2015 Volume 21 Issue 5 Pages 739-744
Protease from lactic acid bacteria is of great importance to flavor and texture quality of fermented foods. An acidic protease from Pediococcus pentosaceus 220 was purified to homogeneity with a 11.5-fold increase in specific activity and 13.4% of recovery by precipitation with ammonium sulfate (20 – 60%, w/v), DEAE-Sepharose CL-6B ionic exchange chromatography, and Sephadex G-75 gel filtration chromatography. The molecular weight of the purified protease was estimated to be 37 kDa by Sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The optimum pH and temperature for protease activities were around pH4.0 and 35°C, respectively. The enzyme was stable at 20 – 40°C and showed pH stability between 4.0 and 7.0. The protease was activated by Ca2+, but inhibited by Zn2+, Mg2+ and Fe3+. The enzyme activity was also strongly inhibited by Sodium dodecyl sulfate and EDTA. It could be deduced that the purified enzyme was an acidic metalloprotease.
Lactic acid bacteria (LAB) have been extensively used in manufacturing various fermented food. Proteolysis is one of the particular physiological trait of LAB and of great importance due to the contribution to the development of organoleptic properties of fermented food and production of bioactive health-beneficial peptides (Savijoki et al., 2006). The proteolytic system of LAB could cause degradation of both sarcoplasmic and myofibrillar proteins (Fadda et al., 2002). The proteolysis by LAB was thought to play an important role in the formation of unique flavor of fermented meat because the peptides and amino acids generated from proteolysis are the major precursors of specific flavor compounds (Hughes et al., 2002; Mcfeeters, 2004). In addition, the proteolytic system of LAB also affected the texture development of fermented fish (Riebroy and Benjakul, 2005).
Due to the contribution of protease produced by microorganism to the formation of texture and flavor of fermented food and its wide industrial application, a number of proteases from several different strains including bacteria and fungi were purified and characterized (Hsiao et al., 2014; Kumar et al., 2008; Kumar et al., 2005; Lee et al., 2010; Li et al., 2014; Mercado-Flores et al., 2003; Shankar et al., 2011; Silva et al., 2011). Pediococcus pentosaceus is kind of LAB widely existed in fermented meat and usually used as starter strains in manufacture of various fermented fish (Asiedu and Sanni, 2002; Glatman et al., 2000; Riebroy et al., 2008; Xu et al., 2010; Yin and Jiang, 2001). However, studies on P.pentosaceus mainly focus on purification and properties of antibacterial compounds such as bacteriocin (Todorov and Dicks, 2009) an pediocin (Todorov and Dicks, 2005). Furthermore, the previous reported tripeptidase and aminopeptidase from P.pentosaceus were intracellular protease (Simitsopoulou et al., 1997; Vafopoulou et al., 1999). To our knowledge, although the proteolytic system of lactic acid bacteria has received special interest, the studies on purification and biochemical properties of an extracellular acidic protease produced from P.pentosaceus have not been reported.
Therefore, the objective of present study was to purify and biochemically characterize of an extracellular protease from P.pentosaceus 220 isolated from fermented fish, providing information to further improve its application as starter culture in meats, and also able to better understand the role of P.pentosaceus in the development of texture and flavor of fermented fish.
Materials P.pentosaceus 220 isolated from traditional fermented fish was provided by School of Food Science and Technology in Jiangnan University.
Preparation of crude enzyme extract P.pentosaceus 220 was inoculated into liquid MRS broth and incubated at 33°C for 20 h with shaking at 100 rpm. The obtained broth was centrifuged at 10000×g for 15 min at 4°C (Sigma Laborzentrifugen, Model 4K15, Osterode,Germany), and the supernatant was collected as crude extracellular enzyme and used for further purification.
Purification of protease Ammonium sulfate was added to the culture supernatant at concentration of 20% to 60% (w/v).The crude enzyme after ammonium sulfate fractionation was loaded onto a DEAE-Sepharose Fast Flow ionic exchange column, previously equilibrated with 50 mmol/L Tris-HCl buffer (pH6.5). The sample was washed onto the column with the same eluting buffer, followed by a linear gradient with 50 mmol/L Tris-HCl buffer (pH6.5) containing 0 to 0.45 mol/L NaCl at a flow rate of 1.0 mL/min. The active fractions pooled from DEAE-Sepharose Fast Flow column was then subjected to gel filtration on a Sephadex G-75 column pre-equilibrated with 50 mmol/L phosphate buffer (pH 6.5) containing 0.1 mol/L NaCl, and eluted with the same buffer at a flow rate of 0.4 mL/min. Each 3 mL of eluted fractions were collected to determine protease activity at pH4.0. Fractions showing high protease activity were pooled and concentrated.
Protease activity assay Protease activity was determined using casein as substrate at 40°C according to the method of Bhaskar et al. (2007). One unit of proteolytic activity (U) was defined as µg tyrosine liberated per min of the enzyme extract. Specific activity was expressed as units per mg protein (U mg−1 protein) of the enzyme extract.
Determination of protein content Protein content was determined by the microbiuret method (Itzhaki and Gill, 1964) using bovine serum albumin (BSA) as a standard. During column chromatography, protein concentration in the fractions was monitored by measuring the absorbance at 280 nm.
Determination of molecular weight Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out in a vertical gel electrophoresis unit (Mini-Protean-3 Cell. Bio- Rad, Richmond, CA, USA) as described by Xu et al. (2010) using 12% resolving gel and 4% stacking gel. Standard molecular weight makers (Sigma, USA) were used to estimate the molecular weight of the purified enzyme.
pH optima and pH stability of protease The optimum pH of protease activity was determined over the pH range of 3.0 to 6.5 in various buffer solutions (Citric acid-sodium citrate buffer of pH 3.0 – 5.5 and phosphate buffer of pH 6.0 – 6.5) at 40°C using casein as a substrate. Relative activity of protease was calculated considering 100% for the maximum activity detected within the pH range.
For the determination of pH stability, the protease was incubated at 35°C for 60 min in different buffer systems (Citric acid-sodium citrate buffer of pH 3.0 – 5.0, phosphate buffer of pH 6 – 8, glycine-NaOH buffer of pH 9.0 – 10.0) at pH3.0 – 10.0. The residual activity was assayed at pH 4.0 and 40°C using casein as substrate. The activity of protease before incubation was taken as 100%.
Temperature optima and thermal stability of protease The optimum temperature of protease activity was determined at different temperature ranging from 20°C to 45°C at pH4.0 using casein as a substrate. Relative activity of protease was calculated considering 100% for the maximum activity detected within the temperature range.
For the determination of thermal stability, the protease in phosphate buffer (pH 6.5) was incubated at temperatures ranging from 20 to 80°C for 60 min. The residual activity was assayed at pH 4.0 and 40°C using casein as substrate. The activity of protease without thermal-treatment was considered as control and expressed as 100% activity.
Effect of metal ions on protease activity Enzymes were incubated with various metal ions (Co2+, Mn2+, Ca2+, Zn2+, Fe3+, Mg2+, K+) using CoCl2, MnSO4, CaCl2, ZnSO4, FeCl3, MgSO4, and KCl at 40°C for 10 min and the remaining activity was measured. A control assay was incubated using distilled water instead of a metal ion solution. The protease activity in the absence of any metal ions was taken as control and expressed as 100% activity.
Effect of enzyme inhibitors on protease activity The effects of inhibitors on enzyme activity were determined using PMSF, iodoacetic acid (IAA), dithiothreitol (DTT), EDTA and SDS. The enzyme was incubated with each inhibitor or additive for 10 min at 40°C, and then remaining activity was measured. The protease activity in the absence of any inhibitor was taken as control and expressed as 100% activity.
Purification of protease Protease produced from P.pentosaceus 220 was purified successively by three steps. Firstly, the crude protease extract was fractionated with ammonium sulfate. The fraction precipitated with 20 – 60% saturation of ammonium sulfate showed high protease activity (data not shown) and was collected. The dialysate of the fraction (20 – 60% saturation) was then subjected to DEAE Sepharose Fast - Flow ion exchange chromatography. As shown in Fig.1, four protein peaks were observed, and most of the enzyme activity was found in the fourth peak, which is the fraction between No. 30 and 45 tube eluted. The active fraction with higher protease activity was pooled and further purified on Sephadex G-75. As shown in Fig.2, two peaks with protein content were observed after separation through Sephadex G-75 but only one single peak with protease activity was obtained. The active peak fraction was collected as purified enzyme. The final purified protease was purified by 11.5 fold with a recovery of 13.4% (Data not shown).
Purification profile of protease from P.pentosaceus 220 on DEAE-Sepharose Fast Flow column.
Purification profile of protease from P.pentosaceus 220 on Sephadex G-75 column.
Determination of the molecular weight The molecular weight of protease from P.pentosaceus 220 was determined using SDS-PAGE. As shown in Fig.3, a single band of the purified enzyme was observed on SDS-PAGE, indicating the homogeneity of the purified enzyme. The molecular weight of the enzyme was calculated to be 37 kDa on the mobility of the bands on SDS-PAGE using a standard curve established with proteins of known molecular weight. The molecular weight of the purified protease from P.pentosaceus 220 was lower than those from Lactococcus lactis ssp. Lactis LB12 (53 kDa) (Guo et al., 2009), Bacillus subtilis (44 kDa) (Yang et al., 2000), Synergistes sp.(60 kDa) (Kumar et al., 2008), Monascus purpureus CCRC31499 (40 kDa) (Liang et al., 2006), but higher than those produced by Bacillus proteolysicus CFR3001 (29 kDa) (Bhaskar et al., 2007), Aspergillus clavatus (30.4 kDa) and Aspergillus oryzae LK-101 (26 kDa) (Lee et al., 2010).
SDS-PAGE pattern of purified protease from P.pentosaceus 220. Land M, Molecular weight makers; Land S, Sample of purified protease.
Effect of pH on the protease activity and stability As shown in Fig.4A, protease from P.pentosaceus 220 exhibited high activity in the range of pH 3.0 – 4.5, and the highest protease activity was found at pH 4.0. Protease activity decreased sharply with increasing pH from 4 to 6.5, and almost no activity was detected at pH 6.5. This clearly indicated that the protease produced by P.pentosaceus 220 was an acidic protease with an optimum pH of around 4.0. The optimum pH of protease from P.pentosaceus 220 was lower than those acidic protease from Synergistes sp. (pH 5.5 – 6.5) (Kumar et al., 2008)), Rhizopus oryzae (pH5.5) (Kumar et al., 2005), Aspergillus clavatus (pH5.5)(Silva et al., 2011), Aspergillus oryzae LK-101 (pH6.5)(Lee et al., 2010), and was close to protease from Aspergillus niger (pH3.5)(Li et al., 2014) and Ustilago maydis (pH4.0) (Mercado-Flores et al., 2003).
pH profile (A) and pH stability (B) of protease from P.pentosaceus 220.
pH stability of protease from P.pentosaceus 220 was shown in Fig.4B. The P.pentosaceus 220 protease is highly stable over acidic pH range, maintaining more than 90% of its original activity in the pH range of 4.0 – 7.0. However, the protease was unstable at pH over 7.0. The residual protease activity decreased sharply with increasing pH from 7.0 to 10.0, and reached its lowest residual activity of 16.7% at pH10. The pH stability of acidic protease from P.pentosaceus 220 was similar to some other acidic protease produced by microorganisms (Kumar et al., 2005; Lee et al., 2010; Li et al., 2014; Silva et al., 2011). The high protease activity and stability in acidic conditions suggested that the protease produced during fermentation contribute to the proteolysis of fish protein.
Effect of temperature on the protease activity and stability Fig.5A illustrated the influence of different temperatures on the P.pentosaceus 220 protease activity. The relative activity increased with increasing temperature from 20°C to 35°C, reaching the relative activity of 100% and 92.5% at 35°C and 40°C, respectively. When the temperature over 40°C, the relative activity declined rapidly and the relative activity was only 59.1% at 45°C. The results showed the optimum temperature of protease from P.pentosaceus 220 was around 35°C. It was in line with several other reports showing that the optimum temperature of acidic protease from bacteria and fungi was in the range of 35 – 40°C (Kumar et al., 2008; Li et al., 2014). But the optimum temperature of acidic protease from P.pentosaceus 220 was lower than that from Aspergillus clavatus (50°C) (Silva et al., 2011) and Aspergillus oryzae LK-101 (50°C) (Lee et al., 2010), and Rhizopus oryzae (60°C) (Kumar et al., 2005).
Temperature profile (A) and thermal stability (B) of protease from P.pentosaceus 220.
As shown in Fig.5B, P.pentosaceus 220 protease was highly stable at temperature range of 20 – 40°C for 60 min of incubation, maintaining more than 92% of its initial activity. But the protease was labile when the temperature exceeding 40°C, and the residual activity was 43.8% and 11.3% after 60 min of incubation at 60°C and 70°C, respectively. It is noted that no activity was detected after incubation at 80°C. The acidic protease from Aspergillus oryzae LK-101 (Lee et al., 2010), Ustilago maydis (Mercado-Flores et al., 2003) and Rhizopus oryzae (Kumar et al., 2005) also displayed higher enzyme activity after incubation at relative low temperature range of 20 – 40°C. The high activity and stability of P.pentosaceus 220 protease at 20 – 40°C suggested that the protease produced during fermentation may participated the proteolysis during fermentation of fish.
Effect of metal ions on protease activity The effect of some metal ions on the activity of protease from P.pentosaceus 220 was shown in Table 1. P.pentosaceus 220 protease was slightly activated by Co2+, Ca2+ and K+ at concentrations of 1 mmol/L and 10 mmol/L. Ca2+ showed obvious enhancement effect at high concentration of 10 mmol/L compared to that of 1 mmol/L. In addition, P.pentosaceus 220 protease was strongly inhibited by Zn2+ and Fe3+ while slightly inhibited by Mg2+ and Mn2+. It was noted that increasing concentration of metal ions could enhanced the inhibitory effect to different extent depended on metal ions. Zn2+ could decreased the protease activity from 69.2% to 23.5% when Zn2+ concentration increased from 1 mmol/L to 10 mmol/L. Metal ions exhibit different influence on activity of protease produced by different microorganisms (Hsiao et al., 2014; Lee et al., 2010; Li et al., 2014; Mnif et al., 2015; Silva et al., 2011).
Effect of inhibitors on protease activity As shown in Table 2, the proteolytic activity of protease from P.pentosaceus 220 was highly inhibited by the chelating agent of EDTA and surfactants of SDS. It suggested that metal ions may be needed for stabilizing the active structure and catalytic role of protease. This was confirmed by the result of higher activated activity in the presence of some metal ions (Table.2). In contrast, there was negligible inhibitory effect of PMSF (serine protease inhibitor) and DTT on protease activity whereas only a small decrease in residual activity in presence of cysteine protease inhibitor of iodoacetic acid. These results indicated that P.pentosaceus 220 protease was a metalloprotease but not a serine protease and cysteine protease. The strong inhibitory effect of EDTA on an acid protease from Aspergillus oryzae LK-101 was also reported (Lee et al., 2010). However, other acidic proteases from Aspergillus oryzae (Li et al., 2014), Synergistes sp.(Kumar et al., 2008), Ustilago maydis (Mercado-Flores et al., 2003) were not sensitive to chelating agent of EDTA.
In this study, an extracellular acidic protease from P.pentosaceus 220 was purified to homogeneity with a 11.5-fold of increase in specific activity and 13.4% of recovery. The enzyme showed high proteolytic activity and high stability at acidic condition and at relative low temperature of 20 – 40°C. It was revealed that the enzyme was a metal-chelator-sensitive acidic protease. The biochemical properties of the protease indicated that the protease produced by P.pentosaceus 220 during fermentation may participated the degradation of protein and contribute to the flavor formation and textural development of fermented fish, and it has potential for application in food processing at acidic conditions to improve quality of food.
Acknowledgements This research was financially supported by the earmarked fund for China Agriculture Research System (CARS-46), BE2013336 and NSF 31171709.