Purifi cation and Characterization of Protease From Bacillus sp. TBRSN- 1

Potato Cyst Nematode (PCN), Globodera rostochiensis, is one of the important potato’s pests and caused economic looses up to 70% in the several centrals of potato plantations in Indonesia. PCN’s shell component of egg shell containing chitin (inner layer) and viteline/ protein (outer layer). The purpose of this research was to purify of protease Bacillus sp. TBRSN-1, isolate from tomato’s rhizosfer in Yogyakarta province. The purifi ed protease could be used for cutting the life cycle of PCN. Results showed that Bacillus sp. TBRSN-1 could produce extracellular protease and purifi cation using DEAE-cellulose ion-exchange chromatography and Sephacryl S-300 gel fi ltration chromatography resulted in specifi c activity 4.31 fold and 1.68% recovery. Analysing using SDS-PAGE 12.5% and molecular weight 48.1 kDa. Km and Vmax values of the protease for casein substrate were 7.83 mg/ml and 4.03 μg/h, respectively. The optimum activity at the temperature


Introduction
Plant-parasitic nematodes cause serious losses to a variety of agricultural crops worldwide. Since the traditional methods based on the use of nematicides and antihelminthic drugs are associated with major environmental and health concerns, the development of biocontrol agents to control nematodes is of major importance (Duncon, 1991). The potato cyst nematode Globodera rostochiensis is the major pests for the potato cultivars in several countries in the world (Margino et al., 2009). Soil treatment with nematocides for controlling Globodera, it is very expensive for farming community. In recent years biological control agents such as chitinolytic and proteolytic bacteria, fungi, actinomycetes were applied to control potato cyst nematode (PCN) eggs shell, for cutting their life cycle. Previous research succeded in controlling PCN up to 60% using mixed cultures inoculum (Margino et al., 2009) and succeded in purifying chitinases of selected bacteria and actinomycetes (Margino et al., 2010, Margino et al., 2012. Because of the fast breeding, easy cultivation and production compared to fungi, nematophagous bacteria have been used extensively as bioinsecticides against nematodes in soil, and levels of control equivalent to those of chemical pesticides development (Zhou et al., 2002). Egg cell of PCN containing vitelin (protein) and chitin layers so that protease can be used for controlling PCN through the namatode's egg.
Proteolytic enzymes are ubiquitous in occurrence, being found in all living organisms, and are essential for cell growth and differentiation. The extracellular proteases are commercial value and find multiple applications for example in biological control (bionematicide). Although there are many microbial sources are available for producing proteases, only a few are recognized as commercial producers, that is strains of Bacillus sp. (Gupta et al., 2002 b ). In this work, we present the purification and characterization protease of Bacillus sp. TBRSN-1, isolated from tomato's rhizosfer.

Microorganism and inoculum preparation
A culture of Bacillus sp. TBRSN-1 previously isolated from soil and identifi ed by standard method for bacterial identifi cation. Stock cultures were maintained in nutrient broth medium (Difco) with 70% glycerol, cultures were preserved at -20 o C. One loopful of bacteria strain (Bacillus sp. TBRSN-1) was transferred to a tube of sterile containing of nutrient broth and allowed to grow overnight at 37 o C (Shafee et al., 2005;Sharmin et al., 2005) before being used to inoculation.

Protease activity
The protease enzyme activity was determined as previously mentioned by Secades and Guijarro (1999) using casein as a substrate. Briefl y, 120 μL of a suitable dilution of enzyme solution was added to 480 μL of casein (2% wt/vol) in reaction buffer, and the mixture was incubated at 30 0 C for 30 min. The reaction was terminated by adding 600 μL of 10% (v/w) TCA and left for 30 min on ice, followed by centrifugation at 15,000 x g, at 4 0 C for 10 min. Eight hundred μL of the supernatant was neutralized by adding 200 μL of 1.8 N NaOH, and the OD value was measured using spectrophotometer at 420 nm (λ 420 ). One units of enzyme activity was defi ned as the amount of enzyme which required to produce an increase in OD value at 420 nm equal to 1.0 in 30 min, at 30 0 C.
The protein content of protease was determined by the method of Lowry et al., (1951) as mentioned in Bradfort (1976) using bovine serum albumin as a standard and during the course of enzyme purifi cation by measuring at OD value at λ280 nm.
The specific activity of the protease protein was expressed in terms of units/ mg protein/ml -1 according the following equation: Specifi c activity = enzyme activity/ protein content (mg/ml -1 ). (Singh et al., 1999) Protease crude enzyme was produced by fermentation. Bacillus sp. TBRSN-1 was cultivated in minimal medium consisting of (g/l): K 2 HPO 4 0.7, KH 2 PO 4 0.3, MgSO 4 .7H 2 O 0.5, FeSO 4 .7H 2 O 0.01, ZnSO 4 0.001, MnCl 2 0.001, skim milk 1%, and distilled water 1 L, pH 7.0. Media were autoclaved at 121 0 C for 20 min. Cultivations were performed on different condition (inoculum concentration, pH, substrate (skim milk) concentration, agitation, temperature, and incubation period) in 250 ml erlenmeyer fl asks with a working volume of 20 ml. The cultures were centrifuged and the supernatants were used for estimation of proteolytic activity.

Purifi cation of the protease
The culture supernatant was first subjected to ammonium sulphate precipitation (Scopes, 1994). Poteins presents in culture broth were extracted by ammonium suplhate 40%, 50%, 60%, 70%, 80%, and 90% (w/v). Extractants were collected by centrifugation at 10,000 g, 4 0 C for 45 min, and the pellet was suspended in 20 mM buffer phosphate, pH 7.0. The 40% (w/v) ammonium sulphte fraction was subjected to gel fi ltration on Sephacryl S-300 column (1.5x 60 cm) equilibrated with 20 mM Tris-HCl, pH 8.0 containing 0.2 M NaCl and 0.02% NaN3. Fractions of 1.5 ml were collected at a fl ow rate of 46 ml/h with the same buffer. Protein content and protease activity were determined. All purifi cation steps were conducted at temperatures not exceeding 4 o C.
Polyacrylamide gel electrophoresis (Laemmli, 1970) S o d i u m D o d e c y l S u l f a t e -P o l y -Acrylamide Gel Electrophoresis (SDS-PAGE) was carried out for the determination of purity and molecular weight of the enzyme as described by Laemmli (1970) using a 5% (w/v) stacking gel and 10% (w/v) separating gel. The molecular weight of the enzyme was estimated using a low molecular weight calibration kit as markers consisting of: phosphorylase b (97 kDa), bovine serum albumin (66 kDa), chicken egg ovalbumin (45 kDa), bovine carbonic anhydrase (29 kDa), and bovine α-lactalbumin (14.2 kDa). Protein bands were visualized by staining with Coomassie Brilliant blue 0.25% (w/v) and nitrate silver 0.1% (w/v). pH optimum and pH stability (Harman et al., 1993) The optimum pH of the purifi ed protease was studied over a pH of 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 9.0, and 10 with casein as a substrate. For the measurement of pH stability, the enzyme was incubated for 30 min at 30 o C in different pH buffers and the residual proteolityc activity was determined under standard assay conditions. The following buffer system were used: 100 mM phosphate buffer, pH 6.0-7.5; 100 mM Tris-HCl buffer, pH 8.0-8.5; and 100 mM glycine-NaOH buffer, pH 9.0-12.

Temperature effect on protease activity and stability
To investigate the effect of temperature, the activity was measured using casein as a substrate at the temperature range from 10 o C to 50 o C in 100 mM phosphate buffer, pH 7.0. Thermal stability was examined by incubating the purifi ed enzyme at different temperatures. Aliquots were withdrawn at desired time intervals to test the remaining activity at pH 7.0 and 30 o C. The non-heated enzyme was considered as control (100%).

Enzyme kinetics determination
K m and V max were determined by the kinetics of Michaelis-Menten Model reactions as described by Wilson and Walker (2005).

Production of protease and precipitation using ammonium sulphate
Bacillus sp. TBRSN-1 was originally isolated from soil around rhizosphere of tomato plant in (Sleman, Yogyakarta) and had high protease and chitinase activities in the culture broth medium. This isolate was also one of the mixed cultures inoculum candidate of bionematicides (data Margino et al., 2009). Production of extracellular protease by new strain Bacillus sp. TBRSN-1 was done in the two liter fermentor, based on the optimization of growth conditions (5%, v/v) of inoculums, substrate concentration (1%, w/v), pH value 7, agitation 150 rpm, temperature at 30 o C, and incubation time 60 hours.

Enzyme precipitation and purifi cation
Present tproteins in the culture fi ltrate were extracted by ammonium sulfate 40, 50, 60, 70, 80, and 90% (w/v). Result showed that 40% saturation was able to produce a maximum protease activity, 324.13 U/mg, and followed 277 U/mg for saturation 50% (w/v) (Figure 1). Furthermore, ammonium sulphate in this 40% saturation level was used for precipitating protein in crude enzyme. The added ammonium sulphate will press out of water molecule from protein and cause the hydrophobic condition of protein compounds (Harris and Angal, 1990). In addition to, ammonium sulphate also leads to the protein precipitation and reduces its solubility. While the solubility of protein decrease, interaction between hydrophobic regions formed agregates, then agregates of proteins which contained of big molecules suddenly precipitated and resulted in more precipitates until its optimum concentration, Figure 1 ( Scope, 1994). Ammonium sulphate purification increased the protease activity 1.46 fold. The precipitation step also decreased the overall protein concentration compared to the protein in the crude enzyme. Icreasesing of the Bacillus sp. TBRSN-1 protease activity using ammonium precipitation 1.46 fold are consistent with published literature, which shows a purifi cation (fold) up to 9,6 (Liao et al., 1998). The prcipitation also gave lower result compared to precipitation of protease Burkholderia strain 2.2 N, using ammonium sulphate 40-60% (w/v) produce protease activity 20 fold (Jewell, 2000). A little bit different result showed by protease activity from Bacillus sp. PS719 which precipitated using ammonium sulphate 80%, resulted in 1.5 fold (Towatana et al., 1999).
The results of ammonium sulphate precipitation in saturation level 40% (w/v) as much as one ml (formerly been dialyzed with 0.05 PBS pH 7.0) applied into the colomn ion exchange chromatography containing DEAE Cellulostein. The 100 fractions were measured in early experiment to find out protein concentration of each fractions (based on the absorpton value at 280 nm). Results showed that two peak of protein but only one had protease activity, that fraction numbers 7-14 and 15-25. After collecting the samples and then be run using SDS-PAGE was found out two lines with a little bit far of their distance (data unshown). Furthermore analysis was done using gel fi ltration chromatography method by Sephacryl S-300 ( Figure 3). This experiment has measured 70 fractions to fi nd out the protein contents of each fraction (based on absorption value at 280 nm). In the gel filtration, one protein peak was observed, which formed by fraction number 51, but fraction numbers 50 and 52 have closed protein peaks and they  had high protease activities. Futhermore, the samples were collected into one tube, then to be freezed-dried for characterization of enzyme. Enzyme purification using Sephacryl S-300 may increased enzyme purity as much as 4.31 times. The yield and purity for each purifi cation steps were summarized at Table 1.
The protease purifi cation using DEAE-Cellulose at Table 1, showed 3.07 fold compared to crude enzyme, its specifi c activity 653.60 U/mg protein, and its recovery was 2.15%. This result a little bit different with purifi cation of extracellular protease from Bacillus subtilis EAG-2, using DEAE-Cellulose, the overal recovery 29% and the purity level was 11 fold (Ghafoor and Hasnain, 2010). The extracellular protease was purifi ed on Sephacryl-300 had purifi cation value 4.3-fold with a recovery 1.68% and a specifi c activity 916.76 U/mg of protein.
All operations were carried out 4 0 C. Only 40% ammonium sulphate was subjected to gel fi ltration on Sephacryl S-300.

Characterization of protease
The characterization of protease were done including SDS-PAGE analysis, molecu;lar weight, pH value and temperature effect.
The character of protease protein produced by Bacillus sp. TBRSN-1 could be  performed on 10% SDS-PAGE ( Figure 4). This fi gure showed that one band of protease protein was found in this experiment. The molecular weight of single protein that resulted in Sephacryl S-300 isolation was determined using relative mobility calibration curve of standard polypeptide ( Figure 5). The molecular weight estimation was determined using regression equation Y= 2.3235 -1.7473 X (with r=0.99), Figure 5. The purifi ed protease was homogenous on SDS-PAGE and its molecular weight was estimated to be 48.1 kDa (Figure 4).
The estimation of protease molecular weight of Bacillus sp.TBRSN-1was about 48,1 kDa ( Figure 5). It was supposed that this band is protease, this was approved by increasing of protease activity and its enzyme purity. Padmapriya and Williams (2012) reported that the purifi ed neutral protease of Bacillus subtilis had molecular weight 50 kDa, while purified serine protease from Bacillus sp. from marine had molecular weight 37 kDa . Towatana et al. (1999) reported that purifi ed an extracellular protease from alkalophilic thermophile Bacillus sp. PS 719 using DEAE-Cellulose ion-exchange chromatography had molecular weight 42 kDa. Yang et al. (2000) reported that Bacillus subtilis isolated from soil in Taiwan, which purifi ed using DEAE-Sepharose ion-exchange chromatography and Sephacryl S-200 gelpermeation chromatography showed that protease had molecular weight 44 kDa.
Based on the several of researches showed that molecular weight of protease from Bacillus sp. TBRSN-1 had the similar types of them.

Effect of pH on enzyme activity
The pH profi le of the purifi ed enzyme was determined using different buffers of varying pH values. The purified enzyme was active in the pH range 3.5 -9.0, with an optimum activity at pH 7.0 ( Fig. 6a) with protease activity was 24.58 U/ml. Similarly, Abdul-Rouf (1990) reported that the optimum pH for all purifi ed 4 proteases enzymes in their reaction mixture was found to be 7.2. Purifi ed protease of Bacillus sp. isolated from soil samples around the Bungalore had specifi c activity at pH neutral (Josephine et al., 2012), other purifi ed protease of Bacillus subtilis also had activity at pH 7.0 (Padmapriya and Williams, 2012).

Effect of temperature on the enzyme activity
The effect of temperature on the activity of protease enzyme was examined at various  (Figure 6b). The protease activity at 10 o C and 40 o C were about 8.75 U/ml and 10.24 U/ml, respectively. The enzyme was completely inactivated after 30 min incubation at 50 o C. While the temperature below or above 30 o C exhibited lower activities of protease. Secades and Guijarro (1999) reported that a novel exoprotease, that was purified from the culture supernatant of Yersinia ruckeri (fi sh pathogen), had more activity in the range of 25 to 42 o C and had an optimum condition at 37 o C. Asker et al. (2013) reported that protease of Bacillus megaterium had the optimum activity at 50 o C, while other purifi ed protease from Bacillus subtilis had optimum activity at 37 o C. This illustration showed that optimum temperature of protease from several Bacillus sp. had the large variation.

Determination of K m and V max value of protease
The result of enzyme kinetic analysis (K m and V max ) are shown in Table 2. Determination of Km and Vmax values were based on the pH condition and optimum temperature that have been procured. In this event, the increasing of substrate saturation will increase enzyme activity to achieve a certain limitations at the certain substrate saturation as well, so that the with the increasing of substrate after optimum limitations will not increase the enzyme activity.
Michaelis Menten's (Km) constant value analysis and maximum speed (Vmax) can be seen at Table 2. According to above computation, there was quantitative relation between the speed (Vi) with substrate saturation (S). Michaelis Menten's (Km) constant was procured about 7.83 mg/ml and maximum speed (V max ) was 4.03 μg/h with regression equation; Y=1942. 7X -248.29 and have correlation value about r = 0.998. Wilson (2005) explained that enzyme activity more higher, if the its K m values was small. The correlation between of reaction velocity (v) with substrate concentration as shown at Figure 7.
Purification of Bacillus sp. TBRSN-1 protease using DEAE-Cellulose and Sephacryl S-300 gel fi ltration, increased a 4.31-fold in specific activity and 1.68% recovery. The molecular weight of the purifi ed protease was estimated to be 48.1 kDa.
The optimum conditions of pure protease activity were on temperature 30 o C, pH 7.0, K m and V max values were 7.83 mg/ ml and 4.03 μg/h, respectively.