Prokaryotic Argonautes for in vivo biotechnology and molecular diagnostics.

Prokaryotic Argonautes (pAgos) are an emerging class of programmable endonucleases that are believed to be more flexible than existing CRISPR-Cas systems and have significant potential for biotechnology. Current applications of pAgos include a myriad of molecular diagnostics and in vitro DNA assembly tools. However, efforts have historically been centered on thermophilic pAgo variants. To enable in vivo biotechnological applications such as gene editing, focus has shifted to pAgos from mesophilic organisms. We discuss what is known of pAgos, how they are being developed for various applications, and strategies to overcome current challenges to in vivo applications in prokaryotes and eukaryotes.

Biochemical and molecular insights on the bioactivity and binding interactions of Bacillus australimaris NJB19 L-asparaginase.

L-asparaginase, an antileukemic enzyme, is indispensable to the treatment of Acute Lymphoblastic Leukemia (ALL). However, the intrinsic glutaminase activity entails various side effects to the patients; thus, an improved version of the enzyme lacking glutaminase activity would be a requisite for effective treatment management of ALL. The present study highlights the biochemical and molecular characteristics of the recombinant glutaminase-free L-asparaginase from Bacillus australimaris NJB19 (BaAsp). Investigation of the active site architecture of the protein unraveled the binding interactions of BaAsp with its substrate. Comparative analysis of the L-asparaginase sequences revealed few substitutions of key amino acids in the BaAsp that could construe its substrate selectivity and specificity. The purified heterologously expressed protein (42 kDa) displayed maximum L-asparaginase activity at 35-40 °C and pH 8.5-9, with no observed L-glutaminase activity. The kinetic parameters, Km and Vmax, were determined as 45.6 µM and 0.16 µmoles min-1, respectively. Furthermore, in silico analysis revealed a conserved zinc-binding site in the protein, which is generally implicated in inhibiting the L-asparaginase activity. However, BaAsp was not inhibited by zinc at 1 mM concentration. Therefore, the findings provide insights on the biochemical and molecular details of BaAsp, which could be valuable in formulating it for alternate antileukemic drug therapy.

A potential type-II L-asparaginase from marine isolate Bacillus australimaris NJB19: Statistical optimization, in silico analysis and structural modeling.

L-asparaginase is a cardinal biotherapeutic drug for treating acute lymphoblastic leukemia, which is highly prevalent in children worldwide. In the current investigation, L-asparaginase producing marine bacterial isolate, Bacillus australimaris NJB19 (MG734654), was observed to be producing extracellular glutaminase free L-asparaginase (13.27 ± 0.4 IU mL-1). Production of L-asparaginase was enhanced by the Box-Behnken design approach that enumerated the significant variables affecting the enzyme production. The optimum levels of the derived variables resulted in 2.8-fold higher levels of the enzyme production (37.93 ± 1.06 IU mL-1). An 1146 bp L-asparaginase biosynthetic gene of Bacillus australimaris NJB19 was identified and cloned in E. coli DH5α, fused with a histidine tag. The in silico analysis of the protein sequence revealed the presence of a signal peptide and classified it as a type II L-asparaginase. Toxic peptide prediction disclosed no toxin domain in the protein sequence, hence suggesting it as a non-toxic protein. The secondary structure analysis of the enzyme displayed a comparable percentage of alpha-helical and random coil structure, while 14.39% and 6.57% of amino acid residues were composed of extended strands and beta-turns, respectively. The functional sites in the three-dimensional structural model of the protein were predicted and interestingly had a few less conserved residues. Bacillus australimaris NJB19 identified in this study produces type-II L-asparaginase, known for its high affinity for asparagine and effectiveness against leukemic cells. Hence, these observations indicate the L-asparaginase, thus obtained, as a potentially significant and novel therapeutic drug.