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Purification and characterization of a novel and robust L-asparaginase having low-glutaminase activity from Bacillus licheniformis: in vitro evaluation of anti-cancerous properties.

Mahajan RV, Kumar V, Rajendran V, Saran S, Ghosh PC, Saxena RK - PLoS ONE (2014)

Bottom Line: Protein was found to be a homotetramer of 134.8 KDa with monomeric size of 33.7 KDa and very specific for its natural substrate i.e. L-asparagine.Kinetic parameters Km, Vmax and kcat of purified enzyme were found as 1.4×10(-5) M, 4.03 IU and 2.68×10(3) s(-1), respectively.However the enzyme had no toxic effect on human erythrocytes and CHO cell lines hence should be considered potential candidate for further pharmaceutical use as an anticancer drug.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Delhi South Campus, New Delhi, Delhi, India.

ABSTRACT
L-asparaginase having low glutaminase has been a key therapeutic agent in the treatment of acute lymphpoblastic leukemia (A.L.L). In the present study, an extracellular L-asparaginase with low glutaminase activity, produced by Bacillus licheniformis was purified to homogeneity. Protein was found to be a homotetramer of 134.8 KDa with monomeric size of 33.7 KDa and very specific for its natural substrate i.e. L-asparagine. The activity of purified L-asparaginase enhanced in presence of cations including Na+ and K+, whereas it was moderately inhibited in the presence of divalent cations and thiol group blocking reagents. The purified enzyme was maximally active over the range of pH 6.0 to 10.0 and temperature of 40°C and enzyme was stable maximum at pH 9.0 and -20°C. CD spectra of L-asparaginase predicted the enzyme to consist of 63.05% α-helix and 3.29% β-sheets in its native form with T222 of 58°C. Fluorescent spectroscopy showed the protein to be stable even in the presence of more than 3 M GdHCl. Kinetic parameters Km, Vmax and kcat of purified enzyme were found as 1.4×10(-5) M, 4.03 IU and 2.68×10(3) s(-1), respectively. The purified L-asparaginase had cytotoxic activity against various cancerous cell lines viz. Jurkat clone E6-1, MCF-7 and K-562 with IC50 of 0.22 IU, 0.78 IU and 0.153 IU respectively. However the enzyme had no toxic effect on human erythrocytes and CHO cell lines hence should be considered potential candidate for further pharmaceutical use as an anticancer drug.

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Effect of chemical parameters on purified L-asparaginase a) Effect of metal ions (100 mM) on enzyme activity; b) Effect of inhibitors (10 mM) on enzyme activity (EDTA: Ethylenediaminetetraacetic acid, EGTA: Ethylene glycol tetraacetic acid, PCMB: p-chloromercuribenzoic acid, PMSF: Phenylmethanesulfonylfluoride, PBA c) Effect of serum and serum components (10 mM) on enzyme activity; d) Substrate (10 mM) specificity of enzyme.100% activity corresponds to 140 U of enzyme. Error bars represent SD of three experiments.
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pone-0099037-g003: Effect of chemical parameters on purified L-asparaginase a) Effect of metal ions (100 mM) on enzyme activity; b) Effect of inhibitors (10 mM) on enzyme activity (EDTA: Ethylenediaminetetraacetic acid, EGTA: Ethylene glycol tetraacetic acid, PCMB: p-chloromercuribenzoic acid, PMSF: Phenylmethanesulfonylfluoride, PBA c) Effect of serum and serum components (10 mM) on enzyme activity; d) Substrate (10 mM) specificity of enzyme.100% activity corresponds to 140 U of enzyme. Error bars represent SD of three experiments.

Mentions: It is evident from the Figure 3a that enzyme activity enhanced considerably in the presence of most the monovalent cations and was maximal in the presence of Na+, K+ and Mg++. However, the other divalent cations had detrimental effect on the enzyme activity. The enzyme retained more than 80% activity in the presence of serine protease inhibitors viz. PMSF & PBA hence indicating that it is not a serine hydrolase. There was decrease of 60% in the activity of L-asparaginase from Bacillus licheniformis RAM-8 in the presence of sulfhydryl inhibitors. Also, dissociating agents urea and EDTA inhibited the activity by 70% (Figure 3b).


Purification and characterization of a novel and robust L-asparaginase having low-glutaminase activity from Bacillus licheniformis: in vitro evaluation of anti-cancerous properties.

Mahajan RV, Kumar V, Rajendran V, Saran S, Ghosh PC, Saxena RK - PLoS ONE (2014)

Effect of chemical parameters on purified L-asparaginase a) Effect of metal ions (100 mM) on enzyme activity; b) Effect of inhibitors (10 mM) on enzyme activity (EDTA: Ethylenediaminetetraacetic acid, EGTA: Ethylene glycol tetraacetic acid, PCMB: p-chloromercuribenzoic acid, PMSF: Phenylmethanesulfonylfluoride, PBA c) Effect of serum and serum components (10 mM) on enzyme activity; d) Substrate (10 mM) specificity of enzyme.100% activity corresponds to 140 U of enzyme. Error bars represent SD of three experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048267&req=5

pone-0099037-g003: Effect of chemical parameters on purified L-asparaginase a) Effect of metal ions (100 mM) on enzyme activity; b) Effect of inhibitors (10 mM) on enzyme activity (EDTA: Ethylenediaminetetraacetic acid, EGTA: Ethylene glycol tetraacetic acid, PCMB: p-chloromercuribenzoic acid, PMSF: Phenylmethanesulfonylfluoride, PBA c) Effect of serum and serum components (10 mM) on enzyme activity; d) Substrate (10 mM) specificity of enzyme.100% activity corresponds to 140 U of enzyme. Error bars represent SD of three experiments.
Mentions: It is evident from the Figure 3a that enzyme activity enhanced considerably in the presence of most the monovalent cations and was maximal in the presence of Na+, K+ and Mg++. However, the other divalent cations had detrimental effect on the enzyme activity. The enzyme retained more than 80% activity in the presence of serine protease inhibitors viz. PMSF & PBA hence indicating that it is not a serine hydrolase. There was decrease of 60% in the activity of L-asparaginase from Bacillus licheniformis RAM-8 in the presence of sulfhydryl inhibitors. Also, dissociating agents urea and EDTA inhibited the activity by 70% (Figure 3b).

Bottom Line: Protein was found to be a homotetramer of 134.8 KDa with monomeric size of 33.7 KDa and very specific for its natural substrate i.e. L-asparagine.Kinetic parameters Km, Vmax and kcat of purified enzyme were found as 1.4×10(-5) M, 4.03 IU and 2.68×10(3) s(-1), respectively.However the enzyme had no toxic effect on human erythrocytes and CHO cell lines hence should be considered potential candidate for further pharmaceutical use as an anticancer drug.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Delhi South Campus, New Delhi, Delhi, India.

ABSTRACT
L-asparaginase having low glutaminase has been a key therapeutic agent in the treatment of acute lymphpoblastic leukemia (A.L.L). In the present study, an extracellular L-asparaginase with low glutaminase activity, produced by Bacillus licheniformis was purified to homogeneity. Protein was found to be a homotetramer of 134.8 KDa with monomeric size of 33.7 KDa and very specific for its natural substrate i.e. L-asparagine. The activity of purified L-asparaginase enhanced in presence of cations including Na+ and K+, whereas it was moderately inhibited in the presence of divalent cations and thiol group blocking reagents. The purified enzyme was maximally active over the range of pH 6.0 to 10.0 and temperature of 40°C and enzyme was stable maximum at pH 9.0 and -20°C. CD spectra of L-asparaginase predicted the enzyme to consist of 63.05% α-helix and 3.29% β-sheets in its native form with T222 of 58°C. Fluorescent spectroscopy showed the protein to be stable even in the presence of more than 3 M GdHCl. Kinetic parameters Km, Vmax and kcat of purified enzyme were found as 1.4×10(-5) M, 4.03 IU and 2.68×10(3) s(-1), respectively. The purified L-asparaginase had cytotoxic activity against various cancerous cell lines viz. Jurkat clone E6-1, MCF-7 and K-562 with IC50 of 0.22 IU, 0.78 IU and 0.153 IU respectively. However the enzyme had no toxic effect on human erythrocytes and CHO cell lines hence should be considered potential candidate for further pharmaceutical use as an anticancer drug.

Show MeSH
Related in: MedlinePlus