SUPPLEMENTARY MATERIAL Purification and biochemical properties of SDS-stable low molecular weight alkaline serine protease from Citrullus Colocynthis Muhammad Bashir Khan, 1,3 Hidayatullah khan, 2 Muhammad Usman Shah, 1 Sanaullah Khan*, 1 1 Department of Bioscience, COMSATS Institute of Information Technology Islamabad, Pakistan. 2 Department of Chemistry, University of Science and Technology, Bannu, Pakistan 3 Present address: Department of Biochemistry, University of Alberta, Canada.. * Author to whom correspondence should be addressed: Tel: +45-53330510, Fax: +92-519235033 Email: sanaullah.khan@comsats.edu.pk Abstract A low molecular weight serine protease from seeds of Citrullus colocynthis was purified to electrophoretic homogeneity with high level of catalytic efficiency (22945 M -1 S -1 ). The enzyme was a monomer with molecular mass of 25 kda estimated by SDS-PAGE. The enzyme was highly active over a ph range of 6.5 to 9.0, with maximum activity at ph 7.5, and was highly stable over a broad temperature range of 20 to 80 C, with an optimum temperature of 50 C. The Km, Vmax and Kcat for casein were 73 μg/ml, 557 U/min/mg and 67/sec respectively. The enzyme was strongly inhibited by PMSF, moderately by soybean trypsin inhibitor, indicating that the enzyme was a serine protease. The enzyme retained 86% and 73% of its initial activity in the presence of Urea and DTT respectively, and its activity was slightly enhanced in the presence of anionic detergent (SDS), indicating that the enzyme was highly active and stable in the presence of these protein perturbing agents. Thus this enzyme is a novel SDS-stable protease with high catalytic efficiency over wide ranges of ph and temperature which is commercially promising for various industrial applications. Keywords: Citrullus colocynthis; serine protease, purification, biochemical characterization, thermostability, detergent stability. 1
Experimental Materials The dried gourds of C. Colocynthis were collected from desert areas of Bannu, Khyber Pakhtunkhwa province (KPK), Pakistan and were identified by Prof. Sultan Mehmood, Department of Botany, University of Science and Technology, Bannu (USTB), KPK, Pakistan. A voucher specimen (BG-135B) has been deposited to USTB herbarium. Sephadex G-50 was purchased from Pharmacia, gelatin from Merck while the rest of chemicals were purchased from Sigma. Seeds extraction The dried gourds were crushed and the seeds were separated. About 30 g seeds were soaked in 50 mm phosphate buffer ph 7.0 for 30 min, air dried and were ground to fine powder. The dried powder was defatted with chilled acetone (-20º C) for 15 min. The homogenate was filtered and air dried. The defatted powder was suspended in 50 mm Tris-HCl buffer ph 8.5 containing 2 M NaCl overnight at 4ºC. The crude extract was filtered by whatman filter paper and centrifuged at 15,000 rpm at 4ºC for 15 min. Protein estimation and enzyme assay Protein concentration was determined at each step of purification by Bradford method using BSA as a standard. The enzyme activity was measured according to the method as previously described (15) with slight modifications. The reaction mixture consisting of 150 µl of enzyme solution, 100 µl of 1% casein (w/v) in 50 mm Tris-HCl buffer ph 8.0 and 1 ml of 50 mm Tris-HCl buffer ph 8.0 was incubated at 37ºC for 30 min. The reaction was stopped by the addition of 200 µl 40% TCA, incubated on ice for 40 min and then centrifuged at 14000 rpm for 10 min. The control was prepared in the same quantity but the enzyme was added to the mixture of substrate and TCA. The TCA soluble fractions were measured at 280 nm. The protease activity was expressed as the difference of absorbance at 280 nm between the control and the sample. One unit of proteolytic activity was defined as the amount of enzyme required to increase the absorbance by 0.001 under the conditions previously described (15). Enzyme purification For differential precipitation, solid ammonium sulfate was added slowly (30 to 70%) to the seed extract with constant stirring at 4 o C. The mixture was allowed to stand for 1 hr at 4 o C and then centrifuged at 10000 rpm for 15 min at 4 o C. The precipitates were dissolved in 50 mm 2
Tris-HCl buffer ph 7.4 and then applied on sephadex G-50 column (2.5 47 cm) equilibrated and eluted with 50 mm Tris-HCl buffer ph 8.5. Elution was performed at flow rate of 12 ml/hr and 3 ml fractions were collected. The enzyme active fractions from sephadex column were pooled and applied on DEAE-Cellulose column (2.5 10 cm) previously equilibrated with 50 mm Tris-HCl ph 8.5. The bound proteins were eluted with linear gradient of 0.0-0.5 M NaCl. These purification steps were repeated twice in order to collect enough amount of proteins for biochemical characterization. Determination of molecular mass and zymography SDS-PAGE consisting of 4% stacking and 12% resolving gel was run according to Laemmli method (16) under both reducing and non-reducing conditions. The relative molecular mass of the enzyme was estimated by comparing its relative mobility with known molecular markers (130-14 kda) on SDS-PAGE. Gelatin zymography was performed on PAGE containing 1% gelatin as a co-polymerized substrate according to the method described previously (17), with slight modifications. After electrophoresis, the gel was washed with 2.5 % Tritron X-100 three times for 20 min and incubated in 50 mm Tris-HCl buffer ph 8.0 containing 0.1 M NaCl and 1 mm CaCl 2 for 5 hr at 37ºC. The gel was then stained with coomassie brilliant blue R-250, and the activity band was observed as white zone on a dark blue background. Characterization of enzyme For the determination of optimum ph, the enzyme activity was assayed in 50 mm phosphate (6.5-7.0) and 50 mm Tris-HCl (ph 7.5 to 10) using casein as a substrate at 37ºC. To determine the optimum temperature, reaction mixture was incubated at various temperatures (20-80 C) under standard assay conditions. For thermal stability determination, the purified enzyme was first incubated at various temperatures ranging from 20ºC to 80ºC for 1 hr. After incubation, casein solution was added to start the reaction under standard assay condition. The effects of metal ions, inhibitors, protein perturbing agents and detergents on the enzyme activity were investigated. The enzyme was incubated with each metals and compounds at different concentrations for 20 min at 30ºC followed by the measurement of residual activity under the standard assay conditions. The substrate specificity of the purified protease towards different native and modified substrates was also examined. The Km and Vmax of the purified enzyme for casein were also measured under standard assay conditions. 3
References Anson ML. 1938. The estimation of pepsin, trypsin, papain, and cathepsin with hemoglobin. J. Gen Physiol. 22: 79-89. Heussen C. Dowdle EB. 1980. Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Anal. Biochem. 102: 196-202. Laemmli UK. 1970. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature (London). 227: 680-685. 4
Table S1. Summary of enzyme activity of C. Colocynthis SS LMW AS-protease at various purification steps measured against casein. Purification Steps Total Protein Total activity Specific Activity Purification Recovery (mg) (Units) (Units/mg ) fold % Crude 270 642 2.4 1 100 (NH 4 ) 2 SO 4 Precipitation 180 560 3.1 1.3 87.4 Sephadex G-50 100 522 5.2 2.1 81.3 DEAE Cellulose 0.31 157 514 216 24.5 5
Table S2. Substrate specificity of C. Colocynthis SS LMW AS-protease for several natural and modified proteins. Substrate Concentration (%) Wavelength (nm) Relative activity (% ) Casein 1 280 100 Haemoglobin 1 280 130 Gelatin 1 280 67 BSA 1 280 53 Azocasein 1 440 33 Azoalbumin 1 440 30 6
Table S3. Effect of various metal ions and compounds on C. Colocynthis SS LMW AS-protease activity Metal ions Concentration (mm) Relative activity (%) None 100 CaCl 2 2 80 MgCl 2 2 110 CuSO 4 2 89 ZnCl 2 2 104 LiCl 2 120 SDS 2.5 (~0.1%) 107 10 (~0.3%) 111 Urea 5 86 10 63 DTT 5 73 EDTA 0.5 73 1 65 PMSF 0.125 90 0.250 75 0.50 61 1 17 2 7 Soybean trypsin inhibitor 0.1 (mg/ml) 82 7
Fig. S1 (A) Optimum ph of C. colocynthis SS LMW AS-protease. The activity of enzyme was assayed in 50 mm phosphate (6.5-7.0) and 50 mm Tris-HCl (ph 7.5 to 10) using casein as a substrate. (B) Optimum temperature (filled circles) and Thermal stability (open circles) of C. colocynthis SS LMW AS-protease. The activity of enzyme was assayed against casein in 50 mm Tris-HCl ph 8.5 at various temperatures (20-80 C). The residual activity of enzyme was determined using standard enzyme assay after 1 hr pre-incubation at various temperatures (26-80 C). (C) Michaelis-Menten enzyme kinetics of SS LMW AS-protease. The activity was measured with casein concentrations (8.5-1088 μg/ml). 8