Characterization of a novel asparaginase from soil metagenomic libraries generated from forest soil
To screen soil metagenomic libraries for novel enzymes with enhanced activities.
To screen soil metagenomic libraries for novel enzymes with enhanced activities. A novel l-asparaginase was identified from forest soil metagenome and its characteristics were studied. The purified protein had a specific activity of 696 IU mg−1 and optimum activity at pH 7 and 35 °C. Enhanced enzyme activities were observed in the presence of Mg2+, Ca2+ and K+. The Km value, 2 mM, and enzyme specificity constant 7.7 mM−1s−1 indicated that the recombinant enzyme has good substrate affinity to l-asparagine compared with commercially-available Escherichia coli asparaginase. The IC50 value of 0.78 µg ml−1 (0.47 IU ml−1) was observed with HL60 cell line and 0.39 µg ml−1(0.23 IU ml−1) with MOLT-3 and MOLT-4 cell lines, which is better than that of commercially-available drugs.
The soil metagenome derived l-asparaginase with enhanced activities could be a potential candidate to develop as a drug in Acute Lymphoblastic Leukemia (ALL) therapy.
KeywordsAcute Lymphoblastic Leukemia Anticancer l-Asparaginase Metagenomics Therapeutic enzyme
The authors are thankful to Prof. M. Radhakrishna Pillai, Director, Rajiv Gandhi Centre Biotechnology for the facilities provided. The authors are grateful to Department of Biotechnology (DBT) and University Grants Commission [UGC SRF, No.20-12/2009(ii) EU-IV], Government of India for the financial support.
Supplementary Table 1—Summary of functional screening of soil metagenomic libraries.
Supplementary Table 2—List of operational taxonomic units (OTU) identified from 16S rDNA library.
Supplementary Fig. 1—Agarose gel electrophoresis of soil metagenomic DNA isolated.
Supplementary Fig. 2—Restriction fragment length polymorphism (RFLP) of 16S rDNA library generated.
Supplementary Fig. 3—Agarose gel electrophoresis of L-asparaginase gene amplified from clone PLD-121.
Supplementary Fig. 4—Phylogeny tree of recombinant asparaginase with homologues sequences.
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