War/d Journa/ of Micrubiob~y & Biotechnology 13, 153-155

Ch~r~~teriz~tiu~ of two ~xtra@luiar

proteases from Leuconostoc oenos

G.C. Roll&n~ M.Ee Farias and k4.C. Marxa De Nadra*

Two extracellular proteolytic activities were characterized from Leuconosfoc oenos isoIated from Argentinian wines.
Both activities were maximal with autoclaved grape juice as substrate. The temperature and pH optima for the two
proteolytic activities were different (30 and 40°C, and pH 4.0 and 5.5, respectively). Both enzymes were
thermostabIe and their activities were unaltered by heating at 7&X2 for 15 min. MetaI ions were not required for
the activities. Neither enzyme appeared to be a se&e protease but both were strongIy inhibited by cysteine and
~-mercaptoethanol, indicating the involvement of d~sulphide bridges.

Key words: Leucanosk oenos, proteases,

Wine is a poor medium for bacteriai growth because of its

limited content of nutrienk. We have already reported the
production of two exh-acellular proteases by each of four
strains of Aeticoeusk oenos (X2L, L2, m and ST) isolated
from Argentinian wines (Rok5n et al. 1993). One enzyme
appears in the early growth phase and the other is maximal
at the end of grow&. The pH and temperature optima for
the production of the two proteases from each strain differ
and the effects of divalent metal ions differ within and
between s&rains.
The proteolyEc a&&es of ~euc~~~s~~c oenus X2L on
different substrates, the effects of inhibitors, pH and
temperature on them and their thermal stability are the
subjects of the present study.

Maktrials and Methods

~et{co~osfucomos strain XzL &ze R&n et al. 1993) was grown in a

basal medium containing IO g yeast extract, 5 g glucose, I,0 m1 3 4 6 6 T 8
Tween 80 and 170 ml grape juice/ I distilled water. The pH was m
adjusted to 4.5 with Cl.1M HCI and the medium was sterilized for
15 min at 1Zl’C. Figure 1. Effect of pH on the proteolytic specific activities pro-
duced by Leuconosfoc oet?os X& using autociaved grape juke
as substrate. O-Protease 1 at WC; *-protease II at 40%.
0.05 M c&ate buffers were used far @-I 2.~ to 6.0 and tD35 M
The aufhors are with the Centro de Referencia para Lacfobacilos phosphate buffers for pH 5.8 to 8.5.
(CERELA)% Chacabuco 145, 4OMl TucumGm, Argentina: fax: 54 81310465.
M.E. Farias and MC” Manta De Nadra are also with the Facultad de
Bioquimica, Quimica y Farmacia, Universldad National de Tucumkn, Dehwzinutian of Pr&eolyfic Adivify
4000 Tucuman, Argontin& *Corresponding author. The method of Hull (194i’), modified by Citti et ul. (I%$), was
@ 1995 Rapid Communkations of Oxford ltd
G. C. Rdlh, A4 E, Fari’as and h&C. A4anca De Nadm

Table 1. Substrate specificity of proteases I and II from f eucon-

ostoc oenos X2L. Results and Discussion
Substrate Activtty (nmoi tyrosine reieased/mS.
~euc#nosf5c oerzos XzL produces two extracellular proteolytic
min) of?
activities during growth at JO’C: protease I occurs in the
Protease I Protease II early growth phase, optimally after ~6 h, and protease II
Grape juice? 7.6 12.0 optimally after about 77 h, at the end of the growth
Grape juice$ 3.3 9.0 (Rollin ef al. 1993). Both proteases, taken at their optimal
Basal medium* 4.8 7.2 time of production, were maximal with autocjaved grape
Albumins 1.3 9.6
juice as substrate (Table 1). With this substrate, proteases I
Gelatins 4.2 9.0
Case@ 0.6 9.0
and II were optimally active at pH 4.0 and 5.5 and (Figure
1) and 30 and 40°C (Figure z), respectively. Their apparent
*Ceils were cultured in basal medium at 3O’r.Z. Protease I was Km values, determined by the usual Lineweaver-Burk plot,
assayed in the culture supernatant taken after 26 h growth and were 5,0 and 3.33 mg protein/ml, respectively.
protease II was assayed after 77 h growth. The values are the Most proteases are optimally active at alkaline pH values
means of duplicate experiments.
tAutoclaved. but there are exceptions. For example, the protease of
*Pr~ipitated with (NH~)~SO~ at 80% saturation and the pellet S~apky~ucucc~s ep~~e~i~is (Teufel & Gbtz 1993) had a pH
suspended in 0.2 M phosphate buffer, pH 7.0* and dialysed over- optimum between 5 and 7 with casein as substrate and the
night against the same buffer. serine protease from Sfa. Qtireu.5 (protease I) was optimal at
§Filtered. pH 4.0 (Drapeau 1978).
Thermostability of the proteases was tested by exposure
of the cell-free culture supematants at different temperatures
for 15 min. Protease I was the~ostable up to 60°C; 10%
inactivation occurred at 70*C and 25% activity still remained
after 1.5 min at 80°C. Protease II was only stable below
50’C; 14% inactivation occurred above 5o°C and only 15%
of the activity remained after incubation at 70’C. The two
proteases are thus different in their thermotolerance. Other
proteases also show thermotolerance: that from Sfa. epider-
midis (Teufel & GGtz 1993) was stable up to 50°C and 20%
of its activity remained after incubation at 8O’C for 25 min.
These results are comparable with those of protease I,
whereas the protease of Sfa. kyicus subsp. kyicus (Ayora ef
al. 1994, which was only stable below .u’C with 40%
activity remaining after 10 min at 65’C, is comparable with
protease II.
The proteolytic activities of the cell-free culture supema-
tants were not affected when stored without any protector
for ten days at 4, - 20 or - Y’o’C. After 30 days, however,
both proteases were inactivated by 40% at all storage
temperatures. Adding 20% glycerol did not alter these
results. Different results were reported by Cowman ef al.
Figure 2. Effect of temperature on the proteolytic specific activi- (1967) for the proteinase of Sfr~foc5cc~ hcfis, which, on
ties produced by Leuconostoc oenos X*L using autoclaved grape storage for 24 h at 3’C, lost 75% activity.
juice as substrate. 0-Protease I at pH4.0; l -protease II at The action of some protease inhibitors is listed in Table
pH 5.5.
2. Metalloproteinases inhibitors, EDTA and o-phenanthro-
line did not affect the enzyme activities, indicating that a
metal ion was not required. The proteases were not inhib-
used to evaluate the two proteolytic activities in cukure supema- ited by iodoacetamide or p-hydroxymercuribenzoate, indi-
tank. Autoclaved grape juice was used as substrate. The reaction cating that sulphydryl groups were not involved in their
was developed at XYC, pH 5, for 6Omin, then stopped by activities. The enzymes were also resistant to PMSF, a
adding 0.72 M kichloroacetic acid. The tyrosine released was
serine protease inhibitor. They were, however, strongly
determined with the phenol reagent, reading at 650 nm, One unit
of protease activity (U) was defined as the amount releasing I inhibited by 10 rnM cysteine and @-mercaptoethanol and
nmol tyrosine per min under the assay conditions. partially inhibited by dithiothreitoi, indicating that disui-
 Profeases from L. oenos

Table 2. Effect of inhibitors on the exWaceMar proteases I and Acknowledgements

II of Leuconostoc oenos X*L.*
This work was partially supported by grants from the
Inhibitor ConcenkaHon Activity (% of control Consejo National de Investigacionas Cientificas y Tkcnicas
On4 value) ofit
(CONICET), and from the Consejo de Investigaciones de la
Protease I Protease II Universidad National de Tucamin (CIUNT), Argentina.

Control 100 100

Cysteine a3 9a References
10 0 0 Ayora, 5, Lindgren, P. 81 G&z, F. 1994 Biochemical properties of
o-Phenanthroline 10 a9 95 a novel metalloprotease from Sfq&y1ococc~ hyicm subsp. hyicus
PMSF 10 109 110 involved in extracellular Iipase processing. Jownul of Bacferiology
p-Mercaptoethanol 129 144 176,3218-3223.
10 0 0 Citti, J.E., Sandine, W.E. & Elliker, P.R. 1963 Some observations
p-Hydroxymercuri- a5 106 on the Hull method for measurement of proteolysis in milk,
benzoate 10 77 a7 Journal of Dairy Science46, 337-342.
1,4-Dithiothreitol 7a a0 Cowman, R.A., Swaisgood, H.E. & Speck, M.L. 1967 Proteinase
10 43 4.5 enzyme system of lactic Streptococci. II. Role of membrane
lodoacetamide 10 112 113 proteinase in cellular function. Journal of Bucteriology 94, 942-
EDTA 10 93 103 948.
Drapeau, G.R. 1978 The primary structure of staphylococcal
*Proteases incubated with inhibitor for 30 min at 30X before protease. Canadian JOI.UXU~ of Biochemistry 56, 534-544.
addition of substrate (autoclaved grape juice) and incubation Hull, M.E. 1947 Studies on milk proteins. II. Calorimetric determina-
then continued for 60 min. tion on the partial hydrolysis of the proteins in milk. JOWUX~ of
tValues are the average of duplicate experiments: 100% values Duiy science 30, 881-884.
were 7.6 and 12.0 U/mg protein for proteases I and II, Rollin, G.C., Farias, M.E. & Manta De Nadra, M.C. I993 Protease
respectively. production by Leucotiostoc oenos strains isolated from wine,
World Journul of Microbiology and Biotechnology 9, 587-589.
Teufel, P. & G6tz, F. 1993 Characterization of an extracellular
phide bonds are probably present at the active sites. From metalloprotease with elastase activity from .%phy/ococca4s epider-
these results alone it is impossible to designate the enzymes tnis. Journal of Bacteriology 175, 4218-4224.

as serine, cysteine, metal or aspartic proteinases.

To our knowledge this is the first characterization of
proteases from Leuconosfoc oenos isolated from wine. (Received in revised form and accepfed 19 Sepfember 1994)

World Journal of Microbiology 6 Biotechnology, Vol If, 1995 155