International Journal of Food Microbiology 98 (2005) 167 – 177

www.elsevier.com/locate/ijfoodmicro

Protease B from Debaryomyces hansenii:

purification and biochemical properties
Tomás Bolumar, Yolanda Sanz, M-Concepción Aristoy, Fidel Toldrá*
Instituto de Agroquı́mica y Tecnologı́a de Alimentos (C.S.I.C.), Apartado de Correos 73, 46100 Burjassot, Valencia, Spain
Received 21 January 2004; received in revised form 10 May 2004; accepted 27 May 2004

Abstract

The protease B (PrB; EC. 3.4.21.48) of Debaryomyces hansenii CECT 12487 was purified by selective fractionation with
protamine sulfate followed by three chromatographic separations. The whole procedure resulted in 324-fold purification with a
recovery yield of 1.0%. PrB was active at neutral-basic pH ranging from 6.0 to 12.0 with an optimum at pH 8.0. The molecular
mass of the denatured enzyme was 30 kDa. Polyclonal-antibodies raised against PrB from Saccharomyces cerevisiae cross-reacted
with the corresponding 30-kDa protein from D. hansenii. The serine protease inhibitor 3,4-DCI and sulphydryl group reagents
markedly reduced the enzyme activity. The K m against N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin was 1.79 mM. The
presence of endogenous inhibitor for PrB was detected in cell-free extracts of D. hansenii although their inhibitory effect was lost
after incubation at 25 8C for 20 h. PrB was able to hydrolyze muscle sarcoplasmic proteins by in vitro assays. This is the first
endopeptidase purified and characterized from the yeast D. hansenii, whose possible contributions to meat fermentation processes
are discussed.
D 2004 Elsevier B.V. All rights reserved.

Keywords: Protease B; Debaryomyces hansenii; N-Succinyl-Leu-Tyr-7-amido-4-methylcoumarin

1. Introduction fermentation (Jones et al., 1997; Flores et al., 1999).

Yeasts are involved in a variety of food fermentation
processes such as baking, brewing and cheese and
sausages making. Nitrogen metabolism in yeast is
mediated by a number of intracellular proteolytic
enzymes that evolved important cellular roles, affect-
ing their physiology and adaptation during food
* Corresponding author. Tel.: +34 96 3900022; fax: +34 96
3636301.
E-mail address: ftoldra@iata.csic.es (F. Toldrá).
0168-1605/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2004.05.021
In this sense, the comprehension of their proteolytic
systems is a key factor for the control of those industrial
processes.
Debaryomyces hansenii is an halo-tolerant yeast
often found in meat and dairy products (Cook, 1995;
Encinas et al., 2000; Petersen et al., 2002; Bintsis et
al., 2003). In recent years, the interest in this specie
has increased as related to its physiology, biochem-
istry and genetic aspects with impact in industrial
fermentations (Nobre et al., 1999; Lépingle et al.,
2000; Strauss et al., 2001; Bolumar et al., 2003a,b). In
168 T. Bolumar et al. / International Journal of Food Microbiology 98 (2005) 167–177
meat and cheese technology applications, several
studies have demonstrated the successful use of D.
hansenii to produce flavorful fermented products
(Olensen and Stahnke, 2000; Van Den Tempel and
Jakobsen, 2000; Martin et al., 2003). However, very
few studies have been focused on the biochemical
basis behind these desirable effects (Besançon et al.,
1995; Durá et al., 2002; Bolumar et al., 2003a,b).
Proteolysis constitutes one of the major biochemical
phenomenon taking place during meat fermentation
that enhances flavor directly by the generation of
small peptides and free amino acids or by their
conversion into volatile aroma compounds. On the
other hand, the peptides and free amino acids
generated constitute nutritional factors that have an
impact on microbial physiology and microbial inter-
actions contributing, to the outcome of the whole
fermentation process (Toldrá et al., 2001).
So far, most of the studies on the proteolytic
system of typically found in meat microorganisms
have been carried out in lactobacilli and, specially, in
Lactobacillus sakei (Sanz and Toldrá, 2002). How-
ever, the metabolic activities of yeasts adapted to the
meat ecosystem are still poorly understood. Cur-
rently, the proteolytic system of Saccharomyces
cerevisiae is the best characterized (Klionsky et al.,
1990; Jones, 1991, 2002; Van Den Hazel et al.,
1996; Jones et al., 1997). This basically consists of
three major protease groups: the vacuolar proteases,
the cytosolic proteosome and the proteases located
along the secretory pathway. The vacuolar proteases
constitutes the higher pool integrated by two major
endopeptidases (PrA and PrB) and several exopepti-
dases (carboxy- and aminopeptidases, Klionsky et
al., 1990). In general, both endopeptidases together
with the proteosome participate in massive protein
degradation. The composition of the proteolytic
system of D. hansenii is, however, scarcely known.
Only two aminopeptidases have been purified from
this yeast, a proline aminopeptidase and an arginine
aminopeptidase (Bolumar et al., 2003a,b), whereas
its endoproteolytic system remains uncharacterized.
The aim of this work was to purify and determine
the biochemical properties of the protease B (PrB)
from D. hansenii. Comparisons of the purified enzyme
with its counterpart from S. cerevisiae as well as
discussions about its possible implication during meat
fermentation are included.
2. Materials and methods
2.1. Yeast strains and growth conditions
The enzyme was purified from D. hansenii CECT
12487, which was isolated from the natural microflora
of fermented sausages (Santos-Mendoza, 2000). For
purification purposes, the microorganism was growth
in 1.17% (w/v) yeast carbon base (Difco, Detroit,
USA) plus 0.1% (w/v) urea (Panreac, Barcelona,
Spain) as nitrogen source. A total of 120 ml of this
medium was inoculated and incubated at 27 8C, for 2
days, with shaking at 110 revolution per minute. This
pre-culture was used to inoculate 400-ml fresh
medium, which was incubated in the same conditions
for 5 days. S. cerevisiae ATCC 18824 was used as
positive control for immunodetection of PrB by
Western analysis, as described below.
2.2. Preparation of cell extract
Cells were harvested at 4080g for 10 min, at 4
8C, washed with 20 mM sodium phosphate, pH
7.5, and then resuspended in an equivalent volume
of the same buffer. This cell-suspension was used
immediately or frozen with liquid nitrogen and
stored at 80 8C. Cell disruption was carried out in
a bead beater (Biospec Products, Washington, NC,
USA). An equivalent volume of glass beads (0.5
mm diameter, Sigma, St. Louis, MO, USA) was
added to the cell-suspension and then four shakings
for 30 s were applied, with 2-min intervals on ice.
Afterwards, non-broken cells and debris were
separated by two centrifugation steps (14,500g,
15 min at 4 8C and 27,000g, 20 min at 4 8C) and
the supernatant constituted the cell extract used for
purification.
2.3. Enzyme standard assay
Protease B was measured by adding 100 Al of
enzyme to 70 Al of McIlvaine buffer (0.1 M citric acid,
0.2 M disodium phosphate), pH 7.5, containing 0.21
mM N-succinyl-leucine-tyrosine-7-amido-4-methyl-
coumarin (N-succinyl-Leu-Tyr-AMC; Sigma). The
reaction mixture was incubated at 37 8C for 10 min.
Fluorescence was measured in a multiscan fluorometer
(Fluoroskan II, Labsystem, Finland) using excitation
 T. Bolumar et al. / International Journal of Food Microbiology 98 (2005) 167–177
and emission wavelengths of 355 and 460 nm,
respectively. Three replicates were measured for each
experimental point. One unit of enzyme activity (U)
was defined as the release of 1 Amol of sub-
strate1000 per hour at 37 8C.
2.4. Enzyme purification
2.4.1. Protamine sulfate fractionation
Protamine sulphate fractionation was used as
described elsewhere (Bolumar et al., 2003a,b). In
this case, 0.03 mg protamine sulfate/mg protein was
added firstly and then 0.08 mg protamine sulfate/mg
protein.
2.4.2. Weak anion exchange chromatography
The supernatant was injected into a HiPrepk 16/10
DEAE column (Amershan Pharmacia Biotech,
Uppsala, Sweden). The column was initially equili-
brated with 25 mM Tris–HCl, pH 6.5, containing 150
mM NaCl, followed by a gradient from 150 to 500 mM
NaCl in 45 min. The flow rate was 4 ml/min and
fractions of 4 ml were collected. The two fractions with
maximum activity were concentrated using a filter
device biomax 10 K NMWL membrane (Millipore,
Bedford, MA, USA).
2.4.3. Gel filtration chromatography
The concentrated fractions were injected onto a
701.6-cm Sephacryl S-300 HR column (Amershan
Pharmacia Biotech, Uppsala, Sweden) previously
equilibrated with 25 mM Tris–HCl, pH 7.5, contain-
ing 0.1 M NaCl. The column was run at a flow rate of
18.5 ml/h. Fraction volume was 4.5 ml. The two
fractions with maximum activity were subjected to the
following purification step.
2.4.4. Hydrophobic interaction chromatography
The sample was injected into a Resourcek PHE
column (1 ml, Amersham Pharmacia Biotech,
Uppsala, Sweden) previously equilibrated with 50
mM phosphate pH 7.0, containing 1 M (NH4)2SO4.
Proteins were eluted applying an initial gradient from
1 to 0.5 M (NH4)2SO4 in 20 min, a second gradient
from 0.5 to 0 M (NH4)2SO4 in 10 min and a final
isocratic step at 0 M (NH4)2SO4 in 5 min. The flow
rate was 1 ml/min and fractions of 1 ml were
collected.
169
2.5. Determination of protein concentration
Protein concentration was determined by the
BCA (bicinchoninic acid) method (Smith et al.,
1985) with the BCA protein assay reagent (Pierce,
Rockford, IL, USA). Bovine serum albumin was
used as a standard.
2.6. Electrophoresis and Western analysis
The purification was monitored by sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-
PAGE), using 12% resolving gels (Laemmli, 1970).
Broad range molecular mass standards were run
simultaneously (Bio-Rad, Hercules, CA, USA). Pro-
teins were visualized by silver staining or transferred
to polyvinylidene difluoride (PVDF) membranes
(Roche, IN, USA). The transference was carried out
by standard procedures in a Mini Trans-Blot electro-
phoretic transfer cell (Bio-Rad). The primary antibody
was a rabbit polyclonal antibody raised against the PrB
of S. cerevisiae BJ 6974, which was kindly supplied
(Moehle et al., 1987). The secondary antibody was
anti-rabbit IgG alkaline phosphatase conjugate
(Sigma). The substrate CDP-start was used for
chemiluminescence detection (Roche).
2.7. Molecular mass determination
The molecular mass of the native enzyme was
estimated by gel filtration using a Sephacryl S-300 HR
column (Amersham Pharmacia Biotech) as previously
described. The column was calibrated using the
following standard proteins: myosin (450 kDa), h-
amylase (200.0 kDa), bovine serum albumin (68.0
kDa), anhydrase (29.0 kDa) and cytochrome c (12.4
kDa). Blue dextran was used to estimate the void
volume. The molecular mass of the enzyme under
denaturing conditions was also determined by SDS-
PAGE as described above.
2.8. Effect of pH and temperature
The protease B activity was assayed against N-
succinyl-Leu-Tyr-AMC in the pH range from 3.0 to
13.0, at 0.5 pH units intervals, using the following
buffers: McIlvaine’s buffer (0.1 M citric acid, 0.2 M
disodium phosphate) for pH values from 3.0 to 8.0,
170 T. Bolumar et al. / International Journal of Food Microbiology 98 (2005) 167–177
Clark and Lub’s borate buffer (0.1 M boric acid in
0.1 M KCl, 0.1 N NaOH) for pH values from 8.0 to
10.0 and Sorensen’s glycine II buffer (0.1 M glycine
in 0.1 N NaCl, 0.1 N NaOH) for pH values from 10.0
to 13.0.
The effect of temperature was determined in the
range 5–75 8C. The substrate solution (200 Al) was
previously equilibrated at each temperature, and then
the reaction was initiated by the addition of the
purified enzyme (100 Al). After incubation, the
reaction was stopped by addition of 100 Al 0.6 M
acetic acid.
2.9. Effect of chemical agents on the activity
The activity of the purified enzyme was assayed in
the presence of different chemical agents (Table 2) to
identify possible inhibitors or activators by the stand-
ard procedure. The effects of several divalent cations
(CaCl2, MnCl2, CoCl2, CuCl2, CdCl2, HgCl2, MgCl2
and ZnCl2) were determined at 0.05–0.5 mM. The
effects of the three salts (NaCl, KI and (NH4)2SO4)
used during the purification procedure was also
determined at 0.1–1.0 M.
All reagents were purchased from Sigma, except for
Pefabloc SC that was from Merck (Darmstadt, Ger-
many) and salts and (NH4)2SO4 that were from
Panreac.
2.10. Determination of kinetic parameters
The kinetic parameters of the purified enzyme were
estimated for N-succinyl-Leu-Tyr-AMC, using con-
centrations ranging from 0.005 to 0.6 mM. Activity
was continuously measured at 37 8C and kinetic
parameters were calculated from Lineweaver-Burk
plots.
2.11. Detection of endogenous protease inhibitors
Cell-free extract was split into three aliquots: the
first was kept at pH 7.5, the second was adjusted to
pH 5.0 and the third was adjusted to pH 5.0 and
supplemented with 1 mM pepstatin A to avoid the
possible activity of aspartic proteases. All were
incubated at 25 8C for 20 h. The activity against N-
succinyl-Leu-Tyr-AMC was measured initially and at
the end of the incubation period. When appropriate,
the measurements were done in the presence of
specific inhibitors of proteases A and B such as 1
mM pepstatin A and 1 mM 3,4-DCI, respectively.
2.12. Proteolytic activity on muscle protein extracts
2.12.1. Extraction of sarcoplasmic muscle proteins
Sarcoplasmic proteins were extracted from Long-
issimus dorsi muscles with 10 volumes of 20 mM
sodium phosphate buffer, pH 7.5 containing 0.02%
azide, as described by Fadda et al. (1999). The
extract was filtered sterilized through a 0.22-Am
pore size membrane (Millipore). The protein con-
tent of the sarcoplasmic extract was approximately
1.9 mg/ml.
2.12.2. Extraction of myofibrillar muscle proteins
Myofibrillar proteins were extracted from the
pellet obtained in the previous step using 10 volumes
of 0.7 M KI containing 0.02% azide, as described by
Fadda et al. (1999). The final myofibrillar extract
was diluted 10 times and filtered sterilized through a
0.22-Am pore size membrane (Millipore). The
protein content of the final myofibrillar extract was
0.4 mg/ml.
2.12.3. Enzymatic mixtures
The enzymatic mixture consisted of 2.5 ml of
sterile sarcoplasmic or myofibrillar protein extract
plus 2.5 ml of the purified PrB. A control protein
extract without the addition of PrB was assayed
simultaneously. The mixtures were incubated at 37
8C. Samples were taken at different times during the
incubation period (0, 2, 5, 10 and 20 days). Protein
hydrolysis was analyzed by SDS-PAGE as described
above.
3. Results
3.1. Purification of PrB
The results of the purification of PrB from the cell
extract of D. hansenii are summarized in Table 1. An
important increase in specific activity of about 20-
fold was already achieved in the initial purification
step by protamine fractionation between 0.03 and
0.08 mg protamine/mg protein. The use of protamine
 T. Bolumar et al. / International Journal of Food Microbiology 98 (2005) 167–177 171

Table 1 3.3. Effect of pH and temperature on the activity

Purification of protease B (PrB) from D. hansenii
Purification step Protein Total Specific Yield Purification The enzyme showed activity at neutral-alkaline pH
(mg) activity activity (%) (fold)
values ranging from 6.0 to 12.0, with an optimum at
(U)a (U/ mg)
pH 8.0. The reaction rate was higher when the
Cell extract 411.75 375.0 0.9 100 1
temperature increased up to 75 8C. Although, the
Resuspended 31.50 562.1 17.8 149.9 19.6
pellet from stability of the enzyme rapidly decreased above 37 8C.
protamine Fifty percent of the enzyme activity was kept after 10
fractionation min incubation at 65 8C.
Weak anion 1.16 77.7 67.0 20.7 73.5
exchange
3.4. Effect of chemical agents
chromatography
Gel filtration 0.16 31.4 196.3 8.4 215.5
chromatography The effects of potential inhibitors or activators on
Hydrophobic 0.01 3.7 370.0 1.0 406.3 PrB activity are shown in Table 2. The serine
interaction protease inhibitor 3,4-DCI and the cysteine protease
chromatography
a
inhibitor p-chloromercuribenzoic acid completely
U=umoles AMC released1000/hour. abolished PrB activity (Table 2). Leupeptin and
iodoacetate, which are inhibitors of serine and
sulfate is a convenient method to purify negatively cysteine proteases, also reduced PrB activity to
charged proteins as it is an easy procedure to initially 68% and 66%, respectively (Table 2). These results
increase the purification level in crude extracts before suggested that serine and cysteine residues are
the application of more sophisticated chromato- important for the catalytic activity. Neither chelating
graphic steps. From the anion-exchange chromatog- agents nor reducing agents significantly affected PrB
raphy on a DEAE column two separated active peaks activity (Table 2). The effects of different divalent
against N-succinyl-Leu-Tyr-AMC were obtained, cations on PrB were also determined. Hg2+ drasti-
eluting at 300 and 400 mM, respectively (Fig. 1A). cally reduced the activity to 0% at both 0.05 and 0.5
The purification was further focused on the second mM. For the rest of the tested divalent cations, only
peak that was the one showing higher activity levels. Cu2+, Cd2+ and Zn2+ concentrations of 0.5 mM
Only the two fractions with maximum activity were caused a remarkable reduction in the activity of
pooled and injected onto the gel filtration column. around 30%.
This chromatographic separation successfully elimi- The presence of KI inhibited the enzyme
nated proteins of low molecular mass (Fig. 1B), activity completely at concentrations over 0.1 M,
which resulted in an important enrichment in specific while NaCl only caused complete inhibition at the
activity (Table 1). From the hydrophobic interaction highest concentrations, 0.5 and 1 M reduced the
column, maximum activity of PrB eluted at 660 mM activity to 50% and 100%, respectively. The
(NH4)2SO4 (Fig. 1C). The whole purification process inhibitory effects of both salts (KI and NaCl) were
yielded 1.0% and 406.3-fold increment in specificity reversible as the activity was recovered after
(Table 1). elimination of the salt by dialysis or dilution.
The inhibitory effect of (NH4)2SO4 was less
3.2. Molecular mass, purity and immunodetection important and 50% of the optimal activity was
retained even at 1 M concentration although the
The SDS-PAGE analysis of the purified sample activity was no longer recovered.
displayed a single band of approximately 30.0 kDa
(Fig. 2A), which corresponds with that reacting with 3.5. Kinetics parameters
the anti-PrB antibody raised against the enzyme of S.
cerevisiae (Fig. 2B). The molecular mass of native The V max and K m values for N-succinyl-Leu-Tyr-
enzyme estimated by gel filtration was approximately AMC were 7.46*10 4 (Amol min 1 mg 1) and 1.76
430 kDa. mM, respectively.
172 T. Bolumar et al. / International Journal of Food Microbiology 98 (2005) 167–177

Fig. 1. Chromatograms from different steps in the purification of PrB from D. hansenii. (A) Weak anion exchange chromatography in a DEAE
column, (B) gel filtration in a Sephacryl S-300 HR column, (C) hydrophobic interaction chromatography in a Resource-PHE column. Protein
was detected by measuring the absorbance at 280 nm (dotted line), PrB activity is expressed in fluorescence units (FU) (solid line) and NaCl or
(NH4)2(SO4) gradient is indicated (long dash line).
 T. Bolumar et al. / International Journal of Food Microbiology 98 (2005) 167–177 173

Fig. 2. (A) SDS-PAGE of the purification steps of PrB from D. hansenii. (1) Cell extract, (2) resuspended pellet from protamine fractionation,
(3) anion exchange chromatography, (4) gel filtration chromatography, (5) purified protein from hydrophobic interaction chromatography and
(6) standard proteins in kDa. (B) Western analysis of active fractions of PrB using polyclonal anti PrB antibodies raised against the enzyme of S.
cerevisiae. (1) Purified fractions of PrB from D. hansenii and (2) cell-free extract from S. cerevisiae.

3.6. Detection of possible endogenous protease higher after incubation at 25 8C for 20 h, suggesting
inhibitors in cell extracts the initial presence of endogenous inhibitors that
could be, at least, partially inactivated along the
The activity against N-succinyl-Leu-Tyr-AMC incubation period (Table 3). The values of the initial
detected in all cell extracts was two- to three-fold activities of the three cell extracts in the control assays
Table 2
were approximately equal to those obtained in the
Effect of chemical agents on the activity of the purified PrB presence of 3,4-DCI plus those obtained in the
Chemicals Relative activitya
presence of pepstatin A. Therefore, the total activity
against N-succinyl-Leu-Tyr-AMC seems to be the
Concentration (mM)
sum of activities from two different class of enzymes,
0.05 0.5 0.1 1 5
b
aspartic protease(s) inhibited by pepstatin A and
Leupeptin 85 68 – – – serine protease(s) inhibited by 3,4-DCI.
Puromycin 88 85 – – –
Bestatin 89 90 – – –
E-64 91 93 – – – 3.7. Proteolytic activity on muscle protein extracts
Pepstatin A 86 95 – – –
Iodoacetate – – 81 66 – The ability of PrB to hydrolyze muscle proteins
3,4-DCI 11 0 0 0 – was determined in vitro by incubation of sarcoplasmic
PMSF – – 100 90 –
Pentabloc SC – – 100 102 –
and myofibrillar protein extracts in the presence of the
p-cloromercuribenzoic – – 0 0 – purified enzyme. The proteolytic changes resulting
EDTA – – – 91 99 from the activity of PrB on sarcoplasmic extracts were
EGTA – – – 86 82 analyzed by SDS-PAGE (Fig. 3). The protein profiles
o-Phenantroline – – 94 90 – revealed a decrease in the intensity of bands of 173,
DTT – – – 104 103
h-mercaptoethanol – – – 86 98
83 and 20 kDa and the appearance of a new band
a corresponding to 133 kDa upon addition of PrB.
Expressed as a percentage of the activity obtained in the
absence of any added chemical agent, which was given a value of Other major changes were the disappearance of
100%. protein bands of 73 and 52 kDa and the appearance
b
(–) Non–determined. of others of 124 and 32 kDa. Myofibrillar proteins
174 T. Bolumar et al. / International Journal of Food Microbiology 98 (2005) 167–177

Table 3
Activity of the cell extract from Debaryomyces hansenii against N-SuccinylLeuTyr-AMC in different conditions to detect potential endogenous
inhibitors
Time Reaction Cell extract at pH=7.0 Cell extract at pH=5.0 Cell extract at pH=5.0+Pepstatin A 1 mM
(hours) mixture Controla Pepstatin 3,4-DCI Control Pepstatin 3,4-DCI Control Pepstatin 3,4-DCI
1 mM 1 mM 1 mM 1 mM 1 mM 1 mM
0 50.4 33.0 18.5 24.5 12.7 13.6 8.1 6.8 4.0
20 100.0 44.9 30.3 62.5 23.1 22.0 21.8 11.6 7.4
a
Expressed as a percentage of the higher activity obtained, which was taken as value 100%.

were not hydrolyzed (data not shown) possibly due to
the presence of KI used in the protein extraction
procedure as demonstrated by inhibition studies
during the characterization of the purified enzyme.
The enzyme did not show exopeptidase activity as it
was not able to hydrolyze substrates of amino-
peptidases such as tyrosine-AMC, leucine-AMC and
arginine-AMC. These results confirmed that the
purified enzyme is an endoprotease type.
4. Discussion
PrB constitutes the first endoprotease purified from
the yeast D. hansenii. The denatured enzyme has a
molecular mass of 30 kDa, which is similar to the
ones (30–33 kDa) reported for the corresponding
protease of S. cerevisiae (Sanada et al., 1979;
Fig. 3. SDS-PAGE of sarcoplasmic protein extracts incubated with
purified PrB of D. hansenii. (1) Standard proteins, (2, 3) control
samples at time 0 and after 20 days of incubation, respectively, (4,
5) samples containing PrB at time 0 and 20 days of incubation,
respectively.
Kominami et al., 1981a; Nowak and Tsai, 1989) and
Candida albicans (Farley et al., 1986). However, the
molecular mass of the native enzyme (430 kDa) from
D. hansenii differs from the ones reported for S.
cerevisiae (34–33.7 kDa) by Kominami et al. (1981a)
and Nowak and Tsai (1989). The differences in
molecular mass between the denatured and native
enzyme from D. hansenii could be due to the possible
oligomeric composition of PrB although its counter-
part form S. cerevisiae has been described as a single-
subunit glycoprotein (Moehle et al., 1987).
The purified protein raised positive reaction using
antibodies against PrB of S. cerevisiae, confirming the
identity of the purified enzyme with its counterpart in
S. cerevisiae. Also, partial exploration of the genome
of D. hansenii has allowed to identified part of the
sequence of the gene (PRB1) likely encoding PrB on
the basis of its homology with that of S. cerevisiae
(Lépingle et al., 2000).
The activity of PrB was optimal at pH 8.0, which
corroborates that is a typical alkaline protease as the
known homologous enzyme for Aspergillus
(Impoolsup et al., 1981). The purified PrBs from
yeasts also have neutral or basic optimal pH (Fujishiro
et al., 1980; Nowak and Tsai, 1989). Most of the
purified PrB showed activities at temperatures above
37 8C, which is a typical characteristic of proteases
from the subtilase family.
On the basis of studies with various inhibitors, PrB
of D. hansenii can be classified as a serine protease
because of its outstanding inhibition by 3,4-DCI. PrB
is considered an endoprotease located in the vacuole
whose primary sequence shows striking homology to
those of the subtilisins, proteinase K and thermitase
(Moehle et al., 1987). Like proteinase K and
thermitase, but unlike the subtilisins, protease B is a
serine protease that normally contains a free cysteine
residue that presumably is near the active site (Nowak
 T. Bolumar et al. / International Journal of Food Microbiology 98 (2005) 167–177
and Tsai, 1989). Cysteine residues are also important
for the catalytic activity of PrB from D. hansenii since
sulphydryl group reagents inhibited it (Table 2). Other
possible homologous enzymes from the fungi, Phy-
comyces spp. and Neurospora crassa, have been
described as well as sulphydryl reagent-sensitive
serine proteases (Fischer, 1979; Abbott and Marluf,
1984).
The presence of endogenous inhibitors in cell-free
extracts of D. hansenii were clearly detected (Table
3), following the strategies that have been proved to
inactivate endogenous protease inhibitors in S. cer-
evisiae, such as long incubation periods at 25 8C and
at acid pH (Fujishiro et al., 1980; Magni et al., 1986).
The existence of these natural inhibitors of PrB has
been described in S. cerevisiae (Fischer and Holzer,
1980; Magni et al., 1986, Schu et al., 1991),
Schizosaccharomyces pombe (Escudero et al., 1993)
and Kluyveromyces lactis (Flores et al., 1999).
Initially, it was proposed that the degradation of the
specific inhibitor of PrB was due to the action of PrA,
which is inhibited by pepstatin A (Jones et al., 2002).
However, our results show that inactivation took place
to the same extent in both extracts adjusted at pH 5,
regardless the presence of pepstatin, suggesting that
this inactivation may be due to the acid pH environ-
ment rather than to the activity of an aspartic protease.
These results are in accordance with those of Magni et
al. (1986). In addition, these assays demonstrated the
presence of at least two type endoproteases (serine
and aspartic proteases) for which natural inhibitors are
initially present. The existence of two major proteo-
lytic activities, PrA and PrB, with their own inhibitors
is documented in S. cerevisiae (Van Den Hazel et al.,
1996). PrA is an aspartic protease inhibited by
pepstatin and PrB a serine protease inhibited by p-
chloromercuribenzoic (PCMB) and Hg2+. In the past,
it was described that the initial site of hydrolysis of the
oxidized B-chain of insulin by PrB of baker’s yeast
and Candida albicans was the Leu-Tyr peptide bond
(Kominami et al., 1981b; Farley et al., 1986). Thus,
the fluorimetric substrate (N-succinyl-Leu-Tyr-AMC)
used in this study could constitute a simple and more
sensitive method to measure this enzyme.
D. hansenii was able to hydrolyze sarcoplasmic
proteins in a previous in vitro assay using cell
suspensions as well as cell-free extracts (Santos et
al., 2001). Moreover, by then nothing was known
175
about the possible enzymes responsible for the
detected hydrolytic changes. In this study, it has been
demonstrated that PrB can be one of the enzymes
involved in this degradation (Fig. 3) and could be
partially responsible for protein breakdown during
meat fermentation. The products resulting from the
degradation of sarcoplasmic proteins by PrB could be
used as nutrients and confer a competitive advantage
to survive in protein rich products to this specie.
In summary, this work reports valuable biochem-
ical data about the properties of the PrB from D.
hansenii, which can be the basis for further studies
focused on its genetic and functional characterization.
The evidence of the existence of intracellular protease
inhibitors in D. hansenii can also contribute to get a
better understanding of the protein metabolism in this
specie. Finally, the functionality of PrB from D.
hansenii in the hydrolysis of muscle sarcoplasmic
proteins should be considered of interest in relation to
its performance as meat starter culture.
Acknowledgements
This work has been supported by grant AGL2001-
1141 from CICYT (Spain). FPU/MEC scholarship to
Tomás Bolumar is fully acknowledged.
Authors wish to thank Dr. Elizabeth Jones from the
Department of Biological Sciences, Carnegie Mellon
University, Pittsburgh (PE, USA) for the kind supply
of the antibody against the PrB of S. cerevisiae.
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