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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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Fluorescence spectroscopy showing the emission spectrum in the range of 300–400nm (excitation wavelength: 292 nm) and unfolding transitions of L-asparaginase at 0M, 3M and 6M guanidine HCl.
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pone-0099037-g006: Fluorescence spectroscopy showing the emission spectrum in the range of 300–400nm (excitation wavelength: 292 nm) and unfolding transitions of L-asparaginase at 0M, 3M and 6M guanidine HCl.

Mentions: The purified L-asparaginase protein showed maximum fluorescence at 323 nm in its native form in the absence of GdHCl, however a shift to 352 nm was observed when 6 M GdnCl was added thus stating its complete unfolding as presented in Figure 6. A higher fluorescence was observed in the protein in its unfolded cold state.


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)

Fluorescence spectroscopy showing the emission spectrum in the range of 300–400nm (excitation wavelength: 292 nm) and unfolding transitions of L-asparaginase at 0M, 3M and 6M guanidine HCl.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0099037-g006: Fluorescence spectroscopy showing the emission spectrum in the range of 300–400nm (excitation wavelength: 292 nm) and unfolding transitions of L-asparaginase at 0M, 3M and 6M guanidine HCl.
Mentions: The purified L-asparaginase protein showed maximum fluorescence at 323 nm in its native form in the absence of GdHCl, however a shift to 352 nm was observed when 6 M GdnCl was added thus stating its complete unfolding as presented in Figure 6. A higher fluorescence was observed in the protein in its unfolded cold state.

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