Purification and characterization of a novel thermo stable L-methioninase from Streptomyces sp . DMMMH 4 and its evaluation for anticancer activity

Article history: Received on: 25/02/2016 Revised on: 21/04/2016 Accepted on: 17/05/2016 Available online: 28/07/2016 L-methioninase has been purified 2.55-fold from the crude extract of Streptomyces sp. DMMMH4. The purification procedure was carried out by heat treatment and gel filtration on Sephadex G-200 column chromatography. SDS-PAGE electrophoresis showed a migrating protein band molecular mass of 47 kDa. The kinetic properties determined for the purified enzyme displayed optimum activity at 70 O C and thermal stability were 70 O C for 30 min. The enzyme showed maximum activity at pH 6 using acetate buffer 0.05M and was relatively stable across a broad range of pH values (5.5-8 pH). The enzyme strongly inhibited by Cr +2 , Fe +2 , Ni +2 , Cd +2 , PMSF, β-mercaptoethanol and SDS while Hg +2 ,Cu +2 and iodoacetate completely inhibited the enzyme activity at a final concentration of 10mM. The purified enzyme exhibited a Km of 0.7, 0.15 and 0.25 mM for L-methionine, DL-ethionine and L-cystine respectively. Cytotoxicity test demonstrate that enzyme was active against liver HepG2, breast MCF-7, lung A549, prostate PC3 and colon HCT116 cancer cell lines and has negligible toxicity toward a normal melanocyte cell line HFB4.


INTRODUCTION
, is a pyridoxal-5′phosphate-dependent enzyme, catalyzes the γ-elimination of Lmethionine to generate α-ketobutyrate, methanethiol, and ammonia as well as the α, β-replacement and β-elimination of Ssubstituted L-cysteines (Tanaka et al., 1983).A major potential therapeutic application of enzymes is in the treatment of cancer.Therefore, much attention has been paid to L-methioninase which have demonstrated antitumor efficacy in vitro as well as in vivo (Kahraman et al., 2011;Tan et al., 1998).L-methioninase is one of few microbial enzymes with high therapeutic value since it was reported as a potent anticancer agent against various types of tumor cell lines Breast, Lung, Colon, Kidney and Glioblastoma (Kokkinakis et al., 2001;Tan et al., 1998).Many human cancer cell lines and primary tumors have an absolute A similar enzyme from a different clone of Pseudomonas putida was isolated and purified by (Ito et al., 1976).The purified enzyme from Pseudomonas putida ICR 3460 was published by (Nakayama et al., 1984) has a molecular weight 43KDa.Furthermore, pure L-methioninase was also obtained from other species of bacteria such as Colstridium sporogens (Kreis and Hession, 1973), Brevibacterium linens (Pinnamaneni et al., 2012).On the other hand, a few studies on the purification and characterization of L-methioninase from fungi such as Aspirgillus flvips reported by (El-Sayed, 2011), the purified enzyme had a molecular mass 47 KDa.In addition, Purification and characterization of this enzyme from the yeast was carried out by (Selim et al., 2015a).To our knowledge, no investigation has yet been performed on purification of this enzyme from Streptomyces species.Although, the only reported on the production of this enzyme by Streptomyces Sp was published through us (Selim et al., 2015b).In the present work, we purified and characterized of a new L-methioninase from Streptomyces sp.DMMMH4 and were to evaluate in-vitro anticancer activity.

Chemicals and reagents
The source of chemicals and reagents used in this study were as follows: L-methionine from (Merk, Germany).Methanethiol used as sodium methanethiolate; Pyridoxal-5phosphate (PLP); 5,5-Dithiobis-2-nitrobenzoicacid (DTNB); Commassi Brilliant Blue G-250 and bovine serum albumin (BSA) were obtained from Sigma-Aldrich (Sigma, St. Louis, USA).Sephadex G-200 and DEAE-cellulose were purchased from Pharmica Biotechnology (Sweden).Cancer and normal cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).All other chemicals were of the highest analytical grade.

Streptomyces strain and production conditions:
Streptomyces sp.DMMMH4 was deposited in the DDBJ/EMBL/GenBank nucleotide sequence databases with accession number: LC021308.This strain was previously isolated from Egyptian soil and chosen as a most potent L-methioninase producer (Selim et al., 2015b).The enzyme production medium used is a modified starch medium.It's contained (g/L): Starch, 20; L-methionine, 3; MgCl 2 anhydrous, 1.5; CaCO 3 , 3 and yeast extract.In addition, growth medium was adjusted to 7 using potassium phosphate buffer 0.075 M.After four days of growth with shaking (150 rbm, New Brunswick, USA) cells were harvested by centrifugation (5500 rpm, Herml, Germany).The clear supernatant was used as a crude enzyme.

Protein determination
Protein concentration was determined according to the method of (Bradford, 1976) and its determination in the purified fractions using method of (Schleif and Wensink, 1981) with bovine serum albumin as standard.

L-methioninase assay
L-Methioninase activity was determined by the method of (Arfi et al., 2003;Laakso et al., 1976) using L-methionine as a substrate.Methanethiol (MTL) produced from substrate reacted with 5,5 dithio-bis-2-nitrobenzoicacid added (DTNB) to form thionitrobenzoic acid was detected spectrophotometrically at 412 nm (Agilent UV/Vis.Cary-100).The assay mixture contained 20mM of L-methionine in 0.1M potassium phosphate buffer pH 7.0, 0.02mM PLP, 0.25mM DTNB and the supernatant in a final volume of 1 ml.One unit (U) of L-methioninase was expressed as the amount of enzyme that releases 1µmole of methanethiol per minute under optimal assay conditions.
In addition, Deaminating activity was carried out by measuring on releasing ammonia from L-methionine according to (Spinnler et al., 2001) using Nessler's reagent.One unit (U) of enzyme activity was expressed as the amount of enzyme that releases one µmol of ammonia per minute under optimal assay conditions.

L-methioninase Purification
The clear supernatant obtained was used as the crude enzyme preparation for the subsequent steps.According to the method of (Selim et al., 2015a), the enzyme was partially purified by heating the supernatant at different temperatures (60-70 O C) for different times (10-25min.)intervals.After cooling on ice the denatured proteins were removed using cooling centrifuge (Sigma, Germany at 5500 RPM) for 15 min.The active fraction was dialyzed overnight against the same volume of 0.05 M potassium phosphate buffer, pH.7 amended with 0.02mM PLP.
The dialyzed supernatant was applied to a (2.5×50 cm) column of Sephadex G-200 pre-equilibrated with 0.05 M potassium phosphate buffer pH 7 containing 0.02mM PLP.The enzyme fractions were eluted with the same buffer at a flow rate 1ml/5min.Eliot (5 ml fractions) was collected separately for the measurement of enzyme activity and protein content.The active fractions were combined and the solution was concentrated and collected prior to confirm the purity of enzyme and determination of molecular mass by (SDS-PAGE).

Determination of purity and molecular mass of L-methioninase
The homogeneity of purified L-methioninase was checked using dissociating polyacrylamide gel electrophoresis (SDS-PAGE) according to a protocol proposed by (Lammeali, 1970) with standard marker proteins.

Statistical analysis
Results are expressed as the mean ± S.D, calculated using excel 2010.

Effect of pH on the enzyme activity and stability
The optimum pH for pure enzyme activity was determined using 0.05 M of different buffers (i.e.) acetate (pH 4.0-6.0);potassium phosphate (pH 6.5-8.0) and Glycine-NaOH (8.5-10).After incubation with different buffers, each reaction enzymatic activity was estimated.The pH stability of the enzyme was determined by preincubating the enzyme solution at different pH values ranging from 4.0 to 10 for 18 h at 4 O C and at room temperature 33 O C. At the end of preincubation time the pH value of enzyme solution was readjusted to pH 7 i.e. (optimum pH used in standard assay method) and then residual enzyme activity was assayed by the standard method.

Effect of temperature on the enzyme activity and stability
The effect of reaction temperature on pure enzyme activity was determined by incubating the reaction mixture at different temperatures ranging (30-90 O C).On the other hand, the thermal stability of the purified enzyme was determined by preincubating the enzyme solution at various temperatures (30-90 O C) for different times (0-100 min) in the absence of substrate.Followed by aliquots was removed, cooled of enzyme solution and the residual activity was measured by the standard assay method as previously mentioned.

Effect of some metal ions and chemical reagents on the enzyme activity
Metal ions with chloride salt and some chemical reagents tested for their effects on pure enzyme activity by incubating enzyme solution with 1 and 10mM final concentrations for 4h before adding the substrate.After preincubation time, enzymatic activity was determined under optimal assay conditions.

Determination of kinetic parameters
In this experiment, the kinetic parameters such as Michalis-Menten constant (Km) and maximum velocity (Vmax) of purified L-methioninase was determined by incubating the enzyme using different concentrations of each substrate in the range of (1-70 mM) under optimum assay conditions.The apparent Km and Vmax of purified enzyme were calculated from a Lineweaver-Burk plot.

In-Vitro anticancer activity assay
To determine the effect of Purified L-methioninase as anticancer activity, we tested the viability of cancer cell lines exposed to various concentrations of pure enzyme.Anticancer activity was expressed by median growth inhibitory concentration (IC 50 ).The antiproliferative activity of pure enzyme was evaluated against liver HepG2, breast MCF-7, lung A549, prostate PC3 and colon HCT116 cancer cell lines as well as the normal cell line (human normal melanocyte, HFB4) in comparison with doxorubicin as reference drug.The cells were grown and maintained in RPMI-1640 medium supplemented with 10% heat inactivated fetal calf serum (GIBCO), penicillin (100 U/ml) and streptomycin (100µg/ml).The cells were maintained at 37 O C in humidified atmosphere containing 5% CO 2 .The cancer cell lines were suspended in medium at cells concentration of x 10 4 cell/well in Corning® 96-well tissue culture plates grown in a 25 cm 2 flask in 5 ml of culture medium, and then incubated for 24 h.The tested enzyme was then added into 96 well plates (six replicates) to achieve eight concentrations of enzyme.Six vehicle controls with media or 0.5% DMSO were run for each 96 well plate as a control.After incubation for 24 h, the numbers of viable cells were determined by the MTT test (Mosmann, 1983).

Purification of L-methioninase
Purification of L-methioninase to homogeneity was accomplished in two successive steps Table (1   This enzyme has been purified from various sources but with several steps, L-methioninase from Brevibacterium linens was purified in five purification steps, including ammonium sulfate precipitation followed by several chromatographic procedures.Purified L-methioninase obtained from Citrobacter freundii by heat treatment at 60 O C followed by separation on DEAE-cellulose column and Sephacryl S-200HR column (Munkhov et al., 2005).Partial purification of this enzyme by heat treatment is consistent with published literature reported for purification from Pseudomonas putida (Tan et al., 1998;Hori et al., 1996).In contrast, Streptomyces sp.DMMMH4 Lmethioninase was purified using only two steps compared to other microbial sources.Accordingly, our results of molecular mass of the enzyme are in agreement with reported by L-methioninases purified from different sources, the molecular mass of Lmethioninase purified from Citrobacter freundii (Munkhov et al., 2005) was found to range from 43.0 to 45.0 kDa per subunit.while, the purified enzyme from Aspergillus flavipes had a molecular mass 47 kDa (El-Sayed, 2011).

Effect of pH on the enzyme activity and stability:
Results in Fig. (3) indicate that somewhat acidic and neutral pH values (6-7 pH) using acetate and potassium phosphate buffers 0.05M were the most favorable for enzyme activity, the maximum activity of enzyme was obtained at pH 6 when acetate buffer 0.05M was used (103.9%).

Fig. 3: Effect of pH on enzyme activity
In addition, when pH 6 adjusted using acetate buffer with different molarities from (0.025-0.20M), the response of enzyme activity was increased with the increase of buffer molarities reaching its maximum value when 0.05M was applied Fig. (4).
Furthermore, the enzyme was found to be stable in the pH range 6-8 and was more labial in the acidic region than in the alkaline region as shown in Table (2).
The optimum pHs for enzyme activity will be studied by many authors, for Aspergillus flavipes neutral to slightly alkaline pH (7-8 pH) was optima for its pure enzyme activity (El-sayed, 2011).While pH 6.5 was the most suitable for Candida tropicalis L-methioninase activity (Selim et al., 2015a).On the other hand, the alkaline range of pH correlated with maximum enzyme activity for bacteria, Pseudomonas putida (Nakayama et al., 1984) & Citrobacter freundii (Mankhov et al., 2005).On the other hand, the stability of the enzyme at different pHs higher than pH 8.5 and lower than pH 5 resulted in a decrease of enzyme activity, (El-sayed, 2011) suggesting that the lower stability of the enzyme at a higher and lower pHs may be attributed to the dissociation of pyridoxal-5-phosphate or unfolding of the enzyme active site.In addition, our data in agreement with (El-sayed, 2011) who mentioned that the isoelectric point of the enzyme was pH 4.8 and the lower enzyme stability in acidic conditions may be due to the closeness of his enzyme to the isoelectric point.The pH stability of the enzyme from other sources was over range from 7 to 8 (Dias and Weimmer, 1998;Tanaka et al,. 1976).

Effect of temperature on the enzyme activity and stability
Data presented in   6), it could be noticed that, the pure enzyme was more stable up to 50 o C for 100 min and not loss of activity followed by a slight decrease of the enzyme stability after 1 hr at 60 O C was occurring.In addition, a severe inhibition of enzyme activity (15%) was noticed when the enzyme was incubated at 90 O C for 100 min.In comparison with enzyme produced by other sources, our enzyme could be consider as a promising agent for its high stability (thermo stable).Pseudomonas putida had optimal activity at 35 O C for different times reported by (Nakayama et al., 1984).Similar ranges were reported for several bacteria such as Brevibacterium Linens (Pinnamaneni et al., 2012), and Citrobacter intermedius (Faleev et al., 1996).In addition, (El-sayed, 2011) reported that, the optimum temperature for L-methioninase activity obtained from Asperigillus flavipes was observed 35 O C.Moreover, maximum activity of L-methioninase from Candida trobicalis was 45 O C and the reaction time increased up to 20 min (Selim et al., 2015a).Thermal stability of the enzyme from different sources was investigated by (Dais and Weimmer, 1998) they showed that L-methioninase from Brevibacterium linins had a thermal stability below 40 O C and also (El-sayed, 2011) finding the enzyme from Aspirgillus Flavipes displayed a relative catalytic stability below a temperature of 40 O C.

Substrate Specificity of Purified L-Methioninase
The ability of enzyme to catalyze the γ-elimination of various Sulfur containing amino acids is presented in Fig. (7).These amino acids were added with equal amounts (20mM) separately to the reaction mixture and incubated under optimum assay conditions.Data obtained from Fig. ( 7) indicate relative activities of the enzyme on these substrates.L-methioninase was found to degrade DL-ethionine with 103.7% more than Lmethionine, followed by cystine and cysteine with 96.4% and 83.2% respectively.The enzyme exhibited high specificity for DLethionine then L-methionine and L-cystine.The relative activity of L-methioninase towards the sulfur containing amino acids may be due to the similar molecular configuration of α &ß carbons (El-sayed, 2011).(Tanaka et al., 1976) mentioned that L-methionine is the preferred substrate for L-methioninase purified from Pseudomonas putida.Furthermore, several derivatives of L-methionine and L-cysteine serve as effective substrate.Studies on the substrate specificity of Candida tropicalis L-methioninase revealed that the enzyme had a relative activity towards various sulfur containing amino acids (Selim et al., 2015a).

Determination of Km and Vmax
L-methionine, DL-ethionine and L-cystine were serve as effective substrates thus we are calculating Km and Vmax for each from a Lineweaver-Burk plot.The Km and Vmax values was found to be (0.respectively as shown in Table (3).The enzyme showed maximal activity at a substrate level of 40, 60 and 50 mM for L-methionine, DL-ethionine and L-cystine respectively; this indicates that the active center of the enzyme became saturated with each substrate at concentrations above 40, 60 and 50 mM.Km and V max were showing a high affinity of enzyme to its substrates exhibiting a low value of Km and confirmed its high therapeutic value of the enzyme.In this respect, L-methioninase from Pseudomonas putida (Esaki and Soda, 1987) was reported to exhibit a Km of 1mM.In addition, The purified enzyme exhibited a Km of 0.7mM from Citrobacter freundii (Munkhov et al., 2005).

Effect of Different Metal Ions and Inhibitors on Lmethioninase Activity
The enzyme activity in the presence of metal ions and some chemical reagents was also determined.The results presented in Table ( 4) demonstrated that L-methioninase activity was inhibited and loss more than 50% of its activity when preincubating with Ni, Cu, Cr, Cd, SDS and iodoacetate at a final concentration of 10 mM.Whereas an increase and the stimulatory effect on enzyme activity found to be occurring with Mg and EDTA at both concentrations (1&10mM) by the relative enzyme activities (102& 100.2% and 101.8& 100.4%, respectively).
Furthermore, insensitivity of the enzyme activity to EDTA (a metallic protease inhibitor) indicated that the functioning of enzyme did not have the absolute requirement of metal ions and ensuring the non-metallic nature of this enzyme.All thiol compounds, DTT, glutathione, and β-mercabtoethanol had an inhibitory effect on enzyme activity by 68.2, 88.8 and 65%, respectively while the enzyme was completely inactivated by a thiol reducing agent iodoacetate, the complete inhibition of enzyme with this agent provides evidence for the presence of -SH group in the active sites of enzyme.Triton X-100 had a significant slight effect on enzyme activity compared to tween (80; 20) as reflected by the relative enzyme activities (92.7 and 100%, respectively).While the effect of sodium dodecyl sulfate as a strong surfactant on the enzyme was remaining 40.5% of its activity at 10 mM.Additionally, enzyme was strongly inhibited by PMSF, β-mercaptoethanol, SDS, and DTT suggests the presence of a cysteine/disulfide bond for maintaining the molecular catalytic folding state of the enzyme, consistent with the results obtained by (Thong et al., 1987) and(Lockwood andCoombs, 1991).(Ferchechi et al., 1986) showed that, the enzyme activity of Brevibacterium linens was stimulated by Na + and K + and strongly inhibited by Zn +2 , Mn +2 , and Cu +2 .(Dais and Weimmer, 1998) founded that iodoacetate inhibited enzyme activity at 10mM while metal chelating agents did not influence enzyme activity.On the other hand, enzyme purified from Aspergillus flavipes was strongly inhibited by DL-propargylglycine, hydroxylamine, PMSF, β-mercaptoethanol, Hg + , Cu 2+ , and Fe 2+ and slight inhibition by Triton X-100 (El-Sayed, 2011).

In-vitro anticancer activity of L-methioninase
The results Table (5) revealed that a good remarkable anticancer activity against cancer cells.L-methioninase was found to be potent anticancer agents had IC 50 values near to the standard drug doxorubicin in various cancer cells with IC 50 values 4.29±0.44µg/ml (0.127 U/ml) in case of lung A549; 7.11±0.82µg/ml (0.21 U/ml) in case of colon HCT116; 6.39±0.71µg/ml (0.19 U/ml) in case of prostate PC3; 5.71 ±0.64µg/ml (0.17 U/ml) in case of breast MCF-7; and 4.33 ±0.46µg/ml (0.13 U/ml) in case of liver HepG-2 versus the standard drug doxorubicin which had IC 50 values 4.11±0.50,5.80±0.65 ,5.60±0.63,2.96 ±0.34 and 3.97±0.45µg/ml respectively, in A549, HCT116 , PC3, MCF-7 and HepG2 cells.It is clear that cancer cell lines were tested are more sensitive to L-methioninase.Additionally, the results revealed that L-methioninase has no toxicity against the growth of normal melanocytes HFB4 cells.
The strong inhibition of these cancer growth by action of L-methioninase, ensure its methionine auxotrophic identity for cancer cells.The efficiency of L-methioninase against various cell lines was reported by many authors (Tan et al., 2010;Sundar andNellaiah, 2013 &Kui-Ying et al., 2015).The sensitivity of cancer cell lines to L-methioninase was investigated by (Hori et al., 1996) they tested L-methioninase produced by Pseudomonas putida against various cancer cell lines and leukemia cell lines are more sensitive to L-methioninase than solid tumor cell lines.(Tan et al., 2010) showed that the rMETase was efficacy in broad series of cancer cell lines.
They reported that rMETase had a mean IC 50 (units/ml) for the following cancer cell types: renal, 0.07; colon, 0.04; lung, 0.12; prostate, 0.01; melanoma, 0.19 and CNS, 0.195.In addition, methionine concentration lower than 10µM is necessary for inhibition of tumor-cell growth accompanied by cell death, this level of methionine depletion can be rapidly attained at 2 units/ml rMETase in vitro (Yoshioka et al., 1998).(El-Sayed et al., 2012) mentioned that the enzyme showed a remarkable activity against prostate (PC3), liver (HEPG2), and breast (MCF7) cancers, with IC 50 0.001U/mL, 0.26 U/ml, and 0.37 U/ml, respectively.Toxicity of cancer cells to L-methioninase by Candida trobicalis against differed among cell lines studied by (Selim et al., 2015a); and they showed the breast cancer cell line was more sensitive (IC 50 of 0.13 U/mL) than liver cancer cell line (IC 50 0.2U/mL).

CONCLUSION
The bacterium Streptomyces has so far never been investigated for L-methioninase purification.The studies presented in this report show that this bacterium may be a future source for larger production of a pure thermo stable L-methioninase.Catalytic parameters of L-methioninase from Streptomyces showed new biochemical prosperities about another Lmethioninase producer.Our data observed that significantly inhibited of the different cancer cell lines and the enzyme did not display toxicity signs as shown using normal melanocytes cell line(HFB4),we can conclude that purified L-methioninase from a new source Streptomyces Sp might be a promising drug for the treatment of different cancer cells.
). Nine eighty percent of L-methioninase activity was obtained from heat treatment at 70 O C for 10 min.Separation of enzyme from other proteins by gel filtration using Sephadex G-200 Fig. (1) resulted in a homogenous enzyme which was purified 2.55 fold with an activity yield of 49.1.

Fig. 1 :
Fig. 1: Purification of L-methioninase using sephadex G-200.On the other hand, when the gel was electrophoresd under denaturing conditions, a single band with an approximate molecular mass of 47 KDa was noted Fig. (2).

Fig. 4 :
Fig. 4: Effect of different molarities on enzyme activity Fig. (5) demonstrated the optimum temperature for the enzyme activity was found to be 70 O C and it has significant activity over rang 60-75 O C. A slight decrease is obtained at higher temperatures (80-90 O C).

Fig. 5 :
Fig. 5: Effect of temperature on enzyme activity

Table 2 :
Effect of pH stability on the pure L-methioninase activity.

Table 3 :
Kinetics of L-methioninase for different substrates.

Table 4 :
Effect of some Metal Ions and chemical reagents.

Table 5 :
In-vitro cytotoxic activity of L-methioninaseas expressed as IC50 values on different cell lines.