IsolatIon and purIfIcatIon of Bacillus thuringiensis var . israelensis IМV В-7465 peptIdase wIth specIfIcIty toward elastIn and collagen

Peptidase of bacillus thuringiensis var. israelensis iМv В-7465 was isolated from culture supernatant using consecutive fractionations by an ammonium sulphate (60% saturation), ion-exchange chromatography and gel-filtration on the tsk-gels toyoperl hW-55 and deae 650(M). specific elastase (442 U∙mg of protein-1) and collagenase (212.7 U∙mg of protein-1) activities of the purified enzyme preparation were 8.0and 6.1-fold, respectively higher than ones of the culture supernatant. Peptidase yields were 33.5 % for elastase activity and 30.1 % for collagenase activity. it was established that the enzyme is serine metal-dependent alkaline peptidase with Mr about 37 kda. Maximal hydrolysis of elastin and collagen occurs at the optimum ph 8.0 and t° 40 and 50 °С, respectively. the purified preparation has high stability at ph in the range of 7.0 to 10.0 and 40-50 °С.


I
t is known that microbial peptidases are widely used in various industries such as pharmaceutical, food, leather, detergent [1].It has been estimated that the annual sales of industrially produced enzymes is about $1 billion, 75% of industrially produced enzymes are hydrolases, and among them 60% are peptidases, most of which (50%) obtained from bacteria of genus Bacillus.The main countries producing peptidase are Denmark (40%), the USA (20%) and Italy (40%).
Enzymes, which are capable of cleaving soluble proteins such as collagen and elastin, the components of connective tissue fibers, take a special place among peptidases of microorganisms.These proteins have firm and resistant to proteolysis structure owing to formation of intermolecular cross-linking.Peptidases with collagenase and elastase activities can be used in the development of drugs for trophic ulcers, pus wounds, burns, as well as in detergents to dissolve protein contaminants.
The peptidases are used in the food industry and for the manufacture of detergents, are isolated in plenty in crude state, but highly purified enzymes are necessary for medicine.
Despite significant progress in the researches on proteolytic enzymes, the needs of different sec-tors of Ukrainian production for preparations with varying degrees of specificity are not being met, especially in medicine and leather industries, which use imported enzymes, which often do not meet quality standards [2].
Previously, based on the screening of microorganisms of different taxonomic groups, we selected Bacillus thuringiensis var.israelensis IMV B-7465 strain which was active towards the insoluble protein substrates, such as collagen and elastin.Since such peptidases could be promising in the development of novel drugs aimed at hydrolysis of collagen and elastin, as well as in detergents for removing insoluble protein contaminants, the aim of our study was to isolate, purify and characterize peptidase of B. thuringiensis var.israelensis IMV B-7465 with collagenase (3.4.24.3) and elastase (3.4.21.11) activities.

Materials and Methods
The strain of Bacillus thuringiensis var.israelensis IMV B-7465, obtained from the water area of Snake Island (Black Sea) and kindly provided by the staff of the Department of Microbiology, Virology and Biotechnology of the Odessa I. I. Mechnikov National University, was the object of the study.The strain is registered as a producer of collagenase doi: http://dx.doi.org/10.15407/ubj88.03.018 peptidase in the Depository of the Zabolotny Institute of Microbiology and Virology, NAS of Ukraine No. IMV B-7465.
B. thuringiensis var.israelensis IMV B-7465 peptidase was isolated from the supernatant, obtained by centrifugation of the culture fluid at 5000 g for 30 min, by ammonium sulfate (60% saturation) precipitation.The precipitate was collected by centrifugation at 5000 g, for 30 min, dissolved in 0.01 M Tris-HCl buffer (pH 7.5) and applied to a column (2.5×40 cm) with anion exchanger TSK DEAE 650 (M) (Toyosoda, Japan).Elution was conducted with 0.01 M Tris-HCl buffer (pH 7.5) in NaCl gradient from 0 to 1.0 M at a flow rate of 0.5 ml/min.Protein fractions which have collagenase and elastase activity were collected, combined and applied to a column (1.8×40 cm) with neutral TSK-gel Toyopearl HW-55 (Toyosoda, Japan).Elution was carried out with the same buffer at a flow rate of 0.85 ml/min.The degree of the enzyme purification was characterized by specific elastase and collagenase activi ty (U•mg of protein -1 ).
Protein content was measured at 280 nm using spectrophotometer SF-26 at all stages of the experiments.Protein concentration was determined by Lowry assay [4].
Total carbohydrate concentration was determined using phenol-sulfuric method [5].For this, 0.5 ml of the examined enzyme solution, 0.5 ml of a 5% phenol solution and 2.5 ml of concentrated sulfuric acid were mixed with intense stirring in tubes and incubated for 30 min at room temperature.Absorbance was measured at 490 nm.The carbohydrate content was assessed by a standard curve for glucose.
For assessment of collagenase activity [6] the mixture containing 10 mg of collagen, 2.5 ml of 0.01 M Tris-HCl buffer (pH 9.0-10.0)and 1 ml of the studied preparation was incubated in a water bath at 37 °C for 3 h.The reaction mixture was then centrifuged at 10 000 g for 5 min and 0.1 ml of supernatant was transferred to tubes containing 0.5 ml of 4% ninhydrin in acetone solution and an equal volume of 0.2 M citrate buffer (pH 5.0).Incubation was carried out in a boiling water bath for 20 min.Then, 5 ml of 50% n-propanol was added to the cooled mixture and kept at room temperature for additional 15 min.The cleavage products were determined at 600 nm using SF-26 spectrophotometer.The equivalent amount of amino acids released during hydrolysis was determined from a standard curve for free L-leucine.One unit of the activity is equivalent to 1 µmol of L-leucine released from collagen during hydrolysis for 3 h at 37 °C.
The basis of the method for determination of elastase activity is colorimetric measurement of the color intensity of a solution containing elastin-congo red as an enzyme substrate [7].The mixture containing 2.0 ml of 0.01 M Tris-HCl buffer (pH 7.5), 5 mg of elastin stained with 0.002% congo red and 1 ml of enzyme solution was incubated at 37 °C for 3 h.The reaction was terminated by putting the tubes with the reaction mixture into an ice bath for 30 min.Non-hydrolyzed elastin was separated by centrifugation at 10 000 g for 5 min.The color intensity was measured at 515 nm on SF-26 spectrophotometer.The amount of enzyme that catalyses hydrolysis of 1 mg of elastin per 1 min was taken as the unit of activity.
SDS-PAGE under denaturing conditions was performed according to Laemmli [8].For this, the studied enzyme preparation was dissolved in a sample buffer (0.5 M Tris-HCl, 2-mercaptoethanol (pH 8.8), 10% SDS, 20% glycerol and 0.001% bromophenol blue), boiled up and then applied to a gel (5-10 µg per well).Electrophoresis was performed in 4% concentrating and 12% separating acrylamide gels at constant amperage 30 mA.The enzyme was detected in the gel by staining with Coomassie G-250.The proteins Fermentas PageRuler™ Prestained Protein Ladder (SM0671) were used as comparative markers.Electrophoregrams were scanned.An increase in the color intensity of peptidase zone was exami ned using Totallab TL100 analysis software.
The effect of pH and temperature on the purified B. thuringiensis var.israelensis IMV B-7465 activity was studied in the temperature range from 20 to 80 °C and pH range from 5.0 to 12.0.Standard 0.01 M phosphate buffer was used to obtain the pH range.To investigate stability, the enzyme was incu-bated in the buffer at pH 7.0-12.0and 40-60 °C for 0, 15, 30, 60 and 120 min, then aliquots were taken to analyze collagenase and elastase activities.
Protein concentration in the peptidase preparation was 0.2 mg/ml.The specific activity was expressed in U per mg of protein.
To compare elastase activity of purified B. thuringiensis var.israelensis IMV B-7465 with commercial preparation, proteinase K (Merck, Germany)

Collagenase activity
Protein isolated from tritirachium album was used.Protein concentration in both preparations was 0.2 mg/ml.All experiments were performed in 5-8 replicates.Statistical processing of the obtained data was performed using Student's t-test [9].The data are presented as mean and standard error (M ± m).Processing of the data presented in graphs was performed using Microsoft Excel 2010 software.P < 0.05 values were considered statistically significant [10].

results and discussion
Peptidase of B. thuringiensis var.israelensis IMV B-7465 was isolated using standard multistage purification procedure: ammonium sulfate fractionation (60% saturation) of the culture supernatant, ionexchange chromatography and gel-filtration.It was shown (Fig. 1) when separating complex enzyme preparation by ion-exchange chromatography on TSK Toyopearl DEAE 650(M) column using a NaCl stepwise gradient from 0 to 1 M, that peptidase with collagenase and elastase activity eluted as one peak at 0. contaminants from the enzyme preparation.Thereat, collagenase and elastase activity increased 3-and 4-fold, respectively compared to the activity of the culture supernatant (Table 1).
The fraction with collagenase and elastase activities was further purified by gel-filtration on a TSK Toyopearl HW-55 column.It was established (Fig. 2) that elution with 0.01 M Tris-HCl buffer (pH 7.5) enabled to obtain the studied enzyme (peak 1) in one fraction.Purification degree was 5.7and 7.4-fold, respectively.Activities in the peaks 2 and 3 were not detected.
Fraction 1, which exhibited collagenase and elastase activity, was further purified by chromatography on Sepharose 6B (Pharmacia, Sweden) column (1.5×25 cm) equilibrated with 0.01 M Tris-HCl buffer (pH 7.5).As a result, only one peak with collagenase and elastase activity was detected (Fig. 3) that indicates homogeneity of the preparation obtained after the second stage of purification.The degree of purification was 6.1-and 8-fold, respectively (Table.1).
The homogeneity of the studied enzyme of B. thuringiensis var.israelensis IMV B-7465 was also confirmed by SDS-PAGE under denaturing conditions (Fig. 4, b).The use of protein-markers in native (Sepharose 6B) (Fig. 4, a) and denaturing con-

Fig. 2. the elution profile of b. thuringiensis var. israelensis iMv b-7465 fraction with collagenase and elastase activities on tsk toyopearl hW-55
Elution volume, ml It is known that the members of genus Bacillus produce mainly peptidases with Mr from 15 to 100 kDa.Mr of bacilli elastases are from 20 to 30 kDa.Thus, the elastases with Mr 29.5 kDa and 25 kDa were isolated from B. licheniformis ZJUEL31410 and Bacillus sp.6644, respectively [11,12].Collagenases from B. megaterium NRC have Mr 25 and 28 kDa [16], from B. cereus -have Mr 38-48 kDa [18,19].
Elastase activity of the purified peptidase of B. thuringiensis var.israelensis IMV B-7465 was found to be 1.5-fold higher than activity of the commercial preparation of proteinase K (Merck, Germany), which was taken as a comparative enzyme.
A small amount of carbohydrates (7%) was found in the composition of the B. thuringiensis var.israelensis IMV B-7465 peptidase.

Fig. 3. the elution profile of peptidase b. thuringiensis var. israelensis iMv b-7465 on sepharose 6b
Elution volume, ml The optimum conditions, exactly pH and temperature ranges, at which enzymes retain their highest activity and stability are important characteristics of any enzyme preparations.Change of environmental conditions greatly affects the structure and function of enzymes that is an example of adap tation at the biochemical level.According to the literature, the majority of bacilli peptidases with collagenase and elastase activities is alkaline and works in the pH range from 5.0 to 11.0.It has been shown (Fig. 5) that the purified B. thuringiensis var.israelensis IMV B-7465 peptidase is active at pH range from 6.0 to 11.0.The pH optimum for collagen and elastin hydrolysis by the enzyme was 8.0 that indicates that the obtained enzyme is alkaline peptidase.
Investigation of the stability of B. thuringiensis var.israelensis IMV B-7465 peptidase showed that after 120 min in the pH range from 7.0 to 9.0, 80-90% of the initial collagenase and elastase activities were retained (Fig. 6).The increase in pH to 11.0 and 12.0 resulted in a decrease in both activities to 50-70%.
The isolated enzyme was active in the temperature range from 30 to 60 °C, at that optimum temperatures for hydrolysis of collagen and elastin by the peptidase were different: 50 °C -for collagen and 40 °C -for elastin (Fig. 7).
The enzyme retained 100% of collagenase and elastase activities during incubation at 40, 50 and 60 °C for 30 min.After incubation for 2 h at 40 and 50 °C, 90 and 80% of the initial activities, respec- tively remained, whereas at 60 °C only 72% was remained (Fig. 8).
The differences in optimum conditions for interactions of enzyme with various substrates can be characterized by either mechanism of the enzymesubstrates binding or changes in the molecule conformation at various temperatures [13].Our results are confirmed by the literature data showing that collagenase peptidases exhibit maximum activities at pH 7.0-8.0 in the temperature range from 25 to 65 °C.The highest activity of collagenase of B. stearothermophilus DPUA1729 reported to be at pH 7.2 and 25 °C [14], B. megaterium NRCat pH 7.5 and 50 °C [15], B. licheniformis F11 -at pH 9.0 and 50 °C [16], B. cereus MBL13 -at pH 8.0 and 40 °C [17], B. cereus AT -at pH 8.0 and 50 °C [18].As it is seen, the majority of bacilli elastases are alkaline enzymes.Optimum conditions for their actions are pH from 8.0 to 10.5 and temperature from 40° to 65 °C [11,19,20].However, among such elastases, neutral enzymes can be also found, e.g.B. licheniformis ZJUEL31410 [10].
Our findings, that B. thuringiensis var.israelensis IMV B-7465 peptidase exhibits sufficiently high stability at 40-60 °C and alkaline pH, are essential for the use of this preparation in leather industry and in detergent compositions.
It is known that elastase (3.4.21.11) and collagenase (3.4.24.3) bacilli enzymes are members of a class of serine or metallopeptidases [10,17].The study on the identification of the functional groups of the catalytic center of B. thuringiensis var.israelensis IMV B-7465 peptidase using group-specific reagents has shown (Fig. 9) that this peptidase belongs to serine peptidases, since a complete inhibition of its activity by PMSF was observed.Since metallochelator EDTA caused the reduction in the activity by 3.7-fold compared to the control, it can be assumed that this enzyme is also metal-dependent.The inhibitory effect of p-CMB (13-fold) may indicate the presence of SH-groups on the surface of the enzyme molecule which are involved in the binding and hydrolysis of insoluble macromolecular protein substrates.

Fig. 1 .
Fig. 1. the elution profile of the complex enzyme preparation of b. thuringiensis var.israelensis iMv b-7465 on tsk toyopearl deae 650(M) with a NaCl stepwise gradient Elution volume, ml 0 40 80 120 160 200 240 280 320 360 400 440 480 520 560 600 640 680 720 2 M NaCl.It enabled to eliminate some protein t a b l e 1. the stages of b.thuringiensis var.israelensis iMv b-7465 peptidase purificationStages of purification collagenase activity, EA -elastase activity.N.А.Nidialkova, l. d. varbanets, v. o.Chernyshenko -PAGE) enabled to determine that the peptidase Mr is about 37 kDa.Since the enzyme homogeneity was shown by gel-filtration on Sepharose 6B and SDS-PAGE, we consider the B. thuringiensis var.israelensis IMV B-7465 peptidase as one enzyme that exhibits specificity for collagen and elastin proteins.Previously we obtained similar results for B. thuringiensis IMV B-7324, which synthesizes peptidase 1 with activity towards elastin and fibrin.

Fig. 6 .
Fig. 6. the dependence of stability of b. thuringiensis var.israelensis iMv b-7465 peptidase on various ph at the hydrolysis of collagen (a) and elastin (b)

Fig. 8 .
Fig. 8. the dependence of stability of b. thuringiensis var.israelensis iMv b-7465 peptidase on various temperatures at the hydrolysis of collagen (a) and elastin (b)