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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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Related in: MedlinePlus

Molecular weight determination of purified enzyme (a) SDS PAGE for steps of purifying L-asparaginase; b) Plot of Ve/Vo against semi-log of molecular weight of proteins on Sephacryl TM S-200 high resolution column (16/60) for α-Lactalbumin (12.4 kDa), carbonic anhydrase (30 kDa), bovine serum albumin (66 kDa), yeast alcohol dehydrogenase (150 kDa), sweet potato β-amylase (200 kDa) and ferritin (450 kDa).
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pone-0099037-g001: Molecular weight determination of purified enzyme (a) SDS PAGE for steps of purifying L-asparaginase; b) Plot of Ve/Vo against semi-log of molecular weight of proteins on Sephacryl TM S-200 high resolution column (16/60) for α-Lactalbumin (12.4 kDa), carbonic anhydrase (30 kDa), bovine serum albumin (66 kDa), yeast alcohol dehydrogenase (150 kDa), sweet potato β-amylase (200 kDa) and ferritin (450 kDa).

Mentions: The stepwise purification of L-asparaginase from Bacillus licheniformis RAM-8 has been presented in Table 1 and Figure 1a. The series of chromatography steps were very effective and gave overall purification of 33-fold. The final protein content of the purified L-asparaginase was 15.25 mg, with the yield of 32.95%. The specific activity of purified enzyme was 697.09 IU/mg of protein. Protein was found to have molecular size of 134.8 kDa (Figure 1b) as determined through gel-filtration chromatography and monomeric size of 33.7 kDa confirmed by SDS-PAGE. The theoretical IEF of enzyme was calculated to be 5.48.


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)

Molecular weight determination of purified enzyme (a) SDS PAGE for steps of purifying L-asparaginase; b) Plot of Ve/Vo against semi-log of molecular weight of proteins on Sephacryl TM S-200 high resolution column (16/60) for α-Lactalbumin (12.4 kDa), carbonic anhydrase (30 kDa), bovine serum albumin (66 kDa), yeast alcohol dehydrogenase (150 kDa), sweet potato β-amylase (200 kDa) and ferritin (450 kDa).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0099037-g001: Molecular weight determination of purified enzyme (a) SDS PAGE for steps of purifying L-asparaginase; b) Plot of Ve/Vo against semi-log of molecular weight of proteins on Sephacryl TM S-200 high resolution column (16/60) for α-Lactalbumin (12.4 kDa), carbonic anhydrase (30 kDa), bovine serum albumin (66 kDa), yeast alcohol dehydrogenase (150 kDa), sweet potato β-amylase (200 kDa) and ferritin (450 kDa).
Mentions: The stepwise purification of L-asparaginase from Bacillus licheniformis RAM-8 has been presented in Table 1 and Figure 1a. The series of chromatography steps were very effective and gave overall purification of 33-fold. The final protein content of the purified L-asparaginase was 15.25 mg, with the yield of 32.95%. The specific activity of purified enzyme was 697.09 IU/mg of protein. Protein was found to have molecular size of 134.8 kDa (Figure 1b) as determined through gel-filtration chromatography and monomeric size of 33.7 kDa confirmed by SDS-PAGE. The theoretical IEF of enzyme was calculated to be 5.48.

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