Protease B From Debaryomyces Hansenii

8/6/2019 Protease B From Debaryomyces Hansenii

http://slidepdf.com/reader/full/protease-b-from-debaryomyces-hansenii 1/11

Protease B from Debaryomyces hansenii:

purification and biochemical properties

Tomas Bolumar, Yolanda Sanz, M-Concepcion Aristoy, Fidel Toldra*

Instituto de Agroquımica y Tecnologıa 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 denaturedenzyme was 30 kDa. Polyclonal-antibodies raisedagainst 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

Yeasts are involved in a variety of food fermentation

processes such as baking, brewing and cheese andsausages 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

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

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; Lepingle et al.,

2000; Strauss et al., 2001; Bolumar et al., 2003a,b). In

0168-1605/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.ijfoodmicro.2004.05.021

* Corresponding author. Tel.: +34 96 3900022; fax: +34 96

3636301.

E-mail address: [email protected] (F. Toldra).

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

www.elsevier.com/locate/ijfoodmicro



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 andJakobsen, 2000; Martin et al., 2003). However, very

few studies have been focused on the biochemical

basis behind these desirable effects (Besancon et al.,

1995; Dura 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 animpact on microbial physiology and microbial inter-

actions contributing, to the outcome of the whole

fermentation process (Toldra 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 Toldra, 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 conditionsfor 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 4080Â g 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 À80 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 (14,500Â g ,

15 min at 4 8C and 27,000Â g , 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) 167–177 168



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-strateÂ1000 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

70Â1.6-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 chromatographyThe 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.

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 electro phoresis (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,




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 10.0to 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, MgCl2and 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




sulfate is a convenient method to purify negatively

charged proteins as it is an easy procedure to initially

increase the purification level in crude extracts before

the application of more sophisticated chromato-

graphic steps. From the anion-exchange chromatog-

raphy on a DEAE column two separated active peaks

against N -succinyl-Leu-Tyr-AMC were obtained,

eluting at 300 and 400 mM, respectively (Fig. 1A).

The purification was further focused on the second

peak that was the one showing higher activity levels.

Only the two fractions with maximum activity were

pooled and injected onto the gel filtration column.

This chromatographic separation successfully elimi-

nated proteins of low molecular mass (Fig. 1B),

which resulted in an important enrichment in specific

activity (Table 1). From the hydrophobic interaction

column, maximum activity of PrB eluted at 660 mM

(NH4)2SO4 (Fig. 1C). The whole purification process

yielded 1.0% and 406.3-fold increment in specificity

(Table 1).

3.2. Molecular mass, purity and immunodetection

The SDS-PAGE analysis of the purified sample

displayed a single band of approximately 30.0 kDa

(Fig. 2A), which corresponds with that reacting with

the anti-PrB antibody raised against the enzyme of S.

cerevisiae (Fig. 2B). The molecular mass of native

enzyme estimated by gel filtration was approximately

430 kDa.

3.3. Effect of pH and temperature on the activity

The enzyme showed activity at neutral-alkaline pH

values ranging from 6.0 to 12.0, with an optimum at pH 8.0. The reaction rate was higher when the

temperature increased up to 75 8C. Although, the

stability of the enzyme rapidly decreased above 37 8C.

Fifty percent of the enzyme activity was kept after 10

min incubation at 65 8C.

3.4. Effect of chemical agents

The effects of potential inhi bitors or activators on

PrB activity are shown in Table 2. The serine

protease inhibitor 3,4-DCI and the cysteine proteaseinhibitor p-chloromercuri benzoic acid completely

abolished PrB activity (Table 2). Leupeptin and

iodoacetate, which are inhibitors of serine and

cysteine proteases, also reduced PrB activity to

68% and 66%, respectively (Table 2). These results

suggested that serine and cysteine residues are

important for the catalytic activity. Neither chelating

agents nor reducing agents significantly affected PrB

activity (Table 2). The effects of different divalent

cations on PrB were also determined. Hg2+ drasti-

cally reduced the activity to 0% at both 0.05 and 0.5

mM. For the rest of the tested divalent cations, only

Cu2+, Cd2+ and Zn2+ concentrations of 0.5 mM

caused a remarkable reduction in the activity of

around 30%.

The presence of KI inhibited the enzyme

activity completely at concentrations over 0.1 M,

while NaCl only caused complete inhibition at the

highest concentrations, 0.5 and 1 M reduced the

activity to 50% and 100%, respectively. The

inhibitory effects of both salts (KI and NaCl) were

reversible as the activity was recovered after

elimination of the salt by dialysis or dilution.The inhibitory effect of (NH4)2SO4 was less

important and 50% of the optimal activity was

retained even at 1 M concentration although the

activity was no longer recovered.

3.5. Kinetics parameters

The V max and K m values for N -succinyl-Leu-Tyr-

AMC were 7.46*10À4 (Amol minÀ1 mgÀ1) and 1.76

mM, respectively.

Table 1

Purification of protease B (PrB) from D. hansenii

Purification step Protein

(mg)

Total

activity(U)a

Specific

activity(U/ mg)

Yield

(%)

Purification

(fold)

Cell extract 411.75 375.0 0.9 100 1

Resuspended

pellet from

protamine

fractionation

31.50 562.1 17.8 149.9 19.6

Weak anion

exchange

chromatography

1.16 77.7 67.0 20.7 73.5

Gel filtration

chromatography

0.16 31.4 196.3 8.4 215.5

Hydrophobic

interaction

chromatography

0.01 3.7 370.0 1.0 406.3

a U=umoles AMC releasedÂ1000/hour.




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).




3.6. Detection of possible endogenous protease

inhibitors in cell extracts

The activity against N -succinyl-Leu-Tyr-AMC

detected in all cell extracts was two- to three-fold

higher after incubation at 25 8C for 20 h, suggesting

the initial presence of endogenous inhibitors that

could be, at least, partially inactivated along the

incubation period (Table 3). The values of the initial

activities of the three cell extracts in the control assays

were approximately equal to those obtained in the

presence of 3,4-DCI plus those obtained in the

presence of pepstatin A. Therefore, the total activity

against N -succinyl-Leu-Tyr-AMC seems to be the

sum of activities from two different class of enzymes,

aspartic protease(s) inhibited by pepstatin A and

serine protease(s) inhibited by 3,4-DCI.

3.7. Proteolytic activity on muscle protein extracts

The ability of PrB to hydrolyze muscle proteins

was determined in vitro by incubation of sarcoplasmicand myofibrillar protein extracts in the presence of the

purified enzyme. The proteolytic changes resulting

from the activity of PrB on sarcoplasmic extracts were

analyzed by SDS-PAGE (Fig. 3). The protein profiles

revealed a decrease in the intensity of bands of 173,

83 and 20 kDa and the appearance of a new band

corresponding to 133 kDa upon addition of PrB.

Other major changes were the disappearance of

protein bands of 73 and 52 kDa and the appearance

of others of 124 and 32 kDa. Myofibrillar proteins

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.

Table 2

Effect of chemical agents on the activity of the purified PrB

Chemicals Relative activitya

Concentration (mM)

0.05 0.5 0.1 1 5

Leupeptin 85 68 – b – –

Puromycin 88 85 – – –

Bestatin 89 90 – – –

E-64 91 93 – – –

Pepstatin A 86 95 – – –

Iodoacetate – – 81 66 –

3,4-DCI 11 0 0 0 – PMSF – – 100 90 –

Pentabloc SC – – 100 102 –

p-cloromercuribenzoic – – 0 0 –

EDTA – – – 91 99

EGTA – – – 86 82

o-Phenantroline – – 94 90 –

DTT – – – 104 103

h-mercaptoethanol – – – 86 98

a Expressed as a percentage of the activity obtained in the

absence of any added chemical agent, which was given a value of

100%. b (–) Non–determined.




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;

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.

cerev isiae (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

(Lepingle et al., 2000).

The activity of PrB was optimal at pH 8.0, which

corroborates that is a typical alkaline protease as the

k n ow n h o mo l o go u s e n zy m e f o r 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

Table 3

Activity of the cell extract from Debaryomyces hansenii against N-SuccinylLeuTyr-AMC in different conditions to detect potential endogenous

inhibitors

Time(hours)

Reactionmixture

Cell extract at pH=7.0 Cell extract at pH=5.0 Cell extract at pH=5.0+Pepstatin A 1 mMControla Pepstatin

1 mM

3,4-DCI

1 mM

Control Pepstatin

1 mM

3,4-DCI

1 mM

Control Pepstatin

1 mM

3,4-DCI

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%.

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.




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 theoxidized 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

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 toTomas 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.

References

Abbott, R., Marluf, G., 1984. Major extracellular proteases of

Neurospora crassa. J. Bacteriol. 159, 505–510.

Besancon, X., Ratomahenina, R., Galzy, P., 1995. Isolation and partial characterization of an esterase EC 3.1.1.1 from a

Debaryomyces hansenii strain. Neth. Milk Dairy J. 49, 97–110.

Bintsis, T., Vafopoulou-Mastrojiannaki, A., Litopoulou-Tzanetaki,

E., Robinson, R.K., 2003. Protease, peptidase and esterase

activities by lactobacilli and yeast isolates from Feta cheese

brine. J. Appl. Microbiol. 95, 68–77.

Bolumar, T., Sanz, Y., Aristoy, M.-C., Toldra, F., 2003a. Purification

and characterization of a prolyl aminopeptidase from Debar-

yomyces hansenii. Appl. Environ. Microbiol. 69, 227–232.

Bolumar, T., Sanz, Y., Aristoy, M.-C., Toldra, F., 2003b. Purifica-

tion and properties of an arginyl aminopeptidase from Debar-

yomyces hansenii. Int. J. Food Microbiol. 86, 141–151.




Cook, P.E., 1995. Fungal ripened meats and meat products.

In: Fermented meats. Campbell-Platt, G., Cook, P.E., Eds.,

Blackie Academic and Professional. Chapman and Hall.

London, UK, pp. 110–129.

Dura, A., Flores, M., Toldra, F., 2002. Purification and character-

ization of a glutaminase from Debaryomyces spp. Int. J. Food

Microbiol. 76, 117–126.

Encinas, J.-P., Lopez-Dıaz, T.-M., Garcıa-Lopez, M.-L., Otero, A.,

Moreno, B., 2000. Yeast populations on Spanish fermented

sausages. Meat Sci. 54, 203–208.

Escudero, B., Parra, F., Suarez-Rendueles, P., 1993. Purification

and characterization of the endogenous inhibitor for protei-

nase B from Schizosaccharomyces pombe. Biochimie 75,

855–859.

Fadda, S., Sanz, Y., Vignolo, G., Aristoy, M.-C., Oliver, G., Toldra,

F., 1999. Characterization of muscle sarcoplasmic and myofi-

brillar protein hydrolysis caused by Lactobacillus plantarum.

Appl. Environ. Microbiol. 65, 3540–3546.Farley, P.C., Shepherd, M.G., Sullivan, P.A., 1986. The purification

and properties of yeast proteinase B from Candida albicans.

Biochem. J. 236, 177–184.

Fischer, E.P., 1979. Limited proteolysis in phycomyces. In: Cohen,

G., Holzer, H. (Eds.), Limited Proteolysis of Microorganism,

U.S. Department of Health, Education, and Welfare. National

Institutes of Health, Bethesda, MD, USA, pp. 139– 144.

publication no. NIH 79-1591.

Fischer, E.P., Holzer, H., 1980. Interaction of proteinases and their

inhibitors from yeast. Activation of carboxipeptidase Y.

Biochim. Biophys. Acta 615, 87–198.

Flores, M.V., Cuellas, A., Voget, C.E., 1999. The proteolytic system

of the yeast Kluyveromyces lactis. Yeast 15, 1437–1448.

Fujishiro, K., Sanada, Y., Tanaka, H., Katunuma, N., 1980.Purification and characterization of yeast protease B. J.

Biochem. 87, 1321–1326.

Impoolsup, A., Bhumiratana, A., Flegel, T.W., 1981. Isolation of

alkaline and neutral proteases from Aspergillus flavus colum-

naris, a soy sauce koji mold. Appl. Environ. Microbiol. 42,

619–628.

Jones, E.W., 1991. Three proteolytic systems of the yeast

Saccharomyces cerevisie. J. Biol. Chem. 266, 7963–7966.

Jones, E.W., 2002. Vacuolar proteases and proteolytic artifacts in

Saccharomyces cerevisiae. In: Guthrie, Christine, Fink, Gerald

R. (Eds.), Guide to Yeast Genetics and Molecular and Cell

Biology, Methods in Enzymology vol. 351. Academic press,

Elselvier Science, CA, USA, pp. 127–150.

Jones, E.W., Webb, G.C., Hiller, M.A., 1997. Biogenesis and

function of the yeast vacuole. In: Pringle, J.R., Broach, J.R.,

Jones, E.W. (Eds.), Molecular and Cellular Biology of the

Yeast Saccharomyces , Cell Cycle and Cell Biology. Cold

Spring Harbor Laboratory Press, Cold Spring Harbor, NY,

USA, pp. 363– 470.

Klionsky, D.J., Herman, P.K., EMR, S.D., 1990. The fungal

vacuole, composition, function and biogenesis. Microbiol.

Rev. 54, 266–292.

Kominami, E., Hoffschulte, H., Holzer, H., 1981a. Purification and

properties of proteinase B from yeast. Biochim. Biophys. Acta

661, 124–135.

Kominami, E., Hoffschulte, H., Leuschel, L., Maier, K., Holzer, H.,

1981b. The substrate specificity of proteinase B from baker’s

yeast. Biochim. Biophys. Acta 661, 136–141.

Laemmli, U.K., 1970. Cleavage of structural proteins during

assembly of the head of bacteriophage T4. Nature 227,

680–685.

Lepingle, A., Casaregola, S., Neuveglise, C., Bon, E., Nguyen,

H.-V., Artiguenave, F., Wincker, P., Gaillardin, C., 2000.

Genomic exploration of the Hemiascomycetous yeast: 14.

Debaryomyces hansenii var. hansenii . FEBS Lett. 487, 82–86.

Magni, G., Drewniak, M., Santarelli, I., Huang, C.Y., 1986.

Reexamination of the activation of yeast proteinase B at pH 5,

loss of inhibition effect of proteinase B inhibitors. Biochem. Int.

12, 557–565.

Martin, A., Cordoba, J.J., Benito, M.J., Aranda, E., Asensio, M.A.,

2003. Effect of Penicillium chrysogenum and Debaryomyces

hansenii on the volatile compounds during controlled ripening

of pork loins. Int. J. Food Microbiol. 84, 327–338.Moehle, C.M., Tizard, R., Lemmon, S.K., Smart, J., Jones, E., 1987.

Protease B of the lysosomelike vacuole of the yeast Saccha-

romyces cerevisiae is homologous to the subtilisin family of

serine proteases. Mol. Cell. Biol. 7, 4390–4399.

Nobre, A., Lucas, C., Leao, C., 1999. Transport and utilization of

hexoses and pentoses in the halotolerant yeast Debaryomyces

hansenii . Appl. Environ. Microbiol. 65, 3594–3598.

Nowak, J., Tsai, H., 1989. Purification and properties of three

endopeptidases from baker’s yeast. Can. J. Microbiol. 35,

295–303.

Olensen, P.-T., Stahnke, L.-H., 2000. The influence of Debaryo-

myces hansenii and Candida utilis on the aroma formation in

garlic spiced fermented sausages and model minces. Meat Sci.

56, 357–368.Petersen, K.M., Westall, S., Jespersen, L., 2002. Microbial

succession of Debaryomyces hansenii strains during the

production of Danish surface-ripened cheeses. J. Dairy Sci.

85, 478–486.

Sanada, Y., Fujishiro, K., Tanaka, H., Katunuma, N., 1979. Isolation

and characterization of yeast protease B. Biochem. Biophys.

Res. Commun. 86, 815–821.

Santos, N., Santos-Mendoca, R.-C., Sanz, Y., Bolumar, T., Aristoy,

M.-C., Toldra, F., 2001. Hydrolysis of pork muscle sarcoplasmic

proteins by Debaryomyces hansenii. Int. J. Food Microbiol. 68,

199–206.

Santos-Mendoza, R.C., 2000. PhD thesis. Aislamiento, seleccion y

caracterizacion de levaduras de embutidos con vistas a su

utilizacion como coadyuvante en el proceso de curado.

Universidad de Valencia, Valencia, Spain.

Sanz, Y., Toldra, F., 2002. Purification and characterization of an

arginine aminopeptidase from Lactobacillus sakei. Appl.

Environ. Microbiol. 68, 1980–1987.

Schu, P., Suarez-Rendueles, P., Wolf, D.H., 1991. The proteinase

yscB inhibitor PB12 gene of yeast and studies on the function

on its protein product. Eur. J. Biochem. 197, 1–7.

Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Garthner,

F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson,

B.J., Klenk, D., 1985. Measurement of protein using bicinchi-

ninic acid. Anal. Biochem. 150, 76–85.




Strauss, M.L., Jolly, N.P., Lambrechts, M.G., Van Rensburg, P.,

2001. Screening for the production of extracellular hydrolytic

enzymes by non-Saccharomyces wine yeast. J. Appl. Microbiol.

91, 182–190.

Toldra, F., Sanz, Y., Flores, M., 2001. Meat fermentation

technology. In: Hui, Y.H., Shorthose, R., Young, O., Koohma-

raie, M., Rogers, R. (Eds.), Meat Science and Applications.

Marcel Dekker, New York, USA, pp. 537–561.

Van Den Hazel, H., Kielland-Brandt, M.C., Winther, J.R., 1996.

Review: biosynthesis and function of yeast vacuolar proteases.

Yeast 12, 1–16.

Van Den Tempel, T., Jakobsen, M., 2000. The technological

characteristics of Debaryomyces hansenii and Yarrowia lip-

olytica and their potential as starter cultures for production of

Danablu. Int. Dairy J. 10, 263–270.


Documents

Protease B From Debaryomyces Hansenii