ISSN 00036838, Applied Biochemistry and Microbiology, 2010, Vol. 46, No. 1, pp. 57–60. © Pleiades Publishing, Inc., 2010.
Effects of Bovine Milk Lactoperoxidase System on Some Bacteria1 (
M. Çankayaa, M. S ¸ i¸secioglua, Ö. Barι¸sb, M. Güllüceb, and H. Özdemira a
b
Department of Chemistry, Faculty of Sciences, Atatiirk University, 25240, Erzurum Departments of Biology, Faculty of Sciences, Ataturk University, 25240, Erzurum, Turkey email:
[email protected] Received February 10, 2009
Abstract—Bovine lactoperoxidase (LPO) was purified from skimmed milk using amberlite CG50H+ resin, CM sephadex C50 ionexchange chromatography, and sephadex G100 gel filtration chromatography. Lac toperoxidase was purified 20.45fold with a yield of 28.8%. Purity of enzyme checked by sodium dodecyl sul phatepolyacrylamide gel electrophoresis method and a single band was observed. Km was 0.25 mM at 20°C, Vmax value was 7.95 µmol/ml min at 20°C (pH 6.0). Antibacterial study was done by disk diffusion method of Kirby–Bauer using Mueller–Hinton agar medium with slight modification. Bovine LPO showed high anti bacterial activity in 100 mM thiocyanate–100 mM H2O2 medium for some bacteria (Brevibacillus centrosau rus, B. choshinensis, B. lyticum, Cedecea davisae, Chryseobacterium indoltheticum, Clavibacter michiganense pv. insidiosum, Kocuria erythromyxa, K. kristinae, K. rosea, K. varians, Paenibacillus validus, Pseudomonas syringae pv. populans, Ralstonia pickettii, Rhodococcus wratislaviensis, Serratia fonticola, Streptomyces viola ceusniger, Vibrio choleraenonO1) respectively, and compared with well known antibacterial substances (levo floxacin, netilmicin). LPO system has inhibition effects on all type bacteria and concentration is really important such as LPO100 mM thiocyanate–100 mM H2O2 system was proposed as an effective agent against many factors causing several diseases. DOI: 10.1134/S0003683810010096 1
Lactoperoxidase (LPO) (donor: hydrogen perox ide oxidoreductase E C. 1.11.1.7) is one of important enzymes in milk, and has an oxidoreductase activity. This enzyme has a crucial role to protect the lactating mammary gland and the intestinal tract of newborn infants against pathogenic microorganisms [1]. Several investigations reported that LPO is a glycoprotein and it is present in milk, saliva, tears [2–4]. LPO consists of a single polypeptide chain containing 612 amino acid resi dues, and molecular weight of bovine milk LPO is about 80 kDa [5, 6]. It contains 15halfcysteine residues and carbohydrate moieties that comprise about 10% of the weight of the molecule [7].
bovine milk LPO. Some of the bacteria used in our study were pathogenic bacteria. MATERIALS AND METHODS Materials. Raw cows’ milk was obtained from Fac ulty of Veterinary, Atatürk University (Erzurum, Tur key). Chemicals used were: sephadex G100, ammo nium sulfate, sodium acetate, CM sephadex C50, sodium phosphate, 2,2’azinobis (3ethylbenzthiaz oline6 sulfonic acid) diammonium salt (ABTS), 3 × 30 cm column, NaCl, Coomassie Brilliant Blue R 250, standard proteins (bovine carbonic anhydrase, bovine albumin, rabbit phosphorylase B, rabbit mus cle myosin) (Sigma, Aldrich, Steinhein^ Germany), and Amberlite CG50 resin (Fluka, Buchs, Switzer land). SDS–PAGE was performed using a MiniPro tein 3 system (BioRad, Germany). Purification of LPO. Bovine milk was centrifuged at 2500 g at 4°C for 15 min to remove fat. Amberlite + CG 50 (N H 4 ) resin (equilibrated with 5 mM sodium acetate pH 6.8) was added in the proportion of 22 g/l to the fresh raw skimmed bovine milk [6, 12]. The supernatant was decanted and the resin was washed with distilled water and 20 mM sodium acetate (pH 6.8). The bound protein was eluted with 0.5 M sodium acetate (pH 6.8). To the greencolored mix ture was gradually added solid ammonium sulfate (I. precipitation; saturation 90%) over a period of
Investigations exhibited that lactoperoxidase is one of the defense systems for organisms. Component of the system is lactoperoxidase, thiocyanate and hydro gen peroxide. The enzyme catalyzes the oxidation of endogenous thiocyanate (SCN–) by hydrogen perox ide (H2O2) producing the antibacterial hypothiocyan ate (OSCN–) [8]. Most of the researches have focused on bacterial inhibition. LPO isolated from goat and camel milk was found to be antibacterial even in the absence of a medium containing thyociyanate–H2O2 [9–11]. The present study was performed to determine the effects of different concentrations of thiocyanate– H2O2 medium on antibacterial properties of purified 1 The article is published in the original.
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30 min while it was being stirred magnetically and the enzyme solution was dialyzed overnight against 5 mM sodium phosphate buffer (pH 6.8). The clear greenish supernatant obtained above was loaded onto a column of CM sephadex C50 (Fluka) (3 × 10 cm) previously equilibrated with 10 mM sodium phosphate buffer (pH 6.8). The columnbounded enzyme was washed with 100 ml of 10 mM phosphate buffer (pH 6.8) con taining 100 mM NaCl. The enzyme was eluted with a linear gradient 100–200 mM NaCl in 10 mM phos phate buffer (pH 6.8) and subjected to ammonium sulfate precipitation (II, saturation 90%). Thereafter the enzyme solution was dialyzed overnight against 5 mM sodium phosphate buffer (pH 6.8). Lactoperox idase enzyme obtained from the CM sephadex C50 column was applied to a column of sephadex Q100 (Fluka) (2.5 × 100 cm). The columnbound enzyme was eluted with 0.1 M phosphate buffer (pH 6.8), and salted out with ammonium sulfate precipitation (III, 90% saturation). The enzyme solution was dia lyzed overnight against 0.5 M sodium phosphate buffer (pH 6.0). Fractions were lyophilized and checked for purity by SDS–PAGE gel [13]. Determination of LPO activity. Lactoperoxidase activities were determined by the procedure of Shin dler and Bardsley [14] with a slight modification and one unit of activity is defined as the amount of enzyme catalyzing the oxidation of 1 μmol of ABTS min–1 at 25°C (molar absorption coefficient; 32400 M–1 cm–1) [14–16], Protein concentration was determined according to the method of Lowry et al. [17]. UVVIS (CHEBIOS (s.r.l.) Optimumone, Italy) spectrophotometer was used for detection of LPO activity and determination of protein concentration. Kinetic studies. To obtain Km and Vmax values at pH 6.0, the enzyme activity was measured at 20°C 412 nm for five different substrates concentrations. For this purpose, 0.2, 0.5, 0.8, 1.1 and 1.5 ml volume from stock solution of the substrates were used. They are completed to the total volume of 2.8 ml with an appropriate buffer solution of the enzyme activity and then added 0,1 ml enzyme and 0,1 ml H2O2. To calcu late Km and Vmax values, one unit of activity is defined as the amount of enzyme catalyzing the oxidation of 1 μmol of ABTS min–1 at 25°C, then Vmax and Km values were obtained from LineweaverBurk graph. Antibacterial studies. Antibacterial activity of bovine lactoperoxidase was done by disk diffusion method of Kirby–Bauer using Mueller–Hinton agar medium with slight modification [18] and compared with well known antibacterial substances such as levo floxacin, netilmicin. For LPO, (200 μl of 100 mM KSCN, 100 μl of 100 mM H2O2 and 200 μl of LPO dis solved in phosphate buffer pH 6.8), (200 μl of 50 mM KSCN, 100 μl of 50 mM H2O2 and 200 μl of LPO dis solved in phosphate buffer pH 6.8), (200 μl of 50 mM KSCN, 100 μl of 20 mM H2O2 and 200 μl of LPO dis
solved in phosphate buffer pH 6.8) and, (200 μl of 10 mM KSCN, 100 μl of 10 mM H2O2 and 200 μl of LPO dissolved in phosphate buffer pH 6.8) were trans ferred into sterile testtubes, respectively. Mixture was allowed to react for 1 min and 30 μl of the each mixture was applied onto sterile disks. Filter sterilized protein was used in the reactions. Control disks were employed for denatured LPO instead of native LPO maintaining the other conditions. Filtersterilized protein was dena tured by using a boiling water bath for 1 h. For the antibacterial substances, BBL SensiDisks ntimicrobial susceptibility test disks which containing levofloxacin 5 μg/disk, netilmicin 30 μg/disk were used and incubated during 16–24 hour at 37°C. Bacterial strains obtained from Department of Biology, Atatürk University were used. Antibacterial effects of LPO–thiocyanatehydro gen peroxide system, levofloxacin and netilmicin against Brevibacillus centrosaurus, B. choshinensis, B. lyticum, Cedecea davisae, Chryseobacterium indolth eticum, Clavibacter michiganense pv.insidiosum, Kocuria erythromyxa, K. kristinae, K. rosea, K. varians, Paeniba cillus validus, Pseudomonas syringae pv. Populans, Ral stonia pickettii, Rhodococcus wratislaviensis, Serratia fon ticola, Streptomyces violaceusniger, Vibrio cholerae nonO1 was determined for LPO. According to Clinical and Laboratory Standards Institute (CLSI), antibac terial zone interpretive criteria were: zone of inhibition for netilmicin ≤12 mm were considered resistance (resistant), between 13–14 mm were considered weak sensitive (intermediate), ≥15 mm were considered sensitive (susceptible). Zone of inhibition for levoflox acin ≤12 mm were considered resistance (resistant), between 13–15 mm were considered weak sensitive (intermediate), ≥16 mm were considered sensitive (susceptible) [20]. RESULTS AND DISCUSSION Specific activity was calculated for crude extract and purified enzyme solution, yielding a purification of 16.9 fold and obtained 8.3 mg (Rz = 0.8) from 11 bovine milk (Table 1). Kinetic parameters as optimum pH, Km and Vmax were calculated from graphics for ABTS substrate on LPO. Optimum pH value was found 6.0 by means of activitypH graphs. Km value at optimum pH was 0.29 mM, and Vmax was 7.05 μmol/ml min. Quality of purified lactoperoxidaes checked by SDSPAGE (figure). As shown in Table 2, LPO–H2O2 (100 mM)–thiy ocyanate (100 mM) system showed much larger inhi bition zones against bacteria than LPO–H2O2 (50 mM)–thiyocyanate (50 mM) system, LPO–H2O2 (50 mM)thiyocyanate (20 mM) system and LPO– H2O2 (10 mM)thiyocyanate (10 mM) system. The lactoperoxidase system is a naturally occur ring, antimicrobial mechanism found in raw milk [20]. LPO can exert antibacterial and antifungal effect on both bacteria and fungus [11, 21]. LPO system in bovine milk has been well established, and reported
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EFFECTS OF BOVINE MILK LACTOPEROXIDASE SYSTEM ON SOME BACTERIA
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Table 1. Purification steps of lactoperoxidase (LPO) from bovine milk Step
Activity, Total Protein, Total EU ml–1 volume, ml mg protein, mg
Crude homogenate
225
1.49
335
506
1.5
100
1.00
31
3.01
93
425
4.6
84
3.00
1.41
273
0.14
38.2
385
10.1
76
6.7
10.23
34
0.79
26.9
348
12.9
69
8.6
Sephadex G100 column
1.69
169
0.1
16.9
286
16.9
56
11.3
Ammonium sulfate and dialyse
6.23
25
0.33
8.3
156
18.9
31
12.6
Ammonium sulfate CM sephadex C50 column Ammonium sulfate
2.25
Total Specific activ Recovery, Purifica activity, ity, EU mg –1 % tion fold –1 EU ml
13.7
Tàble 2. Inhibition zones of LPO–thiocyanate–H2O2 system against some bacteria with disk diffusion method. (I: 100 mM KSCN – 100 mM H2O2, II: 50 mM thiocyanate – 50 mM H2O2, III: 20 mM thiocyanate–50 mM H2O2, IV: 10 mM KSCN– 10 mM H2O2, V : control – denatured LPO) Diameter of inhibition zone, mm* Microorganism
Brevibacillus centrosaurus (Soil) B. choshinensis (Soil) B. lyticum (Soil) Cedecea davisae (Food) Chryseobacterium indoltheticum (Soil) Clavibacter michiganense pv.insidiosum (Plant) Kocuria erythromyxa (Soil) K. kristinae (Food) K. rosea (Soil) K. varians (Food) Paenibacillus validus (Soil) Pseudomonas syringae pv. populans (Plant) Ralstonia pickettii (Food) Rhodococcus wratislaviensis (Soil) Serratia fonticola (Clinic) Streptomyces violaceusniger (Soil) Vibrio choleraenon O1 (Clinic)
LPOsystem
Antibacterial substances
I
II
III
IV
V
LEV
NET
16 17 19 14 20 15 13 14 19 23 16 16 21 22 16 – 23
11 13 – 7 12 10 8 9 14 16 8 8 10 8 8 – 14
8 7 – 7 8 10 – 7 14 16 8 – 10 8 8 – 12
8 7 – 7 8 10 – 7 11 16 8 – 10 8 8 – 10
− – – − – – – – – – – – – – – – –
24 22 23 22 40 22 16 9 18 52 26 8 30 30 36 24 20
30 32 25 13 16 25 24 30 33 21 22 10 20 nt 15 19 28
* Inhibition zone in diameter (mm) around the discs. LPO: 0.9 µg LPO/disk, LEV – levofloksasin (5 µg/disc) and NET − netilmicin (30 µg/disc) were used as positive reference standards antibiotic discs (Oxoid); nt – not tested.
that activation of the system depends on the concen tration of thiocyanate (SCN) and hydrogen peroxide (H2O2). LPO system has the ability to catalyze the oxi dation of SCN by H2O2 with the production of anti bacterial hypo thyociyanate (OSCN–) [22]. LPO has been extensively used in biochemistry as a means to APPLIED BIOCHEMISTRY AND MICROBIOLOGY
radio iodinate proteins model of thyroid [16] and the dairy industry for the preservation of raw milk during transportation to processing plants in chemical indus try [8]. It was reported that most pathogenic bacteria are inhibited by a medium containing thyociyanate– H2O2. LPO–thiocyanate–H2O2 system is an effective
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choleraenonO1. This result showed that immune sys tem of organisms has resistant to defense for the bac teria. Pathogen bacteria or food spoilage bacteria survive and proliferate every where, and they could potentially spread our living places. This mean, we touch all kind of the bacteria. In this work, LPO was purified from bovine milk, and investigated properties against pathogenic bacteria. Antibacterial property at differ ent concentration of LPO–H2O2–thiocyanate system for the some bacteria is sensitive as much as or close to netilmicin, levofloxacin substances.
b
1 2 3 4
REFERENCES SDSPAGE bands of LPO. Standard proteins (a): 1 – rabbit phosphorylase B (97.4 kDa); 2 – bovine albumin (66 kDa); 3 – egg albumin (45 kD); 4 – bovine carbonic anhydrase (29 kDa); (b) – purified LPO from bovine milk (80 kDa).
agent against many of the diseases caused by organ isms in plants and animals [10, 11]. LPO has been purified from different milk sources, such as goat, bovine, buffalo, and camel milk. LPO purified from different milk sources have showed anti bacterial effects with thiocyanate–H2O2 medium on different bacterial. For example, Aeromonas hydro phila, Citrobacter freundi, Escherichia coli, Klebsiella pneumoniae, Proteus mirablis, Pseudomo Shigella dys enteries nas aeruginose, Salmonella enteritidis, S. schot muelleri, S. typhi, Serratia marcescens, Staphylococcus aureus, Shigella sonnei, Klebsiella oxytocica, Staphylo coccus aerogenes, Streptococcus faecalis, Mycobacte rium smegmatis CCM 2067 [9, 11, 15, 23]. In another study, lactoperoxidase was isolated from goat milk and its antibacterial properties were investigated for exploring the possibilities of developing it as an agent ^
to help fight against diseases [10]. U guz and Özdemir [15] showed that bovine lactoperoxidase exhibited antibacterial effects on different pathogenic bacteria at different concentrations of thiocyanate medium. But there is no detailed study regarding pathogenic bacte ria with different concentrations of thiocyanate medi ated BLPO. Levofloxacin and netilmicin are well known anti bacterial and antifungal substances. As shown Table 2, levofloxacin has inhibition effects on Kocuria rosea, Pseudomonas syringae pv. populans and Vibrio cholerae nonO1. But inhibition zone of levofloxacin is weaker than 100 mM LPO system for Kocuria rosea, Pseudomonas syringae pv. populans and Vibrio cholerae nonO1. Netilmicin has inhibition effects on Cedecea davisae, Chryseobacterium indoltheticum, Kocuria vari ans, Pseudomonas syringae pv. populans, Ralstonia pick ettii, Serratia fonticola. But inhibition zone of netilmi cin is weaker than 100 mM LPO system for Kocuria rosea, Pseudomonas syringae pv. populans and Vibrio
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