Biochemical Characterization of Protease Isolated from Different Parts of Choerospondias Axillaris (Lapsi)

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o DOI: 10.4172/2161-1009.1000135 i y B ISSN: 2161-1009 Analytical Biochemistry

Research Article Open Access Biochemical Characterization of Protease Isolated from Different Parts of Choerospondias axillaris (Lapsi) Shrawan Kumar Upadhyay*, Rubin Thapa Magar and Chandan Jung Thapa Department of Biochemistry, Universal Science College, Chakupat, Lalitpur, Nepal

Abstract Proteases were isolated from leaf, bark and root of Choerospondias axillaris, locally called Lapsi. Choerospondias axillaris is a dioecous, deciduous fruit bearing plant with multiple daily uses. The protease was extracted with 0.1 M phosphate buffer of pH 7 and then precipitated successively with TCA and ammonium sulfate. The protease from leaf showed maximum activity at pH 9, temperature at 20°C. While, bark protease showed optimum pH 5 and temperature 60°C. In case of root the optimum pH was 10 and the optimum temperature 35°C. The optimum time of incubation leaf, bark and root was 15 minute. The bell shaped curve was obtained for the effect of enzyme conc. with optimum enzyme

conc. 50 µg for leaf, 30 µg for bark and 50 µg for root. The Km and Vmax value of leaf were 5.61 µM and 185.18 pmol/

min, Km and Vmax value of bark were 2.36 µM and 82.64 pmol/min. While for root, the Km and Vmax values were 1.53 µM and 52.91 pmol/min.

Keywords: Protease; Lapsi; Purification; Characterization TCA precipitation of the crude extracts Introduction Filtrates i.e., crude extracts were then subjected to TCA precipitation [6]. 2.45 M of TCA solution was used to precipitate the crude extract Choerospondias axillaris (locally called “lapsi”) is a large, deciduous so as to make final concentration of TCA 0.2 M. Then samples were fruit-bearing dioecous tree of the family Anacardiaceae. A native of subjected to centrifugation at 10000 rpm for 15 min at 4°C. After the Nepal hills (850-1900 m). The tree has also been reported from centrifugation, the pellets obtained were dissolved on chilled acetone India, China, Thailand, Japan and Vietnam. Lapsi wood is used as light and again centrifuged at 14000 rpm for 5 min at 4°C and repeated twice construction timber and fuel wood; seed stones are used as fuel in brick to remove remaining TCA. Thus obtained pellets were dissolved in 0.1 kilns and the bark has medicinal value for treating secondary burns [1]. M phosphate buffer of pH 7 and were used for protein estimation and Nepal is unique in processing and utilizing lapsi fruits. Lapsi fruit is enzyme assay. consumed fresh, pickled or processed into a variety of sweet and sour fruit products locally called “Titaura” and Candy. The fruits are rich in Ammonium sulfate salt precipitation of TCA precipitated vitamin C content [2]. Though the tree is dioecous, but it is difficult to extracts distinguish male and female plants at the seedling stage [3]. The TCA precipitated extracts were precipitated by ammonium Agrawal et al. [4] were first to observe strong proteolytic activity in sulfate (20-80% w/v). The solutions were kept on for an hour in cold Lapsi’s leaves. Dekhang and Sharma reported optimum pH of 7 for the condition (4°C) and then centrifuged at 10000rpm for 15 min at protease [5]. The protease is not inhibited by phenyl-methane-sulfonyl- 4°C. The precipitates were dialyzed against 0.1M phosphate buffer of fluoride (PMSF) and somewhat about 20-30% inhibited by sodium pH 7 [7]. Following dialysis the fractions of samples were mixed and iodoacetic acid, thus revealing that protease is not a serine protease [5]. these extract (ammonium sulfate precipitated) were used for protein No smaller proteolytic products of BSA could be seen in SDS-PAGE estimation and enzyme assay. using silver staining indicating that the protease is not exopeptidase, Protein estimation of different fractions of the sample Protease activity can be repeatedly precipitated by 0.2 M tricloroacetic acid TCA [6]. The present study was conducted to isolate, purify and by Bradford assay characterize the proteases from different parts of the lapsi plant. Bradford assay was carried out to determine the protein concentration of crude, TCA precipitated and ammonium sulfate Experimental Procedure precipitated extracts of leaf, bark and root [8]. Using the standard Materials calibration curve of BSA the amount of BSA in different fractions was calculated. (Note: molecular weight of BSA 69323.4 Da [9]). Leaf, bark and root of Choerospondias axillaris were collected from Sirutaar area Bhaktapur, Nepal. All other reagents used were of analytical grade. *Corresponding author: Shrawan Kumar Upadhyay, Department of Preparation of the crude extracts Biochemistry, Universal Science College, Chakupat, Lalitpur, Nepal, E-mail: [email protected] Samples were shed dried and grinded to fine powder. 24, 23 and 10 Received April 18, 2013; Accepted May 22, 2013; Published May 25, 2013 g of leaf, bark and root powder respectively were washed with 100 ml of acetone. Then sample was dried to remove remained acetone. The Citation: Upadhyay SK, Magar RT, Thapa CJ (2013) Biochemical Characterization of Protease Isolated from Different Parts of Choerospondias axillaris (Lapsi). acetone washed sample was heated at 70°C on water bath with 0.1 M Biochem Anal Biochem 2:135. doi:10.4172/2161-1009.1000135 phosphate buffer of pH 7; 200 ml for leaf and bark samples and 100 ml for root samples. After 30 min of heat treatment samples were filtered Copyright: © 2013 Upadhyay SK, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits and the filtrates were used as crude extracts. Thus obtained extracts unrestricted use, distribution, and reproduction in any medium, provided the were used for protein estimation and enzyme assay. original author and source are credited.

Biochem Anal Biochem ISSN:2161-1009 Biochem, an open access journal Volume 2 • Issue 3 • 1000135 Citation: Upadhyay SK, Magar RT, Thapa CJ (2013) Biochemical Characterization of Protease Isolated from Different Parts of Choerospondias axillaris (Lapsi). Biochem Anal Biochem 2:135. doi:10.4172/2161-1009.1000135

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Protease assay of different fractions of the samples Statistical Analysis The protease activity of crude, TCA precipitated and ammonium All data were computed from the mean of three independent sulfate precipitated extracts of leaf, bark and root were calculated with experiments and expressed as mean ± SD. Statistical analysis was BSA as a substrate by direct method. A reaction mixture of 200 µl was carried out using GraphPad Prism 6 software. made with BSA (1 mg/ml), extract and 0.1M phosphate buffer of pH 7. The reaction mixture was then incubated. The reaction mixture was Results then quenched with 2.3 ml of Bradford reagent and the absorbance was The protein concentration, purification steps and specific activity measured at 595 nm. The amount of BSA, enzymes, buffer, along with all the fractions of each samples can be summarized in the tables 1-3. incubation time and temperature varied with different experiments that The activity of each fraction of the samples is shown in the figure 1. were done. However 2 controls a) enzymes only and b) BSA only, both without incubation was taken in each case. The calculation performed Optimum pH henceforth is control dependent. Optimal proteolytic activity for leaf was obtained at pH 9 and comparable activities were also seen at pH 3, and pH 6 (Figure 2A), Characterization of protease of 20-80% ammonium while for bark the optimal activity was seen at pH 5 and comparable sulfate precipitated extracts activity was also seen at pH 2 (Figure 2B). In case of root the maximum The 20-80% ammonium sulfate precipitated extract of each sample- activity was seen at pH 10 and comparable activities were seen too at leaf, bark and root were characterized with BSA as a substrate. The effect pH 2, pH 6 and pH 8 (Figure 2C). of pH, temperature, time of incubation, enzyme concentration and the Optimum temperature substrate concentration on protease activity were measured. The optimum temperature of leaf extract was found to be 20°C Assessment of kinetic constants of enzymes of 20-80% (room temperature). The activity gradually decreased with increase in ammonium sulfate precipitated extracts temperature upto 80C and then activity got rised slightly (Figure 3A). The optimal temperature for bark was found to be 60°C (Figure 3B) and The reciprocal of reaction velocity was plotted against the reciprocal for root the optimal temperature was 35°C (Figue 3C). of the corresponding substrate concentration, giving the Lineweaver- Burk plot for the enzyme with the substrate BSA. The Michaelis-Menten Optimum time of Incubation constant (Km) and maximum reaction velocity (Vmax) of enzyme were The proteolytic activity of leaf, bark as well as root were maximum determined from the Lineweaver-Burk plot [10]. at 15 min and then decreased gradually (Figure 4A -4C).

B ra d fo r d A s s a y o f C r u d e e x tr a c ts B r a d fo r d A s s a y o f T C A p p t E x tr a c ts A r a d fo rd BA s s a y o f a m o n iu m s u lfa te p r e c ip ita te d e x tra c ts B C 0 .5 0 .5 Y = 0 .0 0 3 8 0 9 *X + 0 .4 0 .4 0 .4 0 .0 0 2 5 7 1 e e c

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Figure 1: Standard calibration curve of BSA for Bradford assay. (A): crude extracts with 15, 30 & 30 µl of leaf, bark and root samples respectively taken. (B): TCA ppt. extracts with 10, 20 & 20 µl of leaf bark and root samples respectively taken and (C): 50 µl of ammonium sulfate ppt. extracts were mixed with phosphate buffer to maintain the volume 1ml. 5ml Bradford reagent was added in each. After 5 min of incubation absorbance was taken at 595 nm.

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p 1 0 .0 ( ( ( y y y t t t i i 2 0 2 0 i v v v i i 9 .5 i t t t c c c A A A 1 8 1 8 9 .0 0 5 1 0 1 5 0 5 1 0 1 5 0 5 1 0 1 5 p H p H p H Figure 2: Effect of pH on protease activity of ammonium sulfate extract. (A): leaf, (B): bark and (C): root extract. Reaction mixture contained 25 µg BSA (1 mg/ml), 12 µl enzyme (28.56 µg) for leaf, 19 µl enzyme (11.78 µg) for bark and 22 µl enzyme (13.42 µg) for root were taken and volume was maintained to 200 µl buffers of different pH. After 30 min incubation at room temperature direct method was performed.

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Figure 3: Effect of temperature on protease activity. (A) leaf extract, (B) bark extracts and (C) root extracts. The enzymes were incubated at different temperature. 200 µl reaction system was made with 12 µl of enzyme, 50 µg of BSA for leaf, 19 µl of enzyme, 25 µg of BSA for bark and 22 µl of enzyme, 25 µg of BSA for root and appropriate amount of buffer. Direct method was performed after 30 min of incubation. )

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Figure 4: Effect of time of incubation on protease activity. (A) leaf extracts, (B) bark extracts,and (C) root extracts. 200 µl of reaction system containing 50 µg of BSA (1 mg/ml) 12 µl of enzyme for leaf, 25 µg BSA and 19 µl enzyme for bark and 25 µg of BSA and 22 µ enzyme for root extracts were incubated for different time intervals and direct method was performed.

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Figure 5: Effect of enzyme concentration on protease activity. (A): leaf, (B): bark and (C): root. 50 µg of BSA for leaf and 25 µg of BSA for bark and root (1 mg/ml) with varying amount of protease was added. The final volume was made 200 µl by adding phosphate buffer. It was the incubated for 30 min at room temperature and direct method was performed.

Optimum amount of enzyme and Vmax values for root were 1.53 nM and 52.91 pmol/min respectively (Figure 6). The effect of enzyme on protease activity of leaf extract was found to increasing linearly upto 50 µg of enzyme and then decreased (Figure Discussions 5A), while in case of bark the optimal enzyme concentration was In leaf and bark, the decrease in specific activity of TCA precipitated approx. 25µg (Figure 5B). The optimal concentration of enzyme for and again increase in specific activity during (NH ) SO salt precipitation root was found to be 39 µg (Figure 5C). 4 2 4 indicates significant loss of protease activity during TCA precipitation. Effect of substrate concentration on protease activity and However in root, there is successive increase in specific activity from Kinetic constants crude extract to (NH4)2SO4 salt precipitation indicates successive purification of protease. The high optimal temperature of bark protease Lineweaver-Burk plot was used to determine the Km and Vmax values indicates that the bark protease is quite more thermostable than other of lapsi root protease. The mK and Vmax values were determined to be proteases this may be due to presence of higher ammount of cysteine 5.61 nM and 185.16 pmol/min respectively for leaf, while for bark Km residues. Beyound these optimal temperatures the activities were and Vmax values were 2.36 nM and 82.64 pmol/min respectively. The mK decreased due to denaturation of proteases due to heat.

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Figure 6: Effect of substrate concentration on protease activity. (A) leaf, (B) bark and (C) root extracts. The reaction mixture contained 12 µl, 19 µl and 22 µl protease for leaf, bark and root respectively with different amount of substrate (BSA). Phosphate buffer (pH 7, 0.1M) was added to make final vme 200 µl. The reaction mixture was incubated at room temperature for 30 min and direct method was performed.

Volume Protein concentration Total protein Activity (nmol Total activity Specific activity Purification Overall Purification step (ml) (mg/ml) (mg) min-1 ml-1) (nmol min-1 ) (nmol min-1 mg-1 ) factora yield (%)b Crude extract 152 2.06 ± 0.13 313.79 ± 1.33 1.494 ± 0.03 227.15 ± 4.67 0.74 ± 0.01 1 100 TCA ppt. extract 8 2.66 ± 0.56 21.29 ± 0.46 1.74 ± 0.07 13.92 ± 0.56 0.654 ± 0.03 0.88 6.13 20-80% ammonium 9 2.1 ± 1.24 17.494 ± 1.63 1.707 ± 0.01 15.36 ± 0.14 0.883 ± 0.07 1.19 6.76 sulfate ppt. extract Table 1: Summary of protease purification of leaf.

Protein concentration Total protein Activity Total activity Specific activity Overall yield Purification step Volume (ml) Purification factora (mg/ml) (mg) (nmol min-1 ml-1) (nmol min-1 ) (nmol min-1 mg-1 ) (%)b Crude extract 148 1.02 ± 0.66 150.38 ± 3.26 0.663 ± 0.04 98.062 ± 6.49 0.653 ± 0.05 1 100 TCA ppt. extract 3 1.93 ± 1.28 5.784 ± 0.19 0.883 ± 0.005 2.643 ± 0.01 0.458 ± 0.01 0.7 2.7 20-80% ammonium 3.7 0.61 ± 0.56 2.257 ± 0.04 0.469 ± 0.002 1.336 ± 0.008 0.769 ± 0.01 1.18 1.8 sulfate ppt. extract Table 2: Summary of protease purification of bark.

Protein concentration Total protein Activity (nmol Total activity Specific activity Overall yield Purification step Volume (ml) Purification factora (mg/ml) (mg) min-1 ml-1) (nmol min-1 ) (nmol min-1 mg-1 ) (%)b Crude extract 43 1.94 ± 1.2 83.33 ± 1.72 1.096 ± 0.01 4712 ± 0.49 0.566 ± 0.01 1 100 TCA ppt. extract 3 1.35 ± 0.58 4.05 ± 0.09 0.986 ± 0.01 2.957 ± 0.04 0.731 ± 0.01 1.29 6.28 20-80% ammonium 6.2 0.57 ± 0.59 3.52 ± 0.07 0.695 ± 0.01 4.213 ± 0.04 1.225 ± 0.04 2.16 8.94 sulfate ppt. extract Note: aDefined as: purification factor=(specific activity of fraction/specific activity of homogenate). bDefined as: overall yield=total activity of fraction/total activity of homogenate)×100. Table 3: Summary of protease purification of root.

The bell shaped curve obtained for effect of enzyme concentration Chakupat, Lalitpur, Nepal for providing the required facilities to carry out this on protease activity indicated that the enzume concentration inhances research work. the rate of reaction only upto optimal concentration and then started Recommendations to inhibit the rate of reaction due to over saturation of enzyme. The low Biochemical determination of type of protease present in different parts of Km value of proteases indicated that the substrate is tightly bound to Lapsi plant can be done. Phytochemical screening, Antioxidant and Antimicrobial enzume. Activity can be checked in different part of the Lapsi plant. Conclusion References The protease from leaf, bark and root of Choerospondias axillaries 1. Nguyen DD, Nguyen NH, Nguyen TT, Phan TS, Nguyen VD, et al. (1996) The (Lapsi) have been successively purified to homogeneity by TCA and use of a water extract from the bark of Choerospondias axillaris in the treatment of second degree burns. Scand J Plast Reconstr Surg Hand Surg 30: 139-144. (NH4)2SO4 salt and characterizes. All proteases were actively working for up to 15 minutes. The bark protease is quite more thermostable than 2. Shah DJ (1978) Ascorbic acid (Vitamin C) content of Lapsi-pulp and peel at that of leaf and root. Leaf and root proteases were alkaline while bark different stage of maturation. Research Bulletin 2035 B. S. Food Research protease was acidic. The root has more affinity for substrate than other Section, HMGN, Department of Food and Agricultural Marketing Services, Kathmandu. indicated by lowest Km value. 3. Agrawal GK, Khandka DK, Jha B, Thapa A, Tamrakar S, et al. (1991) Studies Acknowledgement on methods for early sex determination of Choreospondias axillaris a fruit The authors are thankful to the authority of Universal Science College bearing Himalayan tree. Biotechnology Letters (Nepal) 1: 23-28.

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4. Agrawal VP, Keshari A, Singh D (1992) Study of Lapsi (Choreospondias 7. McPhie P (1971) Dialysis. Meth Enzymol 22: 23-33. axillaris) protease. Unpublished result. 8. Bradford MM (1976) A rapid and sensitive method for the quantitation of 5. Dekhang RN, Sharma G (2006): Study of Protease from the leaves of microgram quantities of protein utilizing the principle of protein-dye binding. Choreospondias axillaris (Lapsi). Submitted to Universal Science College, Anal Biochem 72: 248-254. Biochemistry Department for the fulfillment of degree requirement of B.Sc. 9. Hilger C, Grigioni F, De Beaufort C, Michel G, Freilinger J, et al. (2001) Biochemistry. Differential binding of IgG and IgA antibodies to antigenic determinants of bovine serum albumin. Clin Exp Immunol 123: 387-394. 6. Singh R (2007) Purification and Characterization of leaves protease from Choerospondias axillaris (Lapsi). Submitted to Universal Science College, 10. Lineweaver H, Burk D (1934) The determination of enzyme dissociation Biochemistry Department for the fulfillment of degree requirement of B.Sc. constants. J Am Chem Soc 56: 658-666. Biochemistry.

Citation: Upadhyay SK, Magar RT, Thapa CJ (2013) Biochemical Characterization of Protease Isolated from Different Parts of Choerospondias axillaris (Lapsi). Biochem Anal Biochem 2:135. doi:10.4172/2161-1009.1000135

Biochem Anal Biochem ISSN:2161-1009 Biochem, an open access journal Volume 2 • Issue 3 • 1000135