Cell Wall, Membrane, and Intracellular Peptidase Activities of Propionibacterium shermanii

Cell Wall, Membrane, and Intracellular Peptidase Activities of Propionibacterium shermanii

STEFAN SAHLSTROM, 1 CRUZ ESPINOSA, THOR LANGSRUD, and TERJE SORHAUG

Department of Dairy and Food Industries Agricultural University of Norway

PO Box 36 1432 As-N/H, Norway

ABSTRACT

A procedure has been developed for the production of spheroplasts from Propionibacterium sbermanii o~ and P. sbermanii ATCC 9614. Thus, it was also possible to produce clean cell wall, membrane, and intracellular fractions. The solubilized cell wall preparation was nearly free from intracellular markers; leakage of aldolase and malate dehydrogenase was below .5% after 90 min in lysozyme. The purity and homogeneity of the membrane fraction were visualized with electron micrographs. Polyacryl- amide gel electrophoresis and a zymogram technique were used to compare peptidases in the pure fractions. Fifteen dipeptides and 2 tr ipeptides were used as substrates. Peptidases of P. sbermanii and P. sbermanii ATCC 9614 were found in the solubilized cell wall (one band and one band), membrane (three bands and two bands), and intracellular fractions (six bands and seven bands). The solubilized cell wall fractions from P. sbermanii oL and P. sbermanii ATCC 9614 contain peptidases with reflectance values of 0 and 57, respectively. The reflectance 57 peptidase has a broad substrate specificity, that of the reflectance 0 peptidase is slightly narrower. Among the five membrane peptidases two appear to be unique for membranes, those with reflectance values of 28 and 75.

An intracellular peptidases of broad specificity with reflectance value of 55 is found in both strains. The enzymes

Received April 22, 1988. Accepted September 6, 1988. 1 Corresponding author.

hydrolyzed most of the 15 dipeptides and the P. sbermanii ATCC enzyme hydrolyzed the 2 tripeptides. The reflectance 24/25 intracellular peptidase of the two strains has the same, limited substrate specificity. The intracellular peptidases for each strain hydrolyzed all the dipeptides used and L-leucine-L- leucine-L-leucine, plus L-alanine-L-leucine-glycine for that from P. sbermanii ATCC 9614.

INTRODUCTION

The proteolyt ic system of propionibacteria is considered to contribute to flavor and texture during the ripening of Swiss type cheese. Knowledge of the properties and cellular locations of the proteinases and peptidases is vital to understand their functions both in bacterial nutri t ion and in cheese mat- uration. Few reports have been published about the proteolyt ic activity of this group of bacteria. Virtanen (19) observed that propionibacteria incubated in milk at 37°C over a long period (2 mo) produced only a small increase in soluble N. With peptones as an added N source, faster growth and greater numbers of cells were observed. This indicates a low endoproteinase activity and an ability to use peptides as the N source. In Propioni- bacterium pentosaceum Berger et al. (1) found an intracellular tripeptidase. Proteolytic activities of propionibacteria were also observed by Searles et al. (17) who measured the increase in trichloroacetic acid soluble N released from casein during growth of propionibacteria. However, Klimovskii et al. (7) found that in the USSR, cheese propionic acid bacteria didonot contr ibute significantly to proteolysis. Lang- stud et al. (9, 10) found that propionibacteria released large amounts of proline when grown in media containing peptides. Enzymatic casein hydrolysates were bet ter substrates than meat

1989 J Dairy Sci 72:342-350 342

PROPIONIBACTERIUM SHERMANI1 PEPTIDASE ACTIVITY 343

hydrolysates or unhydrolyzed casein. The release of proline from peptides coincided with the autolysis of propionibacteria. Floberghagen et al. (5) found 7 to 8 electrophoretic bands with peptidase activity in ultrasonic extracts of Propionibacterium strains. The present report gives protocols for the production of spheroplasts and subsequent electrophoresis experiments to compare the location, electrophoretic mobilities, and peptide specificities of peptidases from the cell wall (CW), cell membrane, and the cell interior of P. sbermanff.

M A T E R I A L S A N D METHODS

Cultures and Growth Conditions

The following strains were used: P. sber- m a n i i a from the Department of Dairy and Food Industries culture collection, and P. sbermanii ATCC 9614, from the American Type Culture Collection, Rockville, MD. Cultures were grown at 30°C in sodium lactate broth (SLB) under semianaerobic conditions (static culture).

Sodium lactate broth contained the following ingredients added to 1000 ml of distilled water: 15 g of 50% sodium lactate (Merck, Darmstadt, Federal Republic of Germany), 10 g of Tryptone (Difco Laboratories, Detroit, MI), 10 g of yeast extract (Difco), .25 g of K2HPO4, .2 g of MgSO4"7H20, and .005 g of MnSO4" H20. The medium was adjusted to pH 7.0 and autoclaved at 121°C for 15 rain.

Production of Spheroplasts and Preparation of Cell Fractions

Bacterial cultures in 1.5 L SLB were har- vested in late logarithmic phase, normally after 36 h of growth. After washing three times in .5 M NaC1, the cells were resuspended in 20% (wt/vol) sucrose at 4°C, with mechanical shaking (G33, New Brunswick Scientific Co., NJ) at 125 rpm for 2 h. Preconditioning the cells in sucrose was done to facilitate the separation of the cell wall material from the cytoplasmic membrane (2). The cells were recovered by centrifugation and then resuspended in Tris buffer pH 7.8 (spheroplast buffer, .01 M Tris, .5 M sucrose, .3 M NaC1, .05 M MgSO4, .01 M KC1) containing 1 mg of lysozyme/ml and were kept at 30°C with

mechanical shaking at 125 rpm. After 90 min of incubation with lysozyme the suspension was centrifuged at 39,000 x g for 10 min, and, the supernatant liquid was retained as the soluble CW fraction (Figure 1). Leakage from the spheroplasts into the soluble cell wall fraction was immediately determined by assaying for aldolase (EC 4.1.2.13) and malate dehydrogenase (MDH, EC 1.1.1.37) (Test kits, Catalog Numbers 123838 and 124940, Bo- ehringer Mannheirn, GrnbH Diagnostica). Molar extinction coefficient for NADH340 = 6.22 - 103 iV/-1 -cm 1 . Sucrose in the CW fraction was removed by gel filtration in PD-10 columns (Pharmacia Fine Chemicals, Uppsala, Swed.) equilibrated with 5 mM sodium phosphate buffer pH 7.5. The main peak at 280 nm was concentrated by freeze drying. Before PAGE the freeze-dried material was solubilized in a small volume of 5 mM sodium phosphate buffer pH 7.5. The pellet containing the 90-rain lysozyme-treated bacteria was resuspended in spheroplast buffer and the incubation with lysozyme (1 mg/ml) continued for 4.5 h. Spheroplasts were recovered by centrifugation, 39,000 × g for 10 min, and then lysed in 5 mM sodium phosphate buffer pH 7.5 at 10x the concentration of the original culture, followed by centrifugation at 50,000 x g for 30 min. The supernatant was concentrated 10x with an Amicon UF cell (Amicon Cor- poration, Scientific Systems Division, Danvers). The molecular cut off for the Diaflo uhrafiher was 10,000 daltons. The resulting liquid was used as the intracellular fraction (Figure 1).

For the preparation of the membrane fraction, separate portions of cells were treated with lysozyme for 20 h, thus almost completely hydrolyzing the cell walls (Figure 1). The resulting spheroplasts recovered by centrifugation were lysed in 5 mM sodium phosphate buffer pH 7.5 containing 2.5 mg/ml deoxy- ribonuclease (DNAse 1: Sigma Chemical Co., St. Louis, MO) and 2.5 mg/ml ribonuclease (RNAse Type l-A, Sigma). After incubation at 37°C for 2 h the mixture was centrifuged at 45,000 × g for 30 min, and the pellet was washed twice with 5 mAd sodium phosphate buffer pH 7.5. A hand-driven Teflon/glass homogenizer (Kebolab AS, Oslo, Norway) was used for resuspension between washings. The washed pellet (1.3 ml) was layered on a linear

Journal of Dairy Science Vol. 72, No. 2, 1989

344 SAHLSTROM ET AL.

CULTURE

I HARVEST WASH

Resuspend in spheroplast buffer

LYSOZYME (lrng/ml) 90 rnin

I Centrifugation I

SPHEROPLASTS Resuspend in spheroplast buffer

LYSOZYME (lmg/rnl) 4,5 h

I Centrifugation !

]

CELL WALL FRACTION (CW)

soluble

CULTURE

I HARVEST WASH

Resuspend in spheroplast buffer

LYSOZYME (lmg/ml) 20 h

Centrifugation I i

SPHEROPLAST OSMOTIC LYSIS DNase + RNase

Centr i fugat ion I

SPHEROPLASTS CELL WALL OSMOTIC LYSIS FRACTION (6 h)

I soluble

Centr i fugat ion I i

Lysed Bacter ia INTRACELLULAR part ic les FRACTION (I)

PARTICULATE FRACTION

I SUCROSE DENSITY GRADIENT (20-80%w/v)

PURIFIED MEMBRANE FRACTION (M)

Figure 1. Flow diagram for preparing cell wall, membrane, and intracellular fractions from Propionibacterum sbermanii.

sucrose gradient (20-80%, 12 ml) and centrifuged in a Beckman swing out rotor (Beckman Scientific Instruments, Fullerton, CA), SW 40, at 60,000 X g for 18 h at 4°C. The gradient was then collected from the bo t tom as approximately 1.0 ml fractions whose peptidase activity and absorbance at 280 nm (A280) were measured (8). The location of membranes within the gradient fractions was determined by electron microscopy of thin sections in Epon 812 (Polaron Equipment Limited, Watford, Engl.). Membrane recovery was achieved by diluting the relevant gradient fractions in 5 mM sodium phosphate buffer pH 7.5 followed by centrifugation at 45,000 x g for 30 rain. Before PAGE, the membrane pellet was solubilized in a small volume of .1% (vol/vol) Triton X-IO0 in 5 mM sodium phosphate buffer. The clean cell fractions were used immediately or stored at --20°C before use. Protein Assay

Protein was determined according to Lowry et al. (13) with bovine serum albumin as standard.

Electrophoresis and Peptidase Zymograms

Polyacrylamide gel electrophoresis was per- formed according to the method of Davis (3) with 8-cm gels of .5 cm D. run at 3mA/gel in .18 M Tris-borate buffer pH 8.4. Loading rates were approximately 100 #g protein in 120 /~1 for cell wall and membrane preparations, or 500 /2g protein in 120 pl for intracellular fractions. Peptidases separated in the gels were visualized after electrophoresis by the method of Lewis and Harris (12). Band mobilities were expressed as reflectance (Rf) values × 100 with phenol red as the front marker. A variety of peptide substrates (Sigma) were used (see Tables 2, 3, and 4).

Enzyme Assay

Dipeptidase activity in fractionated cell wall preparations was assayed with L-Ala-L-Leu by the fluorometric method of Porter et al. (16), using a Perkin Elmer LS-5 fluorescence spec- t rophotometer (Perkin Elmer, Norwalk, CT).


PROPIONIBACTERIUM SHERMANII PEPTIDASE ACTIVITY 345

TABLE 1. Leakage of intracellular enzymes from Propionibacterium sberrnanii a and Propionibacteriurn sberrnanii ATCC 9614 during spheroplast formation. 1

Enzyme activity a

Intracellular Bacterial Cell wall fraction cytosol before Leakage strain Enzyme 90 rain 6 h concentra t ion 90 rain 6 h

( , m o l / m i n ' m l ) - - ( % ) ' ~

P. sberrnanii c~ Aldolase .14 1.16 33.8 .4 3.4 MDH 8.1 100.0 3100 .3 3.2

P. sberrnanii ATCC 9614 Aldolase .11 .15 26.3 .4 .6 MDH 11.8 12.4 3700 .3 .3

Figures are given for a preparation from 1.5 L SLB medium. Results are the mean of two experiments .

2 Activity o f matate dehydrogenase (MDH) and aldolase found in the 90-rain and 6-h cell wall fractions is given as percentage of activity found in the intracellular fraction before concentra t ion with the Amicon UF cell.

RESULTS

Enzyme Activities in Soluble Cell Wall Fractions

L e a k a g e o f i n t r a c e l l u l a r m a r k e r e n z y m e s , a ldo l a se , a n d M D H i n t o t h e 9 0 - r a i n s o l u b l e CW f r a c t i o n d u r i n g s p h e r o p l a s t f o r m a t i o n f r o m cei ls o f P. sbermanii c~ a n d P. sbermanii A T C C 9 6 1 4 w a s < . 5 % o f t h e a c t i v i t y f o u n d in t h e in- t r a c e l l u l a r c y t o s o l b e f o r e c o n c e n t r a t i o n ( T a b l e 1).

T h e s o l u b l e CW f r a c t i o n f r o m P. sberrnanii c~ c o n t a i n e d a p e p t i d a s e ( s ) ( R f = 0) t h a t d id n o t p e n e t r a t e t h e PA-ge l . In P. sberrnanii A T C C 9 6 1 4 o n e p e p t i d a s e w a s r e s o l v e d in t h e z y m o - g r a m ( R f = 57) . T h i s R f 57 f r a c t i o n h a s a l m o s t t h e s a m e s u b s t r a t e s p e c i f i c i t y as t h e R f 0 f r a c t i o n . T h e d i f f e r e n c e s a re t h a t t h e R f 0 f r a c t i o n h y d r o l y z e d t h e p e p t i d e s m o r e a c t i v e l y a n d a l so a t t a c k e d t r i l e u c i n e , b u t n o t L -P he - L - T r p , L -Yrp -L-Ala , a n d L -P ro -L - I l eu ( T a b l e 2) .

Intracellular Peptidases

T h e i n t r a c e l l u l a r f r a c t i o n s o f P. sberrnanii

a n d P. sbermanii A T C C 9 6 1 4 w e r e r e s o l v e d i n t o s ix a n d s e v e n b a n d s o f p e p t i d a s e a c t i v i t y , r e s p e c t i v e l y ( T a b l e 3). T w o f r a c t i o n s h a v e t h e s a m e s u b s t r a t e s p e c i f i c i t y , R f 25 f r o m P. sbermanii A T C C 9614 a n d R f 2 4 f r o m P. sberrnanii o~; t h e o n l y d i f f e r e n c e w a s in t h e i n t e n s i t i e s o f t h e b a n d s . T h e p e p t i d a s e f r o m P. sberrnanii e~ w i t h R f 65 h y d r o l y z e d o n l y 3 o f 15 d i p e p t i d e s a n d t h e s u b s t r a t e s p e c i f i c i t y is

TABLE 2. Hydrolysis of peptides by soluble cell wall fractions of Propionibacterium sbermanii c~ and Propionibacterium sbermanii ATCC 9614.1

Rf Values × 1002

P. sbermanii P. sbermanii ~ ATCC 9614 Substrate in

zymogram 0 57

L-Pro-L-Phe ++++ +++ L-Leu- L-Phe ++++ +++ L-Phe-L-Phe ++++ +++ L-Phe-L-Trp -- ++ L-Lys-L-Phe ++++ +++ L-Ala-L-Leu ++++ ++++ L-Ala-L-Phe ++++ ++++ L-Pro-L-Met ++++ ++++ L-Gly-L-Phe ++++ +++ L-Trp-L-Ala - ++ L-Val-L-Pro -- - L-Pro-L-Ileu -- +++ L-His-L-Phe ++++ +++ L-Pro-L-Leu ++++ ++++ L-Leu-L-Gly ++++ ++++ L-Leu-L-Leu-L-Leu ++++ L-Ala-L-Leu-L-GIy --

1 Soluble cell wall fractions were prepared from cultures grown in sodium lactate broth as described in the text.

2 Peptidases were separated by PAGE and detected in zymograms according to the me thods of Davis (3) and Lewis and Harris (12). Reflectance (Rf) values express the mobil i ty of peptidase bands relative to phenol red as front marker. - = No bands detected; +, ++, +++, ++++ = band intensi ty from very weak to very strong, determined visually.


,N"

<

T A B L E 3 . H y d r o l y s i s o f p e p t i d e s b y i n t r a c e l l u l a r f r a c t i o n s ofPropion ibac ter ium sbermanii c~ a n d Propionibacterium sbermanii A T C C 9 6 1 4 . 1

O x

Q

X e

R f V a l u e s X 1 0 0

. . . . "P. sbermanii c~ P. sbermanii A T C C 9 6 1 4 S u b s t r a t e i n z y m o g r a m 2 4 4 1 4 9 5 5 5 9 6 5 8 2 5 3 2 4 2 4 5 5 5 6 4

o o L-Pro-L-Phe +++ +++ - +++ - - - ++++ - -- - ++++ +

L-Leu-g-Phe - - +++ +++ - ++++ . . . . ++++ -

L-Phe-L-Phe - - - +++ - +++ ++ . . . . +++ +++ . . . . ++ -

L - P h e - L - T r p - - - - - - + + + - - + + + - - . . . . + + + + - - f / ~

L - L y s - L - P h e - - - - + + + + + + - -

L - A l a - L - L e u + + + + - + + + + + + - - - - - - + + + - - - - - - + + + + - - ~ , ++ +++ - _ _ + + + + - ~ :

L-AIa-L-Phe +++ ++

L-Pro-L-Met ++++ ++ ++ +++ -- -- -- ++++ -- +++ -- ++++ -- r~

L - G l y - L - P h e + + + - + + + + - - - - + + + + - - + + + + - - "4

L - T r p - L - A l a - - + + - - + + - - + + + . . . . + + + + + + ~ > _ _ - - - - + + + + + - - - - .

L - V a l - L - P r o - + + + . . . .

L - P r o - L - l i e + + + + - - - - + - - - - - - + + + - - - - - - + + + + - -

L - H i s - L - P h e - - - - - - + + + . . . . . + - - - - + + + - -

L - P r o - L - L e u + + + + - - + + + - - + + + + + + + + - - - + + + - -

L - L e u - L - G I y - - - - + + + . . . . + + - + + + + - . . . . . + + +

L - L e u - L - L e u - L - L e u . . . . . + + + + - - _ _ - - + + - -

L - A I a - L - L e u - L - G I y . . . . . . . . . .

1 T h e i n t r a c e l l u l a r f r a c t i o n s w e r e p r e p a r e d f r o m c u l t u r e s g r o w n i n s o d i u m l a c t a t e b r o t h a s d e s c r i b e d i n t h e t e x t . P e p t i d a s e s e p a r a t i o n a n d d e t e r m i n a t i o n o f

a c t i v i t y w e r e a s d e s c r i b e d i n t h e t e x t . R f = r e f l e c t a n c e .

PROPIONIBACTERIUM SHERMANII PEPTIDASE ACTIVITY 347

quite similar but not identical to that of the peptidase from P. sberrnanii ATCC 9614 with Rf 64. In both strains, an Rf 55 peptidase was found each enzyme with a different substrate specificity. These peptidases are broad spec- trum peptidase which hydrolyzed all dipeptides except L-Val-L-Pro and in P. sberrnanii ~ atso L-Ala-L-Phe was not hydrolyzed. The Rf 55 peptidase in P. sberrnanii ATCC 9 6 1 4 has tripeptidase activity, a difference from the P. sberrnanii a enzyme. An Rf 59 peptidase, which hydrolyzed trileucine as well as L-Leu-L-Phe and L-Lys-L-Phe, is exclusive for P. sberrnanii a.

Pooled together, the intracellular fractions of P. sberrnanii ATCC 9614 are able to hydrolyze all dipeptides and tripeptides tested. Propioni- bac ter ium sberrnanii ATCC 9614 has a peptidase in the soluble CW fraction with Rf 57. Mixing the CW fraction (Rf 57) and the intracellular fraction (Rf 55) before running a zymogram of the mixture with L-Ala-L-Leu as substrate clearly showed two bands of activity. Substrate specificity and low leakage of intraeellular enzymes

independently indicate no contamination of the soluble CW fraction.

Membrane Peptidases

To increase the purity of the membranes, sucrose density gradient centrifugation was used. Two areas of peptidase activity were detected corresponding to regions of A280 (lower peak, fractions 2 to 5, higher peak, fractions 10 to 12, Figure 2). The peptidase activity in Figure 2 was measured without any detergent. The lower peak corresponded to an orange band, which was rich in membrane fragments and vesicles as seen in Figure 3.

The zymograms of the higher peak fractions were similar to those of the lower peak fractions (data not shown), which indicates that the higher peak contains peptidases originating also from the membranes. No membrane fragments or vesicles were detectable with electron microscopy of the higher peak fraction. Es- sentially similar results as presented for P.

40,

o ~oc c cD 0 u) (D

0

LL CD

CE 5¢

3ol

2ot

,oF

80

7 0

50 09 0

0 40

09

30

2 0

10

o 1 = 3 4 5 s r e 9 lO 1, ,2

F r a c t i o n n u m b e r

Figure 2: Sucrose density gradient centrifugation of the particulate fraction obtained after osmotic lysis of Propionibacteriurn sbermanii a spheroplasts; - - sucrose gradient; e - - e absorbance at 280 nm (A280); A - - A peptidase activity with L-Ala-L-Leu as substrate.

Journal of Dairy Science VoL 72, No. 2, 1989

348 SAHLSTRlJM ET AL.

shermani i a in Figures 2 and 3 were obtained for P. shermani i ATCC 9614. The membrane fractions of P. sherrnanii ~ and P. sberrnanii ATCC 9614 contained three and two peptidases, respectively (Table 4). Two of the bands from the P. sbermani i ~ membrane preparation (Rf 24 and 59) were found also in the intracellular preparation (Tables 3 and 4). Thus, the respective enzymes are suspected to be loosely bound to the membrane. One band (Rf 75) appears to be unique for the membrane. The zymograms of the P. sberrnanii ATCC 9614 membrane preparation (Table 4) contained two bands of Rf 28 and Rf 55; substrate specificities and relative levels of hydrolysis were different from those of the intracellular fractions. This indicates that the P.

sbermani i ATCC 9614 membrane peptidases are unique, particularly theft with Rf 28.

DISCUSSION

A procedure to obtain spheroplasts from P. sbermani i cells was developed. After 36 h of incubation in SLB medium, the ceils of the two P. sbermani i strains are only .5 /lm long. Thus, control of the spheroplast production with an ordinary light microscope is impossible. In- direct methods had therefore to be used to evaluate if treatment with lysozyme was effective. The levels of intracellular enzymes (aldolase and malate dehydrogenase) after exposure of spheroplast cell samples to lysis in 5 mM sodium phosphate buffer were measured.

Figure 3. Electron micrograph of a negatively stained membrane fraction from Propionibacterium sbermanii (~. Bar represents .2 ~m.


PROPION1BACTER1UM SHERMANII PEPT1DASE ACTIVITY 349

TABLE 4. Hydrolysis of peptides by membrane fractions of Propionibacterium shermanii c~ and Propionibac- terium sbermanii ATCC 9614.1

Rf Values X 100 P. sbermanii

P. sbermanii c~ ATCC 9614 Substrate in zymograms 24 59 75 28 55

L-Pro-L-Phe ++++ - ++++ +++ ++++ L-Leu- L-Phe -- ++++ +++ -- ++++ L-Phe-L-Phe - - ++++ - +++

- - + + L-Phe-L-Trp -- -- -- - - + + + L-Lys-L-Phe -- ++++ --

L-AIa-L-Leu ++++ -- ++++ -- ++++ L-Ala-L-Phe ++++ -- ++++ -- -- L-Pro- L-Met ++++ -- ++++ +++ ++++ L-Gly-L-Phe -- -- +++ -- ++ L-Trp-L-Ala . . . . . L-VaI-L-Pro . . . . . L-Pro-L-lieu ++++ -- -- + ++++

- - + + L-His-L-Phe - -- -- L-Pro-L-Leu ++++ -- -- ++ +++

- - + + + L-Leu-L-Gly -- -- -- L-Leu-L-Leu-L-Leu -- +++ -- -- -- L-Ala- L- Leu- L-Gly . . . . .

1 The membrane fractions were purified from cultures grown in sodium lactate broth as described in the text. Peptidase activities were determined in supernatant liquids obtained after centrifugation of membranes which had been incubated with .1% (wt/vol) Triton X-100. Peptidase separation and determinations of activity were carried out as described in the text. See Table 2 for abbreviations.

To obta in a pure soluble CW fract ion, the t r e a t m e n t wi th l y sozyme was s topped af ter 90 min; in this way intracellular con t amina t i on was avoided. Prolonged t r ea tmen t wi th lysozyme to 6 h increased leakage of the intracellular enzym es aldolase and MDH, especially for P. sbermanii c~ (Table 1). For the pro- duc t ion of the intracellular f rac t ion the lyso- z y m e t r ea tmen t was s topped when suff ic ient CW material had been hydro lyzed to facil i tate lysis o f the spheroplas ts in 5 mM sodium phospha t e buffer . Lysozyme t r e a t m e n t b e y o n d 6 h gave no increase of lysis measured as tota l activity of aldolase and MDH in the intracellular cytosol . With the purif ied soluble CW fract ion, m e m b r a n e fract ion, and intracellular f ract ion, it was possible to s tudy the mul- t ipl ici ty, specifici ty, and locat ion of pept idases wi th in the p ro teo ly t i c sys tem of P. sbermanii.

This work is part o f a s tudy of the N- utilizing sys tems in propionibacter ia . Because prop ion ibac te r ia in Swiss t ype cheese coexis t wi th lactic acid bacteria, p rop ion ibac te r ia might conta in a comparable or a s u p p l e m e n t o r y sys tem for processing of extracellular p ro te ins and pept ides . For lactic acid bacteria, a m o d e l

has been p roposed to explain the interrela- t ionship be tween the p ro teo ly t i c sys tem and the pep t ide t r anspor t sys tem (14). Thus, CW b o u n d prote inases and pept idases are first in line to process extracellular prote ins . However , no pro te inase act ivi ty was found in the soluble CW fract ions of P. shermanii ~ and P. shermanii

ATCC 9614 at pH 5.3, 6.7, and 8.0 (unpub- lished results), measured wi th a sensitive f luoromet r i c m e t h o d wi th casein as subst ra te (16). Earlier expe r imen t s (7, 17, 19) indicated slow casein degradat ion; however , there was no indica t ion of proteolys is be fo re cell autolysis (9, 10). The present work showed strong pept idase activity in the purif ied CW fract ions, p robab ly involved in pep t ide processing. In Swiss t y p e cheese prote inases f rom lactic acid bacter ia will provide pept ides f rom initial hydrolys is o f p ro te ins to enhance the g rowth of propionibacter ia . In addi t ion to cel l-bound pept idases , m e m b r a n e - b o u n d and intracellular pept idases were found in b o t h strains. The Rf 24 and 59 bands f rom P. sbermanii a membranes and the fast moving Rf 55 band f rom P. sbermanii ATCC 9614 m e m b r a n e s have their coun te rpa r t s among the cy toplasmic pept idase

Journal of Dairy Science Vol. 72, No, 2, 1989

350 SAHLSTROM ET AL.

activities, which indicates that these are loosely bound membrane peptidases. Peptides not comple te ly hydrolyzed during passage through the CW, will possibly be cleaved during membrane t ransport by the membrane peptidases or at least by the intracellular enzymes. In Strep- tococcus cremoris, a t ransport system for amino acids, dipeptides, tr ipeptides, and ol igopept ides has been de tec ted (11, 18). Propionibacterium shermanii a and P. sbermanii ATCC 9614 contain both membrane bound and intracellular peptidases with wide enough specificities to ensure release of most amino acids f rom pept ides t ranspor ted into the cell. However , the peptidases associated with the membrane as well as the intracellular peptidases may have other funct ions than cleavage of entering peptides. An aryl-peptidyl-amidase has been associated with the r ibosomal fract ion of Lactobacillus casei (4). This peptidase was comple te ly solubilized with RNase. The sug- gested func t ion of this enzyme is to hydro lyze specific bonds in the signal sequence o f newly synthesized expor t proteins. These signal sequences are vital for the extrusion mechanism of cell envelope proteins emerging f rom membrane-bound r ibosomes (6). To avoid these peptidases, membrane fract ions were t reated with RNase. It is a reasonable assumption tha t some of the intracellular peptidases cont r ibute to the turnover of endogenous proteins or pept ides (15, 20). Fur ther investigations including purif icat ion o f these peptidases are required to understand their funct ions in the N metabol ism of propionibacter ia .

ACKNOWLEDGMENTS

Elect ron micrographs were provided by Trygve Krekling o f the Agricultural E lec t ron Microscopy Laboratory. Advice by Jens Kolstad on various aspects of this work including f luorescence spect roscopy methods have been invaluable. This research was supported by the Agricultural Research Counci l of Norway through Grant 13.025.2 3, Ni t rogen metabol i sm of propionic acid bacteria.

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Cell Wall, Membrane, and Intracellular Peptidase Activities of Propionibacterium shermanii