Physio-chemical Characterization and Anti-microbial Activity of Serine Protease Inhibitors Purified from the Sophora japonica Seeds

MATERIALS AND METHODS

The work was carried out in Protein Biochemistry Lab, Department of Biochemistry, University of Kashmir, Jammu and Kashmir, India over the period of 2 years from December 2014 to January 2017.

Isolation and purification of protease inhibitors from Sophora Japonica seeds: Mature seeds of locally available legume plants Sophora japonica were selected and procured from the Department of Botany, University of Kashmir. Seeds were shade dried before processing. All extraction processes were carried out at 4°C. Dried seeds (25 g) of the plant were soaked in NaCl (15N) for 24 h. They were then homogenized in ice cold saline-Tris buffer (20 mM Tris, pH 8.0) containing 1 mM sodium metabisulphite at 4°C for 10 min. The filtered homogenate was subjected to centrifugation at 12,00 g for 20 min, the pellet discarded and supernatant retained and stored at 4°C. The proteins were precipitated from the supernatant by 90% saturation of ammonium sulphate and again centrifuged at 12,000 g for 10 min. The pellet formed was dissolved in the minimum volume of double distilled water and dialyzed, against 20 mM Tris buffer, pH 8. Further purification was done on Diethylaminoethyl Cellulose (DEAE-Cellulose) column based ion-exchange chromatography, using Tris-buffer (20 mM, pH 8). The protein was eluted using the linear sodium chloride gradient from 0.05-0.6 M. The eluted fractions were then chromatographed separately on Sephadex G-100 column, using Tris-buffer (0.1 M, pH 8) and NaCl (0.4 M). The eluted fractions were checked for protein concentration by measuring absorbance at 280 nm.

The protein fractions on Sephadex G-100 column resolved into two peak fractions which were named as SJ-pi I (Sophora japonica protease inhibitor I) and SJ-pi II (Sophora japonica protease inhibitor II), respectively.

SDS polyacrylamide gel electrophoresis: Fractions eluted from Sephadex Gel chromatography were checked by SDS PAGE. Molecular weight of purified inhibitors was also determined by SDS-PAGE. For this a protein ladder of known molecular weight was electrophorised along with purified protease inhibitors.

Measurement of trypsin inhibitory activity: The biological activity of inhibitors was measured by the degree of inhibition of tryptic activity using BAPNA as substrate. Different volumes of purified inhibitors were incubated with 10 mg of trypsin (diluted in 1 mM HCl) and with variable volumes of the 0.1 M Tris-HCl buffer, pH 8.0 containing 0.01 M CaCl₂. The final volume was made up to 500 μL. The contents were incubated for 10 min at 37°C and 500 μL of N-Benzoyl-DL-arginine-4-nitroanilide hydrochloride (BAPNA) (8 mM in DMSO) was added. The reaction mixture was incubated for 40 min at 47°C and reaction stopped by the addition of 500 μL of acetic acid (30% v/v). The control, lacking inhibitor was prepared under exactly identical conditions. Blank for each volume of the inhibitor tested was obtained by the addition of acetic acid just before the incubation with the substrate BAPNA¹⁵. The intensity of the yellow color produced was measured by recording the absorbance at 405 nm. Residual trypsin activity was calculated using the following equation:

Residual trypsin activity (%) = Abs at 405 nm in contro l- Abs at 405 nm in sample×100
Inhibition (%) = 100-Residual trypsin activity (%)

Measurement of chymotrypsin inhibitory activity: Inhibition of chymotrypsin activity was determined on acetyl L-Tyrosine ester (ATEE). Again, different volumes of each inhibitor was pre incubated with 10 μg of enzyme solution of 5 min at room temperature in 1 mL of 46 mM Tris 0.5 mM CaCl₂ buffer, pH 8.1 as per the protocol given by Birk et al.¹⁶. The reaction was started by the addition of 3 mL of substrate solution to pre incubated enzyme plus inhibitor solution. Blank was prepared for each volume of extract tested by omitting the enzyme from the reaction mixture. The intensity of yellow color produced was measured by recording the absorbance at 410 nm against an appropriate blank. Control, where chymotrypsin was not pre-incubated with inhibitor was also prepared. Percent inhibition of chymotrypsin activity was calculated by using following relationship:

Effect of pH and temperature on inhibition activity of Protease inhibitors: To determine the pH optima, 50 μL purified protease inhibitors were incubated at 30°C in various pH adjusted buffers e.g., 0.1 M Glycine HCl puffer, pH 2.0 and 3.0.0.1 M NaOAc buffer pH 4.2, 4.6, 5.0 and 5.2, 0.1 M sodium carbonate buffer, pH 10. The inhibitory activities were determined separately in each buffer system.

For determining the temperature optima, 20 μL purified PIs were incubated at various temperatures from 10-80°C, for 10 min and then anti-tryptic activities were determined.

Assaying anti-microbial activity of purified protease inhibitors: The antimicrobial activity of purified protease inhibitors was determined against bacteria (Klebsiella pneumonia, Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Streptococcus pneumonia) and against fungi (Candida albicans and Saccharomyces cerevisiae) by agar well diffusion method¹⁷. Gentamycin was used as standard anti bacterial drug and Nystatin was used as standard anti-fungal drug to compare zone of inhibition. The diameter of inhibition zone was measured in millimeters.

RESULTS

Isolation and purification of protease inhibitors from sophora japonica seeds: Protease inhibitors were purified to homogeneity from seeds of Sophora japonica The crude protein fraction resolved two major peaks and some minor peaks on DEAE cellulose chromatography (Fig. 1). Protein fractions corresponding to the two major peaks showed protease inhibitor activity where as protein fractions corresponding to the minor peaks didn’t show any inhibitory activity against proteases. The pooled major peaks resolved into one major peak subjected to gel filtration chromatography on Sephadex G-100 column (Fig. 2). The fractions under major peaks were found to have anti-protease activity and were named as SJ-pi I (Sophora japonica protease inhibitor I) and SJ-pi II (Sophora japonica protease inhibitor II), respectively. At each purification step specific activity of inhibitors increased over homogenate the results of which were given in Table 1.

Anti-tryptic and anti-chymotryptic activity: The SJ-pi I and SJ-pi II inhibited activity of Trypsin by 91 and 83%, respectively at the molar ratio of 1:1 (Fig. 3). SJ-pi I and SJ-pi II also inhibited activity of chymotrypsin by 89 and 81%, respectively at the molar ratio of 1:1 (Fig. 4).

Table 1:	Purification of protease inhibitors from Sophora japonica seeds
BAPNA was used as substrate to measure activity of Trypsin inhibitor. One inhibitor unit (IU) is defined as amount of inhibitor which suppressed liberation of 1μmol substrate per min by active enzyme

Fig. 1:	Elution profile of crude extract of Sophora japonica on DEAE-cellulose column

Fig. 2:	Elution profiles of crude peak 1 and peak 2 obtained from DEAE cellulose column on Sephadex G-100 column (Series 1 represents absorbance at 280 nm of Peak 1 obtained from DEAE cellulose column and series 2 represents absorbance of Peak 2)

Fig. 3:	Inhibition profile of trypsin activity by purified inhibitors from seeds of Sophora japonica (measured by % inhibition of tryptic activity using BAPNA as substrate)

Fig. 4:	Inhibition profile of chymotrypsin activity by purified inhibitors from seeds of Sophora japonica (measured by inhibition (%) using ATEE as substrate)

Fig. 5:	SDS-polyacrylamide gel electrophoretic pattern of crude homogenate and purified inhibitors. Electrophoresis was carried at a current of 50 mA on 4% stacking and 10% resolving gel. About 50 μg of protein was applied in the wells

Table 2:	Molecular weight and Ve/Vo of standard proteins used in calibration of Sephadex G-100 column

Table 3:	Ve/Vo and molecular weight of protease inhibitors SJ-pi I and SJ-pi II

Determination of molecular weight: The molecular weight of the purified inhibitors was determined by gel filtration chromatography on Sephadex G-100 column, pre-equilibrated with standard proteins as shown in Table 2. The molecular weight of purified inhibitors SJ-pi I and SJ-pi II was found to be 15.1 and 31 kDa, respectively by gel filtration chromatography (Table 3), which was further confirmed by SDS-PAGE (Fig. 5).

Effect of pH and temperature on activity of protease inhibitor: Both the inhibitors were active over a range of pH (6.0-9.0). However the maximum activity of SJ-pi I was found to be at pH 8.0 and SJ-pi II had maximum activity at pH 7.0, Fig. 6. Thermo stability of both inhibitors was determined at determined at temperatures ranging from 10-70°C. The purified inhibitors had the maximum activity in the temperature range of 30-40°C. The SJ-pi I was active even at 50°C.

Fig. 6:	Effect of pH on activity of protease inhibitors purified from seeds of Sophora japonica

The activity of both the inhibitors decreased markedly with further increase in temperature (Fig. 7).

Assaying anti-microbial activity of extracts: Purified inhibitors didn’t show any inhibitory activity against gram negative Klebsiella pneumoniae, Escherichia coli and against fungal strains Candida albicans and Saccharomyces cerevisiae (Fig. 8). However, both inhibitors exhibited anti-bacterial activity against Staphylococcus aureus, Bacillus subtilis and Streptococcus pneumonia (Fig. 9). The inhibition results are given in Table 4.

DISCUSSION

Protease inhibitors (PIs) are ubiquitous small proteins that interfere and limit the proteolytic activity of proteases.

Table 4:	Measurement of zone of inhibition of various bacterial strains

Fig. 7:	Effect of temperature on activity of protease inhibitors purified from seeds of Sophora japonica

They have been purified and characterized from a number of sources including plants animals and micro-organisms. Most of the plant protease inhibitors are proteinaceous in nature and are located mainly in seeds, leaves and tubers, which act as specific defense and regulatory proteins. Naturally occurring protease inhibitors are essential for regulating the activity of their corresponding proteases and play key role in biological processes. PIs act as defense agents against insects and micro-organisms^18-20. Currently PIs are being investigated for their role in the prevention and/or treatment of a diverse set of human pathologies, including cancer, neuro-degenerative and cardiovascular diseases and inflammatory disorders²¹. Many reports are available on the isolation, purification and characterization of protease inhibitor from seeds of legume plants^19,22-25. Two protease inhibitors were purified to homogeneity from seeds of Sophora japonica in three steps by ammonium sulfate precipitation, DEAE cellulose column chromatography and Sephadex G-100 chromatography. The crude homogenate when subjected to ion exchange chromatography resolved into three major peaks and some minor peaks, however the anti-protease activity was only found in two major peaks. Similar results were reported by Rakashanda et al.⁷ for lavatera cashmeriana seeds. The two major peaks with anti-protease activity obtained from DEAE cellulose column were pooled and further subjected to purification on gel filtration chromatography using Sephadex G-100 column. Both the peaks individually resolved into a single major peak each with antiprotease activity, suggesting the presence of two protease inhibitors.

Fig. 8(a-d):	Anti-microbial activity of purified inhibitors against Gram-negative bacterial and fungal strains. A represents SJ-pi I, B represents SJ-pi II and S represents standard drug, (a) Saccharomyces cerevisiae, (b) Candida albicans, (c) Escherichia coli and (d) Klebsiella pneumonia

Fig. 9(a-d):	Anti-microbial activity of purified inhibitors against Gram-positive bacteria, A: SJ-pi I, bB: SJ-pi II and S: Standard drug (Gentamycin), (a) Streptococcus pneumonia, (b) Bacillus subtilis and (c) Streptococcus aureus

The peak fractions were pooled and named as SJ-pi I and SJ-pi II (Sophora japonica protease inhibitor). The results were in agreement with the study carried out by Sharma et al.²⁶ for Albizia lebbeck seeds. The molecular weight of the two purified inhibitors was determined by SDS PAGE and by gel filtration column chromatography. The molecular weight of SJ-pi I was found to be 15.1 kDa and the molecular weight of SJ-pi II was found to be 31 kDa. Both the purified inhibitors were found to consist of a single polypeptide chain as revealed by SDS-PAGE which is consistent with inhibitors purified from Tamarindus indica seeds²⁷ and with the protease purified inhibitors from Prosopisjuliflora seeds²⁸. On the contrary trypsin inhibitor purified from A. kalkora seeds showed the presence of two disulfide linked polypeptide chains²⁹.

The purified protease inhibitors strongly inhibited activity of trypsin and chymotrypsin. The SJ-pi I inhibited 91% trypsin activity and 83% chymotrypsin activity and SJ-pi II inhibited 89% trypsin activity and 81% chymotrypsin activity at the molar ratio of 1:1. This is comparable to the inhibitors purified from lavatera cashmeriana seeds⁷ . Both the inhibitors were active over a range of pH (6.0-9.0). However the maximum activity of SJ-pi I was found to be at pH 8.0 and SJ-pi II had maximum activity at pH 7.0 which is consistent with earlier reports³⁰. The trypsin inhibitor purified from mungbean seeds was found to be active between the pH ranges of 4.0-10.0. In contrast, the trypsin inhibitor purified from pigeon pea seeds had maximum activity between pH 7.0 and 10.0, but when exposed to acidic conditions pH 3.0-5.0 the activity was lost³¹ by 20%. Thermostability of purified inhibitors was determined at different temperatures ranging from 10-70°C. The purified inhibitors had maximum activity in the temperature range of 30-40°C. However, unlike SJ-pi II, SJ-pi I was stable even at 50°C. The activity of both the inhibitors decreased markedly with further increase in temperature. Trypsin inhibitors purified from seeds of Caesalpinia bonduc and Archidendron ellipticum were found to be heat tolerant³² up to 60°C likewise the protease inhibitors purified from Lavatera cashmeriana seeds had the optimum temperature of 30°C however, they were active even upto 60°C. Both SJ-pi I and SJ-pi II had significant antibacterial activity against three tested Gram-positive bacterial strains. However, inhibitors did not exhibit anti-bacterial activity against tested Gram-negative bacterial strains and also they didn’t show any antifungal activity against the tested fungal strains. Earlier reports have also shown proteinase inhibitor purified from Ginkgo biloba seeds did not exhibit any antifungal activity neither it had antibacterial activity against Escherichia coli³³.

CONCLUSION

Two protease inhibitors SJ-pi I and SJ-pi II were purified from seeds of Sophora japonica with molecular weight of 15.1 kDa and 31 kDa respectively. Both inhibitors were active over a range of temperature and pH and were found to inhibit the two most important enzymes of serine proteases family viz trypsin and chymotrypsin, hence these two inhibitors can be grouped into the serine protease inhibitors. However further necessary structural investigations needs to be carried out so as to group them into specific class of serine protease inhibitors. SJ-pi I and SJ-pi II possess significant anti bacterial activity against Gram-positive bacterial strains. These PIs can be ideal candidate for exploration in various therapeutic and biological applications.

SIGNIFICANCE STATEMENT

This study discovers two protease inhibitors, SJ-pi I and SJ-pi II, active at optimum pH and temperature and show promising antibacterial activity. This study will be beneficial in pharmaceutical, agricultural and in industrial field. This study provides a platform to the researchers for future studies of these two protease inhibitors to further characterize them and to elucidate their role in various biological pathways.