Process Biochemistry 46 (2011) 2137–2143

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Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Purification and characterization of a new alkaline serine protease from the

thermophilic fungus Myceliophthora sp.
L.M. Zanphorlin a , H. Cabral b , E. Arantes b , D. Assis c , L. Juliano c , M.A. Juliano c , R. Da-Silva a , E. Gomes a ,
G.O. Bonilla-Rodriguez a,∗
a
UNESP - State University of São Paulo, São José do Rio Preto, SP, Brazil
b
USP - University of São Paulo, Ribeirão Preto, SP, Brazil
c
UNIFESP - Federal University of São Paulo, São Paulo, SP, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: This work reports the purification of a novel alkaline protease enzyme from a putative new thermophilic
Received 22 March 2011 fungus Myceliophthora sp. The molecular weight of the enzyme was determined as 28.2 kDa by using
Received in revised form 24 August 2011 MALDI-TOF MS and it was inhibited by PMSF indicating it is a serine-protease. The optimum pH and tem-
Accepted 24 August 2011
perature were 9.0 and 40–45 ◦ C, respectively. Mg was the only tested cation able to promote an increase
Available online 31 August 2011
of the protease activity. The N-terminal sequence of the purified protease (GVVGVC) presented identity
and homology when compared to other proteases from fungi. This study also provides biochemical infor-
Keywords:
mation about substrate specificity using fluorescence resonance energy transfer (FRET) peptide families
Thermophilic fungus
Myceliophthora sp.
derived from Abz-KLRSSKQ-EDDnp. The results showed that Abz-KLISSKQ-EDDnp is the best substrate
Enzyme among those tested for the purified protease (kcat /Km = 1275.3 mM−1 s−1 ). Also, the specificity data sug-
Protease gest that subsites S1 , S2 , S3 and S1  , S2  , S3  , in general, present a preference for hydrophobic residues with
Serine-protease the exception of Glu in P3 , His in P2  and Arg in P3  . The highest values for the specificity constant kcat /Km
Fluorescent peptides were obtained for P1 , P2 and P2  .
Subsite mapping © 2011 Elsevier Ltd. All rights reserved.

1. Introduction are used in the detergent industry because the pH of cleaning prod-
ucts is usually in the range of 9.0–12.0. Adding these enzymes to
Proteases are the most important industrial enzymes, account- cleaning solutions allows the use of fewer toxic chemicals such as
ing for approximately 60% of the total industrial enzyme market [1]. solvents and corrosive substances, decreasing their environmental
The relevance of this group of enzymes, rich in structural diver- impact [6].
sity and mechanisms of action is reflected in the importance of Currently, the majority of proteases used in detergents are
their applications in industrial processes. They have multiple appli- serine proteases [7] and their catalytic activity depends on the
cations, for example in detergent formulations, textile, food, and interplay of a nucleophile, a general base and an acid. In the two
pharmaceutical industries [2]. Therefore, the industrial demand of largest groups of serine-proteases, the (chymo)trypsin and subtil-
proteolytic enzymes, with appropriate specificity and stability to isin families, the catalytic triad is composed of serine, histidine and
pH, temperature and chemical agents, continues to motivate the aspartate residues which exhibits similar spatial arrangements, but
search for new sources. the order of the residues in the amino acid sequence and tertiary
Many microorganisms secrete proteases to the external envi- structure is different [8,9]. The active site performs the dual role of
ronment in order to degrade proteins; their hydrolysis products binding a substrate and catalyzing a reaction, and thus determines
are used as carbon and nitrogen sources for cell growth [3]. Pro- the specificity of the enzyme. Accordingly, it is possible to obtain
teases with elevated activity and stability in the high alkaline range information about the active site by analyzing the enzyme kinetics
are attractive for bioengineering and biotechnological applications, with different substrates and inhibitors [10].
especially those from bacteria and fungi [4,5]. Alkaline proteases Recently, we isolated a thermofilic fungus Myceliophthora
sp. strain, which produces an extracellular proteolytic enzyme
[11]. The present work reports the purification, biochemi-
cal and biophysical characteristics of the extracellular pro-
∗ Corresponding author at: Departamento de Química e Ciências Ambientais,
tease produced by Myceliophthora sp. using FRET peptides
IBILCE-UNESP, Rua Cristovão Colombo 2265, São José do Rio Preto, SP, CEP 15054-
000, Brazil. Tel.: +55 17 32212361; fax: +55 17 32212356. derived from sequence Abz-KLRSFKQ-EDDnp of the extracel-
E-mail addresses: bonilla@ibilce.unesp.br, gustavo.bonilla@uol.com.br lular serine-protease produced by Myceliophthora sp. Also, we
(G.O. Bonilla-Rodriguez).

1359-5113/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2011.08.014
2138 L.M. Zanphorlin et al. / Process Biochemistry 46 (2011) 2137–2143

investigated the specificity of the subsites S1 , S2 , S3 and S1  , 2.8. Kinetic measurements

S2  and S3  using FRET peptides derived from the sequence
The FRET peptides were assayed in a Shimadzu RF-5301 spectrofluorimeter
Abz-KLRSSKQ-EDDnp (Abz = o-aminobenzoic acid; EDDnp = N-[2,4
at 45 ◦ C in 50 mM glycine buffer, pH 9.0. The enzyme was pre-incubated in the
dinitrophenyl]ethylenediamine; Abz/EDDnp = donor/acceptor flu- assay buffer for 3 min before the addition of substrate. Fluorescence changes were
orescent pair). monitored continuously at 320 nm excitation and 420 nm emission. The enzyme
concentrations for initial rate determinations were chosen at a level intended to
2. Experimental procedures hydrolyze less than 5% of the amount of added substrate over the time course of data
collection. The slope of the generated fluorescence signal was converted to micro-
2.1. Protease production in solid-state fermentation (SSF) moles of substrate hydrolyzed per minute based on a calibration curve obtained
from the complete hydrolysis of each peptide. The kinetic parameters Km and kcat
Erlenmeyer flasks (250 mL) containing media composed by 4.75 g of wheat bran were calculated by nonlinear regression using the GraFit® software (Erithacus Soft-
and 0.25 g of casein and hydrated with 7 mL of distilled water and 3 mL of nutrient ware, Horley, Surrey, UK). Errors were less than 5% for each of the obtained kinetic
solution 0.1% (w/v) (NH4 )2 SO4 , MgSO4 ·7H2 O and NH4 NO3 were inoculated with parameters.
2 mL of a spore suspension and cultivated at 45 ◦ C for 72 h. The fermented material
was mixed with 30 mL of distilled water per 5 g of fermented material, stirred for 2.9. Effects of pH and temperature on enzyme activity
30 min, filtered and centrifuged at 10,000 × g, at 6 ◦ C. The supernatant was used as
a crude enzyme solution. Optimum pH was determined by performing standard activity assays in a pH
range from 3 to 10.5 at 45 ◦ C using suitable buffers: sodium citrate dihydrate, sodium
2.2. Protease activity acetate, sodium phosphate, Hepes, Taps, glycine and Caps. In order to determine
optimal temperature, the enzymatic assay was carried out at different tempera-
Proteolytic activity was assayed as described by Sarath et al. [12] with modifi- tures (25–60 ◦ C), at pH 9.0. The reactions were performed under pseudo first-order
cations. The reaction mixture was made up of 0.2 ml crude enzyme and 0.8 mL of conditions ([S]  Km ) of hydrolysis of substrate Abz-KLRSFKQ-EDDnp. The data were
1% (w/v) casein dissolved in glycine buffer (50 mM, pH 9.0). The reaction was car- fitted with the GraFit® software to the appropriate equation [20].
ried out at 50 ◦ C and stopped after 30 min with 0.5 mL of 15% trichloroacetic acid
(TCA). Test tubes were centrifuged at 15,000 × g for 30 min and the absorbance of 2.10. Effect of inhibitors on enzyme activity
the supernatant was measured at 280 nm using a Cary 100 (Varian) spectropho-
tometer. A control was prepared by adding TCA before the addition of the enzyme The effects of inhibitors on protease activity were studied using PMSF, ben-
solution. One unit of enzyme activity (U mL−1 ) was arbitrarily defined as the amount zamidine, iodoacetic acid, EDTA and E-64. The enzyme was pre-incubated with
of enzyme required to cause an absorbance increase of 0.01 per minute at 280 nm inhibitors for 3 min at pH 9.0 and 45 ◦ C, and then the remaining enzyme activity
under the assay conditions [13]. was estimated using Abz-KLRSFKQ-EDDnp as substrate. The reactions were per-
formed under pseudo first-order conditions of hydrolysis of substrate. The activity
2.3. Purification protocol of the enzyme assayed in the absence of inhibitors was considered as 100%.

The crude enzyme solution was concentrated using a precipitation procedure 2.11. Salt influence on enzyme activity
at 4 ◦ C with ethanol (proportion 1:2). The crude precipitate was collected by cen-
trifugation at 9000 × g for 30 min at 4 ◦ C and then dissolved in 20 mM Tris pH 8.0 The influence of NaCl on the proteolytic activity was investigated up to 0.5 M.
buffer with 0.2 M NaCl. The concentrated crude enzyme solution was then further The protease was pre-incubated with NaCl for 3 min in pH 9.0, at 45 ◦ C, and then the
purified by gel filtration on a Sephacryl S-100 column (1.6 cm × 75 cm) equilibrated remaining enzyme activity was measured using Abz-KLRSFKQ-EDDnp as substrate.
with 20 mM Tris pH 8.0 containing 0.2 M NaCl. Fractions of 3 mL each were collected The reactions were performed under pseudo first-order conditions of hydrolysis of
at a flow rate of 0.15 mL/min and analyzed for protease activity and protein concen- substrate. The activity of the enzyme assayed in the absence of salt was taken as
tration. Fractions exhibiting protease activities were pooled and applied to a Source 100%.
15-Q column (1 cm × 6 cm) that was previously equilibrated with 20 mM Tris, pH
8.5 buffer. Unbound proteins were removed with the equilibration buffer until the 2.12. Effects of surfactants and oxidizing agents on enzyme activity
absorbance at 280 nm reached the baseline. Bound proteins were eluted with a lin-
ear gradient of sodium chloride in the range of 0–2.0 M in the equilibration buffer. The effect of some surfactants (Triton X-100, Tween 20, Tween 80, and SDS)
Fractions of 1 mL each were collected at a flow rate of 1 mL/min and analyzed for and oxidizing agents (DTT and ␤-mercaptoethanol) was studied by pre-incubating
protease activity and protein concentration. the protease with the agent for 3 min, pH 9.0, at 45 ◦ C, prior to activity tests. The
activity of the enzyme without any additive was taken as 100%. The reactions were
2.4. Electrophoresis performed under pseudo first-order conditions of hydrolysis of substrate.

Polyacrylamide gel electrophoresis under denaturing conditions was carried 2.13. Effect of divalent ions on enzyme activity
out to determine the purity and to estimate the molecular weight of the purified
enzyme [14]. Protein bands were visualized after staining with silver nitrate. For The protease was assayed in the presence of different metal ions including Ca2+ ,
the biochemical analysis described below we used the purified protease. Mg2+ , Fe2+ , Cu2+ , Zn2+ , Mn2+ , Hg2+ , and Ni2+ at 5 mM (final concentration). The reac-
tions were performed under pseudo first-order conditions of hydrolysis of substrate.
2.5. Mass spectrometry analysis The activity of the enzyme assayed in the absence of divalent ions was taken as 100%.

Protease molecular mass determination was carried out on a TOF Spec E mass 2.14. Effect of organic solvents on enzyme activity
spectrometer (Micromass, Manchester, UK) operating in linear mode using the
matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) method, In order to determine the effect of organic solvents like methanol, ethanol,
using ␣-cyano-4-hydroxycinnamic acid as the matrix [15]. acetone, butanol and isopropanol (20%, v/v) on proteolytic activity the reactions
were performed under pseudo first-order conditions of hydrolysis of substrate. The
2.6. Determination of protein concentration activity of the enzyme assayed in the absence of organic solvents was taken as 100%.

Protein concentration was determined by the Bradford method [16], using 2.15. Determination of the substrate cleavage sites
bovine serum albumin as a standard.
The scissile bond of hydrolyzed peptides was identified by isolation of the frag-
2.7. FRET peptide synthesis ments using analytical HPLC followed by determination of their molecular mass by
LC/MS using an LCMS-2010 equipped with an ESI-probe (Shimadzu, Japan).
All the FRET peptides used for subsite mapping were synthesized by solid-phase
synthesis and purified as described previously [17,18]. The molecular mass and 2.16. N-terminal sequence
purity of the peptides were confirmed by amino acid analysis and MALDI-TOF using a
Microflex-LT mass spectrometer (Bruker—Daltonics, Billerica, MA, USA). Stock solu- The N-terminal sequence was determined by the Edman method [21] using
tions of peptides were prepared in DMSO, and their concentrations were measured the Protein Sequencer PPSQ-33A (Shimadzu Corporation, Kyoto, Japan). The PTH-
by colorimetric determination of the 2,4-dinitrophenyl group (molar extinction amino acids were separated using HPLC, and they were identified and quantified by
coefficient of 17.300 M−1 cm−1 at 365 nm) using a spectrophotometer Cary 100 analyzing previously quantified standards and comparing retention times and UV
(Varian) [19]. absorption, respectively.
 L.M. Zanphorlin et al. / Process Biochemistry 46 (2011) 2137–2143 2139

Table 1
Parameters of purification of a serine protease from thermophilic fungus Myceliophthora sp.

Step Total protein Total Specific activity Purification Recovery (%)

(mg) activity (U) (U/mg) factor (fold)

Crude Extract 2.49 19.80 8.0 1 100.0

Precipitation 1.25 15.75 12.6 1.6 79.5
Gel filtration 0.03 11.50 383.3 48.2 58.1
Ion exchange 0.01 10.2 1020.0 128.3 51.5

3. Results and discussion performed in pH 9.0 at 45 ◦ C. Proteolytic activity was abolished by

5 mM PMSF (100% inhibition), suggesting that this enzyme would
3.1. Protease purification be a serine-protease. Other protease inhibitors like EDTA, benza-
midine, iodoacetic acid and E-64 had none or weak effect on the
The protease was purified by a three-step procedure which is activity. Serine proteases from thermofilic fungi have been reported
summarized in Table 1. After ethanol precipitation, a Sephacryl S- by several authors such as Khan et al. [29] who reported purified
100 HR column was used to purify the enzyme, obtaining four peaks serine protease from the thermophilic fungus Paecilomyces lilaci-
(Fig. 1a). However, only the first peak (fractions 20–24) presented nus, and by Charles et al. [30] and Hajji et al. [31], who reported
proteolytic activity. Only the fractions 23 and 24 were pooled and purified proteases by thermotolerants fungi Aspergillus nidulans
submitted to an anion exchange column Source 15-Q. HA-10 and Aspergillus clavatus ES1, respectively.
The anion exchange chromatography showed two peaks
(Fig. 1b); the first one (A) eluted in the absence of NaCl containing 3.4. Salt influence on enzyme activity
unbound proteins and the second (B) eluted with a NaCl gradient
of 0–2 M. The proteolytic activity was observed in peak A, indicat- The enzyme was slightly activated 8% in the presence of 250 mM
ing that the enzyme did not bind to the Source 15-Q and, therefore, NaCl, decreasing afterwards. Wang et al. [32] reported that 5 mM
would have a pI higher than 8.5 or maybe it is a glycosylated protein, NaCl had minimal effect on the activity of a protease from A. fumi-
a plausible reason for impaired binding. The enzyme was purified gatus. Here we report that much higher concentrations of NaCl
128.3 fold with a final yield of 51.5% (Table 1), it was homoge- did not significantly enhance catalytic efficiency of the protease
nous on SDS-PAGE, and its molecular weight was estimated to be from Myceliophthora, consistent with Wang et al.’s findings. The
30.1 kDa (Fig. 1). Subsequently we performed a mass spectrometry lack of significant effects of sodium is drastically different from the
(MALDI-TOF) to verify both purity and the molecular weight of the behavior described for other serine proteases, especially for throm-
enzyme (results not shown). The data confirmed the purity of the bin [33], a key enzyme from the blood coagulation cascade, where
protease and its exact molecular weight: 28.2 kDa (data not shown). Na+ exerts an allosteric role and drastically improves the catalytic
The low molecular mass exhibited by the purified protease agrees properties towards its natural substrate fibrinogen.
with other works that report fungal proteases with a Mw lower
than 50 kDa [22,23]. 3.5. Effect of surfactants and oxidizing agents on protease activity

3.2. Effect of pH and temperature on enzyme activity The effect of surfactants and reducing agents on protease activ-
ity is shown in Table 3. The enzyme presented a small loss of activity
The effect of pH on the hydrolysis of Abz-KLRSFKQ-EDDnp is in the presence of the non-ionic surfactant Triton X-100 and it was
shown in Fig. 2a. The protease is more active in the pH range activated in the presence of Tween 20. However, in the presence of
7.0–10.5 with higher activity at pH 9.0 suggesting that it is an
alkaline protease. Purified alkaline proteases from fungi have been
Table 3
described from Clonostachys rosea (pH 9) [24] and Lecanicillium psal- Effect of reducing agents and surfactants on the hydrolysis of Abz-KLRSFKQ-EDDnp
liotae (pH 10) [25]. The temperature dependence was determined by a serine protease from Myceliophthora sp.
from 25 to 60 ◦ C (Fig. 2b). Optimal hydrolysis occurred at 40–45 ◦ C.
Final concentration Relative
Also, at 55 ◦ C, the enzyme retained about 90% activity while at (mM or v/v) activity (%)
60 ◦ C, the activity reduced to 45%. Proteases from fungi show similar
Control – 100.0
results for optimum temperature such as 45 ◦ C for Colletotrichum ␤-Mercaptoethanol 2 mM 91.5
gloeosporioides [26], 50 ◦ C for Trichoderma reesei QM9414 [27] and DTT 2 mM 82.9
Fusarium culmorum [28]. Tween 20 1% 117.2
Triton X-100 1% 88.6
Tween 80 1% 20.0
3.3. Effect of inhibitors on purified enzyme activity
SDS 0.1% 0.0

The effect of some protease inhibitors was investigated on

the hydrolysis of Abz-KLRSFKQ-EDDnp (Table 2). The assay was Table 4
Effect of divalent ions (5 mM final concentration) on the hydrolysis of Abz-KLRSFKQ-
EDDnp by a serine protease from Myceliophthora sp.
Table 2
Effect of different types of inhibitors (5 mM) on the hydrolysis of Abz-KLRSFKQ- Compound Relative activity (%)
EDDnp by a serine protease from Myceliophtora sp. Control 100.0
Mg2+ 122.2
Inhibitors Relative activity (%)
Ca2+ 97.2
Control 100 Ba2+ 94.4
EDTA 95 Mn2+ 91.7
PMSF 0 Zn2+ 77.8
Benzamidine 100 Ni2+ 41.7
E-64 80 Hg2+ 33.3
Iodine-acetic acid 100 Cu2+ 8.3
2140 L.M. Zanphorlin et al. / Process Biochemistry 46 (2011) 2137–2143

Fig. 1. Closed circles: proteolytic activity, open circles: protein profile at 280 nm (a) gel filtration chromatography on Sephacryl S-100 HR. Conditions: 20 mM Tris buffer, 0.2 M
NaCl, pH 8.0; (b) protease ion exchange chromatography on Source 15-Q (fractions 23 and 24 gel filtration). Peak A: the protease elutes in the absence of the saline gradient.
Peak B: elutes during the linear gradient from 0 to 1 M of NaCl. Conditions: 20 mM Tris buffer, pH 8.5. (c) SDS-PAGE 12%. Lane 1, pure protease; lane 2, crude extract and lane
3, molecular weight markers: ␣-lactalbumin (14.4 kDa), trypsin inhibitor (20.1 kDa), carbonic anhydrase (30 kDa), ovalbumin (45 kDa), bovine serum albumin (66 kDa), and
phosphorylase b (97 kDa).

other non-ionic surfactants with a longer chain such as Tween 80, [34]. The increase of activity by Tween 20, was also reported for
it displayed only 20% of proteolytic activity. Also, the protease was the serine protease from C. rosea [23], which exhibited 16% of acti-
completely inactivated by the anionic surfactant SDS. vation by Tween 20. Also, the serine protease from A. clavatus ES1
The activity of alkaline protease from Myrothecium verrucaria [31] presented about 33% of residual activity in the presence of
is only slightly affected by the presence of 2.5% Triton 100 (13%) 0.5% SDS, a higher concentration than the one we tested. On the

a 100 b 100
Relative activity %

80 80
Relative activity %

60 60

40 40

20 20

0 0
7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 20 25 30 35 40 45 50 55 60 65
pH Temperature (ºC)

Fig. 2. The influence of (a) pH and (b) temperature on hydrolysis of Abz-KLRSFKQ-EDDnp by the purified protease.
 L.M. Zanphorlin et al. / Process Biochemistry 46 (2011) 2137–2143 2141

Table 5 Table 7
Effect of organic solvents (20%) on the hydrolysis of Abz-KLRSFKQ-EDDnp by a serine Kinetic parameters for the hydrolysis of the FRET peptide series Abz-KLRSSKQ-
protease from Myceliophthora sp. EDDnp by a serine protease from Myceliophthora sp. for the characterization of its
S1  , S2  , S3  subsite specificity. The assays were performed at 45 ◦ C in 50 mM glycine
Relative activity (%) buffer, pH 9.0. The arrow indicates the cleaved peptide bond.
Control 100.0
Substrate kcat (s−1 ) Km (␮M) kcat /Km
Acetone 0.0
(s−1 mM−1 )
Butanol 0.0
Ethanol 47.2 Abz-KLR↓XSKQ-EDDnp (P1  )
Methanol 38.9 (Reference) Ser 0.61 4.60 133
Isopropanol 91.7 Phe 0.02 0.45 41
Ile 0.02 0.57 34
Gly 0.16 5.70 28
Gln 0.05 2.00 28
other hand, thiol reducing agents such as DTT, which disassoci- Glu 0.17 7.10 24
ate disulfide bonds (34) crucial for stabilizing the tertiary structure His 0.15 7.72 20
of proteins [35]. DTT and ␤-mercaptoethanol did not affect the Arg 0.07 4.81 14
protease activity significantly. Also, the trypsin like protease from Pro No hydrolysis

Abz-KLR↓SXKQ-EDDnp (P2 )
Cordyceps militaris in the presence 1 mM DTT did not suffer signif- Phe 1.42 4.28 332
icant activity loss [36]. In contrast, the activity of serine protease His 0.58 2.24 261
from Cordyceps sinensis had it activity increased in the presence Leu 0.17 1.02 164
2 mM DTT and ␤-mercaptoethanol: 29.8% and 14.5%, respectively (Reference) Ser 0.61 4.60 133
Ala 0.77 7.30 106
[37].
Val 0.15 3.00 51
Gly 0.03 0.66 50
Asn 0.11 2.28 46
Table 6 Arg 0.05 1.20 39
Kinetic parameters for the hydrolysis of the FRET peptide series Abz-KLXSSKQ- Pro No hydrolysis
EDDnp by a serine protease from Myceliophthora sp. for the characterization of its Abz-KLR↓SSXQ-EDDnp (P3  )
S1 , S2 , S3 subsite specificity. The assays were performed at 45 ◦ C in 50 mM glycine (Reference) Lys 0.61 4.60 133
buffer, pH 9.0. The arrow indicates the cleaved peptide bond. Arg 0.04 0.80 52
Substrate kcat (s−1 ) Km (␮M) kcat /Km Val 0.04 1.30 29
(s−1 mM−1 ) Gln 0.11 4.41 24

Abz-KLX↓SSKQ-EDDnp (P1 )
Ile 0.12 0.09 1275
3.6. Effect of divalent ions on enzyme activity
Met 0.78 1.15 676
Trp 0.82 1.28 639
Ala 0.97 3.77 259 Table 4 shows the effect of divalent ions. The addition of MgCl2
Phe 2.16 9.37 230 increased enzyme activity by about 22% compared to the control.
Thr 1.31 7.58 173 Ca2+ , Ba2+ and Mn2+ showed little influence on enzyme activity.
Lys 1.44 8.88 162
(Reference) Arg 0.61 4.60 133
Zn2+ exerted more influence decreasing activity by 22%, whereas
KLPSSK↓Q Pro 0.01 0.08 121 Hg2+ and Ni2+ significantly inhibited the enzyme activity with
Asn 0.41 3.88 105 pronounced inhibition. Also, with addition of Cu2+ , the purified pro-
Ser 0.99 10.63 93 tease was inhibited approximately by 92%. A serine-protease from
Leu 0.38 6.80 56
A. clavatus ES1 was activated in the presence of 5 mM Mg2+ [31], and
Gln 0.16 3.10 53
His 0.17 4.82 35 serine proteases from Scedosporium apiospermum [38] and C. sinen-
Val 0.07 4.81 14 sis [37] were also inhibited in the presence of 5 mM Cu2+ . This metal
Cys 0.04 5.25 7 can react with cysteines promoting their oxidation and formation of
Gly No hydrolysis cystine (Cys–Cys) [39], probably affecting enzyme structure and/or
Abz-KXR↓SSKQ-EDDnp (P2 )
the access to the active site.
Phe 0.04 0.06 683
Pro 0.02 0.07 299
(Reference) Leu 0.61 4.60 133 3.7. Effect of organic solvents on enzyme activity
Val 0.44 5.08 86
Ala 0.25 6.80 37
Ile 0.37 10.70 35
In the presence of acetone and butanol, the enzyme activity was
Tyr 0.05 1.96 25 completely inhibited. Ethanol and methanol caused inhibition of
Thr 0.17 7.19 24 more than 50% in the enzyme: 47.2% and 38.9% of baseline activ-
Gln 0.10 5.50 18 ity, respectively. By contrast, addition of isopropanol only reduced
Gly 0.03 2.30 12
activity to 91.7% (Table 5). These results differ markedly from those
Asp 0.07 8.46 12
Asn 0.02 2.42 7 of the serine protease from Aspergillus fumigatus [26] which pre-
His No hydrolysis sented 78% of its activity in the presence of 50% ethanol.
Abz-XLR↓SSKQ-EDDnp (P3 )
(Reference) Lys 0.61 4.60 133
3.8. Determination of the substrate cleavage sites
Asp 0.71 6.16 115
Ile 0.36 4.02 89
Phe 0.57 6.70 86 FRET peptides derived from Abz-KLRSSKQ-EDDnp: The FRET
Val 0.25 4.67 55 peptide series Abz-XLRSSKQ-EDDnp, Abz-KXRSSKQEDDnp, Abz-
Gln 0.14 5.87 24 KLXSSKQ-EDDnp, Abz-KLRXSKQ-EDDnp, Abz-KLRSXKQ-EDDnp
Leu 0.20 8.52 24
Arg 0.13 5.80 23
and Abz-KLRSSXQ-EDDnp where X is the varying residue, were
Ala 0.07 3.34 22 taken as a reference to explore the specificity of the subsites S3 ,
Asn 0.06 3.39 18 S2 , S1 , and S1  , S2  , S3  , respectively. Assays were performed in
His 0.08 4.90 16 50 mM glycine, pH 9.0 at 45 ◦ C. The kinetic parameters kcat , Km and
Glu 0.07 4.67 16
specificity constant (kcat /Km ) for their hydrolysis by the protease
2142 L.M. Zanphorlin et al. / Process Biochemistry 46 (2011) 2137–2143

Table 8
Amino terminal sequence of protease as compared to other proteases using NCBI Blast Databank.

Protein Sequence NCBI Blast

Serine protease (Myceliophthora sp.) GVVGVC –

Serine protease (Metarhizium flavoviride var. minus) SVVGVQ gb|ACT66133.1|
Serine protease (Metarhizium album) SVVGVQ gb|ACT66132.1|
Serine protease (Metarhizium anisopliae) SVVGVQ gb|ACT66131.1|
Serine protease (Metarhizium majus) SVVGVQ gb|ACT66127.1|
Protease (Coccidioides immitis RS) LVVGVI gb|EAS33583.1|
Trypsin-like protease (Septobasidium carestianum) IVVGVS gb|AAR91723.1|
Protease (Candida glabrata) SVVGVR emb|CAG61409.1|
Serine protease (Monacrosporium haptotylum) GVVGRR gb|ABV46590.1|
Protease (Verticillium dahliae) GVVGAS gb|AAR10769.1|
Protease (Methylocella silvestris BL2) GVVGVC gb|ACK49719.1|
Serine protease (Aedes aegypti) GVIGVC gb|EAT46743.1|

are shown in Tables 6 and 7. The hydrolysis of FRET peptides Acknowledgements

of the six tested series showed a clear preference (analyzed in
terms of the specificity constant kcat /Km ) for hydrophobic residues This work had financial support from FAPESP, CNPq and CAPES
at P1 , P2 and P2  , which also displayed the highest values for (Brazil). The authors thank Austin Vogt from Saint Louis University,
kcat /Km . St. Louis, MO, USA, for his English review
Analyzing the protease behavior in relation to substrate speci-
ficity, Table 6 shows globally a preferential single cleavage occurred
References
at the side of the carbonyl carbon of P1 . The only exception was Pro
in P1 position, that showed a displaced cleavage reaching the C [1] Rao MB, Tankasale AM, Ghatge MS, Desphande VV. Molecular and biotechno-
side of P3  . However, it is relevant to point out that for most sub- logical aspects of microbial proteases. Microbiol Mol Biol R 1998;62:597–634.
strates, the enzyme showed a second point of cleavage (accounting [2] Gupta R, Beg QK, Lorenz P. Bacterial alkaline proteases: molecular approaches
and industrial applications. Appl Microbiol Biotechnol 2002;59:15–32.
for 5–10% of the product concentration) at the C side of P2  position [3] Van Den Hombergh JPTW, Van de Vondervoort PJI, Fraissinet-Tachet L, Visser
(results not shown). For P1 , the protease displayed a greater pref- J. Aspergillus as host for heterologous protein production: the problem of pro-
erence for the hydrophobic residues Ile > Met > Trp, followed by Ala teases. Trends Biotechnol 1997;15:256–63.
[4] Jellouli K, Bougatef A, Manni L, Agrebi R, Siala R, Younes I, et al. Molecular and
and Phe. biochemical characterization of an extracellular serine-protease from Vibrio
The catalytic specificity for Ile was almost tenfold higher than metschnikovii J1. J Ind Microbiol Biotechnol 2009;36:939–48.
for the reference peptide, which has Arg at P1 . Compared to values [5] Wang B, Liu X, Wu W, Liu X, Li S. Purification, characterization, and gene cloning
of an alkaline serine protease from a highly virulent strain of the nematode-
reported by the literature, the serine protease from Myceliophthora
endoparasitic fungus Hirsutella rhossiliensis. Microbiol Res 2009;164:665–73.
sp. was unable to show a preferential cleavage at S1 for trypsin-type [6] Castro HF, Mendes AA, Santos JC. Modificação de óleos e gorduras por
substrates (Arg/Lys) and displayed significant activity only against biotransformação. Quim Nova 2004;27:146–56.
[7] Pushpam PL, Rajesh T, Gunasekaran P. Identification and characterization of
Trp among the characteristic preferred residues Tyr/Phe/Trp/Leu
alkaline serine protease from goat skin surface metagenome. AMB Express
[40]. An alkaline serine protease isolated from the photosynthetic 2011;1:1–3.
bacterium Rubrivivax gelatinosus KDDS1 [41] exhibited a preference [8] Rawlings ND, Barrett AJ. Evolutionary families of peptidases. Biochem J
for Met at this position, but low activity for Trp and Ile. Thus, the 1993;290:205–18.
[9] Polgár L. The catalytic triad of serine peptidases. Cell Mol Life Sci
preference of the alkaline serine protease from Myceliophthora sp. 2005;62:2161–72.
differs from the alkaline serine protease from T. reesei QM9414, [10] Berger A, Schechter I. Mapping the active site of papain with the aid of peptide
classified as being a trypsin-like enzyme [27]. substrates and inhibitors. Philos Trans R Soc B 1970;12:249–64.
[11] Zanphorlin LM, Facchini FDA, Vasconcelos F, Bonugli-Santos RC, Rodrigues
For P2 (Table 6), the highest specificity constant was obtained A, Sette LD, et al. Production, partial characterization and immobilization in
for the hydrophobic residues Phe and Pro, followed by the reference alginate beads of an alkaline protease from a new thermophilic fungus Myce-
peptide (Leu). For P3 the highest preference was for the reference liophthora sp. J Microbiol 2010;48:331–6.
[12] Sarath G, De la Motte RS, Wagner FW. Protease assay methods. In: Beynon RJ,
peptide (Lys) followed by Glu, Ile and Phe, showing poor selectivity. Bond JS, editors. Proteolytic enzymes: a practical approach. IRL Press; 1996. p.
For P1  (Table 7) the preference was for the reference peptide (Ser) 25–54.
followed by the hydrophobic residues Phe and Ile. For P2  (Table 7), [13] Merheb CW, Cabral H, Gomes E, Da-Silva R. Partial characterization of protease
from a thermophilic fungus, Thermoascus aurantiacus, and its hydrolytic activity
the preference was again for the hydrophobic residue Phe followed
on bovine casein. Food Chem 2007;104:127–31.
by the basic residue His; however, at pH 9.0 His predominates in the [14] Laemmli UK. Cleavage of structural proteins during the assembly of the head
uncharged form. In P3  (Table 7), only the reference peptide (Lys) of bacteriophage T4. Nature 1970;227:680–5.
[15] Flensburg J, Belew M. Characterization of recombinant human serum albumin
displayed higher values of kcat /Km .
using matrix-assisted laser desorption ionization time-of-flight mass spec-
trometry. J Chromatogr A 2003;1009:111–7.
3.9. N-terminal sequence [16] Bradford MM. A rapid and sensitive method of the quantification of micro-
gram quantities of proteins utilizing the principle of protein dye binding. Anal
Biochem 1976;72:248–54.
The sequence of 6 residues at the N-terminal of the purified pro- [17] Hirata IY, Cezari MHS, Nakaie C, Boschcov P, Ito AS, Juliano MA, et al. Internally
tease was determined and similar peptides retrieved by BLASTp quenched fluorogenic protease substrates: solid-phase synthesis and fluores-
searches of GenBank. Table 8 shows that the sequenced protease cence spectroscopy of peptides containing ortho-aminobenzoyl/dinitrophenyl
groups as donor–acceptor pairs. Lett Pept Sci 1994;1:299–308.
has identity and conserved residues with some proteases from oth- [18] Korkmaz B, Attucci S, Juliano MA, Kalupov T, Jourdan ML, Juliano L, et al. Mea-
ers fungi. Also, the sequence of this protease has identity with the suring elastase, proteinase 3 and cathepsin G activities at the surface of human
proteases from Methylocella silvestris BL2 (unpublished) and a ser- neutrophils with fluorescence resonance energy transfer substrates. Nat Protoc
2008;3:991–1000.
ine protease from Aedes aegypti [42]. [19] Gouvea IE, Izidoro MA, Judice WAS, Cezaria MHS, Caliendo G, Santagada V,
In conclusion, the protease purified from Myceliophthora sp. et al. Substrate specificity of recombinant dengue 2 virus NS2B-NS3 protease:
presents functional and stability properties that could be interest- influence of natural and unnatural basic amino acids on hydrolysis of synthetic
fluorescent substrates. Arch Biochem Biophys 2007;457:187–96.
ing for applications in food industries or detergent formulations,
[20] Klemencic I, Carmona AK, Cezari MH, Juliano MA, Juliano L, Guncar G, et al.
motivating us to perform tests for specific applications. Biochemical characterization of human cathepsin X revealed that the enzyme
 L.M. Zanphorlin et al. / Process Biochemistry 46 (2011) 2137–2143 2143

is an exopeptidase, acting as carboxymonopeptidase or carboxydipeptidase. [32] Wang S, Chen Y, Wang C, Yen Y, Chern M. Purification and characterization of
Eur J Biochem 2000;267:5404–12. a serine protease extracellularly produced by Aspergillus fumigatus in a shrimp
[21] Edman P. A method for the determination of amino acid sequence in peptides. and crab shell powder medium. Enzyme Microb Technol 2005;36:660–5.
Arch Biochem 1949;22:475. [33] Page MJ, Di Cera E. Role of Na+ and K+ in enzyme function. Physiol Rev
[22] Yang J, Liang L, Zhang Y, Li J, Zhang L, Ye F, et al. Purification and cloning of a novel 2006;86:1049–92.
serine protease from the nematode-trapping fungus Dactylellina varietas and [34] Moreira-Gasparin FG, de Souza CGM, Costa AM, Alexandrino AM, Bracht
its potential roles in infection against nematodes. Appl Microbiol Biotechnol CK, Boer CG, et al. Purification and characterization of an efficient poul-
2007;75:557–65. try feather degrading-protease from Myrothecium verrucaria. Biodegradation
[23] Barata RA, Andrade MHG, Rodrigues RD, Castro IM. Purification and character- 2009;20:727–36.
ization of an extracellular trypsin-like protease of Fusarium oxysporum var. lini. [35] Vieille C, Zeikus GJ. Hyperthermophilic enzymes: sources, uses and molec-
J Biosci Bioeng 2002;94:304–8. ular mechanisms for thermostability. Microbiol Mol Biol R 2001;65:
[24] Li J, Yang J, Huang X, Zhang KQ. Purification and characterization of an extracel- 1–43.
lular serine protease from Clonostachys rosea and its potential as a pathogenic [36] Hattori M, Isomura S, Yokoyama E, Ujita M, Hara A. Extracellular trypsin-
factor. Process Biochem 2006;41:925–9. like proteases produced by Cordyceps militaris. J Biosci Bioeng 2005;100:
[25] Yang J, Huang X, Tian B, Wang M, Niu Q, Zhang K. Isolation and characterization 631–6.
of a serine protease from the nematophagous fungus, Lecanicillium psalliotae, [37] Li H, Hu Z, Yuan J, Fan H, Chen W, Wang S, et al. A novel extracellular protease
displaying nematicidal activity. Biotechnol Lett 2005;27:1123–8. with fibrinolytic activity from the culture supernatant of Cordyceps sinensis:
[26] Dunaevsky YE, Matveeva AR, Beliakova GA, Domash VI, Belozersky MA. Extra- purification and characterization. Phytother Res 2007;21:1234–41.
cellular alkaline proteinase of Colletotrichum gloeosporioides. Biochemistry [38] Larcher G, Cimon B, Symoens F, Tronchin G, Chabasse D, Bouchara J.
(Moscow) 2007;72:345–50. A 33 kDa serine proteinase from Scedosporium apiospermum. Biochem J
[27] Dienes D, Borjesson J, Hagglund P, Tjerneld F, Liden G, Reczey K, et al. Identifica- 1996;315:119–26.
tion of a trypsin-like serine protease from Trichoderma reesei QM9414. Enzyme [39] Rigo A, Corazza A, di Paolo ML, Rossetto M, Ugolini R, Scarpa M. Interac-
Microb Technol 2007;40:1087–94. tion of copper with cysteine: stability of cuprous complexes and catalytic
[28] Pekkarinen AI, Jones BL, Niku-Paavola M. Purification and properties of an alka- role of cupric ions in anaerobic thiol oxidation. J Inorg Biochem 2004;98:
line proteinase of Fusarium culmorum. Eur J Biochem 2002;269:798–807. 1495–501.
[29] Khan A, Williams K, Molloy MP, Nevalainen H. Purification and characterization [40] Barrett AJ, Rawlings ND, Woessner F, editors. Handbook of proteolytic enzymes.
of a serine protease and chitinases from Paecilomyces lilacinus and detection of Elsevier; 2004.
chitinase activity on 2D gels. Protein Expr Purif 2003;32:210–20. [41] Tanskul S, Oda K, Oyama H, Noparatnaraporn N, Tsunemi M, Takada K.
[30] Charles P, Devanathan V, Anbu P, Ponnuswamy MN, Kalaichelvan PT, Hur B. Substrate specificity of alkaline serine proteinase isolated from photosyn-
Purification, characterization and crystallization of an extracellular alkaline thetic bacterium, Rubrivivax gelatinosus KDDS1. Biochem Biophys Res Commun
protease from Aspergillus nidulans HA-10. J Basic Microbiol 2008;48:347–52. 2003;309:547–51.
[31] Hajji M, Kanoun S, Nasri M, Gharsallah N. Purification and characterization of [42] Nene V, Wortman JR, Lawson D, Haas B, Kodira C, Tu Z, et al. Genome
an alkaline serine-protease produced by a new isolated Aspergillus clavatus ES1. sequence of Aedes aegypti, a major arbovirus vector. Science 2007;316:
Process Biochem 2007;42:791–7. 1703–4.