Extracellular l-Asparaginase from a Protease-Deficient Bacillus aryabhattai ITBHU02: Purification, Biochemical Characterization, and Evaluation of Antineoplastic Activity In Vitro

Abstract

An extracellular L-asparaginase produced by a protease-deficient isolate, Bacillus aryabhattai ITBHU02, was purified to homogeneity using ammonium sulfate fractionation and subsequent column chromatography on diethylaminoethyl-Sepharose fast flow and Seralose CL-6B. The enzyme was purified 68.9-fold with specific activity of 680.47 U mg(-1). The molecular weight of the purified enzyme was approximately 38.8 kDa on SDS-PAGE and 155 kDa on native PAGE gel as well as gel filtration column revealing that the enzyme was a homotetramer. The optimum activity of purified L-asparaginase was achieved at pH 8.5 and temperature 40 °C. Kinetic studies depicted that the K m, V max, and k cat values of the enzyme were 0.257 mM, 1.537 U μg(-1), and 993.93 s(-1), respectively. Circular dichroism spectroscopy has showed that the enzyme belonged to α + β class of proteins with approximately 74 % α-helices and 12 % β-sheets. BLASTP analysis of N-terminal sequence K-T-I-I-E-A-V-P-E-L-K-K-I-A of purified L-asparaginase had shown maximum similarity with Bacillus megaterium DSM 319. In vitro cytotoxicity assays with HL60 and MOLT-4 cell lines indicated that the L-asparaginase has significant antineoplastic properties.

Isolation, purification and characterization of an extracellular L-asparaginase produced by a newly isolated <i>Bacillus megaterium</i> strain MG1 from the water bodies of Moraghat forest, Jalpaiguri, India

“…The 31.52-fold purified enzyme had specific activity of 215 U mg -1 protein. The specific activity of asparaginase from B. megaterium MG1 was much higher than that of Bacillus subtilis strain hswx88 and Pectobacterium carotovorum MTCC 1428 (Pradhan et al, 2013) and quite lower than that of B. aryabhattai ITBHU02 and Bacillus licheniformis RAM-8 (Mahajan et al, 2014;Singh et al, 2013). The enzyme has been purified to homogeneity, as observed by the presence of one single band by SDS-PAGE displaying an apparent subunit molecular weight of approximately 47 kDa.…”

“…Protein bands were visualized with Coomassie brilliant blue staining against protein molecular weight markers of 47 kDa,80 kDa,110 kDa,135 kDa,190 kDa,208 kDa (New England BioLabs,USA). Moreover, a gel filtration chromatography using a Hi-Load Superdex-200 16/60 column was equilibrated with 0.05 M Tris-HCl buffer (pH 8.5) containing 0.05 M NaCl and 10% glycerol as described by Singh et al (2013). The column was calibrated with ribonuclease A (13.7 kDa), carbonic anhydrase (29 kDa), ovalbumin (44 kDa), aldolase (158 kDa), and ferritin (440 kDa) (GE Healthcare).…”

“…Mashburn and Wriston (1964) reported anti-tumor activity of the E. coli enzyme. In fact, bacterial Lasparaginases have become a mainstay of multidrug chemotherapeutic regimens for the treatment of malignancies (Singh et al, 2013). The antineoplastic activity of the enzyme lies in the fact that, unlike normal cells, L-asparagine synthetase activity is either absent or extremely low in tumour cells which have a compromised ability to generate L-asparagine endogenously (Sunitha et al, 2010).…”

“…The enzyme has been reportedly isolated from various bacterial origins such as Bacillus subtilis (Pradhan et al, 2013), Bacillus aryabhattai (Singh et al, 2013), Escherichia (Warangkar and Khobragade, 2010), Xanthomonas, Pectobacterium, Photobacterium (Abbas et al, 2010), Aerobacter, Erwinia, Serratia, Streptomyces (Agarwal et al, 2011), and Corynebacterium (Mesas et al, 1990) species; and from fungal origins such as Aspergillus and Candida species (Kumar and Sobha, 2012); and a few from protozoa, for example, Tetrahymena pyriformis (Triantafillou et al, 1988). Though the enzyme is widely distributed, only some of these L-asparaginases possess antineoplastic activity and among the microbial sources, the most commercially notable ones are Escherichia coli, Erwinia carotovora and Serratia marcescens (Kumar and Shobha, 2012).…”

“…Hence, the search for a new serologically different enzyme, having good therapeutic effect, but less or no toxicity, is still on. To obtain a better and alternative source of L-asparaginase, there is an ongoing interest to screen new organisms from different sources (Singh et al, 2013). In this context, we have tried to explore the potent L-asparaginase-producing bacterial strains from water bodies of the Moraghat forest, a territorial forest of Jalpaiguri.…”