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J. Dairy Sci. 87:4097-4103
© American Dairy Science Association, 2004.

Purification and Characterization of Intracellular Proteinase from Lactobacillus casei ssp. casei LLG

J. Y. Shin1,*, W. M. Jeon2, G.-B. Kim1 and B. H. Lee1,3

1 Department of Food Science and Agricultural Chemistry, McGill University, 21,111 Lakeshore Road, Ste-Anne-de-Bellevue, Quebec Canada H9X 3V9
2 Department of Applied Animal Science, Sahmyook University, Seoul, Korea
3 Food Research & Development Centre, Agriculture and Agri-Food Canada, St-Hyacinthe, Quebec, Canada J2S 8E3

Corresponding author: B. H. Lee; e-mail: byong.lee{at}mcgill.ca.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The intracellular proteinase of Lactobacillus casei ssp. casei LLG was isolated in the cytoplasmic fraction with 0.05 M Tris-HCl buffer (pH 7.5). The enzyme was purified by the fast protein liquid chromatography system equipped with ion-exchange and gel filtration chromatographies. This proteinase comprised a single monomeric form and had a molecular weight of about 55 kDa and an isoelectric point near pH 4.9. The optimum pH and temperature for the enzyme activity were determined to be pH 6.5 and 37°C, respectively. The enzyme was inactivated by metal-chelating compounds (EDTA, 1,10-phenanthroline) and less affected by serine proteinase inhibitors (diisopropylfluorophosphate, phenylmethylsulfonyl fluoride). Proteinase activity was increased by Ca++, Mn++, and Co++, and inhibited by Cu++, Mg++, and Zn++. The activity of this enzyme to hydrolyze casein appeared to be more active on ß-casein than {alpha}s1-casein and {kappa}-casein as monitored by polyacrylamide gel electrophoresis.

Key Words: Lactobacillus casei • proteinase • purification

Abbreviation key: LAB = lactic acid bacteria, NSLAB = nonstarter LAB


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In Cheddar cheese, the starter Lactococcus cells normally reach about 109 cfu/g after salting stage. In our previous study and others (Lee et al., 1990a; Trépanier et al., 1992a, 1992b; Demarigny et al., 1996; 1990b; Roy et al., 1997; Beresford et al., 2001), the nonstarter lactic acid bacteria (NSLAB) population that was present at low concentrations (<103/g) was increased to about 108–9cfu/g after about 3 to 4 mo. The NSLAB was always predominated by the Lactobacillus casei group (Lb. casei/Lactobacillus paracasei/Lactobacillus rhamnosus) in good quality of mature Cheddar, and they were judged to improve cheese flavor, but their contribution to cheese flavor and their origins are still unclear. It appears to be a general trend that Lb. casei group predominated in later stage of ripening, regardless of cheese types (in particular for semi- and hard type), and this flora is unlikely unique to each plant and each country (Lee et al., 1990a, 1990b; Linberg et al., 1996; Beresford et al., 2001).

Considering the very hostile environment of low temperature (5 to 6°C), redox potential, water activity, pH, and lactose, but high salt, it is intriguing how Lb. casei grows to such large numbers in cheese. Their energy sources and growth factors have been much debated. Lactobacillus casei seems to grow very well in starter cell debris, and their products released by autolysis (Thomas, 1987), peptides (Nath et al., 1973), and glyco-proteins of milk fat (Williams and Banks, 1997). It has also been shown that NSLAB grow fast in Cheddar cheese (with a generation time of 8.5 d at 6°C), thus low levels of contamination will result in their dominance of total cheese flora (Jordan and Cogan, 1993). Randomly amplified polymorphic DNA was also used to detect the concentrations of NSLAB in Cheddar cheese, confirming the predominance of Lb. paracasei type after 4 to 8 wk of ripening (Fitzsimons et al., 1999).

Several studies have thus been carried out to accelerate Cheddar cheese with selected adjunct live Lb. casei or Lactococcus lactis cultures or shocked/lysis cultures (El Abboudi et al., 1991; Trépanier et al., 1992a, 1992b; McSweeney et al., 1994; Buist et al., 1998; Kang et al., 1998; Madkor et al., 2000).

During cheese ripening, evidence of autolysis and lysis of Lb. helveticus and Lb. casei also occurred naturally by the release of the intracellular enzymes (Dako et al., 1995; Kang et al., 1998; Valence et al., 1998). Thus proteolysis in cheese through both cell envelope-associated proteinase (PrtP or PrtH) and intracellular proteinase, along with more than 10 peptidases (PepN, PepX, PepO, PepQ, PepW, etc.), play a role in development of the desired texture, aroma, and flavor during cheese ripening (Courtin et al., 2002).

The cell-wall-associated proteinases have been reported from Lactococcus lactis ssp. cremoris AC1 (Geis et al., 1985), Lactobacillus delbrueckii ssp. bulgaricus (Kawai et al., 1999), Lactococcus lactis (Buis et al., 1998), and Lactobacillus casei (El Soda et al., 1986; Ezzat et al., 1988; Dhingra and Dutta, 2000). The cell-envelope proteinase genes have also been sequenced from Lactococcus lactis, Lactobacillus paracasei, Lactobacillus helveticus, Lactobacillus delbrueckii, Streptococcus pyogenes, and Streptococcus agalactiae (Siezen, 1999). However, the data on the intracellular proteinases from lactic acid bacteria (LAB) are very limited (Ohmiya and Sato, 1975; Muset et al., 1989; Akuzawa and Okitani, 1995), and their functions are not well known.

Lactobacillus casei ssp. casei LLG expresses a high level of intracellular proteinases and aminopetidases (Arora and Lee, 1994; Habibi-Najafi and Lee, 1994), the combined activity of these enzymes results in the production of cheese flavor compounds (Trépanier et al., 1992a, 1992b; Arora and Lee, 1994; Park et al., 1994; Arora et al., 1995; Christensen et al., 1999). Therefore, purification of the enzyme from LAB and characterization of their properties are considered to be indispensable for elucidation of the mechanisms involved in the processes of texture and flavor formation during cheese ripening and preparation of enzyme-modified cheese (Park et al., 1995; Habibi-Najafi and Lee, 1996).

The intracellular proteinase from Lb. casei ssp. casei LLG was thus purified to determine the specificity on the caseins and clarify the role of this enzyme for the production of accelerated-ripened cheese and enzyme-modified cheese.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Materials
Azocasein, lysozyme from chicken egg white, and mutanolysin from Streptococcus globisporus ATCC 21553 were from Sigma Chemical (St. Louis, MO). Gels, stain dye, and molecular weight markers for the electrophoresis were supplied by Pharmacia (Baie d’Urfe, QC, Canada). All other chemicals were of analytical grade. Ultrafiltration membranes (Centriprep-30) were from Amicon (Beverly, MA). Chromatographic and PD-10 columns were obtained from Pharmacia.

Microorganism and Culture Conditions
Lactobacillus casei ssp. casei LLG used in this study was isolated from a mature Cheddar cheese and tentatively identified by API 50 CHL (Merieux, St-Laurent, QC, Canada). Identification was further achieved by genetic techniques with RFLP and single-strand conformation polymorphism using PCR to amplify specific sequences of aminopeptidase (PepN) genes of Lactobacillus rhamnosus according to our previous method (Lee and Robert, 1997; Belanger, 1998).

Stock culture was maintained in sterile 10% (wt/vol) skim milk (Difco, Detroit, MI), and working culture was revived from frozen culture by 3 consecutive transfers in the same skim milk.

Preparation of Cell-Free Extract
The washed cells were resuspended in appropriate amount of 0.05 M Tris-HCl buffer (pH 7.5) and then disrupted by 3 passages through a French press cell at 18,000 psi (1 psi = 703.0696 kg/m2). The enzyme extract was centrifuged (15,000 x g, 35 min, 4°C) to eliminate cell debris, and the supernatant fraction was used as the intracellular crude enzyme.

Proteinase Assay
The proteinase activity conducted in replicate assays was measured using the chromogenic substrate, azocasein, according to the modified method of Beynon (1992). A portion (100 µL) of 2% (wt/vol) azocasein in 50 mM sodium phosphate buffer (pH 7.0) was mixed with 100 µL of enzyme in an Eppendorf tube. The mixture was incubated at 37°C for 16 h, as the enzyme activity was linear over a 16-h assay. The reaction was terminated by the addition of 500 µL of 12% (wt/vol) TCA solution and mixed thoroughly. After 15 min at room temperature, the mixture was centrifuged at 12,000 x g for 5 min. After 500 µL of supernatant was added to 500 µL of 1.0 M NaOH, the absorbance was measured at 440 nm against a blank without enzyme. One unit of proteinase activity was defined as the amount of the enzyme that results in an increase of 0.01 absorbance per hour at 440 nm.

Protein Determination
Protein content conducted in replicate assays was determined according to Smith et al. (1985). The bicin-choninic acid protein assay reagent supplied with the system (Pierce Chemical Ltd., IL) was used, with BSA as the standard. The protein concentrations in the fractions from the column were measured at 280 nm.

Purification of Enzyme
The active enzyme fractions during the purification were stored at 4°C in the presence of stabilizers (10% glycerol, 100 mM (NH4)2SO4, 1 mM dithiothreitol). The intracellular crude enzyme was fractionated by salting out with ammonium sulfate (40 to 80%) and centrifuged at 10,000 x g for 20 min at 4°C. The precipitate was dissolved in 0.02 M Tris-HCl buffer (pH 8.0), and desalted by Sephadex G-25 gel filtration (PD-10 column, Pharmacia) previously equilibrated with the same buffer. A Pharmacia fast protein liquid chromatography system was used for the purification.

First ion-exchange chromatography.
The desalted crude extract was applied to a preparative Mono Q (HR 16/10) column equilibrated with 0.05 M Tris-HCl buffer (pH 8.0). The enzyme was eluted with a stepwise gradient of NaCl (0 to 1.0 M) in the same buffer at a flow rate of 3 mL/min. Fractions (4 mL) were collected and tested for the activity. Fractions with the highest enzyme activities were pooled, concentrated by ultrafiltration using a Centriprep-30 membrane, and desalted.

Second ion-exchange chromatography.
The concentrated enzyme fraction was applied to the second ion-exchange (Mono Q HR 5/5) column equilibrated with 0.02 M bis-Tris-HCl buffer (pH 6.4). The enzyme was eluted with a stepwise gradient of NaCl (0 to 1.0 M) in the same buffer at a flow rate of 0.75 mL/min, and 1.5-mL fractions were collected. The active fractions were pooled, concentrated by ultrafiltration, and desalted.

Chromatofocusing.
The active fraction was applied to a chromatofocusing Mono P column (20 cm x 0.5 cm) after equilibration of the column with 20 mL of 0.02 M bis-Tris-HCl buffer (pH 6.0). The proteins were eluted with a linear pH gradient from 6.0 to 4.0 at a flow rate of 0.5 mL/min. The pH gradient was generated with a mixture of ampholytes (Polybuffer 7 to 4, Pharmacia), previously adjusted to pH 4.0 with 6 M HCl. The pH value of the fractions was immediately adjusted to near neutral pH with 0.2 M Tris-HCl buffer (pH 7.5) to minimize loss of enzyme activity. The polybuffer was removed from the active fractions by passage through a PD-10 column. The enzyme was concentrated using a Centricon-30 membrane.

Gel filtration chromatograpy.
The enzyme was subjected to a gel filtration (Superose-12 HR 10/30) column equilibrated with 0.05 M Tris-HCl buffer (pH 7.5). Elution was performed at a flow rate of 0.5 mL/min, and 1-mL fractions were collected. The active fractions were pooled and kept frozen at –40°C for further studies.

Hydrolysis of Caseins
Reaction mixture consisted of 4 volumes of different caseins ({alpha}s1-, ß-, and {kappa}-CN) in 0.05 M phosphate buffer, pH 7.0, and 1 volume of enzyme solution and incubated at 37°C. Samples (10 µL) were taken at 0, 0.5, 2, 6, and 12 h. The protein samples were diluted (4:1) in sample buffer containing 62.5 mM Tris-HCl, pH 6.8, 2% (wt/vol) SDS, 10% (vol/vol) glycerol, 0.005% (wt/vol) bromo-phenol blue, 5% (vol/vol) 2-ß-mercaptoethanol, and heated for 4 min at 95°C before applying to the gels.

Polyacrylamide Gel Electrophoresis
The purity of the enzyme preparation was examined by native and SDS-PAGE using the Phast System (Pharmacia). The SDS-PAGE was performed according to the method of Laemmli (1970). The molecular weight of the subunit of the purified enzyme was determined by SDS-PAGE using 12.5% homogeneous PhastGel. The protein samples were mixed 1:1 with sample buffer (20 mM Tris-HCl, pH 8.0) containing 2 mM EDTA, 5% SDS, 10% ß-mercaptoethanol and 0.001% (wt/vol) bromophenol blue, boiled for 4 min and applied to the gels. The low molecular weight proteins were used as standards for the predicting of molecular mass of the pure protein. The gels were stained with Coomassie blue-R 250.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Purification of Enzyme
Cell-free extracts obtained from the cytoplasmic fraction of Lb. casei ssp. casei LLG were subjected to ion-exchange (Mono Q HR 16/10 and Mono Q HR 5/5) columns, chromatofocusing (Mono P), and finally gel filtration column (Suprose 12 HR 10/30). The elution profiles during purification are shown in Figure 1Go.



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  		Figure 1. Purification of the intracellular proteinase from Lactobacillus casei ssp. casei LLG, protein concentration ( — ), enzyme activity (•••), and NaCl or pH gradient (---). A) Mono Q HR 16/10, B) Mono Q HR 5/5, C) Mono P, and D) Superose 12 HR 10/30.

 
The fractions eluted at 0.10 to 0.12 M NaCl gradient of Mono Q HR 16/10 ion-exchange chromatography were subjected to Mono Q HR 5/5 ion-exchange column. Then the fractions eluted at 0.16 to 0.18 M NaCl gradient of Mono Q 5/5 column were subjected to Mono P isoelectrofocusing column. The fractions eluted at 4.4 to 4.9 pH gradient of Mono P column were subjected again to Superose 12 HR 10/30 gel filtration column. The scheme of purification is summarized in Table 1Go. The enzyme was purified through 4 consecutive purification steps and appeared as a single band on SDS-PAGE (Figure 2Go). The molecular weight of enzyme was estimated to be 55 kDa by SDS-PAGE and gel filtration.


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  		Table 1. Summary of purification steps of proteinase from Lactobacillus casei ssp. casei LLG.
 


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  		Figure 2. SDS-PAGE of the purified intracellular proteinase of Lactobacillus casei ssp. casei LLG on 12.5 % polyacrylamide gel. Lane 1: molecular weight marker, Lane 2: purified enzyme.

 
Effects of pH and Temperature
The effects of pH and temperature on the proteinase activity of purified enzyme were determined with azocasein. The appropriate amount of enzyme and substrate prepared in 0.05 M acetate (pH 5.5 and 6.0), 0.05 M sodium phosphate (pH 6.5 to 7.0), 0.05 M Tris-HCl (pH 7.5 to 8.0) buffers were incubated at 37°C, and the activity was measured. The appropriate amount of enzyme and substrate prepared in 0.05 M phosphate buffer (pH 6.5) were incubated at different temperatures (25 to 55°C), and the activity was measured. The optimal pH was about 6.5, but at the pH less than 6.0 and higher than 7.5, the enzyme activity was sharply decreased (Figure 3Go). The optimal temperature of the purified enzyme was about 37°C (Figure 4Go). The relative activity at 25°C was less than 45%, and about 20% of the activity was retained at 55°C.



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  		Figure 3. Effect of pH on the activity of intracellular proteinase from Lactobacillus casei ssp. casei LLG.

 


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  		Figure 4. Effect of temperature on the activity of intracellular proteinase from Lactobacillus casei ssp. casei LLG.

 
Effect of Divalent Cations and Inhibitors
The effect of divalent cations on the proteinase activity of purified enzyme was determined by preincubation with a final concentration of 1 mM of cations in 0.05 M sodium phosphate buffer (pH 6.5) for 30 min at 37°C before addition of substrate. The effect of inhibitors on the activity of purified enzyme was determined by preincubation with a final concentration of 0.1, 1.0, and 10 mM of inhibitors for 30 min at 37°C before addition of substrate. Inhibition was expressed as a percentage of the activity without effector (control). The enzyme activity was strongly inhibited by Cu++, Mg++, and Zn++ at 1.0 mM, but Ca++, Co++, and Mn++ had no inhibitory effect. The activity was almost inhibited after the proteinase was treated with EDTA and 1,10-phenanthroline, but not strongly inhibited by phenylmethylsulfonylfluoride and diisopropylfluorophosphate treatment. The sulfhydryl group reagents, iodoacetic acid, and iodoacetamide had no significant effect on the proteinase activity (Table 2Go).


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  		Table 2. Effect of divalent cations and inhibitors on the proteinase activity.
 
Hydrolysis of Caseins
The actions of intracellular proteinase of Lactobacillus casei ssp. casei LLG on {alpha}s1-CN, ß-CN, and {kappa}-CN were observed by SDS-PAGE. This enzyme hydrolyzed all caseins but seemed to be more active on ß-CN than {alpha}s1-CN and {kappa}-CN (Figure 5Go). In particular, ß-CN was almost completely hydrolyzed by incubation for 12 h with this intracellular proteinase.



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  		Figure 5. SDS-PAGE on the action of intracellular proteinase from Lactobacillus casei ssp. casei LLG on {alpha}s1-, ß-, and {kappa}-CN.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The proteolytic system (peptidases and proteinases) of lactic acid bacteria used in cheese making plays an important role in the texture and flavor of the cheese, and it is well known that Lactococcus species are the most important starter used in cheese manufacture (Beresford et al., 2001). Despite the extensive works on the role of cell-envelope associated proteinase, the data on intracellular proteinase from lactic acid bacteria are limited.

The intracellular proteinase Lb. casei ssp. casei LLG after several steps of purification showed that the purified enzyme is a monomeric enzyme with the apparent molecular mass of 55,000 as estimated by SDS-PAGE. Intracellular proteinases from other lactic acid bacteria were reported that Lc. lactis ssp. cremoris had a molecular mass of 140 kDa (Ohmiya and Sato, 1975), and Lc. lactis ssp. lactis NCDO 763 (Muset et al., 1989), and IAM 1198 (Akuzawa and Okitani, 1995) had a molecular mass of about 93 and 12 kDa, respectively. The sizes of intracellular proteinase from LAB appeared to be different among strains, and their function may also be different in cell. Maximum activity of intracellular proteinase from Lb. casei ssp. casei LLG was obtained at 37°C and pH 6.5. The enzyme properties were pH 6.5 to 7.0 and 30°C for Lc. lactis ssp. cremoris (Ohmiya and Sato, 1975), pH 7.5 and 45°C for Lc. lactis ssp. lactis NCDO 763 (Muset et al., 1989), pH 5.5 to 6.0 and 30°C for Lc. lactis ssp. lactis IAM 1198 (Akuzawa and Okitani, 1995).

The enzyme was activated by Ca++, Mn++, and Co++, but inhibited by Cu++, Mg++, and Zn++ that are in agreement with those of Lc. lactis ssp. cremoris (Ohmiya and Sato, 1975). This enzyme was also inactivated by metal chelating compounds (EDTA, 1,10-phenanthroline) and less affected by serine proteinase inhibitors (diisopropylfluorophosphate, phenylmethylsulfonylfluoride) and SH-groups reagents (iodoacetamide, iodoacetic acid). These results indicate that this proteianse is a metalloenzyme characteristic, which agrees with those of Lc. lactis ssp. cremoris (Ohmiya and Sato, 1975) and Lc. lactis ssp. lactis NCDO (Muset et al., 1989) but does not agree with that of Lc. lactis ssp. lactis IAM 1198 (Akuzawa and Okitani, 1995).

Lactobacillus spp. are the major portion of the NSLAB in some cheeses. During ripening of Cheddar cheese, the intracellular proteolytic enzymes of Lactobacillus casei appeared to play an important role in protein breakdown after the death and lysis of the cells. Lactobacillus casei has the peptidolytic, esterolytic, and proteolytic activities (Roy et al., 1997), thus the use of Lactobacillus as adjunct cultures has gained much attention recently in the production of accelerated ripened cheese and enzyme-modified cheese (Arora et al., 1995; Park et al., 1995; Habibi-Najafi and Lee, 1996).

This enzyme hydrolyzed all milk casein fractions, but hydrolyzed ß-CN more preferentially. On the basis of degradation patterns of {alpha}s1-, ß- and {kappa}-CN, 2 proteinase specificity classes may be possible as in Lactococci, but their comparisons are meaningless as they are unrelated enzymes. This proteinase probably involves later stage in the proteolysis of cheese curd after cell lysis. Intracellular proteinases are probably important in the turnover of denatured protein, activation of zymogens, and termination of newly synthesized proteins (Muset et al., 1989).


   FOOTNOTES
 
* Current address: Dept. of Food and Biotechnology, Chungcheong University, Chungpuk, Korea. Back

Received for publication February 15, 2002. Accepted for publication July 22, 2003.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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