PURIFICATION AND CHARACTERIZATION OF BROMELAIN-LIKE CYSTEINE PROTEASE FROM Billbergia pyramidalis (Sims) Lindl.

Transcription

1 CHAPTER-IV PURIFICATION AND CHARACTERIZATION OF BROMELAIN-LIKE CYSTEINE PROTEASE FROM Billbergia pyramidalis (Sims) Lindl. 4.1 INTRODUCTION Proteases are a unique class of enzymes, because of their immense physiological and commercial importance. Ranging from cellular level to the organism level, the plant proteases execute wide variety of functions, hence they are envisaged to have extensive use in biotechnological and industrial applications (Hedstrom, 2002). They take part in both synthetic and degradative processes. These proteases are ubiquitously found in microbes, plants and animals, as they are physiologically essential for all the biotic components of the ecosystem (Rao et al., 1998). Plant proteolytic enzymes have received tremendous importance because of their broad specificity, stability, reaction rate and sustainability to various ph and temperature conditions (Uhlig, 1998).The catalytic performance of plant proteases are influenced by certain factors like the source, climatic conditions for growth and various methods implemented for their extraction and purification (Westermeier et al., 1998; Brueske et al., 1980). Protein purification is essential for characterization of a protein and also in predicting its structure and its interaction with other molecules. Separation techniques used for purification exploit different properties of proteins like size, solubility, binding affinity, physico-chemical properties, and biological activity. The objective of a protein purification scheme is to retain high fold purification and yield. The present objective is to purify bromelain-like cysteine protease from B. pyramidalis using multistep purification procedure and also to characterize the purified enzyme using various biochemical methods and to determine its structure by spectroscopic studies. 60

2 4.2 MATERIALS AND METHODS Materials CM cellulose, Sephadex G-150 and Bovine serum albumin, were procured from Sigma Aldrich (USA). Standard protein molecular weight markers purchased from Bio-Rad Laboratories (USA). All the other chemicals are of analytical grade and are procured from Hi-Media Laboratories (Mumbai, India). All the reagents were prepared using deionized (Millipore) water Extraction of crude enzyme Crude enzyme was extracted from the leaves of B. pyramidalis. Leaf tissue was weighed (100 grams) and macerated in a mortar and pestle using liquid nitrogen to produce a fine powder and subsequently the enzyme was extracted using optimized homogenization medium (250 ml), containing 0.5M Sucrose, 2.5mM DTT, 5 mm CaCl 2 solution and 25mM Tris - HCl buffer with ph 7.0 respectively. The process of extraction was done by cold maceration and filtered using Whatman No.1 filter paper, prior to centrifugation at 10,000 g at 4-6 C for 10 min. The supernatant thus obtained was further analyzed and purified using biochemical methods.minor modifications were made to the existing enzyme extraction procedures as reported by Gautam et al., (2010) and Sean et al., (1986) to enhance the protease activity Protease Assay Activity of the protease was evaluated using (GDU) gelatin digestion unit analytical method with minor modifications (Moodie, 2001) as described earlier in Chapter II Protein Estimation Concentration of Protein present in the enzyme was determined by the method described by Lowry et al., (1951) using crystalline BSA as standard. 61

3 Specific Activity of the enzyme Specific activity of the enzyme was determined by dividing the enzyme units with the protein concentration and expressed as U/mg protein Yield of Purified Enzyme Yield of each purified fraction is the activity in percentage obtained, by dividing the total activity of the particular fraction with the total activity of the crude enzyme Fold Purification Fold purification of each purified fraction was obtained by dividing the specific activity of that fraction with the specific activity of crude enzyme Purification of Protease The purification protocol includes different sequential steps like - the salting out proteins by ammonium sulfate precipitation and separation of proteins by gel filtration and ion exchange chromatography. Later on, at every step the proteolytic activity, protein concentration, specific activity, yield and fold purification are determined. A state of purity was determined at every successive stage of purification by performing SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). The purified enzyme was stored at 4 C for further analysis Ammonium sulphate ((NH 4 ) 2 SO 4 ) Precipitation This was performed according to the method described by Devakate et al The crude plant extract was subjected to ammonium sulfate precipitation by adding varying concentrations of ammonium sulfate with gentle stirring for 30 min to obtain 0 20%, 20 40%, 40 60% and 60 80% saturation and the contents were centrifuged at 10,000 rpm for 15 min at 4 C and the precipitates of the four fractions were collected. Fractionation of proteins using ((NH 4 ) 2 SO 4 is a simple and effective means to acquire the desired concentration of the protein of interest, by simultaneously excluding the unwanted proteins. 62

4 Dialysis of Proteins The salt precipitated protein was dissolved in minimum quantity of 0.1M Tris-HCl buffer at ph 7.0 and was transferred to pretreated dialysis membrane bag (Hi-Media, cut off value kda). The salts from the above purification step are removed by the process of dialysis using 0.01M Tris HCl buffer (ph 7.0) and dialyzed for 24 h at 4 C with three frequent changes in the buffer solution for every 8 h and the dialyzed samples were analyzed to determine the protein content and enzyme activity. The fraction with high enzymatic activity was subjected to further purification Sephadex Gel filtration Chromatography The active sample of ammonium sulfate fractionation after dialysis (2.5 ml) was loaded onto a column of Sephadex G-150 (0.6 X 44cm) pre-equilibrated with 0.1 M Tris HCl buffer, ph 7.0, for 3 bed volumes. Then the column was eluted at a flow rate 20 ml/ h using the same buffer and 3.0 ml fractions were collected. The enzymatically active fractions were collectively pooled and dialyzed again for further Ion exchange chromatography CM-cellulose Ion Exchange Chromatography The enzymatically active protein fraction of gel filtration was loaded on to CM-cellulose bed of 1.5 cm thickness and was equilibrated with 0.5 M Tris-HCl buffer, ph 8.0, and eluted with eluting buffer containing 25mM Tris HCl buffer, ph 7.0 and 25mM NaCl. The eluate containing bound proteins were collected as 3ml fractions with a linear gradient of increasing ionic strength of sodium chloride (25mM 150 mm) as described by Gautam et al., Fractions with high proteolytic activity were pooled and subjected to further purification by AKTA FPLC purifier (Amersham Pharmacia Biotech) using Mono S column to increase the purity of protease Cation Exchange Chromatography using AKTA FPLC system Fast protein liquid chromatography (FPLC) is an automated liquid chromatography system intended for purification of proteins, peptides and other biomolecules. The active fraction from ion exchange chromatography was re-chromatographed using 63

5 Mono S 5/50 GL column in AKTA FPLC system (Amersham Pharmacia Biotech). Mono S is a strong cation exchanger chromatography column, equilibrated with 25mM Tris HCl buffer (ph 7.0). 500 µl of sample with high proteolytic activity was loaded and fractions were eluted at a flow rate of 0.8ml/min by adding the start buffer (25mM Tris-HCl, ph 7.0) followed by NaCl gradient from M in the same buffer. Elution pattern of the protease was monitored and the absorbance was measured at 280 nm. Enzyme activity of the eluted fraction was analyzed, and stored at -20ºC for further studies High Pressure Liquid Chromatography (HPLC) HPLC is used for identifying, purifying, and quantifying the components of a mixture. The purity of active Mono S fraction was checked by using AGILENT 1100 series HPLC system. The purified Mono S fraction was applied to Agilent TC C18 (4.6 x 250 mm) and eluted with 0.1% TFA in filtered acetonitrile as a solvent system. The elution pattern of protease was observed and the absorbance was measured at 280 nm BIOCHEMICAL CHARACTERIZATION OF PURIFIED PROTEASE The pure protease obtained after a multistep purification process was further subjected to characterization to determine its biochemical properties, like molecular weight determination by SDS-PAGE, zymogram analysis, optimum ph and temperature conditions for maximum protease activity, stability of protease at altered ph and temperature, effect of various metal ions, inhibitors, activators, enzyme kinetic parameters and substrate specificity analysis, as described under the following sections SDS PAGE To determine the purity and the molecular weight of the purified protein, SDS polyacrylamide gel electrophoresis was carried out using a 4% stacking gel and 12% separating polyacrylamide gel as described by Laemmli (1970); Schagger and Von Jagow (1987). The protein sample was mixed with sample loading buffer containing 0.5M Tris HCl of ph 6.8, 20 % (v/v) glycerol and 4 % (w/v) SDS. The protein sample was loaded into the wells and then subjected to electrophoresis with a constant voltage supply of 64

6 50mv. On completion of electrophoretic run, the gel was stained with 0.02% Comassive Brilliant Blue-250 dissolved in methanol and acetic acid. Later destained with a mixture of acetic acid and methanol solution for several hours till the bands are clearly visible. The standard proteins like phosphorylase b (molecular weight 97.4 kda), bovine serum albumin (molecular weight 66.2 kda), ovalbumin (molecular weight 45.0 kda), carbonic anhydrase (molecular weight 31.0 kda), soybean trypsin inhibitor (molecular weight 21.5 kda), and lysozyme (molecular weight 14.4 kda) are used as molecular weight markers to generate the calibration curve Zymogram Analysis Zymogram is an electrophoretic representation of hydrolytic enzymes on the basis of substrate degradation. Activity of protein separated by reducing SDS-PAGE was detected by zymogram analysis with minor modifications in the method reported by Westergaard et al., (1980) and Farrokh et al., (2014). Within the polyacrylamide gel matrix, the substrate (gelatin) was copolymerized for detection of enzymatic activity. Samples are normally prepared by the standard SDS-PAGE treatment buffer, under non-reducing conditions, i.e., without subjecting it to the heat treatment and sample buffer devoid of β-mercaptoethanol. Electrophoretic run was performed for 3 5 h with a constant voltage supply of 80 V. During electrophoresis, the SDS causes the protease to denature and become inactive. On completion of electrophoretic run, the gel was washed with 2.5% Triton X-100 solution for 3 x 10 min. Incubating the gel with Triton X-100 removes SDS and allows the protease to partially renature and recover its activity. Later the gel was rinsed with deionized water and subsequently, the gel was further incubated for 1h at 37ºC in an appropriate activation buffer or zymogram developing buffer containing 2.5% Triton X-100, 5mM Tris ph 7.0 and 3mM Ca 2+ and 2µM Zn 2+ ion source to enhance the protease activity. The unstained gel was overlaid on an agarose gel soaked in 1% gelatin substrate solution for 60 min at 37ºC and then washed twice with deionized water. The agarose gel was then, stained using 0.02% Coomassie brilliant blue R-250 for 2-3 h and then transferred to destaining solution till the gelatinolytic activity of the protease was visualized as transparent clear bands against the blue background. 65

7 Effect of ph on Activity and Stability of Purified Protease The effect of ph on activity of purified enzyme was studied by incubating the purified protease at different ph levels ranging from ph at 37 C using gelatin as substrate. The buffers used are 0.1 M concentration of citric acid buffer (ph 3.0 and 4.0), sodium acetate buffer (ph 5.0 and 6.0), Tris-HCl buffer (ph 6.0 and 7.0) sodium phosphate buffer (ph 8.0) and glycine NaOH buffer (ph 9.0 and 10.0). The effect of ph on protease stability was also determined simultaneously by pre incubating the purified enzyme in respective buffer solutions for 2 h and the residual protease activity was assayed under standard conditions Effect of temperature on activity and thermal stability of Purified Protease The optimum temperature required for maximum protease activity of purified protease was determined by conducting the assay at various temperatures ranging from 20ºC to 80ºC.The enzyme mixture was incubated at different temperatures for 30 min using gelatin as substrate. Thermal stability of the protease was assayed at optimum temperature (30 ºC to 60 ºC) obtained from the above analysis, by pre-incubating the protease for a period of 2h at different time intervals (30, 60, 90, and 120 min) and the residual protease activity was measured Effect of Metal ions on the Activity of Purified Protease The effect of various metal ions on the activity of purified enzyme was determined by incubating the enzyme with various metal ion sources (5 mm) viz, Ca 2+, Mg 2+, Cu 2+, Zn 2+, Mn 2+, Fe 3+, Na + and Hg 2+ for 30min at 25 ºC and relative protease activities were measured under standard assay conditions by taking the activity of the control sample without metal ion as 100% Influence of Inhibitors on Protease Activity The effect of inhibitors on proteolytic activity of the purified protease was determined by pre-incubating the protease with various inhibitors like Trypsin inhibitor, EDTA 66

8 (Ethylene diammine tetra acetic acid), PMSF (Phenylmethanesulfonyl fluoride), IAM (Iodoacetamide) and DEPC (Diethylpyrocarbonate) at various concentrations (2mM, 5mM, and 10mM) for 30 min at 37 ºC. The relative protease activity was assayed under standard conditions by taking the activity of the control sample without inhibitor as 100% Effect of Activators and Reducing agents on the Activity of Purified Protease The effect of activators and reducing agents on the activity of protease was determined by incubating the protease with various protease activators like sodium benzoate, glutathione, sodium metabisulphite salt and reducing agents like DTT, β- mercaptoethanol at 2mM, 5mM and 10mM concentrations for 30 min at 37ºC. The proteolytic activity was determined under standard assay conditions and relative activity was measured by taking the activity of the control sample as 100% Determination of Kinetic Parameters and Substrate Specificity of Purified Protease Substrate specificity analysis and rate of the enzymatic reaction was measured using gelatin and casein as substrates. The purified enzyme was incubated in reaction mixture containing 0.1M Tris-HCl buffer at ph 7.0 with different concentrations of substrate ranging from 3mM to 100mM for 20 min at 30 C and assayed for protease activity. A graph was plotted by taking the reciprocal of activity data versus reciprocal of substrate concentration. The maximum reaction velocity Vmax, Michaelis-Menten K M constant and the reaction specificity Vmax/K M were determined using Lineweaver-Burk plot Statistical Analysis All assays in this study were carried out in triplicates and the results are presented as the mean of three replicates ± SD. The data of estimates of protease activity were subjected to one way analysis of variance (ANOVA) using Graph pad Prism software Version 6.0 to assess the significance of the various effects. A value of p<0.05 was considered as statistically significant. 67

9 4.2.5 Spectroscopic analysis of purified protease Organic molecular groups and functional groups, involved in the compound are identified using FTIR and NMR Spectroscopy Fourier Transform Infrared (FTIR) Spectroscopy FTIR is useful for identification of organic molecular groups and compounds. The functional groups, side chains and cross-links involved in the compound, give rise to characteristic vibrational frequencies in the infra-red range by absorbing the light in the infra-red region of the electromagnetic spectrum. FTIR absorption spectra of the purified protease was performed by using a Perkin Elmer FTIR spectrometer. Approximately 0.5 mg of dry protein sample was taken along with 1% of potassium bromide (KBr) and was compressed to form a pellet. The spectrum was scanned from 400 to 4000 cm -1, at a resolution of 4 cm -1 and the data was analyzed by using PE-GRAMS/ software, as stated by Liao and Chen (2004), Liao et al. (2004) and Devakate et al. (2009) H and 13 C NMR The NMR studies of purified compound were performed by using Bruker AMX-400 spectrometer. 1 H and 13 C NMR data were acquired at and MH Z respectively. The chemical shifts were expressed in δ (ppm) using D 2 O as solvent and acetonitrile as internal reference. 68

10 4.3 RESULTS AND DISCUSSION Purification of Bromelain-like Cysteine Protease The protease from B.pyramidalis was purified using 250 ml of crude enzyme. The specific activity of the crude enzyme was found to be U/mg. The purification results are summarized in Table 4. Table 4.1: Purification of bromelain like cysteine protease from B. pyramidalis. S.no Steps of Purification Total Protein (mg) Total Activity (GDU Units) Specific Activity (U/mg) Fold purification Yield (%) 1. Crude Ammonium salt precipitation (40% 60% saturation) Dialysis Gel filtration by Sephadex G-150 Ion exchange chromatography using CM cellulose Re-chromatographed on Mono S column (FPLC- AKTA purifier) Initially the crude enzyme was subjected to precipitation with 0-20% saturation of ammonium sulphate, the precipitated protein showed negligible protease activity. The fraction collected at % saturation was found to have the higher protease activity and protein content as compared to other saturation levels. 69

11 The specific activity of the protease was found to be U/mg with 78.63% yield at this stage. Further increase in the salt concentration resulted in decrease in specific activity of protease. After concentration by ammonium sulfate fractionation (40-60%) and dialysis, the specific activity increased to U/mg with 56.35% yield and 1.74 fold purification. Figure 4.1 shows the elution profile of the dialyzed fraction by Sephadex G-150 column chromatography, the enzyme was eluted as a single peak in the fractions After gel filtration chromatography 29.71% yield and 4.05 fold purity was achieved. The enzymatically active protein fractions were pooled and loaded on cation exchange chromatography column with CM-cellulose bed. The fractions were collected in a step wise gradient and the elution profile of cation exchange chromatography was shown in Figure 4.2. At this purification step, the specific activity of protease was found to be U/mg and the purity of protease increased to fold with a yield of 21.95%. The active fraction was pooled and re-chromatographed on Mono S 5/50 GL column in AKTA FPLC system. Rechromatography of the proteolytic fraction from Ion exchange column yielded single peak (Figure 4.3). The protein fraction of the Mono S column was found to exhibit higher enzyme activity with an overall yield of 16.58% and fold purification and the purified enzyme was coded as PSA BP -07. Different strategies have been executed for the extraction and purification of enzyme. These include aqueous two phase systems, reversed micellar systems, precipitation and various chromatographic techniques. For the purification of the bromelain from pineapple fruit, Devakate et al., 2009 employed precipitation and chromatographic methods and reported 3.3 times high fold purification than the fold purity obtained from precipitation. Kumar et al., 2011 employed affinity based RMS extraction method to purify the enzyme bromelain from pineapple waste and reported a yield purification of fold. There are various reports on the use of Reverse micellar system (RMS) for bromelain extraction from pineapple plant in the literature 5.2 purification fold was reported by Hebbar et al., (2008) and 4.54 purification fold by Hemavathi et al., (2007). Similarly an integrated approach, combining RMS with ultrafiltration was described by Hebbar et al., 70

12 (2012) in isolating the protease bromelain from pineapple core and reported to have 8.9-fold purification after ultrafiltration. Figure 4.1: Sephadex gel column chromatography profile of enzyme Figure 4.2: CM-Cellulose Ion Exchange Column chromatography profile of enzyme 71

13 Figure 4.3: Mono S 5/50 GL column elution profile using AKTA FPLC system High Pressure Liquid Chromatography analysis The purified protease PSA BP-07 was further analyzed by HPLC, the enzyme was eluted as sharp peak with retention time at 3.02 min (Figure4.4). The results inferred that chromatographic techniques had greatly maintained the homogeneity and structural integrity of the plant protease. 72

14 Figure 4.4: HPLC analysis of purifiied protease, PSA BP Biochemical Characterization of Purified Protease, PSA BP SDS-PAGE for Molecular Weight Determination The state of purity was determined at every successive stage of purification by using 12 % SDS-PAGE. The electrophoretic pattern of all the purified fractions obtained from ammonium sulfate fractionation, Sephadex G-150 gel chromatography, CM cellulose ion exchange chromatography and re-chromatographed mono S column fraction along with the crude enzyme is shown in Figure 4.5. From the electrophoretic pattern, it is evident that the crude and ammonium sulphate fraction contain many protein bands and were not appropriately resolved. Gel filtration chromatographic fraction showed 3 protein bands, Ion exchange distinct chromatographic fraction showed 2 distinct protein bands, and protein fraction of the Mono S column showed single band equivalent to the single peak observed in Mono S column elution profile, indicating that the protease was purified to apparent homogeneity. The relative mobilities of standard molecular weight protein markers in lane 1 are compared with that of the purified protease and the molecular weight of the 73

15 protein was measured by using a standard calibration curve of log molecular weight. The purified protease separated as single band on SDS PAGE exhibited a molecular weight of 36.8 kda, this clearly shows that it is a single polypeptide and confirms the purity of the protein. Figure 4.5: SDS-PAGE Lane 1- Crude enzyme Lane % Ammonium sulfate precipitation fraction Lane 3- Sephadex Gel column chromatography fraction Lane 4- CM- Cellulose Ion exchange chromatography fraction Lane 5- Mono S column fraction (Purified protease, PSA BP-07) Lane 6- standard bromelain Lane 7- Standard protein marker 74

16 Zymogram analysis The purified protease PSA BP-07 was subjected to zymogram analysis.the zymography (activity staining) studies of the protease PSA BP-07 fraction also showed a single clear hydrolyzed portion on the stained agarose gel (Figure 4.6). The presence of proteolytic activity was visualized as a clear band of lysis against a deep blue background. Figure 4.6 Zymogram of purified protease in comparison with pure protein band on SDS-PAGE. Lane 1: Standard protein molecular weight markers on SDS-PAGE gel Lane 2: Purified protease PSA BP-07 on SDS-PAGE gel Lane 3: Activity staining of purified protease PSA BP-07 on agarose gel (zymogram) Effect of ph on Activity and Stability of Purified Protease, PSA BP-07 The effect of ph on the activity of protease, PSA BP-07 was studied and the results are shown in Figure 4.7. Significant differences were observed in the activity and stability of protease incubated at different ph levels. The protease PSA BP-07 showed activity at various ph levels between ph but maximum protease activity was observed at ph 75

17 7.0. Similar reports were given by Murachi and Neurath (1960) that the ph optima for stem bromelain lies, around ph 7 0. Sunantha et al., (2012) reported optimum ph 8.0 for bromelain enzyme activity. The ph stability profile was studied by pre incubating the enzyme in various levels of ph ranging from for 2 h and the results are shown in Figure 4.8. Significant differences were observed in the stability of protease PSA BP-07 at different ph with increase in time period. The purified enzyme showed stability at ph range ( ). At ph 7.0 the protease retained 100% activity for 2 h. The residual activity of 72% and 52% was observed at ph values 6.0 and 8.0 respectively. The stability of the protease was low at ph 5 and ph 8. Similar reports were reported on stability of stem bromelain from pine apple by Kumar et al., (2011) which is found to be highly stable at ph range of Figure 4.7: Effect of ph on Activity of Purified Protease, PSA BP-07 The values are represented as the mean of three replicates ± SD (p<0.05) 76

18 Figure 4.8: Effect of ph on Stability of Purified Protease, PSA BP-07 The values are represented as the mean of three replicates ± SD (p<0.05) Effect of Temperature on Activity and Stability of purified protease PSA BP-07 Significant differences were observed in the activity of protease PSA BP-07 at various temperatures (Figure 4.9). The protease PSA BP-07was found to be active over a broad range of temperature 30 to 50 ºC, with optimum activity at 40 ºC. An abrupt decrease in protease activity was observed with further increase in temperature. Jutamongkon and Charoenrein, (2010) also reported 40 ºC as the optimum temperature for fruit bromelain and Suh et al., (1992) reported ºC as optimum temp for stem bromelain enzyme. The thermal stability profile of protease PSA BP-07 was studied by pre incubating the enzyme at different temperatures ranging from C for 2 h and the results are shown in Figure Significant differences were observed in the stability of purified protease at different temperatures with increase in time period. The enzyme retained 100% stability at 40 C even after 2 h of incubation. Whereas, protease showed 72% stability at 30 C, 52% stability at 50 ºC and 18% stability at 60 ºC after 2 h of incubation. At 50 C and 60 C the enzyme retained 72 % and 45% of its initial activity up to 1 h and gradually 77

19 lost its activity on further incubation. These results are in accordance with the characteristics of pineapple bromelain, which is found to be active between 40 ºC and 60 ºC (Okino et al., 2010; Corzo et al., 2012). Figure 4.9: Effect of Temperature on Activity of Purified Protease, PSA BP-07 The values are represented as the mean of three replicates ± SD (p<0.05) Figure 4.10: Effect of Temperature on Stability of Purified Protease, PSA BP-07 The values are represented as the mean of three replicates ± SD (p<0.05) 78

20 Effect of metal ions on Activity of Purified Protease, PSA BP-07 The impact of various metal ions on activity of protease PSA BP-07 was studied and the results are shown in Figure Significant differences were observed in the activity of purified protease with different metal ions. It was observed that the divalent metal ions have positively influenced the activity of protease. Ca 2+ has increased the protease activity to 32%, followed by Mg 2+ (12%) and Zn 2+ (6%), However, Hg 2+, Cu 2+, Fe 3+ and Na + strongly reduced the proteolytic activity of enzyme. Among the metal ions Hg 2+ found to be a potent inhibitor of the protease PSA BP-07. This clearly confers the thiol nature of the enzyme. Similar results were reported by Wang et al., 2009 that Ca 2+ ions at 60 ºC had greatly enhanced the activity and stability of bromelain from pineapple. Enhanced proteolytic activity was found with Ca 2+ ions as it maintains and stabilizes the confirmation of enzyme molecule. Kaul et al., (2002) also reported similar stimulatory effect of Mg +2 and Ca +2 on enzyme Papain from Carica papaya which belongs to cysteine endopeptidase family. Reduced bromelain enzyme activity by Fe 3+, Cu 2+ ions was also reported by Liang et al., (2011). Figure 4.11: Effect of metal ions on Activity of Purified Protease, PSA BP-07 The values are represented as the mean of three replicates ± SD (p<0.05) 79

21 Effect of inhibitors on Activity of Purified Protease, PSA BP-07 The effect of various concentrations of inhibitors on proteolytic activity of protease PSA BP-07 was studied and the results are shown in Figure Significant differences were observed in the activity of protease PSA BP-07. EDTA, PMSF and trypsin inhibitor did not show any effect on activity of protease. Whereas, Iodoacetamide (IAM) and Diethylpyrocarbonate (DEPC) inhibited the enzyme by 84%, and 80% at 2mM concentration and complete loss in enzyme activity was observed with increase in concentration to 10mM. The results indicate that the purified protease PSA BP-07 belongs to cysteine protease family, as complete loss in proteolytic activity of enzyme was observed with sulfhydryl modifying reagent (IAM) and histidyl modifying reagent (DEPC). This clearly infers that the purified protease contains histidine at the active site along with cysteine, as proposed for papain (Dunn, 1989). As EDTA did not show significant effect on the enzyme activity it excludes probability of the protease being a metalloenzyme. These results are in accordance with previous studies on cysteine proteases (Shaha et al., 2013 Shamkant et al., 2012, and Nagarathnam et al., 2010). Figure 4.12: Effect of inhibitors on Activity of Purified Protease, PSA BP-07 The values are represented as the mean of three replicates ± SD (p<0.05) 80

22 Effect of Activators and Reducing agents on Activity of Purified Protease, PSA BP-07 The effect of various activators and reducing agents on activity of protease PSA BP-07 was studied and the results are shown in Figure L-cysteine hydrochloride monohydrate and sodium metabisulfite enhanced the enzyme activity to 42% and 63% with increasing concentrations, whereas sodium benzoate exhibited 102 % proteolytic activity. Zhao et al., (2011) also reported that metabisulfite would be the finest activator in enhancing the bromelain enzyme activity as compared to other activators. However the protease is sufficiently active even without the activator. Addition of activator reactivates the enzymatic activity when enzyme loses an appreciable amount of its activity by undue storage or preparation conditions. The effect of the reducing agents β- mercaptoethanol and dithiothreitol (DTT) on the activity of protease PSA BP-07 was increased to 54% and 16% at 2mM concentration. Further incubation with higher concentrations resulted in loss of enzyme activity. As DTT is a thiol group protectant it conferred protective effects against oxidation and it also enhanced the protease activity. The results are in accordance with the earlier reports by Ma and shi (1997) and Takagi et al., (1990) on enhanced activity and stability of protease by the addition of DTT. The enhanced proteolytic activity in the presence of sulfhydryl reagents or reducing agents, clearly confirmed the thiol nature of the protease PSA BP-07. Figure 4.13: Effect of activators and reducing agents on Activity of Purified Protease, PSA BP-07 The values are represented as the mean of three replicates ± SD (p<0.05) 81

23 Determination of Kinetic Parameters and Substrate Specificity Substrate specificity analysis and rate of the enzymatic reaction was measured using gelatin and casein as substrates. The Michaelis-Menten (K M ) constant, maximum reaction velocity Vmax, and the reaction specificity Vmax/K M were determined by plotting the activity data at optimum ph and temperature, as a function of substrate concentration according to Lineweaver Burk plot (Figure 4.14 and 4.15). The enzyme kinetic constants K M and V max are good estimates to determine substrate specificity of an enzyme. K M value for the protease PSA BP-07 varied with different substrates, indicating the specificity of the enzyme towards the substrate. The apparent K M and V max of the enzyme for gelatin was found to be mg/ml and for casein. The most effective substrate was determined by calculating V max /K M, which is known as enzyme catalytic power. The catalytic power of protease PSA BP-07 towards gelatin was found to be 5.50 and 2.20 for casein. The summary of kinetic constants K M and V max for hydrolysis of casein and gelatin substrates by the purified protease was shown in Table 4.2. From the enzyme kinetic studies, the protease PSA BP-07 has shown a lower K M and high V max with gelatin, signifying that the protease has strong affinity and high catalytic efficiency towards gelatin when compared to casein. Table 4.2 Substrate specificity analysis - Enzyme kinetics constants S.no Substrate K M (mg/ml) V max (U/mg/min) 1. Gelatin Casein From the results gelatin was found to be the suitable substrate for protease activity when compared to casein. Corzo et al., (2012) reported the enzyme kinetic studies of fruit bromelain from A.comosus exhibiting highest results of Vmax and lower results of K M towards the substrates azocasein and azo albumin. Similar results were stated by Silva et al., (2014) by determining the kinetic parameters of bromelain from Ananas erectifolius. 82

24 Figure 4.14 Linearization of the kinetic data of hydrolysis of gelatin to determine the constants by the method of Lineweaver-Burk (LB). LB double reciprocal plot of protease PSA BP-07 from B. pyramidalis. The intercept on x- axis related to -1/K M and the intercept on y-axis is related to 1/Vmax. Data presented is an average of 3 replicates ± S.D. 83

25 Figure 4.15 Linearization of the kinetic data of hydrolysis of casein to determine the constants by the method of Lineweaver-Burk. LB double reciprocal plot of protease PSA BP-07 from B. pyramidalis. The intercept on x-axis relates to -1/K M and the intercept on the y-axis relates to 1/Vmax. Data presented is an average of 3 replicates ± S.D. 84

26 4.3.4 Spectroscopic analysis of purified protease, PSA BP-07 Organic molecular groups and functional groups, involved in the compound are identified using FTIR and NMR Spectroscopy FTIR Spectroscopy The dry protein sample was used for the analysis of FTIR spectroscopy, by employing standard KBr pellet or disc method. The IR absorption spectrum of the purified protease PSA BP-07 was shown in Figure The absorption spectrum shows strong peak at , , cm 1 attributed to the O H stretching vibration and peaks at , cm 1 assigned to C O stretching vibration, thereby indicating the presence of carbonyl compounds in the protein. The C-N stretch vibration frequencies observed at , , cm 1 confirmed the presence of aliphatic groups (CH 2 - S-) and C-S stretch at cm 1 confirms the existence of thiol or thio ether group in the protein. Depending on the fingerprint characters of the peaks positions, shapes and intensities, the other functional groups present in the protein are identified and listed in the Table 4.3. Presence of C O group and C-N stretching in the protein sample confirms the presence of amino acids and presence of amine group in their side chain. As there is no absorbance in between the region cm -1, indicates that there is no cyanide groups present in the protein. Similar functional groups were also observed in bromelain proteases from Ananas comosus with very slight variation in IR bands both in terms of intensity and frequency (Chandrasekar et al., 2012; Swaroop et al., 2013). 85

27 Table 4.3 Identified functional groups of protein from FTIR spectra. S.no Frequency Range Functional groups cm 1 (C-Br) alkyl halide stretch , , cm 1 C-N stretch cm 1 CH 2 - S-) C-S stretch , cm 1 (C-Cl) alkyl halides stretch cm 1 (C-H ) aliphatic stretch cm 1 1º and 2º amines N-H wag , , cm 1 nitrosamine N-N stretch , , cm 1 N O Bend cm 1 (C-CHO) aliphatic bending , cm 1 (N-O) symmetric stretch , cm 1 C O stretching vibration , , cm 1 (C=O) transition metal carbonyls stretch cm 1 (COOH ) Carboxylic acid stretch , , cm 1 (O H) stretching vibration 86

28 Figure4.16: The Infrared absorption spectrum of the purified protease, PSA BP

29 NMR Spectrum The interpretations of structural information for the protease PSA BP-07 was annotated mainly by NMR spectroscopy. The 1 H NMR and 13 C NMR spectrum of protease PSA BP-07 was shown in Figure 4.17 and The 1 H chemical shifts values at δ 0.978, 3.660, and ppm can be attributed to CH 3, CH 2 and OH of carbohydrate backbone in the spectra clearly indicated the presence of carbohydrate moiety in the protease. The shifts at δ , δ 59.7, δ 27.7 can be assigned to C-OH, CH 2, CH 3 in the 13 C spectra and shifts at δ 3.274, δ 3.573, δ 3.711, δ 7.31 in 1 H spectra confirms the presence of cysteine in the purified protease. Figure 4.17: 1 H NMR spectrum of the purified protease, PSA BP

30 Figure 4.18: 13 C NMR spectrum of the purified protease, PSA BP CONCLUSION The protease extracted from the leaves of Billbergia pyramidalis was purified to homogeneity by (NH 4 ) 2 SO 4 precipitation (40-60% saturation), Sephadex G-150 gel filtration chromatography, CM cellulose ion-exchange chromatography and Mono S column chromatography. The purified protease was found to exhibit higher enzyme activity with an overall yield of 16.58% and fold purification. The purified enzyme was coded as PSA BP -07. The purity of the protein was analyzed by HPLC analysis, which has shown a sharp peak at retention time of 3.02 min. The purified protease migrated as a single band when it was subjected to SDS-PAGE and the molecular mass of protease was found to be ~37 kda. Later the purified enzyme was subjected to zymogram analysis and the proteolytic activity of the protein is indicated by the white band over blue background on an agarose gel. 89

31 The optimum temperature required for enzyme activity was found to be 40 C and was 100% stable for 2 h. The optimum ph for enzyme activity was found to be 7.0 and was stable at a ph range 6.0 to 8.0. The divalent metal ions Mg 2+, Ca 2+ enhanced relative activity of the enzyme while Hg 2+ and Na 2+ inhibited the enzyme activity. Strong inhibition of enzyme activity Hg 2+ clearly confers the thiol nature of the enzyme. Iodoacetamide (IAM) and Diethylpyrocarbonate (DEPC) completely inhibited the proteolytic activity at 10mM concentration inferring that the purified protease contain histidine at the active site along with cysteine. The enhanced proteolytic activity in the presence of activator sodium metabisulfite and reducing or sulfhydryl reagents DTT clearly established the thiol nature of the protease PSA BP-07. These results indicate that the purified protease, PSA BP -07 belongs to cysteine protease family. The apparent Km and Vmax of the enzyme for gelatin was found to be mg/ml and U/mg/min respectively. Organic molecular groups and functional groups, involved in the compound are identified using FTIR and NMR Spectroscopy. IR band at cm -1 confirmed the existence of Thiol group in the protein and the presence of C O and C-N stretching groups in the protein sample confirmed the presence of amino acids and amide group in their side chain. 1 H and 13 C NMR spectra also confirmed the presence of cysteine and carbohydrate moiety in the purified protein sample. Considering the properties of the reported enzyme PSA BP -07 with regard to activity, substrate specificity, stability and sustainability to various ph and temperature conditions, the protease from B. pyramidalis may turn out to be an essential plant protease with extensive use in food, biotechnological and pharmaceutical industries. 90