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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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CD spectra of purified L-asparaginase a) Far UV CD spectra of L-asparaginase at 0.1 mg/ml in 0.1 M tris HCL (pH-8.4); b) Melting temperature of enzyme (T222nm).c). Near UV CD spectra of purified L-asparaginase 1.0 mg/ml in 0.1 M Tris HCL (pH-8.4).
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pone-0099037-g005: CD spectra of purified L-asparaginase a) Far UV CD spectra of L-asparaginase at 0.1 mg/ml in 0.1 M tris HCL (pH-8.4); b) Melting temperature of enzyme (T222nm).c). Near UV CD spectra of purified L-asparaginase 1.0 mg/ml in 0.1 M Tris HCL (pH-8.4).

Mentions: CD spectrum of purified L-asparaginase gave negative elipticities at 208 and 222 nm (Figure 5a). The k2D analysis of the far UV CD spectrum from 240 to 200 nm predicted apha helix to be 63.05% and beta sheets to be 3.29% and Tm of protein was found to be 58°C which state its reason for steep decent in the activity when enzyme was tested above 60°C (Figure 5b). The near UV CD spectra of the protein gave negative elipticities in the range of 260 to 290 as presented in Figure 5c.


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)

CD spectra of purified L-asparaginase a) Far UV CD spectra of L-asparaginase at 0.1 mg/ml in 0.1 M tris HCL (pH-8.4); b) Melting temperature of enzyme (T222nm).c). Near UV CD spectra of purified L-asparaginase 1.0 mg/ml in 0.1 M Tris HCL (pH-8.4).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0099037-g005: CD spectra of purified L-asparaginase a) Far UV CD spectra of L-asparaginase at 0.1 mg/ml in 0.1 M tris HCL (pH-8.4); b) Melting temperature of enzyme (T222nm).c). Near UV CD spectra of purified L-asparaginase 1.0 mg/ml in 0.1 M Tris HCL (pH-8.4).
Mentions: CD spectrum of purified L-asparaginase gave negative elipticities at 208 and 222 nm (Figure 5a). The k2D analysis of the far UV CD spectrum from 240 to 200 nm predicted apha helix to be 63.05% and beta sheets to be 3.29% and Tm of protein was found to be 58°C which state its reason for steep decent in the activity when enzyme was tested above 60°C (Figure 5b). The near UV CD spectra of the protein gave negative elipticities in the range of 260 to 290 as presented in Figure 5c.

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