Purification and characterization of two extracellular proteinases from Arthrobacter nicotianae 9458

FEMS Microbiology Letters 170 (1999) 327^333

Puri¢cation and characterization of two extracellular proteinases from Arthrobacter nicotianae 9458 Emanuele Smacchi a , Patrick F. Fox b , Marco Gobbetti c; * a

c

Institute of Dairy Microbiology, Agricultural Faculty of Perugia, S. Costanzo, 06126 Perugia, Italy b Department of Food Chemistry, University College Cork, Cork Ireland Istituto di Produzioni e Preparazioni Alimentari, Facoltaé di Agraria di Foggia, Via Napoli 25, 71100 Foggia, Italy Received 4 September 1998; received in revised form 19 November 1998; accepted 23 November 1998

Abstract Two extracellular serine proteinases with molecular masses of about 53^55 and 70^72 kDa, were purified from Arthrobacter nicotianae 9458 and characterized. The enzymes differed with respect to temperature optimum, 55^60 and 37³C, respectively, tolerance to low values of pH and temperature, heat stability, sensitivity to EDTA and sulfhydryl blocking agents, and hydrophobicity. Both proteinases were optimally active in the pH range of 9.0 and 9.5, had considerable activity at pH 6.0 on Ks1 - and L-caseins, and tolerated NaCl over 5%. Specificity on casein fractions was generally similar and L-casein was more susceptible to hydrolysis than Ks1 -casein. The proteinases of Arthrobacter spp. may play a significant role in ripening of the smear surface-ripened cheeses. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Arthrobacter ; Proteinases; Smear surface-ripened cheese; L-Casein

1. Introduction Bacterial smear surface-ripened cheeses, such as Limburger, Muënster, Saint-Paulin, Appenzeller, Trappist, Tilsiter, Taleggio and Quartirolo, can be loosely de¢ned as cheeses in which bacteria are present in large numbers on the surface of the cheese and play a signi¢cant role in determining the ¢nal characteristics and attributes of the cheese. The diffusion into the interior of the cheeses of low molecular weight compounds, produced through the combined action of various extracellular hydrolases * Corresponding author. Tel.: +39 753-2387; Fax: +39 753-2387

synthesized by the smear micro£ora, is required for the development of the characteristic qualities of these cheeses. At the end of ripening, the surface micro£ora of bacterial smear surface-ripened cheeses is dominated by acid-sensitive bacteria, such as Brevibacterium, Arthrobacter, Micrococcus and Corynebacterium spp. [1]. Extensive studies have been made on extracellular proteases of Brevibacterium linens [2^5]. Arthrobacter spp. are Gram-positive chemo-organotrophs with a respiratory metabolism, never fermentative, widely distributed among the bacterial population of the soil. However, species of the genus Arthrobacter are major components of the smear micro£ora of the surface-ripened cheeses [1,6^8] and

0378-1097 / 99 / $19.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 5 6 2 - X

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also of mould surface-ripened cheeses, such as Brie and Camembert [9]. Despite their presence at high cell numbers on the surface of several cheeses, no studies on the enzymology of dairy Arthrobacter spp. have been reported. To our knowledge, only a few strains from soil have been studied. A serine protease secreted by Arthrobacter aureus [10] and a protease from another Arthrobacter sp. [11] have been characterized. In this article, we report the puri¢cation and characterization of two extracellular proteinases from Arthrobacter nicotianae 9458, a typical species present in the bacterial smear of surface-ripened cheeses.

Prepacked fast protein liquid chromatography (FPLC) columns of DEAE-Sephacel anion exchanger XK26, Sephacryl 200, Phenyl-Sepharose, Mono-Q HR5/5 and Superose 12 HR 10/30 were obtained from Pharmacia-Biotech, Uppsala, Sweden. Inhibitors, Na-caseinate and protein molecular mass standards were purchased from Sigma (St. Louis, MO).

two peaks containing the proteinases were each run separately, with the same conditions, in the following chromatographic steps. Active fractions were pooled, dialyzed against 50 mM Tris-HCl bu¡er, pH 7.5, concentrated 10-fold by freeze-drying and subjected to gel ¢ltration on Sephacryl 200 (70U2.0 cm internal diameter). Elution with 50 mM Tris-HCl bu¡er, pH 7.5, containing 0.1 M NaCl was at a £ow rate of 42 ml h31 . Active fractions were pooled, dialyzed against distilled water, freeze dried, resuspended in 50 mM Tris-HCl bu¡er, pH 7.5, containing 1.7 M (NH4 )2 SO4 and applied to a Phenyl-Sepharose hydrophobic interaction column (25U1.0 cm internal diameter). After application of the sample, the protein was eluted at a £ow rate of 24 ml h31 with a linear (NH4 )2 SO4 gradient from 1.7 to 0 M in 50 mM Tris-HCl bu¡er, pH 7.5. Active fractions were pooled, dialyzed, freeze dried and resuspended in 20 mM potassium phosphate bu¡er (KPi), pH 7.0. Proteinases were ¢nally puri¢ed on a FPLC Mono-Q HR5/5 column by eluting with a linear NaCl gradient from 0 to 0.8 M and from 0.8 to 1.0 M in the same bu¡er at a £ow rate of 60 ml h31 . Protein amounts during puri¢cation were determined using a Bio-Rad Protein Assay Kit (Bio-Rad Laboratories).

2.2. Enzyme puri¢cation

2.3. Enzyme assays

Six liters of a 72-h culture of A. nicotianae 9458 in nutrient broth, supplemented with yeast extract (0.5%, w/v), were harvested by centrifugation at 16 000Ug for 15 min at 4³C. Cells were in late exponential growth phase. As shown by microscope observations, cells were of medium-short rod forms and no coccoid cells, which appear in stationary phase culture, were present. The cell-free supernatant was freeze-dried (MOD E1PTB; Edwards, Milan, Italy), resuspended in distilled water, concentrated to one-sixtieth of the initial volume by ultra¢ltration using a Sartocon Mini SM 17521 unit, ¢tted with 10-kDa cut-o¡ cellulose triacetate membranes (Sartorius, Goettingen, Germany) and dialyzed for 24 h at 5³C against 50 mM Tris-HCl bu¡er, pH 7.5. The sample was applied to a DEAE-Sephacel anion exchange XK26 column and proteins were eluted at a £ow rate of 48 ml h31 with a linear NaCl gradient (0^0.5 M) in 50 mM Tris-HCl bu¡er, pH 7.5. The

Proteinase activity during puri¢cation steps was routinely assayed using £uorescein isothiocyanate (FITC)-labeled casein (0.2% (w/v) ¢nal concentration) in 50 mM Tris-HCl bu¡er, pH 7.5, according to the method of Twinning [12]. Activity in chromatographic fractions was measured after incubation at 37³C for 2 h. The incubation mixture contained 0.02% NaN3 . The £uorescence of the control sample (to which puri¢ed enzyme was added after incubation of the substrate) was less than 5% of that measured in the enzyme-containing sample. One unit of proteinase activity was de¢ned as the increase in £uorescence of 1 unit per min and £uorescence was measured using HPLC monitor (Shimadzu, Kyoto, Japan). Activity on Na-caseinate (2 mg ml31 ¢nal concentration) was determined in 50 mM KPi bu¡er pH 6.5, at 37³C for di¡erent times of incubation in the presence of 0.05% (w/v) NaN3 . pH 6.5 was chosen

2. Materials and methods 2.1. Chemicals

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since this is the usual pH of smear surface-ripened cheese. According to their speci¢c activity, proteinase 1 (P1) and proteinase 2 (P2) were diluted proportionally to have the same activity on £uorescent casein and used at these concentrations in the various assays on Na-caseinate. Urea-polyacrylamide gel electrophoresis (urea-PAGE) was performed according to the method of Andrews [13]. Gels were stained by the method of Blakesley and Boezi [14]. Analyses on Na-caseinate as substrate were performed to have a comparison between the substrate speci¢city of the two proteinases in conditions which approach those of the cheese ripening. The e¡ect of pH on activity of the two enzymes was examined at 37³C using Na-acetate, pH 5^5.5, Na-phosphate, pH 6^7, Tris-HCl, pH 7.5^8.5 or Nacarbonate, pH 9^11. Final bu¡er concentration in all assays was 100 mM. The e¡ect of pH was assayed both on £uorescent casein (after 2 h incubation) and Na-caseinate (after 4 and 14 h incubation). The temperature dependence of the two extracellular proteinases was determined in the temperature range 5^ 55³C for P1, and in the range 5^65³C for P2 at pH 9.5 and 9.0, respectively. Proteinase activity was measured using FITC-labeled casein. For heat-stability, portions of the enzyme solution were heated in capillary glass tubes at 50^65³C for P1 and at 65^ 71³C for P2, for several min. The e¡ect of divalent cations or inhibitors on activity of the two enzymes

329

was determined after preincubation of the enzyme solution, for 20 min at 30³C, with chemical reagents (¢nal concentrations, 1 and 5 mM). The reaction was initiated by adding FITC-labeled casein and the activity was measured after incubating at 37³C for 5 h. To assay the capacity of Ca2‡ , Zn2‡ , Ni2‡ and Co2‡ to reactivate the apoenzymes, an aliquot of a solution of each enzyme was preincubated at 5³C for 30 min in presence of EDTA at a ¢nal concentration of 10 mM; the solution was then dialyzed. Residual activity was then assayed in presence of divalent cations at ¢nal concentration of 1 or 5 mM using FITClabeled casein as substrate. To determine the sensitivity to NaCl, solutions of 1% Na-caseinate, containing 0, 1, 2, 5 or 10% NaCl in 50 mM KPi bu¡er, pH 6.5 (with 0.05% (w/v) NaN3 ), were incubated at 37³C for 4 h in presence of the enzyme solutions. The mixtures were subsequently run on urea-PAGE. 2.4. Molecular mass measurement The relative molecular mass of the puri¢ed proteinases was estimated by FPLC gel ¢ltration on a Superose 12 HR 10/30 column (50 mM KPi running bu¡er (pH 7.0) containing 0.15 M NaCl) and by sodium dodecyl sulfate (SDS)-PAGE according to the procedure of Laemmli [15]. Aliquots of 4, 0.4 and 0.2 Wg of protein were loaded onto the gel for

Table 1 Puri¢cation of extracellular proteinases 1 and 2 from Arthrobacter nicotianae 9458 Enzyme and puri¢cation step Proteinase 1 Ultra¢ltrated retentate of cell-free supernatanta DEAE-Sephacel Sephacryl 200 Phenyl-Sepharose Mono-Q Proteinase 2 Ultra¢ltrated retentate of cell-free supernatanta DEAE-Sephacel Sephacryl 200 Phenyl-Sepharose Mono-Q a

Total protein (mg)

Total activity (units)

Speci¢c activity (units mg31 )

Puri¢cation factor (fold)

Activity yield (%)

110.0

1780.3

16.2

1.0

100

10.1 3.1 1.6 0.4

1319.2 902.5 712.1 283.2

130.6 291.1 445.0 708.0

8.1 18.0 27.5 43.7

74 51 40 16

110.0

1780.3

16.2

1.0

100

6.2 4.8 1.4 0.5

856.3 750.1 432.3 316.7

138.1 156.2 308.8 633.4

8.5 9.6 19.1 39.1

48 42 24 18

The enzyme activity on the ultra¢ltrated retentate corresponds to the sum of both the proteinase activities.

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cell free supernatant, proteinase P1 and proteinase P2, respectively. Standards were provided by Sigma: L-galactosidase (112 kDa), bovine albumin (66 kDa), egg albumin (45 kDa), pepsin (34.7 kDa), trypsinogen (24 kDa) and L-lactoglobulin (18.4 kDa).

3. Results 3.1. Enzyme puri¢cation Results of the puri¢cation protocols of the two proteinases from A. nicotianae 9458 are summarized in Table 1. Chromatography of the cell-free supernatant on DEAE-Sephacel resolved two peaks with proteinase activity. Proteinase 1 (P1) and proteinase 2 (P2) were separately treated in the subsequent chromatography steps. P1 and P2 were puri¢ed 44and 39-fold with a recovery of 16 and 18% of total enzyme activity, respectively. On hydrophobic interaction chromatography, P2 was eluted at 0 M (NH4 )2 SO4 , compared with 0.5 M (NH4 )2 SO4 for P1. The cell-free supernatant of A. nicotianae 9458 after 72 h of cultivation contained few proteins

Fig. 1. SDS-PAGE of the puri¢ed proteinases from Arthrobacter nicotianae 9458. Lanes : 1, reference proteins (see Section 2); 2, cell-free supernatant; 3, proteinase P1; and 4, proteinase P2.

Fig. 2. Urea-PAGE of Na-caseinate hydrolysates produced by the extracellular proteinases P1 (A) and P2 (B) of Arthrobacter nicotianae 9458. Lanes : 1, Na-caseinate standard; and 2^5, hydrolysis products at 2, 4, 6 and 8 h, respectively.

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(Fig. 1). Proteinase activity was already detected in the supernatant after 24^36 h of incubation when cells were in the early exponential growth phase. After the ¢nal anion exchange chromatography, P1 and P2 were homogeneous by SDS-PAGE. Molecular massses of P1 and P2 were about 53^55 and ca. 70^72 kDa, respectively. The same values were estimated by gel ¢ltration on Superose 12 (data not shown). 3.2. Enzyme characterization The speci¢city of the extracellular proteinases P1 and P2 from A. nicotianae 9458, on Na-caseinate, were generally similar (Fig. 2). Urea-PAGE showed that Ks1 -, and especially L-casein, were rapidly degraded, at pH 6.5 and 37³C, by both proteinases. The extent of hydrolysis increased markedly up to 8 h of incubation where hydrolysis of L- and Ks1 caseins especially by P1 was almost complete. Hydrolysis of Na-caseinate showed a major peptide band with a low electrophoretic mobility for both P1 and P2, whereas at 4^8 h of incubation, peptide bands with high electrophoretic mobility were also present in the gels. The pH optima for P1 and P2 on £uorescent casein did not di¡er much and were determined to be pH 9.5 and 9.0, respectively; activities on £uorescent casein at pH 6.5 were 15 and 60% of the maximum, respectively. Increasing the pH from 6.0 to 9.0 enhanced the hydrolysis of Na-caseinate (Fig. 3). However, after 4 h of incubation, both proteinases caused considerable hydrolysis of Ks1 - and L-caseins at pH 6.0. The pH did not modify the substrate speci¢city since in all cases L-casein was hydrolyzed preferentially. At pH 9.5 and 9.0, the temperature optima were 37³C and 55^60³C for P1 and P2, respectively. More than 40 and 15% of maximum activity was expressed at 15³C by P1 and P2, respectively. P1 retained about 70 and 10% of maximum activity after exposure for 5 min at 52 and 60³C, respectively. Heating C

Fig. 3. Urea-PAGE of Na-caseinate hydrolysates produced by the extracellular proteinases P1 (A) and P2 (B) of Arthrobacter nicotianae 9458 after 4 h incubation. Lanes: 1, Na-caseinate standard ; and 2^5, hydrolysis products at pH 6.0, 7.0, 8.0 and 9.0, respectively.

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Table 2 E¡ect of chemical reagents and divalent cations on the activity of the extracellular proteinases 1 and 2 from Arthrobacter nicotianae 9458 Reagenta or cation

EDTA 1,10-Phenanthroline PMSF NEM Iodoacetic acid DTT Ca2‡ Fe2‡ Cu2‡ Zn2‡ Hg2‡ Mn2‡ Mg2‡ Ni2‡ Co2‡

Proteinase activity (%)b 1 mM of reagent/cation

5 mM of reagent/cation

P1

P2

P1

P2

9 83 7 88 90 98 91 84 33 8 16 72 94 28 46

87 91 6 85 87 80 90 97 35 13 23 68 83 17 40

8 80 4 80 14 117 101 7 4 5 3 24 107 8 11

70 98 2 89 46 104 79 3 2 2 1 22 85 3 6

a EDTA, ethylenediaminetetracetic acid; PMSF, phenylmethylsulfonyl £uoride ; NEM, N-ethylmaleimide ; DTT, dithiothreitol. b Control taken as 100%.

at 70³C for 1 min completely and irreversibly inactivated the enzyme. P2 retained more than 80 and 50% of maximum activity after treatment at 65 and 70³C for 5 and 1 min, respectively. Treatment at 85³C for 1 min was necessary for complete inactivation (data not shown). As shown in Table 2, activities of P1 and P2 were strongly reduced by the serine proteinase inhibitor PMSF. The metal chelator EDTA had an high inhibitory e¡ect on P1 (at both 1 and 5 mM), but inhibited to a moderate extent, and only at 5 mM, P2 activity. Iodoacetic acid showed higher inhibitory e¡ect on both enzymes, especially P1, at concentration of 5 mM. Several divalent cations such as Zn2‡ , Hg2‡ , Ni2‡ , Cu2‡ and Co2‡ inhibited both the enzymes at 1 mM. At 5 mM, Fe2‡ and Mn2‡ also became strongly inhibitory. After treatment with 10 mM EDTA, no reactivation of the apoenzyme was achieved by incubation with any cations (data not shown). As determined by urea-PAGE, the activity of P1 and P2 was not inhibited, at pH 6.5 and 37³C, by concentrations of NaCl up to 5%, but was moder-

ately reduced in presence of 10% NaCl (data not shown).

4. Discussion Arthrobacter spp., together with B. linens, are the main microorganisms found on several bacterial smear and mould surface-ripened cheeses [6,9]. Several authors reported that growth of B. linens was essential for the development of the characteristic £avors, colors, aromas and textures of smear surface-ripened cheeses [6,16]. Despite their presence at high cell numbers in the smear, the role of the extracellular enzymes by Arthrobacter spp. has probably been underestimated with respect to B. linens. The two extracellular proteinases (P1 and P2) of A. nicotianae 9458 showed a rather similar substrate speci¢city on Na-caseinate, but di¡ered in several properties. P2 had an higher temperature optimum, 55^60³C, but had relatively low activity at 15³C. P1 had 40% of maximum activity at cheese ripening temperature (V15³C). Both proteinases had a similar pH optimum (9.0^9.5), but relative activity on £uorescent casein at pH 6.5 was 60 and 15% of the maximum activity for P1 and P2, respectively. However, urea-PAGE showed considerable hydrolysis of Na-caseinate by both enzymes at pH 6.0. Di¡erences in the structure of the two proteinases were con¢rmed by a di¡erent sensitivity to EDTA and to sulfhydryl blocking agents. The di¡erent behavior during puri¢cation on Phenyl-Sepharose also indicated the di¡erent degree of hydrophobicity of the two enzyme molecules. The endoserine protease secreted by A. aureus [10] isolated from soil di¡ered greatly from both proteinases of A. nicotianae 9458. It had a lower molecular mass (22 kDa), was active over a broad pH range but had an optimum at pH 7.0 and, although not stable at elevated temperatures, had an optimum at 70³C. Similar to P2, it was not a¡ected by EDTA or sulfhydryl blocking agents. Serine proteinases have been puri¢ed in several B. linens strains. Properties varied, perhaps because of wide inter-strain di¡erences. It has been reported that B. linens produced ¢ve extracellular serine proteinases (three of them with molecular masses ranging from 37 to 44 kDa), all of which had a high pH optimum, pH 11, and temperature

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optima of 40^55³C [2]. Rattray et al. [4] puri¢ed a dimeric 126-kDa extracellular proteinase from B. linens ATCC 9174 with pH and temperature optima of 8.5 and 50³C, respectively, which agreed with other partially puri¢ed enzymes from the same species [3]. Proteinases P1 and P2 from A. nicotianae 9458 had temperature optima of 37³C and 55^60³C, respectively, and had a slightly more alkaline pH optimum than the proteinases of B. linens. Proteinases from smear cheese micro£ora, such as coryneforms, micrococci and yeasts, generally show alkaline pH optima. Nevertheless, most of these enzymes have considerable activity at cheese pH of 6.0^7.0 which are still compatible with cell growth in the cheese surface [17]. The proteolytic action of various strains of B. linens during growth in media containing bovine casein has been described [18]; L-casein was hydrolyzed faster and to a greater degree than Ks1 -casein. Similarly, both P1 and P2 seemed to be more active on L- than on Ks1 -casein. Hydrolysis of L-casein by the proteinases of lactic acid bacteria and chymosin is very limited in cheese, probably because intermolecular hydrophobic interactions mask suitable cleavage sites [19]. Therefore the activity of Arthrobacter enzymes should be important. Tolerance to or activation by NaCl is a prerequisite for the activity of bacterial enzymes in the smear of the surface-ripened cheeses. The proteinases P1 and P2 of A. nicotianae 9458 and the proteinase of B. linens [4], are quite insensitive to NaCl. Mixed starter cultures from smear cheese micro£ora have been developed and Arthrobacter spp. speci¢cally showed a positive e¡ect in cheese proteolysis [20]. Our results show the secretion of two di¡erent proteinases by A. nicotianae 9458 which are relatively active on Ks1 - and especially on L-casein at the pH, temperature and NaCl concentration of cheese ripening. Further studies on cheesemaking are in progress in our laboratory to con¢rm the promising role of Arthrobacter spp. proteinases.

[3]

[4]

[5]

[6]

[7] [8]

[9]

[10]

[11]

[12] [13] [14]

[15]

[16]

[17]

[18]

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