Purification and characterization of thermophilic alkaline protease

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A bacterium producing thermophilic alkalophilic protease was purified using ammonium sulphate precipitation, dialysis and gel filtration chromatography techniques with 6.8 fold purification and 24% recovery. Molecular weight of the protease was found to be 32 KDa using SDS-PAGE. It exhibited a broad range of pH (5.0 to 12.0) and temperature (30 to 90ËšC) for its activity but the maximal optima were pH 10.0 and temperature and 65ËšC. PMSF completely inhibited the activity which indicated it as serine type protease. Ca2+, Mg2+, Mn2+ and K+ found to increase the activity. This enzyme could hydrolyze a variety of protein substrates. azocasein, BSA, gelatin and egg albumin. Kinetic studies using casein as a substrate followed Michaelis - menten type with 0.55 mg/ml of Km and 1428 µmol min-1 of Vmax, which shows high affinity and catalytic property of the protease enzyme. The RV.B2.90 protease also exhibited remarkable stability a factor that makes it as potential candidate for wide industrial applications.

Keywords: Bacillus cereus, alkalophilic, thermophilic, purification, characterization

Microbial proteases are one of the major groups of enzymes forming 65% of enzyme market approximately. Among proteases, alkaline proteases have drawn much interest of scientists due to its tremendous application in industrial sector since its discovery in 19141. Even though a lot of proteolytic enzymes have been identified and characterized, enzymes with high alkaline and thermo stability are still in demand. For Bioengineering and Biotechnological applications enzymes with higher stability and activity are of much interest. Many microbes like Bacteria, moulds and yeasts are involved in protease production. Bacillus is the major alkaline protease producing organism for commercial applications2. Alkaline proteases are active over a wide range of high pH with a metal in the active site or a serine residue. Serine proteases are the most important group of alkaline protease used in industry.3

An alkaline thermophilic serine protease producing Bacillus cereus RV.B2.90 (Genbank accession number HQ197382) was identified and characterized previously by Vijayalakshmi et al4. It is essential to purify and characterize a given enzyme in detail for potential application. This paper describes the purification and characterization of an alkalophilic, thermophilic protease from Bacillus cereus RV.B2.90.

Materials and Methods

Microorganism­ used and production media -The Bacillus sp used was isolated from soil sample collected from Kerala state, India. The isolate was sub cultured on nutrient agar slopes containing 0.1% casein and preserved at 4ËšC. The production medium was composed of (g/l): peptone, 5.0; yeast extract, 1; sodium chloride 0.5; glucose 1; KH2PO4, 1; K2HPO4, 1; MgSO4, 0.5; pH 8.0 and grown in 250 ml Erlenmeyer flask with a working volume of 100 ml. incubated for 24 hrs under static condition at 37ËšC.

Partial purification of the protease - The broth was centrifuged at 15000 rpm for 20 min at 4ËšC. The cell free extract was used for partial purification and characterization of enzyme. All the experiments were carried out under cold conditions (4ËšC). A 0.5 ml of culture supernatant was added to 2 ml of Tris - Hcl pH 8.0 containing 2.0% (w/v) casein solution, incubated at 37ËšC for 10 min, undigested protein was removed from the reaction by adding 2.5 ml of 0.1M trichloroacetic acid (TCA) and incubated for 30 min followed by centrifugation at 10,000 rpm for 10 min at 4ËšC. To 2.5 ml of clear supernatant, 3 ml of 0.5M Na2CO3 and 0.5 ml of folin ciocalteu reagent were added, mixed well and incubated for 10 min. Absorbance was measured at 660 nm. The standard curve was plotted using tyrosine. One unit of enzyme activity is defined as the amount of enzyme required to liberate 1µmol of tyrosine per min under the defined assay conditions5.

Ammonium sulphate precipitation - The culture supernatant containing extracellular protease was analyzed for pH, total proteins and subjected to ammonium sulphate precipitation. Ammonium sulphate was added to the culture supernatant in small quantities with constant stirring. Each precipitate fraction was separated by centrifugation at 10000 rpm for 15 min and dissolved in 0.1 M Tris-HCl (pH 8.0). The fractions in buffer were dialyzed twice for 6-8 hrs against the same buffer6.

Gel Permeation Chromatography, protein and protease estimations - The dialyzed material was applied to Sephadex G-75 column (2 x 20 cm) which was previously equilibrated with 0.1 M Tris - HCl (pH 8.0). Before loading the sample the column was washed with 250 ml of the same buffer and 2 ml fractions were collected at a flow rate of 40 ml h-1. Both protein and enzyme activity were estimated. Protein concentrations were determined7 with bovine serum albumin (BSA) as a standard. For column chromatography, the concentrations were estimated by measuring their absorbance at 280 nm. The consecutive samples showing presence of protein and enzyme activity were pooled together, lyophilized and dissolved in 0.1 M Tris-HCl (pH 8.0) for further tests8. Protease activity was measured by caseinolytic activity by using 100µl of each fraction5

Polyacrylamide gel electrophoresis

During purification, protein samples were analyzed by performing SDS-PAGE according to Lemmali et al9 using 12% Polyacrylamide gel. Protein bands were detected by Coomassie brilliant blue (0.1%) staining.

Characterization of the enzyme

Effect of pH on protease activity - The activity of partially purified enzyme was determined at different pH values. The enzyme fractions were pre incubated in different buffers, citrate (pH 4.0 - 6.0), Phosphate (pH 7.0), Tris Hcl (pH 8.0 - 10.0), carbonate-bicarbonate (pH 11.0 - 13.0) buffers for 1 hr at 65ËšC. These were used for enzyme assay10.

Effect of temperature and CaCl2 on enzyme activity and stability - The effect of temperature on enzyme activity was determined by incubation at various temperatures ranging from 30ËšC to 90ËšC for 15 min at pH 10.0. Thermal stability was determined by incubating the partially purified enzyme at different temperatures ranging from 30ËšC to 90ËšC for 15 min, in the presence and absence of 2 mM of CaCl2. The activity was measured under standard assay conditions11.

Effect of various metal ions - The effect of metal ions on protease activity was determined by incubating the enzyme (0.1 mg in Tris-HCl pH 10.0) at 1,5 and 10 mM concentration of various metallic salts including CaCl2, MgCl2, CoCl2, LiCl2, KCl, NaCl, MnCl2, NiSO4, CuSO4, AgSO4 and ZnSO4 at 65ËšC. Activity without addition of any metal was taken as 100%12.

Enzyme Kinetics - Enzyme at fixed concentration (100 µg) was added to different concentration of substrate (1 - 50 mg/ml) and incubates at 65ËšC pH 10.0. The kinetic parameters Km and Vmax were determined from Lineweaver-Burk plot between 1/[S] and 1/ [V].

Enzyme specificity for various protein substrates - The proteolytic assay was done with different protein substrates including egg albumin, gelatin, casein, azocasein and bovine serum albumin in 100 mM Tris-HCl buffer (pH - 8.0). Five hundred micro liters of 1% solution of different substrates were diluted with Tris-HCl buffer and mixed with 100µg of enzyme solution. The mixture was incubated at 37ËšC for 15 min, the reaction was stopped by adding 1.1 M of trichloro acetic acid and the peptide concentration in the supernatant was estimated by measuring the absorbance at 280 nm13.

Results and discussion

Purification and molecular weight determination of protease - Protease from Bacillus cereus RV.B2.90 was purified by a combination of ammonium sulphate precipitation and gel filtration chromatography (Table 1). Ammonium sulphate precipitation yielded 2.96 fold purification with 93% recovery. Precipitate was dialyzed overnight against the same buffer which yielded 3.27fold purification with 85% recovery. The dialyzed enzyme was further purified by applying to Sephadex G-75 column (2 x 20 cm) and eluted with 0.1M Tris-HCl buffer of pH - 8.0. The figure shows (Fig. 1) the fractionation pattern of partially purified protease by gel filtration chromatography. The active peak showed 151 U/mg with 6.88 fold purification and 24% recovery. Appearance of a single band in SDS-PAGE indicated it as a monomer protein with a molecular weight of 32 KDa. This coincides with the earlier reports that Bacillus proteases are less than 50 KDa14.

Effect of pH - The effect of pH was studied at different pH ranging from 4 to 12 at 60˚C. Enzyme displayed a broad range of pH 4.0 - 12.0, but an optimum of 8.0 - 11.0. Highest activity (560 U/ml) at pH 10.0 was taken as 100%. This was in accordance with the previous reports15, 16, 6 that showed maximum activity at pH 10.0 for Bacillus cereus, Bacillus mojavensis and Bacillus subtilis respectively. In contrast another study17 showed optimum activity at pH 8.0 for Bacillus cereus. It retained 90% (526 U/ml) of its activity at pH 12.0, this indicated its stable nature towards alkalinity. The activities were 453 U/ml and 510 U/ml at pH 6.0 and 7.0 respectively. Even though it was active in acidic pH 4.0 and 5.0, it showed a progressive reduction in activity with 308 U/ml and 392 U/ml respectively. Similarly Subba Rao et al14 reported reduction of activity in acidic pH range by Bacillus sps. A report18 showed 60% of activity between pH 7.0 to 13.0 by V. fluvialis. In our study the enzyme had significantly high stability at very high pH range which could make it more suitable for use in detergent formulations19.

Effect of Temperature on enzyme activity and stability - Effect of temperature on activity was studied in different temperatures ranging from 30 to 90ËšC at pH 10.0 using casein as a substrate. Highest activity found at 65ËšC was taken as 100% (1182 U/ml). This was in accordance with a previous report20 showing highest activity at 65ËšC for a Bacillus sps. At 60 and 70ËšC it showed 97% (1098 U/ml) and 90% (1059 U/ml) of its relative activities respectively. At 90ËšC, it showed a steep decline and could retain only 34% (400 U/ml) activity. At 35 and 40ËšC, it showed 58% (802 U/ml) and 63% (858 U/ml) activity. The enzyme was active even at low temperatures. Optimum temperatures for thermophilic Bacillus HS08 was 65°C21 and for Bacillus subtilis PE-11 and Bacillus mojavensis was 60°C6,22. In contrast, Ogino et al23 reported optimum temperature be 40ËšC and complete disappearance of activity at 60ËšC by a Bacillus sps.

To determine the heat stability, enzyme in Tris-HCl buffer (pH 10.0) was incubated with and without 2mM Ca2+ at different temperatures (30 to 90ËšC) for 30 min and then the residual activity was measured. Highest activity at 65ËšC without Ca2+ was taken as 100% (1182 U/ml). At 90ËšC in the presence of Ca2+ 44% (467 U/ml) of its original activity was retained. Between 30 and 35ËšC, the enzyme activity was similar both in the presence and absence of Ca2+. Ghorbel et al17 have reported similar findings for Bacillus cereus. Addition of Ca2+ increases the activity and stability at higher temperature. Johnvesly and Naik24 reported the enhancement of activity with the addition of Calcuim for a Bacillus sps.

In the detergent industry alkaline proteases that remain active over a wide range of temperatures including lower temperatures would be a definite advantage so that washing of stains and other materials without exposing the dress materials to a high temperature could be achieved. The B.cereus RV.B2.90 protease is promising in this regard as it was retaining 60% of the maximum activity at 35ËšC which is rare. It was absolutely stable even up to a temperature of 90ËšC with 40% of its original activity.

Effect of Metallic ions - The effect of various metal ions with different concentrations (1, 5 and 10 mM) on 100 µg of partially purified enzyme with 1 mM Tris-HCl buffer in pH 10.0 at 65ËšC was determined. Mg2+, K+, Mn2+ and Ca2+ increased the activity from 400 U/ml to 655 U/ml, 488 U/ml, 483 U/ml and 444 U/ml respectively. These ions were found to protect the enzyme from denaturation and maintained its active conformation at high temperatures25. At 10 mM of K+, Ca2+ and Mn2+ the activity was reduced. Mg2+ at 10 mM had no inhibitory effect. Zn2+ and Cu2+ ions had inhibitory effects on protease activity, at even 1mM concentration. Zn2+ at 10 mM completely inhibited the activity. Similarly previous report26 showed inhibitory effect of these metal ions for Bacillus cereus. Zinc concentrations higher than 0.1mM have been reported to suppress protease enzymes. The basis is said to be blocking of the active site of the enzyme by zinc hydroxide. Patel et al27 also showed inhibition of protease by Cu2+; Co2+ at 1mm concentration had no significant effect but at 5 and 10 mm was found to decrease the original activity by 30 and 70% respectively. Na2+ at 1 and 5mM concentration increased the activity, but at 10 mM the original activity was decreased to 22% similar to earlier report28 on decrease in activity with increased salt concentration for Bacillus pumilus. The present study indicates that Bacillus RV.B2.90 is moderately halo-tolerant protease producer. Hence the isolate could find application in bacterial protease containing detergent additives.

Enzyme kinetics

The enzyme was characterized for Km and Vmax values against casein substrate at different concentrations (1 - 50 mg/ml). The Km value was found to be 0.55 mg/ml and Vmax was 1428 µmol min-1 which indicated its high affinity and efficient catalytic property on the substrate compared to previous studies showing high Km values for proteases as 6.6 mg/ml10, 2.0 mg/ml30, 1.8 mg/ml29 and 0.597 mg/ml14.

Effect of inhibitors - To determine nature of the enzyme 100 µg of enzyme was pre incubated with various types of inhibitors at 1, 5 and 10mM concentrations for 30 min at 65ËšC. Enzyme without inhibitors was taken as 100%. Enzyme was completely inhibited in the presence of PMSF at 5 and 10 mM concentration which is a serine protease inhibitor. DTT and EDTA had no significant effect on enzyme activity. It retained 79% of activity in the presence of EDTA, which suggested that the enzyme had no requirement of metallic cofactors28. This property of stability in the presence of chelating agents like EDTA used extensively in detergents is again advantageous. This result indicated that it is a serine protease. There are similarly reports of serine type proteases in Bacillus cereus31, Bacillus sps24, Bacillus clausii32 and Bacillus horikoshii33. In contrast Xu et al26 reported a metalloprotease from Bacillus cereus.

Effect of enzyme on various protein substrates - The capacity of the protease enzyme to hydrolyze various natural and modified substrates like casein, BSA, gelatin, egg albumin and azocasein was studied. The substrates at 1% in 300 µl of assay buffer were mixed with 100 µg of enzyme, incubated at 37ËšC for 30 min followed by addition of 1.1 M of trichloroacetic acid to stop the reaction and allowed to stand for 30 min at room temperature. The undigested protein was removed by centrifugation and the released peptides were monitored. It showed higher specificity to azocasein (362 U/ml), similar to previous report, by Singh et al34 for a serine protease by Bacillus sphaericus. Next to azocasein, casein showed more specificity (264 U/ml), followed by egg albumin (194 U/ml), BSA (182 U/ml) and gelatin (158 U/ml). Similarly Yossan et al25 and Adinarayana et al6 showed less specificity towards egg albumin, BSA and gelatin by Bacillus megaterium and Bacillus subtilis respectively.

Conclusion

The Bacillus RV.B2.90 produced highly alkalophilic thermophilic protease enzyme. The enzyme was purified by gel permeation chromatography to 6.88 fold. It was a monomeric protein and the molecular weight was 32 KDa as determined by SDS-PAGE. Maximum activity was at pH 10.0 and 65ËšC. Mg2+, K+, Mn2+ and Ca2+ were found to increase the activity. As it was inhibited by PMSF it is a serine type protease. Km value was found to be low (0.55 mg/ml) indicating its high affinity towards the substrate, also it could hydrolyze a variety of substrates other than casein. Due to its high temperature and pH optima, high stability, non inhibition by chelating agents and a broad temperature range of activity the organism and the enzyme are potent candidates for application in a wide variety of industries especially in detergent formulations.

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