Purification and Characterization of a Heat-Stable Protease from Pseudomonas sp. B-25

Purification and Characterization of a Heat-Stable Protease from Pseudomonas sp. B - 2 5 R. K. M A L I K and D. K. M A T H U R Division of Dairy Microbiology National Dairy Research Institute Karnal -- 132001, India

ABSTRACT

Pseudomonas sp. B--25, a psychrotrophic organism isolated from refrigerated butter samples, produced an extracellular heat-stable neutral metalloprotease. This protease was purified to homogeneity from a cell-free broth culture b y precipitation with ammonium sulfate, acetone fractionation, and gel filtration through Sephadex G - 1 0 0 . The protease was active over a wide pH range (pH 6.0 to 10.0) and had o p t i m u m activity at 65°C. The enzyme was heat stable, as it retained about 26% activity even after heat treatment at 70°C for 10 rain in buffer. It was inhibited by heavy metal ions, but manganese 2+ had a slight stimulatory effect. The enzyme was inhibited by metal chelating agents b u t not significantly by diisopropylfluorophosphate. It had a molecular weight of 41,200. The purified protease had comparatively high content of lysine, arginine, glycine, alanine, and methionine and low content of asparatic acid, threonine, serine, isoleucine, and phenylalanine. INTRODUCTION

Practices in the dairy industry often cause large volumes of raw milk and milk products to remain stored at refrigeration temperatures for extended periods during which psychrotrophic bacteria can grow (3, 18). Several workers (7, 9, 16, 17, 23) have isolated proteolytic enzymes from such psychrotrophs as Pseudomonas,

Enterobacter, Flavobacterium, Achromobacter, Lactobacillus, Micrococcus, and Alcaligenes. Many such organisms produce proteases (1, 6, 8, 22).

Received December 20, 1982. 1984 J Dairy Sci 67:522-530

heat

stable

Prominent among the psychrotrophic bacteria producing heat-resistant proteases are the Pseudomonads. Continued action of proteases from these organisms has been implicated in spoilage of sterile or refrigerated dairy products. However, few studies have been made on purification and characterization of these heat stable proteases. Alichanidis and Andrews (1) purified an extracellular heat stable protease from P. fluorescens A R - 1 1 that had a pHoptimum at 6.5 and a molecular weight of 38,400. It was 50% inactivated after heating in buffer at 150°C for 8.5 s. The heat stable neutral metalloprotease from Pseudomonas sp. MC 60 (4, 5) had a molecular weight of 48,500 and a half-life at 149°C of 7.4 s when heated in buffer containing Ca a+ and Zn 2+ or in milk. In a recent study on the purification and characterization of a heat stable protease from P. fluorescens B - 5 2 (22), the enzyme had a molecular weight of 46,900. The protease was active over a wide pH range (pH 6.0 to 10.5) and had optimum activity at 45 to 50°C. It had a half-life of 37.5 s at 150°C in milk. In our paper, as part of a study on proteases of psychrotrophic bacteria, the purification and characterization of an extracellular protease produced by Pseudomonas sp. B - 2 5 are described. MATERIALS AND METHODS Culture

Pseudomonas sp. B - 2 5 was isolated from a refrigerated butter sample and maintained on slopes of tryptone dextrose yeast extract agar at 5°C. For maximum enzyme production the culture was inoculated (2.5% vol/vol) into several 500-ml Erlenmeyer flasks containing 100 ml sterile TYEP medium (tryptone, 1.0%; yeast extract, .25%; K2HPO4, .1%; KH2PO4, .1%) pH 7.0 and incubated at 22°C for 48 h on a gyratory shaker (200 rpm),

522

PROTEASE FROM PSEUDOMONAS SP. B--25

523

Enzyme Purification

Protein Determination

The culture was centrifuged in a Sharpies continuous centrifuge at 20,000 rpm to remove the cells. The supernatant containing the extracellular protease was treated with ammonium sulfate (45 to 70% saturation), and after standing for 18 h, the precipitate was collected by centrifugation at 7,000 x g for 20 min in a refrigerated centrifuge. It was redissolved in .05 M phosphate buffer (pH 7.5) and dialyzed against the same buffer for 18 h. The dialyzed enzyme was fractionated with chilled acetone (1:1, vol/yol). The acetone precipitated enzyme was chromatographed on a column (55 x 2.5 cm) of Sephadex G - 1 0 0 with the same buffer for further purification. Fractions of 5 ml were collected at a flow rate of 25 ml/h and analyzed for protein and protease activity. Enzyme fractions with high specific activity were pooled and concentrated by ultrafiltration in an Amicon (Amicon Corp., Lexington, MA) ultrafiltration cell with a U M - 10 membrane.

The protein concentration of enzyme samples was determined by Folin assay (14) with bovine serum albumin (Sigma) as a standard.

Enzyme Assay

Protease activity was determined according to the method of Keay and Wildi (12) with modifications. To 1 ml of the substrate (1% casein in .05 M phosphate buffer, pH 7.0) was added 1.0 ml of suitably diluted enzyme, and the reaction mixture was incubated at 37°C for 10 min. The reaction was terminated by adding 2.0 ml of .4 A4 trichloroacetic acid (TCA). The precipitated proteins then were filtered through Whatman No. 1 filter paper. To I mI of the filtrate obtained after TCA precipitation, 5.0 ml of .4 M sodium carbonate solution was added followed by 1.0 ml of 1 N Folin's reagent. The solution was incubated at 37°C for 20 min for color development. The intensity of the biue color developed was measured at 660 nm in a Systronics Spectrophotometer 106 (MK.II).

Polyacrylamide Gel Electrophoresis

Electrophoresis was at pH 7.5 on 10% polyacrylamide gels in the presence of .1% (wt/vol) sodium d o d e c y l sulfate (SDS) (25). Ultracentrifugal Analysis

Ultracentrifugal analysis was in a Spmco Model E Ultracentrifuge (Beckman Instruments) with a Schlieren optical system at 25°C. Sedimentation velocity was measured at 52,000 x g. Optimum pH

One percent casein solutions were prepared in .05 M citrate (pH 5.5 to 6.0), phosphate (pH 6.5 to 7.5), Tris-HC1 (pH 8.0 to 8.8), carbonatebicarbonate (pH 9.2 to 10.5), glycine-NaOH (pH 11.0), and KC1-NaOH (pH 11.5) buffers. The purified enzyme was diluted in the above buffers and assayed separately in each substrate solution of the same pH. Optimum Temperature

Protease activity of the purified enzyme was assayed at temperatures ranging from 15 to 90°C. Before addition of the enzyme, the substrate (1% casein, pH 7.5) was tempered at the respective temperatures for 5 min. Heat Stability

Aliquots of the enzyme solution in .05 M phosphate buffer (pH 7.5) were exposed to temperatures ranging from 40 to 75°C for 10 and 30 min. The heat-treated enzyme samples immediately were transferred to a chilled water bath, and residual protease activity was determined at 37°C.

Unit of Activity

Michae|is-Menten Constant

Protease activity of the enzyme was expressed in units, the amount of enzyme required to release TCA soluble fragments giving blue color equivalent to 1 /~g of tyrosine under conditions of the assay.

One percent casein solution was diluted with phosphate buffer to obtain .25 to 10 mg/ml final concentration of the substrate in the reaction mixture. The enzyme concentration was k e p t constant at 10 #g/ml, and protease Journal of Dairy Science Vol. 67, No. 3, 1984

524

MALIK AND MATHUR

activity was determined under the specified conditions. Michaelis-Menten constant (Km) was calculated by the double reciprocal method (13).

.600

25 • 2(

~

•

Prote=se ectivity Protein

4.80

-3BO

Effect of Metal ions and Chemical Reagents

The enzyme solution (100 /~g/ml) was preincubated at 370C for 30 rain at pH 7.5 with different metal ions at a concentration of 1 mM and with various chemical reagents at a final concentration of 1 x 10 - 2 , 1 x 10 - 3 , or 1 x 10--aM. The residual protease activity of the enzyme subsequently was assayed after dilution of the enzyme to 10/~g/ml. Molecu lar Weight

The molecular weight of the purified enzyme preparation was determined by the gel filtration method (2) with Sephadex G - 1 0 0 column (53 x 2.5 cm). Amino Acid Composition

A sample of the enzyme was hydrolyzed in vacuo in 6 M He1 for 24 h at 1100C and amino acid composition determined by a JEOL Automatic Amino Acid Analyzer (Model No. JLC--6 AH). RESULTS Protease Purification

The (NH4)2SO4 precipitated enzyme (45 to 70% saturation) on dialysis yielded 69.3% recovery and 16.58-fold purification of the enzyme. Acetone precipitate of the dialyzed enzyme afforded 38.37-fold increase in specific activity and 53.8% recovery of the protease. when the acetone precipitated enzyme was chromatographed on a column of Sephadex G - 1 0 0 , the enzyme was eluted on a single peak between the 25th and 36th fractions concomitant with the major protein peak (Figure 1). The other minor protein peaks were devoid Of any proteolytic activity. Results from a typical purification procedure (summarized in Table 1) show that the enzyme was purified some 55-fold with a total yield of 35% of the original activity. The purified enzyme was homogeneous by polyacrylamide gel electrophoresis at pH 7.5 in the presence of .1% SDS as only one stained Journal of Dairy Science Vol. 67, No. 3, 1984

Ir~

1~0

i~o

180

220

260

300

Elution" volum¢ ( m l )

Figure 1. Elution profile of the acetone precipitated protease of Pseudomonas sp. B-25 through Sephadex G-IO0. Column, 2.5 × 53 cm; sample load, 45 mg of enzyme protein in 15 ml; eluant, .05 M phosphate buffer (pH 7.5); pressure head, 30 cm; flow rate, 25 ml/h.

band was evident (Figure 2), and on ultracentrifugal analysis the enzyme protein sedimented as a single component (Figure 3) with no perceptible evidence of heterogeneity. Optimum pH and Temperature

Protease activity increased as pH of the substrate was raised from 6.0 to 8.8 (Figure 4). At higher pH, enzyme activity exhibited a sharp decline. Although the enzyme was not functional at pH 5.4 and 6.0, protease activity at pH 11.5 was only 16% of maximum. Minimum enzyme activity was at pH 8.8, at 65°C at pH 7.5 (Figure 5). A sharp decline in enzyme activity was noted at temperatures higher than 65°C. Heat Stability

Heat stability of purified protease was examined at pH 7.5 on exposure to different temperatures for 10 rain and 30 min (Figure 6). The purified enzyme was t00% stable at 40 and 45°C for 30 and 10 min, respectively. Enzyme activity, however, declined at higher temperatures. Michaelis-Menten Constant

The K m of P s e u d o m o n a s sp. B--25 protease (Figure 7) was 4.0 mg/ml. Effect of Metal Ions and Chemical Reagents

The effect of various cations on protease activity was studied at a concentration of 1 x

525

PROTEASE FROM PSEUDOMONAS SP. B--25 TABLE 1. Purification scheme for the extracellular protease of Pseudomonas sp. B--25.

Purification step

Culture supernatant Ammonium sulfate precipitation (45 to 70% saturation) Acetone fractionation (1 : 1 vol/vol) Gel filtration (Sephadex G-IO0)

Total enzyme

Total protein

Specific activity

(units)

(mg)

250,000

3,240.0

77.16

1.00

100.0

173,250

135.4

1,279.54

16.58

69.3

134,400

45.4

2,960.35

38.37

53.8

87,600

20.7

4,231.88

54.85

35.0

Purification

Recovery

(-fold)

(%)

10--3M. Although mercury inhibited the enzyme to the maximum 97.6%, nickel and copper also caused strong inhibition (Table 2). Enzyme activity was inhibited to the extent of 19.1, 16.7, 14.3, and 11.1%, in the presence of K +, Co ++, Pb ++, or Ag +. Manganese had a slight stimulatory effect. Among the chemical reagents (Table 3), diisopropylfluoro-phosphate (DFP) completely inactivated the enzyme at 10 mM concentration, although the inhibition was only 12.2% in the presence of 1 mM DFP. Among the chelating agents, 1 x 10 - 2 M 1,10 phenanthroline exhibited 92.7% inhibition, and ethylenediaminetetracetate (EDTA) at 1 × 10 - 3 M concentration caused 61% loss of enzyme activity. Inhibition by the oxidizing agents KMnO4 (1 × 10 - 3 M) and iodine (1 x 10--2M) also was more than 60%. Sodium thioglycollate and K4Fe(CN)6, however, slightly stimulated protease activity. Molecu lar Weight

Figure 2. Electrophoretic mobility of purified extracellular protease of Pseudomonas sp. B 25 on SDS polyacrylamide gel. Sample, 15 #g enzyme protein in .05 ml glycerol; buffer, .05 M phosphate buffer (pH 7.5) with .2% SDS; current, 4 mA per gel for 2 h; stain, Commassie brilliant blue R-250.

The molecular weight of the purified extracellular protease was estimated by Sephadex G - 1 0 0 gel filtration technique by plotting the log molecular weight of the standard proteins against Ve/Vo and extrapolating the corresponding ratio of the enzyme protein (Figure 8). The molecular weight of the enzyme was 41,200. Amino Acid Composition

Acid hydrolyzate of the enzyme yielded 356 amino acid residues (Table 4). The enzyme protein had an abundance of glycine residues, Journal of Dairy Science Vol. 67, No. 3, 1984

526

MALIK AND MATHUR

Figure 3. Oltracentrifugal pattern of purified extracellular protease of Pseudomonas sp. B - 2 5 . Sample, .3% enzyme protein in .05 M phosphate buffer (pH 7.5); speed, 30,000 rpm; temperature, 25°C; instrument, Beckman Spinco Model E Analytical Ultracentrifuge. The photographs were taken at zero time and at subsequent 4-min intervals.

70, f o l l o w e d b y alanine, 56, a n d asparatic acid, 46. A l t h o u g h cystine, p r o l i n e , a n d t y r o s i n e were d e t e c t e d , o n l y f o u r residues of m e t h i o n i n e were in t h e e n z y m e m o l e c u l e .

DISCUSSION

An

heat

stable

protease

of

geneity by a three-step purification scheme (Table 1; Figure 1). T h e p u r i f i e d protease, a l t h o u g h active over a wide pH range (pH 6.0 t o

I001

IOO

~

extracellular

P s e u d o m o n a s sp. B--25 was p u r i f i e d to h o m o -

80

.

\

g 6oi

-~ so g

40I 20 20 I

sl0

7'0

8'-0

9'.0

10'-0

11'.0

12'-0

pH Figure 4. Effect of pH on activity of purified extracellular protease of Pseudomonas sp. B - 2 5 . One percent casein solutions were prepared at different pH with buffers of .05 M ionic strength. Protease activity of the enzyme (10 /~g/ml) was assayed separately in each case. Journal of Dairy Science Vol. 67, No. 3, 1984

I

I

I

10 20 30 40 50 60 70 80 90 Ternperoture (C)

Figure 5. Effect of temperature on activity of purified extracellular protease of Pseudomonas sp. B--25. Protease activity was assayed at different temperatures by the enzyme solution containing 10 ~g protein/ml.

PROTEASE FROM PSEUDOMONAS SP. B--25

100~

O

m

i

n

-o---o--

10

rain

52 7

0,2~

Incubatkm

Incubation

0"2[~

~ 8o >

g

g

sff

_l> 0.12 ~ l.O'

O,OE

m

2O

0-0~ " 35

/.0 /.5 50 65 60 65 70 75 Temperature(C)

Figure 6. Heat stability of protease of Pseudomonas sp. (100 /zg/ml) was preincubated at the indicated temperatures, tease activity was assayed at of the enzyme to 10 ~g/ml.

purified extracellular B--25. The enzyme for 10 and 30 rain and the residual pro37°C after dilution

10.0) was o p t i m u m at pH 8.8. In this respect, the B--25 protease is similar to the protease f r o m P. fluorescens R - 1 2 (10, 22). The opt i m u m t e m p e r a t u r e for the protease of a Streptornyces sp. was 6 0 ° C (19). However, the t e m p e r a t u r e o p t i m a of proteases vary considerably f r o m one organism to another. The d f f e r e n t t e m p e r a t u r e s in the literature are 35°C in Pseudornonas sp. MC 60 (12), 4 5 ° C in

TABLE 2. Effect of metal ions on activity of purified extracellular protease of Pseudomonas sp, B--25. Salta

Residual activity (%)

None MnC12 AgC1 Pb (CH3 COO) 2 CoC12 KC1 CuSO 4 NiC1~ HgC12

100.0 108.3 88.9 85.7 83.3 80.9

59.5 40.5 2.4

aFinal concentration of metal salts in the purified extracellular protease (10 #g/ml) was 1 mM in .05 M phosphate buffer (pH 7.5). Residual protease activity was assayed after preincubation of the enzyme at 37°C for 30 rain.

-1'.0

1!0

2'.0

3'.o

4'.0

[-~-~] ( rag/ml ) Figure 7. Lineweaver-Burk plot of the reaction volocity versus substrate concentration for purified extracellular protease of Pseudomonas sp. B-25. The reaction velocity was measured at 37°C after 10 min at a constant enzyme concentration (10 #g/ml) and substrate concentration ranging from .25 to 10 mg/ml in .05 M phosphate buffer (pH 7.5).

P. fluorescens (1), and 45 to 50°C in P. fluorescens B - 5 2 (22). Purified e n z y m e was stable at 4 5 ° C for 10 min b u t lost 14% of its original activity after 30 min (Figure 6). A b o u t 26% activity was present even after h e a t t r e a t m e n t at 70°C for 10 min. Proteinase of P. putrefaciens (24) was, however, inactivated w h e n heated at 6 0 ° C for 3 min. A 36% loss of activity of P. aeruginosa A T C C 10145 ~rotease was n o t e d after exposure for 15 s at 72 C, whereas heating for 30 min at 63°C resulted in o n l y 6% loss, and boiling for 2 min c o m p l e t e l y inactivated the e n z y m e (11). Studies on h e a t inactivation of the extracellular protease of P. fluorescens A R - 1 1 showed that for 50% inactivation, 25-s exposure at 130°C, 17 s at 140°C, or 8.5 s at 1 5 0 ° C was required (1). The K m of the purified extracellular protease for casein was 4.0 m g / m l at pH 7.5 and 37°C, suggesting a wide specificity of the e n z y m e t o w a r d different substrates. The K m of the protease f r o m P. fluorescens A R - - 1 1 was .13 mM (1). A l t h o u g h m e r c u r y at 1.0 m M c o n c e n t r a t i o n caused a b o u t 98% inactivation o f the protease, inhibition by Cu ++ or Ni ++ was f r o m 40 to 60%. Similarly, inhibitory effect of heavy metal ions on the protease of P. putrefaciens Journal of Dairy Science Vol. 67, No. 3, 1984

528

MALIK AND MATHUR

TABLE 3. Effect of some chemical reagents on the activity of purified extracellular protease of Pseudomonas sp. B--25. Reagents I

None 2-Mercaptoethanol Sodium dodecyl sulfate (SDS) Diisoproplyfluorophospbate (DFP)

Concentration

Residual activity

04)

(%)

"1")( 10 - 3 1 × 10 - 4 I × 10 - a 1 × 10 - 2 1 × 10 - 3

100.0 92.7 92.7 92.7 87.8 87.8

1 X 10 -2

Iodine

Thiomersal

1× 1× 1× 1X 1×

10 - 3 10 -2 10 - 3 10 - 4 10 - 3

×

10 -4

8-Hydroxy-quinoline 5-sulfonic acid

1 × 10 - 3

N-Ethylmaleimide

1

1,10-Phenanthroline KMnO 4 Ethylenediaminetetraacetate (EDTA)

0

82.9 39.0 82.9 87,8 82.9 82.9 75.6

1 × 10 -4

90.2

1× 1× 1× 1X 1×

63.4 7.3 39.0 80.5 39.0 58.5

10 - 3 10 - 2 10 - 3 10 - 4 10 - 3

1 × 10 -4

1Residual protease activity of the purified extracellular protease (10 #g/ml) in .05 M phosphate buffer (pH 7.5) was assayed after preincubation with various chemical reagents (concentration indicated) at 37°C for 30 min.

2-]

~ A

2-~1.~

~psinogen A

Ve --Vo I.E

rose of PseuclProt omonos, sp.B-25"~bumin Bovine serum o[humin

1-~ t.'.1

/.~2

'~ t,'-/., /,'.5 ~-8 Log molecular weight

"2"7

/,!8

Figure 8. Molecular weight determination of purified extracellular protease of Pseudomonas sp. B--25. The column (2.5 X 53 cm) was calibrated with ribonuclease A (13,700), chymotrypsinogen A (25,000), pepsin (34,700), ovalalbumin (43,000), and bovine serum albumin (67,000). Sample load, 5 mg standard protein in 1.0 ml and 600 tag enzyme protein in 3 ml buffer; eluant, .05 M phosphate buffer (pH 7.5); pressure head, 30 cm; flow rate, 25 ml/h. Journal of Dairy Science Vol. 67, No. 3, 1984

(24) and P. fluorescens B--52 (22) was r e p o r t e d . Manganese ions e x h i b i t e d a slightly s t i m u l a t o r y e f f e c t o n t h e activity o f Pseudomonas sp. B - 2 5 protease. H o w e v e r , an i n h i b i t o r y e f f e c t o f t h e cations o n the p u r i f i e d fractions, viz. I--1, I--2, and I I I - 1 o f t h e p r o t e a s e o b t a i n e d f r o m an o b l i g a t o r y p s y c h r o p h i l i c b a c t e r i u m N R C 1004 was r e p o r t e d (20). Purified p r o t e a s e was i n h i b i t e d b y m e t a l chelating agents E D T A (61%), 1,10 p h e n a n t h r o l i n e (37%), and 8 - h y d r o x y q u i n o l i n e - 5 sulfonic acid (24%). It was n o t a f f e c t e d significantly b y 1 mad D F P , t h e i n h i b i t o r o f alkaline proteases. The extracellular p r o t e a s e o f Pseudomonas sp. B--25 t h u s m a y b e classified as a neutral m e t a l l o e n z y m e , n o t w i t h s t a n d i n g the fact t h a t t h e p r o t e a s e has an o p t i m u m pH o f 8.8, w h i c h is slightly t o w a r d alkaline. T h a t t h e e n z y m e was n o t a f f e c t e d b y D F P also suggests the possible absence o f serine residue in t h e active c e n t e r o f t h e e n z y m e . T h i o l r e a g e n t s like p a r a c h l o r o m e r c u r i b e n z o a t e (p--CMB), m e r c a p t o e t h a n o l , N - e t h y l m a l e i m i d e , and t h i o m e r s a l

PROTEASE FROM PSEUDOMONAS SP. B--25 TABLE 4. Amino acid composition of purified extracellular protease of Pseudornonas sp. B--25.

Amino acid

Residues per molecule 1

Lysine Histidine Argininc Asparatic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine

19 8 10 46 22 26 27 0 70 56 0 24 4 11 21 0 12

t Based on molecular weight 41,200 of the protease.

caused slight inhibition of the protease indicating the possibility of sulfhydryl groups in the active center of the enzyme. Presence of oxidizable amino acids at the active site of the e n z y m e is indicated because protease activity was inhibited (61%) in the presence o f I × 10--2M KMnO4. Similar results also have been r e p o r t e d for Bacillus subtilis K - 2 6 (21). The molecular weight o f P s e u d o r n o n a s sp. B - 2 5 protease as determined by gel filtration is 41,200 (Figure 8). Gel filtration chromatography suggested a single h o m o g e n e o u s entity. Considerable similarity was observed for the molecular weights r e p o r t e d for m a n y Pseud o m o n a d proteases (1, 4, 8, 10, 22). However, a molecular weight of a p p r o x i m a t e l y 23,000 was n o t e d for a h e a t stable protease of Pseud o m o n a s fluorescens P--26 (15). A m i n o acid analysis of the e n z y m e indicated similarities of amino acid c o m p o s i t i o n of this e n z y m e and proteases r e p o r t e d by others (4, 22). A m o u n t s of lysine, arginine, glycine, alanine, and m e t h i o n i n e are slightly higher in the test e n z y m e c o m p a r e d to o t h e r proteases, and asparatic acid, threonine, serine, isoleucine, and phenylalanine c o n t e n t are less. A high c o n t e n t of low m o l e c u l a r weight a m i n o acids such as glycine m a y be a prerequisite for heat

529

stability, because small side chains w o u l d m i n i m i z e stearic hindrance and allow structural flexibility. ACKNOWLEDGMENTS

Thanks are due to I. S. Verma, Director, National Dairy Research Institute, Karnal, for providing necessary facilities to this investigation. We are also t h a n k f u l to N. S. R e d d y , D e p a r t m e n t of F o o d Science and T e c h n o l o g y , O k a y a m a University, Japan, for a m i n o acid analysis and M.V.R. Rao, D e p a r t m e n t of Chemistry, Delhi University, Delhi, for ultracentrifugal analysis. Technical assistance of V. B. Sabharwal also is a c k n o w l e d g e d gratefully.

REFERENCES

1 Alichanidis, E., and A. T. Andrews. 1977. Some properties of the extracellular protease produced by the psychrotrophic bacterium Pseudomonas fluorescens strain AR--11. Biochim. Biophys. Acta 485:424. 2 Andrews, P. 1964. Estimation of the molecular weights of proteins by Sephadex gel filtration. Biochem. J. 91:222. 3 Andrey, J. Jr., and W. C. Frazier. 1959. Psychrophiles in milk held two days in farm bulk cooling tanks. J. Dairy Sci. 42:1781. 4 Barach, J. T., and D. M. Adams. 1977. Thermostability at ultrahigh temperatures of thermolysin and a protease from a psychrotrophic Pseudomonas. Biochim. Biophys. Acta 485:417. 5 Barach, J. T., D. M. Adams, and M. L. Speck. 1976. Stabilization of a psychrotrophic Pseudomonas protease by calcium against thermal inactivation in milk at UHT. Appl. Environ. Microbiol. 31 :875. 6 Bengtsson, K., L. Gardhage, and B. Isakasson. 1973. Gelation in UHT treated milk, whey and casein solution. The effect of heat-resistant proteases. Milchwissenschaft 28:495. 7 Chung, B. H., and R. Y. Cannon. 1971. Psyehrotrophic sporeforming bacteria in raw milk supplies. J. Dairy Sci. 54:448. 8 Gebre-Egziabher, A., E. S. Humbert, and G. Blankenagel. 1980. Heat-stable proteases from psychrotrophs in milk. J. Food Prot. 43:197. 9 Ingraham, J. L., and J. L. Stokes. 1959. Psychrophilic bacteria. Bacteriol. Rev. 23:97. 10 Juan, S. M., and J. J. Cazzulo. 1976. The extracellular protease from Pseudomonas fluorescens. Experientia 32:1120. 11 Juffs, H. S., and H. W. Doelle. 1968. Some properties of the extracellular proteolytic enzymes of the milk spoiling organism Pseudomonas aeruginosa ATCC 10145. J. Dairy Res. 35:395. 12 Keay, L., and B. S. Wildi. 1970. Proteases of genus Bacillus. Neutral proteases. Biotech. Bioeng. 12:179. Journal of Dairy Science Vol. 67, No. 3, 1984

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13 Lineweaver, H., and D. Burk. 1934. The determination of enzyme dissociation constants. J. Am. Chem. Soe. 56:658. 14 Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265. 15 Mayerhofer, H. J., R. T. Marshall, C. H. White, and M. Lu. 1973. Characterization o f a heat stable protease of Pseudomonas fluorescens P26. Appl. Microbiol. 25:44. 16 Mickels, R., N. L. Fish, and T. J. Claydon. 1967. Some chemical and flavor characteristics of a milk proteolysate of Pseudomonas fluorescens. J. Dairy Sci. 50:172. 17 Moreno, V., and F. V. Kosikowsky. 1973. Peptides, amino acids and amines liberated from B-casein by micrococcal cell free preparations. J. Dairy Sci. 56:39. 18 Morse, P. M., H. Jackson, C. H. MeNaughton, A. G. Leggatt, G. B. Landerkin, and C. K. Johns. 1968. Investigation o f factors contributing to the bacterial count of bulk tank milk. IIl. Increase in count from cow to bulk tank, and effects of refrigerated storage and preliminary incubation. J. Dairy Sei. 51:1192. 19 Nakanishi, T., Y. Matsumura, N. Minamiura, and T.

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20

21 22

23

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