protease From S. obliqua Larvae
TABLE 3. Effect of Various Protease Inhibitors on the S. obliqua Alkaline
Protease*
Inhibitor
Concentration
(mM)
Target
protease(s)
Trypsin
0.04
1.00
1.00
0.02
Serine protease
Trypsin-like
Chymotrypsin
Trypsin-like
100 ± 0
100 ± 0
0
100 ± 0
SBTI
TLCK
TPCK
Ovomucoid
Alkaline protease
Inhibition (%)
75 ± 7
100 ± 0
38 ± 5
8±2
*The reaction mixture in a total volume of 3.0 ml contained 5 mg of enzyme protein and the indicated amount of inhibitors in 0.1 M Tris-HCl buffer, pH8.0. After
pre-incubation for 30 min at 37°C, residual activity was determined against BApNA
as described in Materials and Methods. Each value represents the ± SD of three
determinations.
which at the concentrations sufficient to completely inhibit trypsin activity resulted in only a
marginal inhibition in the case of the alkaline protease from S. obliqua. TLCK acts as a specific inhibitor of trypsin (Shaw et al., 1965) by binding
to the histidine residue within the active site. The
effect of TLCK on the protease of S. obliqua (Table
3) demonstrates that the histidine is also present
at the active site of the larval protease. The proteolytic activity of the larval midgut of Helicoverpa
armigera was also found to be inhibited by TLCK
(Johnston et al., 1991). On the other hand, TPCK
a specific inhibitor of chymotrypsin was found to
be partially inhibitory (38%). Such type of nonspecific inhibition exerted by TPCK is also found
in other lepidopteran larvae of Greater wax moth
(Hamed and Attias, 1987). The proteinaceous protease inhibitor SBTI caused 75% inhibition to the
S. obliqua protease and 100% inhibition to bovine
trypsin. In spite of the trypsin like characteristics,
the S. obliqua protease was not affected by ovomucoid. Lack of inhibition of trypsin-like proteases
by ovomucoid has been reported earlier (Houseman et al.,1989; Johnston et al., 1991). Applebaum’s prediction that adaptation of the proteases
in lepidoptera to an extremely alkaline environment may affect their interactions with “classical”
trypsin and chymotrypsin inhibitors is apparently
substantiated by these observations.
DISCUSSION
Like most of the lepidopterans, Spilosoma obliqua
(Arctiidae: Lepidoptera) larvae goes through six larSeptember 2002
9
val molts before a pupation stage. The most voracious among these larval instars is the fifth instar
larva. Taking into consideration the amount of gut
protein and protease activity that is available during the fifth instar larval stage (Anwar and Saleemuddin, 2001), we purified and characterized a
protease from these larval guts using ammonium
sulfate fractionation, DEAE-cellulose chromatography, and Hb-sepharose affinity chromatography.
The procedure adapted for the purification of the
protease was effective in eliminating nearly all the
contaminating proteins and the final preparation
obtained was found to be electrophoretically pure
with an overall yield of fifteen percent. Similar purification schemes have been employed for protease
purification by other investigators (Smith and Turk,
1974; DeMartino and Croall, 1983). The molecular weight of the purified alkaline protease determined by gel electrophoresis in the presence of SDS
was estimated at 120 kDa, while the estimated
molecular weight by gel chromatography was
found to be 90 kDa. The observed differences in
the molecular weights as determined by electrophoresis and gel chromatography can be attributed
to the inherent differences in the methods, as one
is under denaturing conditions and the other is in
native conditions. It is also noteworthy to mention that the purified alkaline protease had about
11% carbohydrate content in its composition, and
the proteins containing high carbohydrate content
have been shown to exhibit anomalous behavior
in SDS gels (Leach et al., 1980).
The purified protease was found to be optimally
active at pH 11.0. It is now well established that a
large number of proteases present in the insect guts
have alkaline pH optima. These include, those from
S. littoralis, pH 11.0 (Ishaaya et al, 1971), S. litura,
pH 9.0, 10.5, and 11.0 (Ahmad et. al., 1976, 1980),
Heloithis zea, pH 11.0 (Klocke and Chan, 1982),
G. mellonella, pH 10.5 and 11.2 (Hamed and Attias,
1987), Helicoverpa armigera, pH 9.5 and 10.0
(Johnston et. al., 1991), Phtorimaea operculla, pH >
9.0 (Christeller et. al., 1992), Manduca sexta, pH
8.5 (Samuels et. al., 1993), and Helothis virescens,
pH 10.0–11.0 (Johnston et al., 1995). While several investigators have been able to purify more
10
Anwar and Saleemuddin
than one protease from the larval guts, we have
not looked for the presence of other proteases in
the total gut homogenate. However, lack of significant multiplicity of peaks in the DEAE-cellulose
chromatographic profile indicates a major contribution of the purified protease towards the digestion of leaf protein in the larval guts. The purified
alkaline protease exhibited a temperature optimum
of 50°C. The alkaline proteases from other lepidopterans also appear to be thermostable and the
temperature optima of 60°, 55°, and 50°C, respectively (Ahmad et al., 1980). Protease activity from
the digestive tract of Acheta domesticus was found
to increase from 20° to 45°C in a study performed
by Teo and Woodring (1988). The Km value of the
purified S. obliqua was found to be 2.0 ´ 10–6 M,
suggesting a moderate affinity towards BApNA. The
Km value for the B. mori alkaline protease using
BApNA as a substrate has been reported to be 8.27
´ 10–7 M (Euguchi and Kuriyama, 1985), which is
about 40-fold higher than S. obliqua.
Although both “trypsin-like” and “chymotrypsinlike” proteases have been located in the lepidopteran
guts, the S. obliqua protease appears to be more
“trypsin like.” The enzyme readily hydrolyzed typical trypsin substrates BApNA, BAEE, and BAME. It
is also interesting to note that each milligram of the
purified enzyme was more active than the same
amount of trypsin, indicating a high degree of substrate hydrolysis by S. obliqua protease. High activity
against BAEE by trypsin-like digestive protease from
C. fumifera has been reported (Milne and Kaplan,
1993). The ability of the S. obliqua digestive protease to hydrolyze various protein substrates was also
investigated and the rates of hydrolysis of these substrates were in the order of casein>hemoglobin>
bovine serum albumin>ovalbumin>gelatin (Anwar
and Saleemuddin, 2000).
The effect of various proteolytic inhibitors on
the purified alkaline protease from S. obliqua demonstrates that the purified enzyme is indeed
trypsin-like also in terms of its susceptibility to specific inhibitors. Inhibition by TLCK and SBTI confirms the trypsin-like activity. While TLCK is known
to act as a specific inhibitor of trypsin and trypsinlike proteases by binding to the histidine residue
within the active site of the enzyme, the inhibition of the S. obliqua protease by TLCK indicates
that a histidine residue might be at its active site.
However, the partial inhibition by TPCK, a specific inhibitor of chymotrypsin might indicate that
the specificity pocket allows the partial binding of
chymotrypsin with the substrates. This suggests that
S. obliqua alkaline protease might be some what
different than trypsin. This was demonstrated by
the lack of any significant inhibition by ovomucoid, which is highly inhibitory towards bovine
trypsin. It has been observed earlier that insect proteases are sensitive to proteinaceous plant protease
inhibitors, but may not be affected by those from
animal sources (Johnston et al., 1991).
The loss of sensitivity towards protease inhibitors from animal sources may also be related to
the adaptation of lepidopteran proteases to function in highly alkaline environments (Applebaum,
1985). The inhibition, although lower than the alkaline protease activity by TPCK, is an interesting
example of lepidopteran protease having some
characteristics of both trypsin and chymotrypsin.
Johnston et al. (1991) have shown that partially
purified protease from the larvae of H. armigera
was found to be sensitive to chymostatin, a typical
chymotrypsin inhibitor. Although both trypsin and
chymotrypsin-like proteases have been purified
from the lepidopteran guts (Johnston et al., 1995),
examples of those with the characteristics of both
have also been reported. However, caution must
be exercised in arriving at conclusions on the basis of inhibition by various inhibitors in view of
their sensitivity to alkaline environments (Peterson
et al., 1995), especially in the case of lepidopteran
proteases, which are optimally active at alkaline
pH. Successful development of protease inhibitors
that can be used as pest control agents requires
the knowledge of the sensitivity of these inhibitors towards each member within the family of insect species (Zhu and Baker, 1999).
ACKNOWLEDGMENTS
We thank Dr. L.D. Tiwari, Senior Scientist at the
Indian Agricultural Research Institute, Pusa, New
Archives of Insect Biochemistry and Physiology
Purification of Protease From S. obliqua Larvae
11
Delhi, India, for useful discussions and Dr. Khowaja
Jamal Department of Zoology, AMU, Aligarh, India, for the initial culture of S. obliqua.
Euguchi M, Kuriyama K. 1985. Purification and characterization of membrane bound alkaline proteases from midgut
tissue of the silkworm Bombyx mori. J Biochem (Tokyo)
97:1437–1445.
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Archives of Insect Biochemistry and Physiology