- Email: [email protected]
Activity of Bacillus polymyxa protease on components of the plasmin system in milk
ARTICLE IN PRESS
International Dairy Journal 16 (2006) 586–592 www.elsevier.com/locate/idairyj
Activity of Bacillus polymyxa protease on components of the plasmin system in milk N.K. Larson, B. Ismail, S.S. Nielsen, K.D. Hayes Department of Food Science, Purdue University, West Lafayette, IN 47907, USA Received 6 May 2005; accepted 30 September 2005
Abstract The activity of a metalloprotease produced by Bacillus polymyxa toward the plasmin system was studied in both buffer and milk. Chromogenic assays were carried out to monitor effects of the protease on plasmin activity in a buffer system. Among the plasmin system components, plasminogen was most affected by the protease. Hydrolysis of plasminogen was identified and visualized using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and casein–SDS–PAGE, respectively. Results confirmed that B. polymyxa protease cleaved bovine plasminogen, generating a plasmin-like activity. Kinetic parameters of the protease showed that its activity on plasminogen can be physiologically relevant where significant impact on plasminogen activation in milk can occur. This finding was further confirmed when, during refrigerated storage, a significant increase (Po0:01) in plasmin-like activity was observed in protease-treated pasteurized milk. Results of this study confirmed the interaction of the B. polymyxa protease with the plasmin system by acting as a plasminogen activator. r 2006 Published by Elsevier Ltd. Keywords: Bacillus polymyxa protease; Plasminogen activator; Plasmin; Milk
1. Introduction Proteolysis in milk has gained much interest from researchers due to its complexity and versatile effects on quality. Proteolysis can have beneficial effects where it may be essential for desirable qualities in dairy products, such as flavor development and texture changes during ripening of cheese (Nielsen, 2002). Conversely, uncontrolled proteolysis can have a detrimental effect on dairy product quality, such as poor curd formation (Srinivasan & Lucey, 2002), gelation of stored UHT milk (Kohlmann, Nielsen, & Ladisch, 1991), and degradation in stored casein intended to be used as functional ingredients in food (Nielsen, 2002). Proteolysis in milk has been shown to be caused by native proteases and proteases produced by psychrotrophic microorganisms (Fairbairn & Law, 1986; Grufferty & Fox, 1988).
Corresponding author. Tel.: +1 765 496 2864; fax: +1 765 496 7953.
E-mail address: [email protected] (K.D. Hayes). 0958-6946/$ - see front matter r 2006 Published by Elsevier Ltd. doi:10.1016/j.idairyj.2005.09.020
Plasmin (E.C. 3.4.21.7) (PL), an alkaline serine proteinase, is the principle native proteolytic enzyme in milk, where it hydrolyzes mostly as1-CN, as2-CN and b-CN (Bastian & Brown, 1996; Grufferty & Fox, 1988). The activity of PL has been shown to significantly affect the quality of fluid milk and dairy products (Bastian & Brown, 1996). PL exists in milk primarily in its zymogen form, plasminogen (PG), which can be converted into active PL by plasminogen activators (PAs) (Grufferty & Fox, 1988). The conversion of PG to PL is mediated by at least two types of PA, tissue-type (t-PA), which are associated with casein, and urokinase-type (u-PA), which are associated with somatic cells (Bastian & Brown, 1996). Other than PG, PL and PA the PL system also includes plasminogen activator inhibitors (PAIs) and plasmin inhibitors (PIs), which are present mainly in milk serum (Weber & Nielsen, 1991), and their effects on PL and PA, respectively, change depending mainly on pH and heat treatment (Precetti, Oria, & Nielsen, 1997; Richardson, 1983). In the US, extended refrigerated storage of milk on the farm, in transport, at the dairy plant, and in supermarkets
ARTICLE IN PRESS N.K. Larson et al. / International Dairy Journal 16 (2006) 586–592
have led to a total age of the milk before consumption of 20–21 days (Cousin, 1982). Refrigerated storage of raw and pasteurized milk for long periods has resulted in quality problems for the dairy industry. These problems are primarily related to the growth and the metabolic activities of psychrotrophic bacteria. In refrigerated raw milk, Gram-negative psychrotrophs are responsible for spoilage; of these, Pseudomonas species are predominant. Pasteurized milk, on the other hand, is spoiled primarily by Gram-negative psychrotrophs that recontaminate the milk after pasteurization, or by Gram-positive psychrotrophs that survive pasteurization. Among microorganisms that survive pasteurization, sporeforming Bacillus species dominate (Cousin, 1982; Sørhaug & Stepaniak, 1997). Ternstrom, Lindberg, and Molin (1993) found that approximately one-third of refrigerated pasteurized milk samples were spoiled by Gram-positive bacteria; of those, 77% were spoiled by Bacillus polymyxa and B. cereus. As Bacillus species grow in milk, they secrete heatresistant extracellular proteases that are thought to deleteriously affect the quality of the milk. Matta and Punj (1998) isolated and partially characterized a protease from B. polymyxa B-17; the protease was classified as a neutral metalloproteinase and was found to be active over a pH range of 5.5–10.0, with maximum activity at pH 7.5 and 50 1C. The enzyme retained 35% activity even after treatment at 70 1C for 10 min (Matta & Punj, 1998). The B. polymyxa protease (BPP), referred to as ‘‘Milcozyme’’ in the literature, has been used as a microbial rennet substitute (Jarmul, Reps, Poznanski, & Zlazowska, 1983). However, Milcozyme was found to be an unacceptable rennet substitute because of a lower yield and softer curd than produced with rennet (Reps, Poznanski, Rymaszewski et al., 1974). Studies have shown that cheeses made with Milcozyme blends (Jarmul & Reps, 1987) or Milcozyme alone (Poznanski, Reps, Kowalewska, Maszewski, & Jedrychowski, 1974; Reps, Poznanski, Kowalewska, & Roskosz, 1974) had the highest rate of proteolysis compared to those produced by rennet. Recent research has confirmed that a heat-stable metalloprotease produced by Pseudomonas fluorescens M3/6, which was characterized and shown to have activity on a-, b- and k-caseins (Kohlmann, Nielsen, & Ladisch, 1991; Kohlmann, Nielsen, Steenson, & Ladisch, 1991), enhanced activation of PG by stimulating PA (Frohbieter et al., 2005). This finding, coupled with the finding that BPP was related to high rate of proteolysis in cheese (Jarmul & Reps, 1987; Poznanski et al., 1974; Reps, Poznanski, Kowalewska, et al., 1974), suggest that the PL system and the metalloprotease system may be functionally interactive and cooperative in extra-cellular proteolysis. A preliminary experiment showed that BPP acted as a PA in modified Tris buffer (MTB). This observation, along with the suggested PL system and metalloproteases interactions, have led to the hypothesis that BPP interacts with the PL system to induce activation of PG. Therefore, the objective
587
of this study was to determine the activity of BPP toward the PL system in both buffer and milk. 2. Materials and methods 2.1. Reagents and sources Bovine PG (product #416), SpectrozymesPL (Spec PL; product #251), and human high molecular weight 2-chain u-PA (product #124) were purchased from American Diagnostica, Inc., Greenwich, Ct. BPP (product #P-6141) was purchased from Sigma Chemical Co., St. Louis, MO, USA. Bovine PL (product #602 370) was purchased from Roche Diagnostics, Indianapolis, IN, USA. All above reagents were diluted to appropriate concentrations in MTB (0.05 M Tris, 0.1 M NaCl, 0.01% Tween 80, pH 7.6). A micro BCA Protein assay kit (product #23235) was purchased from Pierce (Rockford, IL, USA). Laemmli buffer (product #161-0737), pre-stained broad-range molecular weight standards (product #161-0318), 40% acrylamide/bis solution (2.6% Crosslinking (C)), and 10 Tris/ Glycine/SDS (product #161-0732) were purchased from Bio-Rad Laboratories (Richmond, CA, USA). 2.2. Protein content and activity measurement B. polymyxa protease was dissolved in Tris–HCl buffer (0.05 M, pH 7.5; 1 and 1.3 mg mL1) and kept frozen at 20 1C. Protease samples were thawed and analyzed for specific activity in triplicate. To determine the amount of BPP to be used in chromogenic assays and the amount required for addition to milk, the specific activity of the enzyme was measured. The specific activity of BPP was measured with an azocasein assay (Kohlmann, Nielsen, & Ladisch, 1991), and expressed as a function of protein content, which was determined using a micro BCA Protein assay kit, following the manufacturer’s instructions. One unit of azocasein activity (proteolytic activity) was defined as the amount of enzyme required to produce an increase in absorbance of 0.01 h1 at 366 nm. The specific activity of BPP was determined to be 4.7 103 (Units mg1 protein). 2.3. Protease activity on PL and PG in buffer Chromogenic assays as outlined by Lu and Nielsen (1993) were carried out in triplicate to measure the effect of BPP on PL and PG. The amounts and reagents used to prepare each assay are presented in Table 1. MTB was added to all samples to yield a final volume of 100 mL. A volume of MTB (6.6 mL) was replaced with BPP for BPPtreated samples. Effects of the protease were tested with SpecPL, PL, and PG; appropriate blanks were included for all treatments. All samples (100 mL) were prepared in 96well microtiter plates and incubated at 37 1C for 1 h. After incubation, absorbance was read at 405 and 490 nm using an enzyme-linked immunosorbent assay (ELISA) plate
ARTICLE IN PRESS N.K. Larson et al. / International Dairy Journal 16 (2006) 586–592
588
Table 1 Reagents and volumes (mL) used to determine the effect of Bacillus polymyxa protease on bovine PL and PG Sample
MTBa
PGb
PLc
BPPd
SpectrozymesPL
1 2 3 4 5 6 7 8
93.4 68.4 86 61 85 10 3.4 54.4
0 0 14 14 0 0 0 14
0 0 0 0 15 15 15 0
6.6 6.6 0 0 0 0 6.6 6.6
0 25e 0 25e 0 75f 75f 25e
a
MTB (0.05 M, pH 7.6). Bovine PG (0.032 mg mL1). c Bovine PL (8 mU mL1). d BPP (1.3 mg mL1). e 3.2 mM Spec PL. f 1.6 mM Spec PL. b
Table 3 Casein–SDS–PAGE and SDS–PAGE gel formulations Ingredients
Casein–SDS–PAGE SDS–Page 15% Stacking 15% resolving gel resolving gel gel
Deionized distilled water, mL Lower buffera, mL Upper bufferb, mL Acrylamide (40%), mL Glycerol (87%), mL Caseinc, mL TEMEDd, mL APSe, mL Total volume, mL
2.2
3
2
2
2.4 0.6 0.8 5 30 8
2.4 0.6 5 30 8
9
3.75 1.35
9 53 14.1
a
Lower Buffer ¼ 1.5 M Tris–HCl buffer, pH 8.8, 0.4% SDS. Upper Buffer ¼ 0.5 M Tris–HCl buffer, pH 6.8, 0.4% SDS. c 2% casein-Hammersten in lower buffer:DDW, 1:3, v/v. d TEMED ¼ N, N, N0 , N0 -tetramethylethylenediamine. e APS ¼ 10% Ammonium persulfate. b
Table 2 Reagents and volumes (mL) used to determine the effect of BPP on bovine PG and human u-PA Sample
MTBa
PGb
u-PAc
BPPd
Spec PLe
1 2 3 4 5 6
75 50 35 65 40 25
25 25 25 0 25 25
0 0 15 0 0 15
0 0 0 10 10 10
0 25 25 25 25 25
a
MTB (0.05 M, pH 7.6). Bovine PG (0.032 mg mL1). c Human u-PA (1.5 IU mL1). d BPP (1 mg mL1). e 3.2 mM Spec PL. b
reader (Vmax Kinetic Microplate Reader, Molecular Devices Co., Menlo Park, CA, USA). 2.4. Protease activity on PG and human u-PA in buffer The coupled chromogenic assay outlined by Lu and Nielsen (1993) was used for measuring the effect of BPP on PG and human u-PA. The amounts and reagents used are presented in Table 2. MTB was added to all samples to make up a final volume of 100 mL. Effects of the protease were tested with SpecPL, PG, and u-PA; appropriate blanks were included for all treatments. All samples (100 mL) were prepared in 96-well microtiter plates and incubated at 37 1C for 1 h. After incubation absorbance was read at 405 and 490 nm using the ELISA plate reader.
10 mM) mixed in a 1:1:1 ratio (v/v) were placed separately in micro-centrifuge tubes and incubated for 1 h at 37 1C. Samples (10 mL) were mixed 1:2 (v/v) with Laemmli buffer under non-reducing conditions and placed in a boiling water bath for 5 min. Cooled samples and pre-stained, broad-range molecular weight standards were loaded into wells of a discontinuous acrylamide gel consisting of 15% acrylamide resolving gel and 4% acrylamide stacking gel (Table 3). Running conditions of the gel and the staining and destaining procedures were performed as outlined by Fajardo-Lira, Oria, Hayes, and Nielsen (2000). 2.6. Visualization of PL-like activity upon hydrolysis of bovine PG by BPP Casein–SDS–PAGE was used to visualize the protein bands with PL-like activity in bovine PG samples incubated with BBP for 1 h at 37 1C. EDTA (10 mM) was added in 1:1 ratio (v/v) to the samples after incubation, to inhibit the activity of BPP, for clearer distinction of the zones of clearance caused by the PG hydrolysate. The casein SDS–PAGE method followed was that of Fajardo-Lira et al. (2000) with the following modifications. Samples (10 mL) were mixed 1:2 (v/v) with Laemmli buffer under non-reducing conditions and held at room temperature for 30 min. Samples and pre-stained, broad-range molecular weight standards then were loaded into wells of a discontinuous casein-acrylamide gel (pre-ran for 1 h at 200 V) with a 15% acrylamide resolving gel and 4% acrylamide stacking gel (Table 3). For comparison purposes, a sample of PG with BPP incubated with EDTA (10 mM) in 1:1 ratio (v/v) for 1 h at 37 1C was loaded on the gel after mixing with Laemmli buffer as indicated above.
2.5. Hydrolysis of bovine PG in the presence of BPP 2.7. Dose dependency of protease activity on PG Samples of BPP (1 mg mL1), bovine PG (1 mg mL1), BPP with bovine PG in 1:1 (v/v) mixture, and BPP with bovine PG and Ethylenediamine tetraacetic acid (EDTA;
Reactions were carried out to test a dose-dependent effect of BPP on PG. In triplicate, reaction mixtures were prepared
ARTICLE IN PRESS N.K. Larson et al. / International Dairy Journal 16 (2006) 586–592
in a 96-well microtiter plate to a total volume of 100 mL. A blank containing MTB (75 mL) and 3.2 mM SpecPL (25 mL) was prepared, as well as a control containing MTB (62 mL), 3.2 mM SpecPL (25 mL) and 0.032 mg mL1 bovine PG (13 mL). The same reagents as in the control were mixed with five levels of 0.13 mg mL1 BPP (10, 20, 30, 40, and 50 mL) that replaced an equal amount of MTB in each of five wells representing the treatment mixtures. After incubation at 37 1C for 1 h, absorbance was read at 405 and 490 nm using the ELISA plate reader. 2.8. Effect of BPP on the kinetic parameters of PG activation Assays were carried out to calculate the Michaelis–Menten constant (KM) for activation of bovine PG by BPP. The following scheme describes the reactions that took place in these assays: KM
kcat
PG þ BPP 2 BPPPG 2 BPP þPLlike enzyme;
ð1Þ
589
were incubated at 4 1C for up to 18 h and were analyzed for PL activity after 0, 6, 12 and 18 h of incubation. Prior to determination of PL activity, the milk samples were ultracentrifuged at 100,000 g for 60 min at 4 1C. The resultant casein pellet (3 g) was reconstituted with purified water (Barnstead/Thermolyne Model D4631, Dubuque, IA, USA) to a total volume of 30 mL. To disrupt the ionic bridges formed by calcium ions, trisodium citrate was added to obtain a 0.1 M solution. To help dissociate PL from the casein micelle, e-amino caproic acid (0.1 g), a lysine analog, was added. The slurry was stirred in a water bath (Precision Model 260, GCA Co., Chicago, IL, USA) for 15 min at 45 1C; samples then were cooled to 22 1C and the pH was adjusted to 4.6 with concentrated hydrochloric acid to remove caseins. After holding for 15 min at 22 1C, samples were transferred to 50-mL tubes and centrifuged (1000 g, 21 1C, 15 min). The supernatant was recovered and the pH was adjusted to 7.5 using 8 M sodium hydroxide. The resultant supernatant was used for PL analyses, which were carried out following the colorimetric method described by Fajardo-Lira and Nielsen (1998).
PLlike enzyme þ Substrate2PLlike enzymeSubstrate ! PLlike enzyme þ pNA þ tripeptide,
2.10. Statistical analysis ð2Þ
where pNA is the p-nitroanilide group released from the chromogenic substrate, KM is the Michaelis–Menten constant for PA, and kcat is the rate constant for PG activation. Analysis of the reaction and calculation of the desired kinetic parameters were completed following the model described by Nishino, Yamauchi, Horie, Nagumo, and Suzuki (2000), with the modifications outlined by Ripple, Nielsen, and Hayes (2004). Reaction mixtures in triplicate were prepared by mixing 120 mL of BPP (1.67 106 mM) with varying volumes (0, 30, 60, 90, 120 mL) of bovine PG (1.81 104 mM), a constant volume (150 mL) of 3.2 mM Spec PL, and the necessary amount of MTB to make up a volume of 600 mL. Kinetic reactions were carried out at 37 1C in a spectrophotometer equipped with a temperaturecontrolled cuvette holder. The reaction progress was monitored by reading the absorbance at 405 and 490 nm every 2 min for up to 1 h. 2.9. Effect of BPP on PL activity in milk Fresh milk was obtained from the Purdue University Dairy Research Center and processed the same day. The milk was transported in sterile containers on ice to maintain low bacterial counts. Upon arrival, milk was pooled and pasteurized (74.5 1C for 15 s) using a pilot-scale plate heat exchanger (FT 74 Armfield, Jackson, NJ, USA). Then, in duplicate, milk was divided into eight 200 mLsamples, four were inoculated each with 1 mL of BPP (3.3 104 mg protein mL1), and the other four were left untreated. Milk samples (protease-treated and untreated)
Analyses of variance and regression analysis were carried out utilizing SAS for Windows, version 6.0 (SAS, 2000) to determine effects of BPP on components of the PL system. Significant differences between means were determined (Pp0:05) following PROC GLM and Tukey–Kramer multiple means comparison test. 3. Results and discussion 3.1. Protease activity on PL system components in buffer B. polymyxa protease did not have a significant effect on SpecPL or on PL (Fig. 1). However, in the presence of BPP, PG was activated, which was indicated by a significant increase in absorbance (bars 4 and 8, Fig. 1). Thus, BPP could have behaved as a PA that caused the conversion of PG into a product that has a PL-like activity. Alternatively, the purchased PG could contain traces of uPA; therefore, to confirm that BPP had essentially converted PG into a product that has a PL-like activity rather than stimulating the trace amounts of u-PA present in the sample, the effect of BPP on u-PA was tested. Results from the PG and u-PA coupled assay showed that BPP and u-PA exerted almost an additive effect on PG when placed together in one sample (Fig. 2). The absorbance value (1.309) of the sample that had BPP together with PG (0.816) plus that of the sample containing u-PA and PG (0.493) was a little less than the absorbance value (1.353) of the sample that had BPP, u-PA and PG, which might indicate a minor stimulation, if any, of contaminating u-PA, to result in a slight production of PL. These results had further supported the hypothesis that
ARTICLE IN PRESS N.K. Larson et al. / International Dairy Journal 16 (2006) 586–592
590
Absorbance at 405 nm
0.800
A
0.600
B
B
6
7
0.400
0.200 C 0.000
1
C 2
C
C
3
4
C 5
8
Sample
Fig. 1. Effect of BPP (6.6 mL of 1.3 mg enzyme mL1) on bovine PG and PL. Uppercase letters represent significantly different values (Pp0:05). Sample: 1 ¼ BPP; 2 ¼ BPP+Spec PL; 3 ¼ Bovine PG; 4 ¼ Bovine PG+Spec PL; 5 ¼ Bovine PL; 6 ¼ Bovine PL+Spec PL; 7 ¼ BPP+ Bovine PL+Spec PL; 8 ¼ BPP+Bovine PG+Spec PL. Values are means of three replicates.
Fig. 3. SDS–PAGE visualization of breakdown of bovine PG induced by BPP after incubation at 37 1C for 1 h. Lane 1, Mr standards; Lane 2, bovine PG; Lane 3, bovine PG+BPP+EDTA; Lane 4, bovine PG+BPP; Lane 5, BPP.
Absorbance at 405 nm
1.6 A
1.4 1.2 1
B
0.8 0.6
C
0.4 0.2
D
D
1
2
D
0 3
4
5
6
Sample
Fig. 2. Effect of BPP (10 mL of 1 mg enzyme mL1) on bovine PG and human u-PA. Uppercase letters represent significantly different values (Pp0:05). Sample: 1 ¼ Bovine PG; 2 ¼ Bovine PG+Spec PL; 3 ¼ Bovine PG+human u-PA+Spec PL; 4 ¼ BPP+Spec PL; 5 ¼ BPP+Bovine PG+Spec PL; 6 ¼ BPP+human u-PA+Bovine PG+Spec PL. Values are means of three replicates.
BPP did in fact convert PG into a product with PL-like activity. 3.2. SDS–PAGE and casein–SDS–PAGE of PG and BPP SDS–PAGE and casein–SDS–PAGE were performed to identify and visualize the protein with the PL-like activity. SDS–PAGE showed that BPP hydrolyzed PG into at least four distinctive fragments at molecular masses ranging from 50 to 29 kDa (Fig. 3, lane 4 compared with lanes 2 and 5). During incubation of PG with BPP, the presence of EDTA hindered the hydrolysis of PG by BPP (Fig. 3 lane 3 compared to lanes 2 and 4). This observation confirmed that BPP is a metalloprotease (Matta & Punj, 1998), which hydrolyzed PG to yield a fragment with PL-like activity as observed by the casein–SDS–PAGE. A new zone of clearance at around 37 kDa and a more intense zone of clearance at around 50 kDa was observed when EDTA was not present during the incubation of PG with BPP (Fig. 4, lane 3 compared to lane 4). Casein–SDS–PAGE (Fig. 4, lane 4 compared to lane 2) also showed that this hydrolysis
Fig. 4. Casein SDS–PAGE visualization of PL-like activity induced after incubation of bovine PG with BPP for 1 h at 37 1C. Lane 1, Mr standards; Lane 2, bovine PG; Lane 3, bovine PG+BPP+EDTA; Lane 4, bovine PG+BPP with EDTA added after incubation; Lane 5, BPP; lane 6, bovine PL.
fragment, which was about 37 kDa (Fig. 3, lane 4) caused a zone of clearance against the dark background of undegraded casein, indicating PL-like activity that was not observed in lanes 2, 3 and 5. PL can exist in varying molecular weights ranging from 90 to 26 kDa, with the most common being in the range of 50–48 kDa (Bastian & Brown, 1996; Grufferty and Fox, 1988; Choi, Hahm, Maeng, & Kim, 2005). PL cleaves the first 77 residues of bovine PG to give Arg-PG that will later on be cleaved by PA to yield the active PL, which in turn will cleave itself into various molecular mass chains. A PL standard (Fig. 4, lane 6) showed three distinctive zones of clearance at around 90, 50 and 37 kDa, with the one at around 50 kDa being the most significant. Bovine PG (Fig. 4, lane 2) showed some contamination with PL; however, it was clear that incubation with BPP caused more distinctive zones of clearance to appear similar to the zones of clearance observed for the PL standard (Fig. 4, compare lanes 4 and 6). This observation confirms that BPP acted as a PA and caused activation of PG. As observed in lane 5 of Fig. 4, a
ARTICLE IN PRESS N.K. Larson et al. / International Dairy Journal 16 (2006) 586–592
zone of clearance had spread as the enzyme moved down in the gel during the electrophoresis run indicating that BPP can hydrolyze casein, as suggested by Matta and Punj (1998). Thus, the presence of EDTA, which was added after incubation of PG with BPP, inhibited BPP and therefore aided in clear distinction of the zones of clearance caused by PL-like activity. 3.3. Dose dependency of protease activity on PG Results confirmed the dependency of activation of PG on the amount of BPP present in the sample (Fig. 5). Considering the specific amounts of BPP tested in this study, the degree of PG activation increased linearly with higher amounts of BPP present in the reaction mixture (R2 ¼ 0:9911). 3.4. Effect of BPP on the kinetic parameters of PG activation A rate value was calculated for each concentration of PG, and was used to construct a Lineweaver-Burk plot (Fig. 6). This plot was used to determine kinetic parameters corresponding to the first step in the coupled PG-activation
Absorbance 405 nm
1 0.8
y = 0.1409x + 0.0043 R2 = 0.9911
0.6 0.4 0.2 0 0
1
2
3
4
5
6
7
BPP (µg)
591
scheme, which is activation of PG by BPP. All LineweaverBurk plots are slightly non-linear because of the wellcharacterized effect of PL activity generated during the reaction on PG (Wohl, Summaria, & Robbins, 1980). The average KM and Vmax for BPP were 0.06 mM and 0.07 103 mM min1, respectively, and kcat/KM was 3.4 mM1 min1. The concentration of bovine PG in milk ranges between 3.5 and 14 mg mL1 (Bastian & Brown, 1996), which is equivalent to a range of 0.03–0.18 mM assuming the molecular mass of bovine PG to be 80 kDa. Thus, the activation reaction in milk can proceed at greater than 12 Vmax. However, the velocity of the reaction mediated by BPP suggested that the reaction might need more time than PA to produce PL-like activity. BPP could thus be physiologically relevant when given enough time for it to react with PG. Therefore, when milk is held for several hours after pasteurization, BPP, if present in high enough amounts, will have a significant impact on the PL-like activity in milk, as will be discussed in the following section. 3.5. Effect of BPP on PL activity in milk Over refrigerated storage, PL activity significantly increased (Po0:01) in both control and BPP-treated samples of milk (Fig. 7). However, the increase was more pronounced and was significantly (Po0:01) higher in the BPP-treated sample than the control. This confirms that BPP in milk, as in buffer, acted as a PA and converted PG into a product with a PL-like activity. The amount of BPP added to the milk, based on its kinetic parameters, caused the increase in PL-like activity to be higher than the increase observed in the control sample. Therefore, refrigerated pasteurized milk in the market may undergo a similar increase in PL-like activity, although to a variable extent depending on the level of B. polymyxa in milk and the amount of protease it produced.
Fig. 5. Dose-dependency of activation of bovine PG by BPP. Values are mean of three determinations, with error bars representing standard deviations. 0.45 0.40
Plasmin (µg ml-1 milk)
1.5E+08 y = 850.85x + 2E+07
.
1/ k1 (min mM-1)
2
R = 0.9124 1.0E+08
5.0E+07
0.35 0.30 0.25 0.20 0.15 0.10 0.05
-6.0E+04
0.00
0.0E+00 0.0E+00
0 6.0E+04
1.2E+05
1/[PG] mM-1
Fig. 6. Lineweaver-Burk plot for BPP (3.33 107 mM). [PG] ¼ concentration of bovine PG. Values represent means of three determinations, with error bars representing standard deviations (Note: error bars are hidden by the data points).
5
10
15
20
Storage time (h) Fig. 7. PL activity (mg mL1) of milk incubated with (dashed line) or without (solid line) BPP (3.3 104 mg protein mL1) for up to 18 h at 4 1C. Lines are means of two determinations with error bars representing standard errors (Note: Error bars for the control are hidden behind data points).
ARTICLE IN PRESS 592
N.K. Larson et al. / International Dairy Journal 16 (2006) 586–592
4. Conclusion BPP was found to be yet another factor that contributes to the complexity of the proteolysis reactions taking place in milk and dairy products. Results confirmed the interaction between microbial proteases, BPP in this case, with the PL system, which therefore can together functionally contribute to extra-cellular proteolysis. The more information revealed about the factors affecting the PL system, the better control is possible of the required conditions for storage or processing of milk for goodquality dairy products. BPP, in both buffer and milk systems, activated PG; thus, together with PAs that are already present in milk, BPP caused an increase in PL or PL-like activities over storage. Bacillus polymyxa protease induces PL-like activity, and at the same time, can hydrolyze casein; thus, as in the case of PL, the presence of BPP in milk has to be controlled to avoid detrimental effect on dairy products. On the other hand, BPP, under controlled conditions, may contribute to beneficial effects of PL, which might aid in flavor development and texture changes during ripening of cheese. Therefore, BPP, since it is an approved coagulant, can be added to cheese-milk in specific amounts and under controlled conditions to enhance the quality and shorten the ripening time. Now that it is confirmed that BPP affects the PL system in general, and PG activation in particular, more research is still needed to test the effects of processing conditions, such as pH, temperature, and calcium chloride addition, on BPP activity, and thus optimize the conditions under which a specified amount of BPP may be added to cheese-milk. References Bastian, E. D., & Brown, J. B. (1996). Plasmin in milk and dairy products: An update. International Dairy Journal, 6, 435–457. Choi, N., Hahm, J., Maeng, P. J., & Kim, S. (2005). Comparative study of enzyme activity and stability of bovine and human plasmins in electrophoretic reagents, b-mercaptoethanol, DTT, SDS, Titron X100, and urea. Journal of Biochemistry and Molecular Biology, 38, 177–181. Cousin, M. A. (1982). Presence and activity of psychrotrophic microorganisms in milk and dairy products: A review. Journal of Food Protection, 45, 172–207. Fairbairn, D. J., & Law, B. A. (1986). Proteinases of psychrotrophic bacteria: Their production, properties, effects and control. Journal of Dairy Research, 53, 139–143. Fajardo-Lira, C., & Nielsen, S. S. (1998). Effect of psychrotrophic microorganisms on the plasmin system in milk. Journal of Dairy Science, 81, 901–908. Fajardo-Lira, C., Oria, M., Hayes, K. D., & Nielsen, S. S. (2000). Effect of psychrotrophic bacteria and of an isolated protease from Pseudomonas fluorescens M3/6 on the plasmin system of fresh milk. Journal of Dairy Science, 83, 2190–2199. Frohbieter, K., Ismail, B., Nielsen, S. S., & Hayes, K. D. (2005). Effects of Pseudomonas fluorescens M3/6 bacterial protease on plasmin system and plasminogen activation. Journal of Dairy Science, 88(10), 3392–3401.
Grufferty, M. B., & Fox, P. F. (1988). Milk alkaline proteinase. Journal of Dairy Research, 55, 609–630. Jarmul, I., & Reps, A. (1987). Use of a mixture of pepsin with a microbiological preparation ‘Milcozyme’ in cheese production. Acta Academiae Agriculturae ac Technicae Olstenensis, Technologia Alimentorum, 22, 19–27. Jarmul, I., Reps, A., Poznanski, S., & Zlazowska, H. (1983). Free volatile fatty acids in cheese made using a mixture of pepsin with microbial rennet substitutes. Zeszyty Naukowe Akademii Rolniczo Technicznej w Olsztynie, Technologia Zywnosci, 18, 79–89. Kohlmann, K. L., Nielsen, S. S., & Ladisch, M. R. (1991). Purification and characterization of an extracellular protease produced by Pseudomonas fluorescens M3/6. Journal of Dairy Science, 74, 4125–4136. Kohlmann, K. L., Nielsen, S. S., Steenson, L. R., & Ladisch, M. R. (1991). Production of proteases by psychrotrophic microorganisms. Journal of Dairy Science, 74, 3275–3283. Lu, D. D., & Nielsen, S. S. (1993). Assays for native plasminogen activators in bovine milk. Journal of Dairy Science, 76, 3362–3368. Matta, H., & Punj, V. (1998). Isolation and partial characterization of a thermostable extracellular protease of Bacillus polymyxa B-17. International Journal of Food Microbiology, 42, 139–145. Nielsen, S. S. (2002). Plasmin system and microbial proteases in milk: Characteristics, roles and relationship. Journal of Agricultural and Food Chemistry, 50, 6628–6634. Nishino, T., Yamauchi, T., Horie, M., Nagumo, T., & Suzuki, H. (2000). Effects of a fucoidan on the activation of plasminogen by uPA and tPA. Thrombosis Research, 99, 623–634. Poznanski, S., Reps, A., Kowalewska, J., Maszewski, J. R., & Jedrychowski, L. (1974). Proteolytic activity of coagulating preparations of microbial origin depending on kind of substrate. Milchwissenschaft, 29, 742–746. Precetti, A. S., Oria, M. P., & Nielsen, S. S. (1997). Presence of bovine milk of two protease inhibitors of the plasmin system. Journal of Dairy Science, 80, 1490–1496. Reps, A., Poznanski, S., Rymaszewski, J., Jakubowski, J., Smietana, Z., Kowalewska, J., et al. (1974). The characteristics of different milkcoagulating enzymes. XIX International Dairy Congress, 1E, 323–324. Reps, A., Poznanski, S., Kowalewska, J., & Roskosz, D. (1974). Characteristics of ripening in cheeses made using microbial rennet substitutes. Roczniki Instytutu Przemyslu Mleczarskiego, 16, 5–20. Richardson, B. C. (1983). The proteinases of bovine milk and the effect of pasteurization on their activity. New Zealand Journal of Dairy Science, 18, 233–245. Ripple, K. M., Nielsen, S. S., & Hayes, K. D. (2004). Effects of native and denatured whey proteins on plasminogen activator activity. Journal of Dairy Science, 87, 2344–2350. SAS Institute Inc. (2000). PC SAS, Version 6. Cary, NC, USA: SAS Institute Inc. Srinivasan, M., & Lucey, J. A. (2002). Effects of plasmin on the formation and rheological properties of rennet-induced skim milk gels. Journal of Dairy Science, 85, 1070–1078. Sørhaug, T., & Stepaniak, L. (1997). Psychrotrophs and their enzymes in milk and dairy products: Quality aspects. A review. Trends in Food Science and Technology, 8, 35–40. Ternstrom, A., Lindberg, A. M., & Molin, G. (1993). Classification of the spoilage flora of raw and pasteurized bovine milk, with special reference to Pseudomonas and Bacillus. Journal of Applied Bacteriology, 75, 25–34. Weber, B. A., & Nielsen, S. S. (1991). Isolation and partial characterization of a native serine-type protease inhibitor from bovine milk. Journal of Dairy Science, 74, 764–771. Wohl, R. C., Summaria, L., & Robbins, K. C. (1980). Kinetics of activation of human plasminogen by different activator species at pH 7.4 and 37 1C. Journal of Biological Chemistry, 255, 2005–2013.
International Dairy Journal 16 (2006) 586–592 www.elsevier.com/locate/idairyj
Activity of Bacillus polymyxa protease on components of the plasmin system in milk N.K. Larson, B. Ismail, S.S. Nielsen, K.D. Hayes Department of Food Science, Purdue University, West Lafayette, IN 47907, USA Received 6 May 2005; accepted 30 September 2005
Abstract The activity of a metalloprotease produced by Bacillus polymyxa toward the plasmin system was studied in both buffer and milk. Chromogenic assays were carried out to monitor effects of the protease on plasmin activity in a buffer system. Among the plasmin system components, plasminogen was most affected by the protease. Hydrolysis of plasminogen was identified and visualized using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and casein–SDS–PAGE, respectively. Results confirmed that B. polymyxa protease cleaved bovine plasminogen, generating a plasmin-like activity. Kinetic parameters of the protease showed that its activity on plasminogen can be physiologically relevant where significant impact on plasminogen activation in milk can occur. This finding was further confirmed when, during refrigerated storage, a significant increase (Po0:01) in plasmin-like activity was observed in protease-treated pasteurized milk. Results of this study confirmed the interaction of the B. polymyxa protease with the plasmin system by acting as a plasminogen activator. r 2006 Published by Elsevier Ltd. Keywords: Bacillus polymyxa protease; Plasminogen activator; Plasmin; Milk
1. Introduction Proteolysis in milk has gained much interest from researchers due to its complexity and versatile effects on quality. Proteolysis can have beneficial effects where it may be essential for desirable qualities in dairy products, such as flavor development and texture changes during ripening of cheese (Nielsen, 2002). Conversely, uncontrolled proteolysis can have a detrimental effect on dairy product quality, such as poor curd formation (Srinivasan & Lucey, 2002), gelation of stored UHT milk (Kohlmann, Nielsen, & Ladisch, 1991), and degradation in stored casein intended to be used as functional ingredients in food (Nielsen, 2002). Proteolysis in milk has been shown to be caused by native proteases and proteases produced by psychrotrophic microorganisms (Fairbairn & Law, 1986; Grufferty & Fox, 1988).
Corresponding author. Tel.: +1 765 496 2864; fax: +1 765 496 7953.
E-mail address: [email protected] (K.D. Hayes). 0958-6946/$ - see front matter r 2006 Published by Elsevier Ltd. doi:10.1016/j.idairyj.2005.09.020
Plasmin (E.C. 3.4.21.7) (PL), an alkaline serine proteinase, is the principle native proteolytic enzyme in milk, where it hydrolyzes mostly as1-CN, as2-CN and b-CN (Bastian & Brown, 1996; Grufferty & Fox, 1988). The activity of PL has been shown to significantly affect the quality of fluid milk and dairy products (Bastian & Brown, 1996). PL exists in milk primarily in its zymogen form, plasminogen (PG), which can be converted into active PL by plasminogen activators (PAs) (Grufferty & Fox, 1988). The conversion of PG to PL is mediated by at least two types of PA, tissue-type (t-PA), which are associated with casein, and urokinase-type (u-PA), which are associated with somatic cells (Bastian & Brown, 1996). Other than PG, PL and PA the PL system also includes plasminogen activator inhibitors (PAIs) and plasmin inhibitors (PIs), which are present mainly in milk serum (Weber & Nielsen, 1991), and their effects on PL and PA, respectively, change depending mainly on pH and heat treatment (Precetti, Oria, & Nielsen, 1997; Richardson, 1983). In the US, extended refrigerated storage of milk on the farm, in transport, at the dairy plant, and in supermarkets
ARTICLE IN PRESS N.K. Larson et al. / International Dairy Journal 16 (2006) 586–592
have led to a total age of the milk before consumption of 20–21 days (Cousin, 1982). Refrigerated storage of raw and pasteurized milk for long periods has resulted in quality problems for the dairy industry. These problems are primarily related to the growth and the metabolic activities of psychrotrophic bacteria. In refrigerated raw milk, Gram-negative psychrotrophs are responsible for spoilage; of these, Pseudomonas species are predominant. Pasteurized milk, on the other hand, is spoiled primarily by Gram-negative psychrotrophs that recontaminate the milk after pasteurization, or by Gram-positive psychrotrophs that survive pasteurization. Among microorganisms that survive pasteurization, sporeforming Bacillus species dominate (Cousin, 1982; Sørhaug & Stepaniak, 1997). Ternstrom, Lindberg, and Molin (1993) found that approximately one-third of refrigerated pasteurized milk samples were spoiled by Gram-positive bacteria; of those, 77% were spoiled by Bacillus polymyxa and B. cereus. As Bacillus species grow in milk, they secrete heatresistant extracellular proteases that are thought to deleteriously affect the quality of the milk. Matta and Punj (1998) isolated and partially characterized a protease from B. polymyxa B-17; the protease was classified as a neutral metalloproteinase and was found to be active over a pH range of 5.5–10.0, with maximum activity at pH 7.5 and 50 1C. The enzyme retained 35% activity even after treatment at 70 1C for 10 min (Matta & Punj, 1998). The B. polymyxa protease (BPP), referred to as ‘‘Milcozyme’’ in the literature, has been used as a microbial rennet substitute (Jarmul, Reps, Poznanski, & Zlazowska, 1983). However, Milcozyme was found to be an unacceptable rennet substitute because of a lower yield and softer curd than produced with rennet (Reps, Poznanski, Rymaszewski et al., 1974). Studies have shown that cheeses made with Milcozyme blends (Jarmul & Reps, 1987) or Milcozyme alone (Poznanski, Reps, Kowalewska, Maszewski, & Jedrychowski, 1974; Reps, Poznanski, Kowalewska, & Roskosz, 1974) had the highest rate of proteolysis compared to those produced by rennet. Recent research has confirmed that a heat-stable metalloprotease produced by Pseudomonas fluorescens M3/6, which was characterized and shown to have activity on a-, b- and k-caseins (Kohlmann, Nielsen, & Ladisch, 1991; Kohlmann, Nielsen, Steenson, & Ladisch, 1991), enhanced activation of PG by stimulating PA (Frohbieter et al., 2005). This finding, coupled with the finding that BPP was related to high rate of proteolysis in cheese (Jarmul & Reps, 1987; Poznanski et al., 1974; Reps, Poznanski, Kowalewska, et al., 1974), suggest that the PL system and the metalloprotease system may be functionally interactive and cooperative in extra-cellular proteolysis. A preliminary experiment showed that BPP acted as a PA in modified Tris buffer (MTB). This observation, along with the suggested PL system and metalloproteases interactions, have led to the hypothesis that BPP interacts with the PL system to induce activation of PG. Therefore, the objective
587
of this study was to determine the activity of BPP toward the PL system in both buffer and milk. 2. Materials and methods 2.1. Reagents and sources Bovine PG (product #416), SpectrozymesPL (Spec PL; product #251), and human high molecular weight 2-chain u-PA (product #124) were purchased from American Diagnostica, Inc., Greenwich, Ct. BPP (product #P-6141) was purchased from Sigma Chemical Co., St. Louis, MO, USA. Bovine PL (product #602 370) was purchased from Roche Diagnostics, Indianapolis, IN, USA. All above reagents were diluted to appropriate concentrations in MTB (0.05 M Tris, 0.1 M NaCl, 0.01% Tween 80, pH 7.6). A micro BCA Protein assay kit (product #23235) was purchased from Pierce (Rockford, IL, USA). Laemmli buffer (product #161-0737), pre-stained broad-range molecular weight standards (product #161-0318), 40% acrylamide/bis solution (2.6% Crosslinking (C)), and 10 Tris/ Glycine/SDS (product #161-0732) were purchased from Bio-Rad Laboratories (Richmond, CA, USA). 2.2. Protein content and activity measurement B. polymyxa protease was dissolved in Tris–HCl buffer (0.05 M, pH 7.5; 1 and 1.3 mg mL1) and kept frozen at 20 1C. Protease samples were thawed and analyzed for specific activity in triplicate. To determine the amount of BPP to be used in chromogenic assays and the amount required for addition to milk, the specific activity of the enzyme was measured. The specific activity of BPP was measured with an azocasein assay (Kohlmann, Nielsen, & Ladisch, 1991), and expressed as a function of protein content, which was determined using a micro BCA Protein assay kit, following the manufacturer’s instructions. One unit of azocasein activity (proteolytic activity) was defined as the amount of enzyme required to produce an increase in absorbance of 0.01 h1 at 366 nm. The specific activity of BPP was determined to be 4.7 103 (Units mg1 protein). 2.3. Protease activity on PL and PG in buffer Chromogenic assays as outlined by Lu and Nielsen (1993) were carried out in triplicate to measure the effect of BPP on PL and PG. The amounts and reagents used to prepare each assay are presented in Table 1. MTB was added to all samples to yield a final volume of 100 mL. A volume of MTB (6.6 mL) was replaced with BPP for BPPtreated samples. Effects of the protease were tested with SpecPL, PL, and PG; appropriate blanks were included for all treatments. All samples (100 mL) were prepared in 96well microtiter plates and incubated at 37 1C for 1 h. After incubation, absorbance was read at 405 and 490 nm using an enzyme-linked immunosorbent assay (ELISA) plate
ARTICLE IN PRESS N.K. Larson et al. / International Dairy Journal 16 (2006) 586–592
588
Table 1 Reagents and volumes (mL) used to determine the effect of Bacillus polymyxa protease on bovine PL and PG Sample
MTBa
PGb
PLc
BPPd
SpectrozymesPL
1 2 3 4 5 6 7 8
93.4 68.4 86 61 85 10 3.4 54.4
0 0 14 14 0 0 0 14
0 0 0 0 15 15 15 0
6.6 6.6 0 0 0 0 6.6 6.6
0 25e 0 25e 0 75f 75f 25e
a
MTB (0.05 M, pH 7.6). Bovine PG (0.032 mg mL1). c Bovine PL (8 mU mL1). d BPP (1.3 mg mL1). e 3.2 mM Spec PL. f 1.6 mM Spec PL. b
Table 3 Casein–SDS–PAGE and SDS–PAGE gel formulations Ingredients
Casein–SDS–PAGE SDS–Page 15% Stacking 15% resolving gel resolving gel gel
Deionized distilled water, mL Lower buffera, mL Upper bufferb, mL Acrylamide (40%), mL Glycerol (87%), mL Caseinc, mL TEMEDd, mL APSe, mL Total volume, mL
2.2
3
2
2
2.4 0.6 0.8 5 30 8
2.4 0.6 5 30 8
9
3.75 1.35
9 53 14.1
a
Lower Buffer ¼ 1.5 M Tris–HCl buffer, pH 8.8, 0.4% SDS. Upper Buffer ¼ 0.5 M Tris–HCl buffer, pH 6.8, 0.4% SDS. c 2% casein-Hammersten in lower buffer:DDW, 1:3, v/v. d TEMED ¼ N, N, N0 , N0 -tetramethylethylenediamine. e APS ¼ 10% Ammonium persulfate. b
Table 2 Reagents and volumes (mL) used to determine the effect of BPP on bovine PG and human u-PA Sample
MTBa
PGb
u-PAc
BPPd
Spec PLe
1 2 3 4 5 6
75 50 35 65 40 25
25 25 25 0 25 25
0 0 15 0 0 15
0 0 0 10 10 10
0 25 25 25 25 25
a
MTB (0.05 M, pH 7.6). Bovine PG (0.032 mg mL1). c Human u-PA (1.5 IU mL1). d BPP (1 mg mL1). e 3.2 mM Spec PL. b
reader (Vmax Kinetic Microplate Reader, Molecular Devices Co., Menlo Park, CA, USA). 2.4. Protease activity on PG and human u-PA in buffer The coupled chromogenic assay outlined by Lu and Nielsen (1993) was used for measuring the effect of BPP on PG and human u-PA. The amounts and reagents used are presented in Table 2. MTB was added to all samples to make up a final volume of 100 mL. Effects of the protease were tested with SpecPL, PG, and u-PA; appropriate blanks were included for all treatments. All samples (100 mL) were prepared in 96-well microtiter plates and incubated at 37 1C for 1 h. After incubation absorbance was read at 405 and 490 nm using the ELISA plate reader.
10 mM) mixed in a 1:1:1 ratio (v/v) were placed separately in micro-centrifuge tubes and incubated for 1 h at 37 1C. Samples (10 mL) were mixed 1:2 (v/v) with Laemmli buffer under non-reducing conditions and placed in a boiling water bath for 5 min. Cooled samples and pre-stained, broad-range molecular weight standards were loaded into wells of a discontinuous acrylamide gel consisting of 15% acrylamide resolving gel and 4% acrylamide stacking gel (Table 3). Running conditions of the gel and the staining and destaining procedures were performed as outlined by Fajardo-Lira, Oria, Hayes, and Nielsen (2000). 2.6. Visualization of PL-like activity upon hydrolysis of bovine PG by BPP Casein–SDS–PAGE was used to visualize the protein bands with PL-like activity in bovine PG samples incubated with BBP for 1 h at 37 1C. EDTA (10 mM) was added in 1:1 ratio (v/v) to the samples after incubation, to inhibit the activity of BPP, for clearer distinction of the zones of clearance caused by the PG hydrolysate. The casein SDS–PAGE method followed was that of Fajardo-Lira et al. (2000) with the following modifications. Samples (10 mL) were mixed 1:2 (v/v) with Laemmli buffer under non-reducing conditions and held at room temperature for 30 min. Samples and pre-stained, broad-range molecular weight standards then were loaded into wells of a discontinuous casein-acrylamide gel (pre-ran for 1 h at 200 V) with a 15% acrylamide resolving gel and 4% acrylamide stacking gel (Table 3). For comparison purposes, a sample of PG with BPP incubated with EDTA (10 mM) in 1:1 ratio (v/v) for 1 h at 37 1C was loaded on the gel after mixing with Laemmli buffer as indicated above.
2.5. Hydrolysis of bovine PG in the presence of BPP 2.7. Dose dependency of protease activity on PG Samples of BPP (1 mg mL1), bovine PG (1 mg mL1), BPP with bovine PG in 1:1 (v/v) mixture, and BPP with bovine PG and Ethylenediamine tetraacetic acid (EDTA;
Reactions were carried out to test a dose-dependent effect of BPP on PG. In triplicate, reaction mixtures were prepared
ARTICLE IN PRESS N.K. Larson et al. / International Dairy Journal 16 (2006) 586–592
in a 96-well microtiter plate to a total volume of 100 mL. A blank containing MTB (75 mL) and 3.2 mM SpecPL (25 mL) was prepared, as well as a control containing MTB (62 mL), 3.2 mM SpecPL (25 mL) and 0.032 mg mL1 bovine PG (13 mL). The same reagents as in the control were mixed with five levels of 0.13 mg mL1 BPP (10, 20, 30, 40, and 50 mL) that replaced an equal amount of MTB in each of five wells representing the treatment mixtures. After incubation at 37 1C for 1 h, absorbance was read at 405 and 490 nm using the ELISA plate reader. 2.8. Effect of BPP on the kinetic parameters of PG activation Assays were carried out to calculate the Michaelis–Menten constant (KM) for activation of bovine PG by BPP. The following scheme describes the reactions that took place in these assays: KM
kcat
PG þ BPP 2 BPPPG 2 BPP þPLlike enzyme;
ð1Þ
589
were incubated at 4 1C for up to 18 h and were analyzed for PL activity after 0, 6, 12 and 18 h of incubation. Prior to determination of PL activity, the milk samples were ultracentrifuged at 100,000 g for 60 min at 4 1C. The resultant casein pellet (3 g) was reconstituted with purified water (Barnstead/Thermolyne Model D4631, Dubuque, IA, USA) to a total volume of 30 mL. To disrupt the ionic bridges formed by calcium ions, trisodium citrate was added to obtain a 0.1 M solution. To help dissociate PL from the casein micelle, e-amino caproic acid (0.1 g), a lysine analog, was added. The slurry was stirred in a water bath (Precision Model 260, GCA Co., Chicago, IL, USA) for 15 min at 45 1C; samples then were cooled to 22 1C and the pH was adjusted to 4.6 with concentrated hydrochloric acid to remove caseins. After holding for 15 min at 22 1C, samples were transferred to 50-mL tubes and centrifuged (1000 g, 21 1C, 15 min). The supernatant was recovered and the pH was adjusted to 7.5 using 8 M sodium hydroxide. The resultant supernatant was used for PL analyses, which were carried out following the colorimetric method described by Fajardo-Lira and Nielsen (1998).
PLlike enzyme þ Substrate2PLlike enzymeSubstrate ! PLlike enzyme þ pNA þ tripeptide,
2.10. Statistical analysis ð2Þ
where pNA is the p-nitroanilide group released from the chromogenic substrate, KM is the Michaelis–Menten constant for PA, and kcat is the rate constant for PG activation. Analysis of the reaction and calculation of the desired kinetic parameters were completed following the model described by Nishino, Yamauchi, Horie, Nagumo, and Suzuki (2000), with the modifications outlined by Ripple, Nielsen, and Hayes (2004). Reaction mixtures in triplicate were prepared by mixing 120 mL of BPP (1.67 106 mM) with varying volumes (0, 30, 60, 90, 120 mL) of bovine PG (1.81 104 mM), a constant volume (150 mL) of 3.2 mM Spec PL, and the necessary amount of MTB to make up a volume of 600 mL. Kinetic reactions were carried out at 37 1C in a spectrophotometer equipped with a temperaturecontrolled cuvette holder. The reaction progress was monitored by reading the absorbance at 405 and 490 nm every 2 min for up to 1 h. 2.9. Effect of BPP on PL activity in milk Fresh milk was obtained from the Purdue University Dairy Research Center and processed the same day. The milk was transported in sterile containers on ice to maintain low bacterial counts. Upon arrival, milk was pooled and pasteurized (74.5 1C for 15 s) using a pilot-scale plate heat exchanger (FT 74 Armfield, Jackson, NJ, USA). Then, in duplicate, milk was divided into eight 200 mLsamples, four were inoculated each with 1 mL of BPP (3.3 104 mg protein mL1), and the other four were left untreated. Milk samples (protease-treated and untreated)
Analyses of variance and regression analysis were carried out utilizing SAS for Windows, version 6.0 (SAS, 2000) to determine effects of BPP on components of the PL system. Significant differences between means were determined (Pp0:05) following PROC GLM and Tukey–Kramer multiple means comparison test. 3. Results and discussion 3.1. Protease activity on PL system components in buffer B. polymyxa protease did not have a significant effect on SpecPL or on PL (Fig. 1). However, in the presence of BPP, PG was activated, which was indicated by a significant increase in absorbance (bars 4 and 8, Fig. 1). Thus, BPP could have behaved as a PA that caused the conversion of PG into a product that has a PL-like activity. Alternatively, the purchased PG could contain traces of uPA; therefore, to confirm that BPP had essentially converted PG into a product that has a PL-like activity rather than stimulating the trace amounts of u-PA present in the sample, the effect of BPP on u-PA was tested. Results from the PG and u-PA coupled assay showed that BPP and u-PA exerted almost an additive effect on PG when placed together in one sample (Fig. 2). The absorbance value (1.309) of the sample that had BPP together with PG (0.816) plus that of the sample containing u-PA and PG (0.493) was a little less than the absorbance value (1.353) of the sample that had BPP, u-PA and PG, which might indicate a minor stimulation, if any, of contaminating u-PA, to result in a slight production of PL. These results had further supported the hypothesis that
ARTICLE IN PRESS N.K. Larson et al. / International Dairy Journal 16 (2006) 586–592
590
Absorbance at 405 nm
0.800
A
0.600
B
B
6
7
0.400
0.200 C 0.000
1
C 2
C
C
3
4
C 5
8
Sample
Fig. 1. Effect of BPP (6.6 mL of 1.3 mg enzyme mL1) on bovine PG and PL. Uppercase letters represent significantly different values (Pp0:05). Sample: 1 ¼ BPP; 2 ¼ BPP+Spec PL; 3 ¼ Bovine PG; 4 ¼ Bovine PG+Spec PL; 5 ¼ Bovine PL; 6 ¼ Bovine PL+Spec PL; 7 ¼ BPP+ Bovine PL+Spec PL; 8 ¼ BPP+Bovine PG+Spec PL. Values are means of three replicates.
Fig. 3. SDS–PAGE visualization of breakdown of bovine PG induced by BPP after incubation at 37 1C for 1 h. Lane 1, Mr standards; Lane 2, bovine PG; Lane 3, bovine PG+BPP+EDTA; Lane 4, bovine PG+BPP; Lane 5, BPP.
Absorbance at 405 nm
1.6 A
1.4 1.2 1
B
0.8 0.6
C
0.4 0.2
D
D
1
2
D
0 3
4
5
6
Sample
Fig. 2. Effect of BPP (10 mL of 1 mg enzyme mL1) on bovine PG and human u-PA. Uppercase letters represent significantly different values (Pp0:05). Sample: 1 ¼ Bovine PG; 2 ¼ Bovine PG+Spec PL; 3 ¼ Bovine PG+human u-PA+Spec PL; 4 ¼ BPP+Spec PL; 5 ¼ BPP+Bovine PG+Spec PL; 6 ¼ BPP+human u-PA+Bovine PG+Spec PL. Values are means of three replicates.
BPP did in fact convert PG into a product with PL-like activity. 3.2. SDS–PAGE and casein–SDS–PAGE of PG and BPP SDS–PAGE and casein–SDS–PAGE were performed to identify and visualize the protein with the PL-like activity. SDS–PAGE showed that BPP hydrolyzed PG into at least four distinctive fragments at molecular masses ranging from 50 to 29 kDa (Fig. 3, lane 4 compared with lanes 2 and 5). During incubation of PG with BPP, the presence of EDTA hindered the hydrolysis of PG by BPP (Fig. 3 lane 3 compared to lanes 2 and 4). This observation confirmed that BPP is a metalloprotease (Matta & Punj, 1998), which hydrolyzed PG to yield a fragment with PL-like activity as observed by the casein–SDS–PAGE. A new zone of clearance at around 37 kDa and a more intense zone of clearance at around 50 kDa was observed when EDTA was not present during the incubation of PG with BPP (Fig. 4, lane 3 compared to lane 4). Casein–SDS–PAGE (Fig. 4, lane 4 compared to lane 2) also showed that this hydrolysis
Fig. 4. Casein SDS–PAGE visualization of PL-like activity induced after incubation of bovine PG with BPP for 1 h at 37 1C. Lane 1, Mr standards; Lane 2, bovine PG; Lane 3, bovine PG+BPP+EDTA; Lane 4, bovine PG+BPP with EDTA added after incubation; Lane 5, BPP; lane 6, bovine PL.
fragment, which was about 37 kDa (Fig. 3, lane 4) caused a zone of clearance against the dark background of undegraded casein, indicating PL-like activity that was not observed in lanes 2, 3 and 5. PL can exist in varying molecular weights ranging from 90 to 26 kDa, with the most common being in the range of 50–48 kDa (Bastian & Brown, 1996; Grufferty and Fox, 1988; Choi, Hahm, Maeng, & Kim, 2005). PL cleaves the first 77 residues of bovine PG to give Arg-PG that will later on be cleaved by PA to yield the active PL, which in turn will cleave itself into various molecular mass chains. A PL standard (Fig. 4, lane 6) showed three distinctive zones of clearance at around 90, 50 and 37 kDa, with the one at around 50 kDa being the most significant. Bovine PG (Fig. 4, lane 2) showed some contamination with PL; however, it was clear that incubation with BPP caused more distinctive zones of clearance to appear similar to the zones of clearance observed for the PL standard (Fig. 4, compare lanes 4 and 6). This observation confirms that BPP acted as a PA and caused activation of PG. As observed in lane 5 of Fig. 4, a
ARTICLE IN PRESS N.K. Larson et al. / International Dairy Journal 16 (2006) 586–592
zone of clearance had spread as the enzyme moved down in the gel during the electrophoresis run indicating that BPP can hydrolyze casein, as suggested by Matta and Punj (1998). Thus, the presence of EDTA, which was added after incubation of PG with BPP, inhibited BPP and therefore aided in clear distinction of the zones of clearance caused by PL-like activity. 3.3. Dose dependency of protease activity on PG Results confirmed the dependency of activation of PG on the amount of BPP present in the sample (Fig. 5). Considering the specific amounts of BPP tested in this study, the degree of PG activation increased linearly with higher amounts of BPP present in the reaction mixture (R2 ¼ 0:9911). 3.4. Effect of BPP on the kinetic parameters of PG activation A rate value was calculated for each concentration of PG, and was used to construct a Lineweaver-Burk plot (Fig. 6). This plot was used to determine kinetic parameters corresponding to the first step in the coupled PG-activation
Absorbance 405 nm
1 0.8
y = 0.1409x + 0.0043 R2 = 0.9911
0.6 0.4 0.2 0 0
1
2
3
4
5
6
7
BPP (µg)
591
scheme, which is activation of PG by BPP. All LineweaverBurk plots are slightly non-linear because of the wellcharacterized effect of PL activity generated during the reaction on PG (Wohl, Summaria, & Robbins, 1980). The average KM and Vmax for BPP were 0.06 mM and 0.07 103 mM min1, respectively, and kcat/KM was 3.4 mM1 min1. The concentration of bovine PG in milk ranges between 3.5 and 14 mg mL1 (Bastian & Brown, 1996), which is equivalent to a range of 0.03–0.18 mM assuming the molecular mass of bovine PG to be 80 kDa. Thus, the activation reaction in milk can proceed at greater than 12 Vmax. However, the velocity of the reaction mediated by BPP suggested that the reaction might need more time than PA to produce PL-like activity. BPP could thus be physiologically relevant when given enough time for it to react with PG. Therefore, when milk is held for several hours after pasteurization, BPP, if present in high enough amounts, will have a significant impact on the PL-like activity in milk, as will be discussed in the following section. 3.5. Effect of BPP on PL activity in milk Over refrigerated storage, PL activity significantly increased (Po0:01) in both control and BPP-treated samples of milk (Fig. 7). However, the increase was more pronounced and was significantly (Po0:01) higher in the BPP-treated sample than the control. This confirms that BPP in milk, as in buffer, acted as a PA and converted PG into a product with a PL-like activity. The amount of BPP added to the milk, based on its kinetic parameters, caused the increase in PL-like activity to be higher than the increase observed in the control sample. Therefore, refrigerated pasteurized milk in the market may undergo a similar increase in PL-like activity, although to a variable extent depending on the level of B. polymyxa in milk and the amount of protease it produced.
Fig. 5. Dose-dependency of activation of bovine PG by BPP. Values are mean of three determinations, with error bars representing standard deviations. 0.45 0.40
Plasmin (µg ml-1 milk)
1.5E+08 y = 850.85x + 2E+07
.
1/ k1 (min mM-1)
2
R = 0.9124 1.0E+08
5.0E+07
0.35 0.30 0.25 0.20 0.15 0.10 0.05
-6.0E+04
0.00
0.0E+00 0.0E+00
0 6.0E+04
1.2E+05
1/[PG] mM-1
Fig. 6. Lineweaver-Burk plot for BPP (3.33 107 mM). [PG] ¼ concentration of bovine PG. Values represent means of three determinations, with error bars representing standard deviations (Note: error bars are hidden by the data points).
5
10
15
20
Storage time (h) Fig. 7. PL activity (mg mL1) of milk incubated with (dashed line) or without (solid line) BPP (3.3 104 mg protein mL1) for up to 18 h at 4 1C. Lines are means of two determinations with error bars representing standard errors (Note: Error bars for the control are hidden behind data points).
ARTICLE IN PRESS 592
N.K. Larson et al. / International Dairy Journal 16 (2006) 586–592
4. Conclusion BPP was found to be yet another factor that contributes to the complexity of the proteolysis reactions taking place in milk and dairy products. Results confirmed the interaction between microbial proteases, BPP in this case, with the PL system, which therefore can together functionally contribute to extra-cellular proteolysis. The more information revealed about the factors affecting the PL system, the better control is possible of the required conditions for storage or processing of milk for goodquality dairy products. BPP, in both buffer and milk systems, activated PG; thus, together with PAs that are already present in milk, BPP caused an increase in PL or PL-like activities over storage. Bacillus polymyxa protease induces PL-like activity, and at the same time, can hydrolyze casein; thus, as in the case of PL, the presence of BPP in milk has to be controlled to avoid detrimental effect on dairy products. On the other hand, BPP, under controlled conditions, may contribute to beneficial effects of PL, which might aid in flavor development and texture changes during ripening of cheese. Therefore, BPP, since it is an approved coagulant, can be added to cheese-milk in specific amounts and under controlled conditions to enhance the quality and shorten the ripening time. Now that it is confirmed that BPP affects the PL system in general, and PG activation in particular, more research is still needed to test the effects of processing conditions, such as pH, temperature, and calcium chloride addition, on BPP activity, and thus optimize the conditions under which a specified amount of BPP may be added to cheese-milk. References Bastian, E. D., & Brown, J. B. (1996). Plasmin in milk and dairy products: An update. International Dairy Journal, 6, 435–457. Choi, N., Hahm, J., Maeng, P. J., & Kim, S. (2005). Comparative study of enzyme activity and stability of bovine and human plasmins in electrophoretic reagents, b-mercaptoethanol, DTT, SDS, Titron X100, and urea. Journal of Biochemistry and Molecular Biology, 38, 177–181. Cousin, M. A. (1982). Presence and activity of psychrotrophic microorganisms in milk and dairy products: A review. Journal of Food Protection, 45, 172–207. Fairbairn, D. J., & Law, B. A. (1986). Proteinases of psychrotrophic bacteria: Their production, properties, effects and control. Journal of Dairy Research, 53, 139–143. Fajardo-Lira, C., & Nielsen, S. S. (1998). Effect of psychrotrophic microorganisms on the plasmin system in milk. Journal of Dairy Science, 81, 901–908. Fajardo-Lira, C., Oria, M., Hayes, K. D., & Nielsen, S. S. (2000). Effect of psychrotrophic bacteria and of an isolated protease from Pseudomonas fluorescens M3/6 on the plasmin system of fresh milk. Journal of Dairy Science, 83, 2190–2199. Frohbieter, K., Ismail, B., Nielsen, S. S., & Hayes, K. D. (2005). Effects of Pseudomonas fluorescens M3/6 bacterial protease on plasmin system and plasminogen activation. Journal of Dairy Science, 88(10), 3392–3401.
Grufferty, M. B., & Fox, P. F. (1988). Milk alkaline proteinase. Journal of Dairy Research, 55, 609–630. Jarmul, I., & Reps, A. (1987). Use of a mixture of pepsin with a microbiological preparation ‘Milcozyme’ in cheese production. Acta Academiae Agriculturae ac Technicae Olstenensis, Technologia Alimentorum, 22, 19–27. Jarmul, I., Reps, A., Poznanski, S., & Zlazowska, H. (1983). Free volatile fatty acids in cheese made using a mixture of pepsin with microbial rennet substitutes. Zeszyty Naukowe Akademii Rolniczo Technicznej w Olsztynie, Technologia Zywnosci, 18, 79–89. Kohlmann, K. L., Nielsen, S. S., & Ladisch, M. R. (1991). Purification and characterization of an extracellular protease produced by Pseudomonas fluorescens M3/6. Journal of Dairy Science, 74, 4125–4136. Kohlmann, K. L., Nielsen, S. S., Steenson, L. R., & Ladisch, M. R. (1991). Production of proteases by psychrotrophic microorganisms. Journal of Dairy Science, 74, 3275–3283. Lu, D. D., & Nielsen, S. S. (1993). Assays for native plasminogen activators in bovine milk. Journal of Dairy Science, 76, 3362–3368. Matta, H., & Punj, V. (1998). Isolation and partial characterization of a thermostable extracellular protease of Bacillus polymyxa B-17. International Journal of Food Microbiology, 42, 139–145. Nielsen, S. S. (2002). Plasmin system and microbial proteases in milk: Characteristics, roles and relationship. Journal of Agricultural and Food Chemistry, 50, 6628–6634. Nishino, T., Yamauchi, T., Horie, M., Nagumo, T., & Suzuki, H. (2000). Effects of a fucoidan on the activation of plasminogen by uPA and tPA. Thrombosis Research, 99, 623–634. Poznanski, S., Reps, A., Kowalewska, J., Maszewski, J. R., & Jedrychowski, L. (1974). Proteolytic activity of coagulating preparations of microbial origin depending on kind of substrate. Milchwissenschaft, 29, 742–746. Precetti, A. S., Oria, M. P., & Nielsen, S. S. (1997). Presence of bovine milk of two protease inhibitors of the plasmin system. Journal of Dairy Science, 80, 1490–1496. Reps, A., Poznanski, S., Rymaszewski, J., Jakubowski, J., Smietana, Z., Kowalewska, J., et al. (1974). The characteristics of different milkcoagulating enzymes. XIX International Dairy Congress, 1E, 323–324. Reps, A., Poznanski, S., Kowalewska, J., & Roskosz, D. (1974). Characteristics of ripening in cheeses made using microbial rennet substitutes. Roczniki Instytutu Przemyslu Mleczarskiego, 16, 5–20. Richardson, B. C. (1983). The proteinases of bovine milk and the effect of pasteurization on their activity. New Zealand Journal of Dairy Science, 18, 233–245. Ripple, K. M., Nielsen, S. S., & Hayes, K. D. (2004). Effects of native and denatured whey proteins on plasminogen activator activity. Journal of Dairy Science, 87, 2344–2350. SAS Institute Inc. (2000). PC SAS, Version 6. Cary, NC, USA: SAS Institute Inc. Srinivasan, M., & Lucey, J. A. (2002). Effects of plasmin on the formation and rheological properties of rennet-induced skim milk gels. Journal of Dairy Science, 85, 1070–1078. Sørhaug, T., & Stepaniak, L. (1997). Psychrotrophs and their enzymes in milk and dairy products: Quality aspects. A review. Trends in Food Science and Technology, 8, 35–40. Ternstrom, A., Lindberg, A. M., & Molin, G. (1993). Classification of the spoilage flora of raw and pasteurized bovine milk, with special reference to Pseudomonas and Bacillus. Journal of Applied Bacteriology, 75, 25–34. Weber, B. A., & Nielsen, S. S. (1991). Isolation and partial characterization of a native serine-type protease inhibitor from bovine milk. Journal of Dairy Science, 74, 764–771. Wohl, R. C., Summaria, L., & Robbins, K. C. (1980). Kinetics of activation of human plasminogen by different activator species at pH 7.4 and 37 1C. Journal of Biological Chemistry, 255, 2005–2013.