Purification and Partial Characterization of an Aminopeptidase from Micrococcus freudenreichii ATCC 407

System. Appl. Microbio1. 12, 112-118 (1989)

Purification and Partial Characterization of an Aminopeptidase from Micrococcus freudenreichii ATCC 407 TARUN BHOWMIK and ELMER H. MARTH* Department of Food Science and The Food Research Institute, University of Wisconsin, Madison, Wisconsin 53706, USA Received January 16, 1989

.Summary An aminopeptidase capable of hydrolyzing L-lysine-p-nitroanilide was purified from the cell-free extract of Micrococcus freudenreichii ATCC 407. The purified enzyme had a molecular weight of 43,000 and activity optima of pH 6.5 and 37°C. The aminopeptidase activity was stimulated by Ca ++ and strongly inhibited by ethylenediaminetetraacetate (EDTA), 1,10 phenanthroline, HgCI 2, iodoacetamide, and dithiothreitol. Activity of the enzyme also was markedly inhibited by sodium chloride at pH 5.0. The enzyme had a broad substrate specificity but was inactive on peptides having proline as the N- or Cterminal amino acid.

Key words: Micrococcus freudenreichii - Enzyme - Aminopeptidase - Purification - Cheese ripening

Introduction The cheese ripening process often is long and complicated; one way to reduce the cost of productibn is to reduce the time needed to ripen the product. During ripening several chemical changes occur including degradation of protein, fat, and carbohydrates. Proteolysis is important in the ripening process and is caused by action of cheese microflora, milk proteases, and added rennet or other coagulant. Attempts have been made to reduce the ripening time through accelerated proteolysis, and different ways to achieve this are described in the literature (Law and Wigmore, 1982; Sood and Kosikowski, 1979). Proteases and peptidases from the secondary cheese flora, which includes micrococci, playa significant role during the later part of the ripening process. Presence of micrococci in raw milk and cheese has been reported (Alford and Frazier, 1950; Marth, 1963; Nath and Ledford, 1972; Prasad et a1., 1983). Reports also are available regarding use of micrococci to improve and enhance the flavor of Cheddar cheese as well as to reduce the time needed to ripen this product (Alford and Frazier, 1950; Robertson and Perry, 1961). It has been suggested that proteolytic and lipolytic enzymes from micrococci are important in the ripening process (Marth, 1963; Sood and Kosikowski, 1979). * To whom reprint requests should be addressed.

There are several reports which indicate the ability of some micrococcal species/strains to synthesize extracellular proteolytic enzymes (Husain and McDonald, 1958; Prasad et a1., 1986). Recently, we investigated the presence of intracellular protease and amino/imino peptidase enzymes in nine different strains of micrococci (Bhowmik and Marth, 1988). Our investigation indicated the presence of protease and peptidases (hydrolyzing the substrates L-Iysine-p-nitroanilide, L-Ieucine-p-nitroanilide, Lproline-p-nitroanilide, L-alanine-p-nitroanilide and Lmethionine-p-nitroanilide) in all the strains tested. It also was found that Micrococcus freudenreichii ATCC 407 had a relatively greater amount of amino/imino peptidase enzyme activity than did the other strains. This paper describes the purification and partial characterization of an aminopeptidase which was produced by M. freudenreichii ATCC 407 and which can hydrolyze Llysine-p-nitroanilide. , Materials and Methods Organism and Growth Conditions

M. freudenreichii ATCC 407 obtained from the American Type Culture Collection, Rockville, MD, was grown in tryptone broth (Alford and Frazier, 1950) for 24 h at 30°C with continu-

Aminopeptidase from M. freudenreichii ous agitation, and pH of the broth was maintained at 6.5 by addition of 1 N HCI using a pH controller (type 45 AR Chemtrix Inc., Hillsboro, OH). Preparation of Cell-Free Extracts

Cells were harvested by centrifugation at 6000 x g for 10 min at 4°C and washed twice, once with cold saline (0.85%) solution and then with deionized water. The pellet was suspended in buffer containing 0.01 M Tris-HCI, 1.5 mM CaCl z and 1.0 mM phenyl methyl sulfonyl fluoride (PMSF), pH 7.5, mixed with glass beads (0.1 mm in diameter) and homogenized for 15 min as described previously (Bhowmik and Marth, 1988). The supernatant fluid obtained after centrifugation of the homogenized cell suspension was used as the cell-free extract. Enzyme Assay

For determination of aminopeptidase activity, the method of El Soda and Desmazeaud (1982) was followed using L-lysine-pnitro anilide (16.4 mM) as substrate. The specific activity was calculated according to the method of Exterkate (1975) and expressed as !l moles of p-nitroanilide hydrolyzed/min/mg of protein at 3rc. Protein Determination

Protein concentrations were determined by the method of Lowry et al. (1951), using bovine serum albumin as the standard.

113

Biotech. Inc., Piscataway, NJ) previously equilibrated with TrisHCI (0.01 M) plus CaCl z (1.5 mM), and buffered at pH 7.5. The enzyme was eluted in 5.6-ml fractions with the same buffer and the fractions (no. 19 to 25) containing active enzyme were pooled and concentrated by ultrafiltration (PM 10 membrane, Amicon Corp.). (e) Second DEAE-cellulose chromatography. The concentrated enzyme solution obtained from the previous step was reapplied to a column (1 X 15 cm, flow rate 30 mllh) of DEAEcellulose, previously equilibrated with buffer containing 0.01 M Tris-HCI and 1.5 mM CaCl z at pH 7.5. The enzyme was eluted in 3.0-ml fractions by a step-wise gradient of sodium chloride (0.1 M to 0.5 M) in the same buffer. The fractions with enzyme activity (no. 23 to 25) were pooled and kept frozen for further studies. Polyacrylamide Gel Electrophoresis

Polyacrylamide gel electrophoresis was done according to the method of Fling and Gregorson (1986) using an 8 to 25% gradient of monomer concentration in the presence of sodium dodecyl sulfate (SDS). Protein was stained with coomassie blue. Native polyacrylamide gel electrophoresis was done according to the method of Owens and Haley (1976) using 8% running gel. One part of the gel was stained with coomassie blue and the other part was incubated in L-lysine-p-nitroanilide solution for detection of enzyme activity. Substrate Specificity

Purification of the Aminopeptidase

All of the following purification steps were done at 4°C. Protein concentration of each fraction was monitored by measuring the absorbance at 280 nm and enzyme activity was followed by measuring at 410 nm, the absorbance of the liberated p-nitroaniline from L-lysine-p-nitroanilide used as a substrate. (a) First diethylaminoethyl (DEAE) cellulose chromatography. The crude cell-free extract containing about 580 mg of protein was applied to a DEAE-cellulose (Sigma Chemical Co., St. Louis, MO) column (1.5 x 80 cm, flow rate 30 mllh) preyiously equilibrated with buffer containing 0.01 M Tris-HCI,plus 1.5 mM CaCl z, pH 7.5. The column was washed with the same buffer and then the enzyme was eluted using a sodium chloride gradient (0 to 1.0 M NaCI) and the eluent was collected in 5.5-ml fractions. The fractions with enzyme activity (no. 90 to 110) were pooled and concentrated in an ultrafiltration cell using a PM 10 membrane (Amicon Corp.). (b) First hydroxylapatite chromatography. The concentrated enzyme solution obtained from the previous step was applied to a column (2 x 10.5 cm, flow rate 25 mIIh) of hydroxylapatite (BioRad Chemical Div., Richmond, CAl previously equilibrated with 0.01 M sodium phosphate buffer, pH 6.8. The enzyme was eluted in 4.0-ml fractions using a step-wise gradient of sodium phosphate buffer containing 0.01 M to 0.20 M of phosphate at pH 6.8. The fractions with enzyme activity (no. 35 to 38) were pooled. (c) Second hydroxylapatite chromatography. The enzyme solution obtained from the previous step was applied to a column (2 x 9 cm, flow rate 17 mllh) of hydroxylapatite, previously equilibrated with 0.15 M sodium phosphate buffer, pH 6.8. The enzyme was eluted in 3.0-ml fractions by a step-wise gradient of sodium phosphate containing 0.15 M to 0.25 M of phosphate at pH 6.8. The fractions with enzyme activity (no. 15 to 20) were pooled. (d) Gel permeation chromCltography. The enzyme solution obtained from the previous step was applied to a column (2 X 50 cm, flow rate 34 mllh) of Sephadex G-200 (Phatmacia LKB

Substrate specificity of the purified enzyme was determined for aminopeptidase, dipeptidase and protease activity. Aminopeptidase activity was measured as described previously (see enzyme assay section) using L-leucine-p-nitroanilide, L-proline-p-nitroanilide, L-alanine-p-nitroanilide and L-methionine-p-nitroanilide as substrates. Proteolytic activity was measured by the method of Redina (1971) and Lin et al. (1969) with 0.2% N, N dimethyl casein dissolved in 0.05 M Tris-HCI buffer, pH 7.5. Dipeptidase activities were determined as in the proteolytic enzyme assay using Lys-Lys, Gly-Tyr, Ala-His, Lys-Phe, Lys-Gly, Lys-Ala, Pro-Gly, and Pro-Phe (all in 1.0 mM concentration) as substrates. All the substrates were obtained from Sigma Chemical Co., St. Louis, MO.

Results

Purification of the Aminopeptidase

Results of the enzyme purification procedures are summarized in Table 1. Fig. 1, 2 and 3, respectively, give results of fractionation of the aminopeptidase by the first DEAE-cellulose chromatography, the second hydroxylapatite chromatography and the second DEAE-cellulose chromatography (data from the first hydroxylapatite chromatography and gel permeation chromatography are not given). The electrophoretic pattern of enzymes obtained at each purification step is in Fig. 4. The enzyme from the crude cell-free extract was purified about 10-fold with a yield of 18% and exhibited a major protein band with some minor bands on polyacrylamide gel electrophoresis after the final purification step. The electrophoretic pattern of enzyme obtained after the final purification step in native PAGE after protein stain is in

114

T. Bhowmik and E. H. Marth

Steps

Crude cell-free extract First DEAE-cellulose chromatography First hydroxylapatite chromatography Second hydroxylapatite chromatography Gel permeation chromatography (Sephadex (G-200) Second DEAE-cellulose chromatography a

b

0

co

-

2

.D ~

0

,

Sp. actb

Yield (%)

Purification (fold)

581 296 100 36 11

141543 58097 49087 50944 35024

243 1.96 486 1396 3127

100 41 35 36 25

1.0 0.80 2.0 6.0 13.0

2

6149

2549

18

10.0

/

0.4

10

1.2

- 1.0

r---------------,

0

..

0.6~

"i o 5 :::U

0

JJ 0.4~

Z

0.3

0

<0 N

0 .2

en

0.2

D

0.1

00

r-----;r---------. 2.0

- 0.1

0 .0

0

10

2

N

1.0

...."It

0.5

0.0 60

01-......................-'--"-.;;;a::.....-.1........---1

20

30

Fraction No.

30

40

0.0 50

40

50

Fig. 3. Chromatography of the enzyme fraction obtained by gel permeation chromatography on second DEAE-cellulose column. Symbols: (0-0) protein (280 nm); (6---6) aminopeptidase activity (410 nm); (_. -' -) NaClgradient (A: 0.10 M; B: 0.20 M; C: 0.30 M; D: 0040 M; E : 0.50 M).

Molecular Weight o

o ---....... o 10

20

Fig. 5. It shows one major band which also had enzyme activity after incubation with L-Iysine-p-nitroanilide (data not shown).

1.5

co

..;

D

Fraction No.

Fig. 1. Chromatography of the cell-free extract on DEAE-cellulose column. Symbols: (0-0) protein (280 nm); (0---0) aminopeptidase activity (410 nm); (_. -' -) NaCl gradient (0-1.0 M).

3

..,....

Q

c(

c(

- 0.2

0

0.5

0.4

0.3

Fracllon No.

o

Table 1. Purification of an aminopeptidase from M. freudenreichii ATCC 407

- 0.0

I'~

-

1

I

II II II I' I I I I I I I I

-

3

iii

Total act'

Total activity expressed as !! mol p-nitroanilide hydrolysed/min. Specific activity expressed as !! mol p-nitroanilide hydrolysed/min/mg protein.

4

N

Total protein (mg)

Fig. 2. Chromatography of the enzyme fraction obtained from first hydroxylapatite chromatography on second hydroxylapatite column. Symbols: (0-=-0) protein (280 nm); (0---0) aminopeptidase activity (410 nm); (- . - . -) sodium phosphate gradient (A: 0.15 M; B: 0.20 M; C: 0.25 M).

The molecular weight of the enzyme, averaged from five different trials, was estimated to be about 43,000 using SDS-polyacrylamide gel electrophoresis (Fig. 4).

Effect of pH on Enzyme Activity The optimum pH for enzyme activity was determined with 0.01 M potassium phosphate buffer in the range of pH 5.0 to 8.5. The enzyme had an optimum pH at 6.5 (Fig. 6).

. Effect of Temperature on Enzyme Activity The effect of temperature (5-55°C) on enzyme activity was determined using 0.01 M potassium phosphate buffer, pH 6.5. The optimum temperature for activity of the enzyme was 37°C (Fig. 7).

Aminopeptidase from M. freudenreichii

a b

1

2

3

5

6

7

8

115

9

c

d e

f

Fig. 4. SDS-PAGE of cell-free extract and enzyme fraction obtained from different purification steps. Lanes (1) MW standard (a: 94,000; b: 67,000; c: 43,000; d: 30,000; e: 20.1,000; f: 14.2,000) (2) crude cell-free extract; (3) after first DEAE-cellulose chromatography; (4) after first hydroxylapatite (HA) chromatography; (5) fraction no. 16 after second HA chromatography; (6) fraction no. 17 after second HA chromatography; (7) fraction no. 18 after second HA chromatography; (8) after gel permeation chromatography (Sephadex G-200); (9) after second DEAE-cellulose chromatography. Each sample was loaded on the basis of protein content (50 !1g).

Fig. 5. Native PAGE of an aminopeptidase obtained after the final purification step. The gel was stained with coomassie blue. Enzyme activity was observed in band position E. 120 r - - - - - - - - - - - - , 100

'#

~80

~

">

~60 III

4>

~40

Table 2. Effect of metal ions and reducing agent on enzyme activity Metal ion/Reagent

Conc (mM)

B 4>

a:

20

ReI. act. (%J" 0L..-&.-'-~L..-L_'_"'__'__'__'_1;I...J

None CaClz

MnCh ZnCl z Diothiothreitol

Dithiothreitol (10 mM) + Iodoacetamide

a

b

0.0 0.1 1.0 2.5 0.1 1.0 2.5 0.1 1.0 2.5 0.1 1.0 2.5 0.1 1.0 2.5 10.0 0.1 1.0 10.0

100 135 147 150 96 99 95 89 86 69 85 6 0 93 88 80 65 85 b 79 44

Rate of hydrolysis of L-Lys-p-nitroanilide in the absence of metal ion/reducing agents was takeQ,as 100. Relative activity was calculated on the basis that the rate of hydrolysis of L-Lys-p-nitroanilide in the presence of dithiothreitol (10 mM) was 100.

o

10

20

30

Tempera~ure

40

50

60

(Cl

Fig. 6. Effect of pH on aminopeptidase activity. 1~ r----------;

100

•

~ 40

-.

;;

a:

~

o L--oo-'--'-......-'-"""-'---'---L....IiL.J

o

10

20

30

40

Temperature (C)

Fig. 7. Effect of temperature on aminopeptidase activity.

50

60

T. Bhowmik and E. H. Marth

116

Table 3. Effect of different inhibitors on enzyme activity Inhibitor

Cone (mM)

None EDTA

0.0 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0 1.0 5.0

1,10 Phenanthroline HgCh PMSF

Inhibition' (%)

a

99

100 100 73 100 100

94

100 100

a a

, The rate of hydrolysis of L-Lys-p-nitroanilide in the absence of any inhibitor was taken as the control, i.e., 0% inhibition.

Effect of Metal Ions and Inhibitors on Enzyme Activity

The effect of metal ions on aminopeptidase activity was studied using dialysed purified enzyme (dialysed against 0.01 M Tris-HCI buffer, pH 7.5, for 24 h at 4°C) in 0.01 M Tris-HCI buffer, pH 6.5. Enzyme activity was enhanced loS-fold by CaCl2 (2.5 mM) while MnCl 2 and ZnCl 2 had an inhibitory effect and MgCl z apparently had no effect on enzyme activity (Table 2). The effect of enzyme inhibitors was studied using 0.01 M potassium phosphate buffer, pH 6.5, and results are in Table 3. Total inhibition of enzyme activity was caused by the metal complexing agents, ethylenediaminetetraacetic acid (EDTA), 1,10 phenanthroline and mercuric chloride but phenyl methyl sulfonyl fluoride (PMSF) had no effect on enzyme activity. Effect of Sodium Chloride on Enzyme Activity

The aminopeptidase activity was determined using 0.01 M potassium phosphate buffer, pH 5.0 and 6.5, containing different percentages of salts; results are in Fig. 8. The presence of 5% sodium chloride at pH 5.0 caused about 90% inhibition of enzyme activity, but at pH 6.5 it caused about 35% inhibition.

,..

">

100

60

u

•

60

~

•

.

40

O!

20

.

~

i

\\

.......

0

The hydrolytic action of the aminopeptidase on various substrates was examined and results are in Table 3. The enzyme had broad substrate specificity, but could not degrade peptides having proline as the N- or C-terminal amino acid. No proteolytic activity on N, N dimethyl casein was detected. Discussion An aminopeptidase from M. freudenreichii ATCC 407 was purified by several steps. The pooled fractions with enzyme activity obtained after the final purification step were exposed to electrophoresis in the non-denatured conTable 4. Relative activity of aminopeptidase on various substrates A. Aminopeptidase Substrate Lys-p-NA Leu-p-NA Pro-p-NA Ala-p-NA Met-p-NA B. Dipeptidase Substrate

C. Protease Substrate

..... .. ... ... ....

0

Substrate Specificity

Lys-Lys Gly-Tyr Ala-His Lys-Phe Leu-pro Lys-Gly Lys-Ala Pro-gly Pro-Phe Pro-Leu

120

t

Effect of Reducing Agent on Enzyme Activity The aminopeptidase activity was determined using 0.01 M potassium phosphate buffer, pH 6.5, containing different concentrations of dithiothreitol, and the effect of iodoacetamide was studied by assaying the enzyme activity in buffer containing 0.01 M phosphate and 10 mM dithiothreitol, pH 6.5, added with various concentrations of iodoacetamide. Presence of dithiothreitol caused inhibition of enzyme activity and addition of iodoacetamide in the presence of dithiothreitol caused a significant reduction of enzyme activity (Table 2).

5

10

15

20

NaCI (%)

Fig. 8. Effect of sodium chloride on aminopeptidase activity at pH 6.5 (0-0) and at pH 5.0 (6-' -6).

N,N dimethyl casein

ReI. act. (%)' 100 25

a

14 10

ReI. act. (%)b 100 64

35

10

a

15

6

a a

a

ReI. act. (%)

a

, The rate of hydrolysis of L,L-ys-p-nitroanilide was taken as 100. b The rate of hydrolysis of Lys-Lys was taken as 100.

Aminopeptidase from M. (reudenreichii dition and subsequently were stained for protein as well as incubated with substrate solution for detection of enzyme activity on gel. Results showed a major band after coomassie blue stain (Fig. 5) and also a yellow band at the same position indicating aminopeptidase activity (data not shown). Pooled fractions ' with enzyme activity obtained from each step were analysed by SDS polyacrylamide gel electrophoresis. After the final purification step, i. e., after the second DEAE-cellulose chromatography, there was one major protein band and some minor protein bands. The minor bands could not be removed under our experimental conditions. We have not further investigated the origin of these minor bands, which could be minor impurities associated with the enzyme because of the presence of calcium chloride in the eluting buffer or could be subunits of the enzyme. Although the extent of purification of the enzyme was reduced after the second DEAE-cellulose chromatography, the enzyme was most pure at this step as evidenced by SDS-polyacrylamide gel electrophoresis. This reduction could be the result of some enzyme denaturation during purification. It also can be noted that low molecular weight-protein bands « 43,000) present in the crude cellfree extract were absent after the second DEAE-cellulose chromatography, but the enzyme was purified only about 10-fold. This low degree of purification probably resulted because of the very high specific activity in the crude cellfree extract. This high specific activity could be the result of hydrolysis of lysine-p-nitroanilide by some other enzymes present in the crude cell-free extract as this substrate is not very specific. There is no report about any intracellular aminopeptidase purified from M. freudenreichii ATCC 407 or any other micrococci associated with cheese. However, there are some reports about purification and characterization of aminopeptidases of lactic acid bacteria. The molecular weight of the aminopeptidase in this study has been estimated from the major protein band (Fig. 4) to be about 43,000. The aminopeptidase of Streptococcus thermophilus (Rabier and Desmazeaud, 1973 ), Streptococcus diacetylactis (Desmazeaud and Zevaco, 1979), Lactobacillus lac tis (Eggimann and Bachmann, 1980) and Bifidobacterium breve (Cheng and Nagasawa, 1984) had molecular weights of 62,000, 85,000 ± 6,000, 78,000 to 81,000 and 61,000, respectively. Our aminopeptidase was active over a broad pH range between 6.0 to 8.5, and had an optimum pH at 6.5. This was similar to that of the aminopeptidases of S. thermophilus (Rabier and Desmazeaud, 1973), S. diacetylactis (Desmazeaud and Zevaco, 1979), L. lactis (Eggimann and Bachmann, 1980) and B. breve (Cheng and Nagasawa, 1984). The optimum temperature for activity by our enzyme was 37°C, activity decreased markedly above 40 °C and at 55°C activity was almost completely lost. In contrast, the aminopeptidase of L. lactis (Eggimann and Bachmann, 1980) had optimum activity at pH 4.5 and was stable up to 60 °C but aminopeptidase;; from L. casei (El Soda et aI., 1978) and S. thermophilus (Rabier and Desmazeaud, 1973) were unstable at 40 °C.

117

The aminopeptidase seems to be a metalloenzyme as its activity was strongly stimulated by calcium chloride and inhibited by metal-complexing agents such as EDTA, HgCI1 and 1,10 phenanthroline. Furthermore, there was no enzyme activity in a cell-free extract lacking metal ions such as those from CaCl2 or MgCl 2 (data not shown). We also observed that enzyme activity inhibited by EDTA, HgCl1 or, 1,10 phenanthroline could not be reactivated by addition of CaCI1 or MgCl 2 (data not shown). The aminopeptidases of S. thermophilus (Rabier and Desmazeaud, 1973), S. diacetylactis (Desmazeaud and Zevaco, 1979), L.lactis (Eggimann and Bachmann, 1980) and B. breve (Cheng and Nagasawa, 1984) also were found to be metalloenzymes. The aminopeptidase of M. freudenreichii ATCC 407 was strongly inactivated at pH 5.0 rather than at pH 6.5 when in the presence of 5% sodium chloride. This probably resulted from the change in ionic environment in and/ or around the active site of the enzyme. No such report on aminopeptidases of lactic acid bacteria is available. The aminopeptidase activity of the micrococcus was sensitive to the presence of dithiothreiotol (DIT) as well as iodoacetamide, and these results suggest the possible involvement of the disulfide bond in or near the active site of the enzyme. The presence of sulfhydryl groups in the aminopeptidase from B. breve (Cheng and Nagasawa, 1984) has been suggested. The aminopeptidase can hydrolyze a broad spectrum of substrates, but was inactive on the substrates having proline as N- or C-terminal amino acid. Peptidase enzymes are important in the cheese ripening process for conversion ot\ large peptides derived from casein through rennet (chyni9sin) and/or plasmin action to low molecular weight peptides and amino acids which contribute to a desirable flavor in the cheese (Law, 1984; Nisser, 1981). Major sources of peptidase enzymes are starter bacteria and the secondary cheese flora. Micrococci are found in large numbers in cheese and may playa role during the ripening process. It has been reported that addition of M. freudenreichii to cheesemilk caused increased flavor production at the early stage of ripening of Cheddar cheese made from pasteurized milk (Alford and Frazier, 1950). The aminopeptidase from M. freudenreichii may have been responsible for this observation and so We investigated this enzyme in some detail. The purified aminopeptidase enzyme retained about 50% of its initial activity at pH 5.0, which is close to the pH values encountered in Cheddar cheese undergoing the ripening process. Inhibition of enzyme activity at pH 5.0 and in the presence of 5% sodium chloride, however, may be modified by the real cheese environment. The broad spectrum hydrolytic activity of this aminopeptidase may be very beneficial during the cheese ripening process. In a previous study (Bhowmik and Marth, 1988), we reported the intracellular peptidase activity toward different substrates which are used for measuring aminopeptidase activity, but it was not certain whether the activity resulted from the presence of only one or several peptidase enzymes. From results of this study it appears that there might be several peptidase enzymes which may have several sub-

118

T.Bhowmik and E. H. Marth

strate specificities as evidenced by the inability of this enzyme to hydrolyze peptides containing proline as the Nor C-terminal amino acid. However, further research is needed to confirm this, and future studies will be done to evaluate the contribution of this organism during the cheese ripening process. Acknowledgement. Research supported by the College of Agricultural and Life Sciences, University of Wisconsin, and by the W. V. Price Cheese Research Institute.

References Alford, ]. A., Frazier, W. c.: Occurrence of micrococci in Cheddar cheese made from raw and from pasteurized milk. J. Dairy Sci. 33,107-114 (1950) Alford, ]. A., Frazier, W. c.: Effect of micrococci on the development of flavor when added to Cheddar cheese made from pasteurized milk. J. Dairy Sci. 33,115-120 (1950) Bhowmik, T., Marth, E. H.: Protease and peptidase activity of Micrococcus species. J. Dairy Sci. 71, 2358-2365 (1988) Cheng, C. c., Nagasawa, T.: Purification and some properties of an aminopeptidase from Bifidobacterium breve. Jpn. J. Zootech. Sci 56, 257-266 (1984) Desmazeaud, M.]., Zevaco, c.: Isolation and general properties of two intracellular aminopeptidases. Milchwissenschaft 34, 606-610 (1979) Eggimann, B., Bachmann, M.: Purification and partial characterization of an aminopeptidase from Lactobacillus lactis. App!. Environ. Microbio!. 40, 876-882 (1980) El Soda, M. M., Desmazeaud, M. M., Bergere, ]. L.: Peptide hydrdolases of Lactobacillus casei: Isolation and general properties of various peptidase activities. J. Dairy Res. 45, 445-455 (1978) El Soda, M. M., Desmazeaud, M. ].: Les peptide hydrolases des Lactobacillus du gronpe thermobacterium. 1. Mise en evidence de ce activities chez Lactobacillus helveticus, L. acidophilus, L. lactis et L. bulgaricus. Can. J. Microbiol. 28, 1181-1188 (1982) Exterkate, F. A.: An introductory study of the proteolytic system of Streptococcus cremoris strain HP. Neth. Milk Dairy J. 29, 303-318 (1975)

Fling, S. P., Gregorson, D. S.: Peptide and protein molecular weight determination by electrophoresis using a high molarity Tris buffer system without urea. Anal. Biochem. 155, 83-88 (1986) Husain, I., McDonald, 1. ].: Characteristics of an extracellular proteinase from Micrococcus freudenreichii. Can. J. Microbiol. 4, 237-242 (1958) Law, B. A.: Microorganisms and their enzymes in the maturation of cheese. Prog. Ind. Microbiol. 19,250-252 (1984) Law, B. A., Wigmore, A. S.: Accelerated cheese ripening with food grade proteinases. J. Dairy Res. 49, 137-146 (1982) Lin, Y., Means, G. E., Finney, R. E.: The action of proteolytic enzymes on N, N dimethyl proteins. Basis for a micro assay for proteolytic enzymes. J. BioI. Chern. 244, 789-793 (1969) Lowry, O. H., Rosebrough, N. ]., Farr, A. L., Randall, R. ].: Protein measurement with the folin phenol reagent. J. Bio!. Chern. 193,265-275 (1951) Marth, E. H.: Microbiological and chemical aspects of Cheddar cheese ripening. A review. J. Dairy Sci. 46, 869-890 (1963) Nath, K. R., Ledford, R. A.: Caseinolytic activity of micrococci isolated from Cheddar cheese. J. Dairy Sci. 10, 1424-1427 (1972) Owens, ]. R., Haley, B. E.: A study of adenosine 3'-5' cydic monophosphate binding sites of human erythrocyte membranes using 8-azidoadenosine 3-5 cydic monophosphate, a photo affinity probe. J. Supramol. Struc. 5, 91-102 (1976) Prasad, R., Malik, R. K., Mathur, D. K.: Isolation and screening of ~-caseinolytic Micrococcus spp. from Cheddar cheese. Asian J. Dairy Res. 2, 67-72 (1983) Prasad, R., Malik, R. K., Mathur, D. K.: Purification and characterization of extracellular caseinolytic enzymes of Micrococcus sp. MCC-315 isolated from Cheddar cheese. J. Dairy Sci. 69, 633-642 (1986) Rabier, D., Desmazeaud, M. ].: Inventaire des differentes activites peptidasiques intracellulaires de Streptococcus thermophilus. Biochimie 55, 389-404 (1973) Redina, G.: Experimental Methods in Modern Biochemistry, pp. 215-218. Philadelphia PA, W. B. Saunders 1971 Robertson, P. S., Perry, K. D.: Enhancement of flavor of Cheddar cheese by adding a strain of Micrococcus to the milk. J. Dairy Res. 28, 245-253 (1961) Visser, S.: Proteolytic enzymes and their action on milk proteins. Neth. Milk Dairy J. 35, 65-88 (1981)

Professor Dr. Elmer H. Marth, Department of Food Science, University of Wisconsin-Madison, 1605 Linden Drive, Madison, Wisconsin 53706, U.S.A.