Bacillus sonorensis L. Asparaginase: Cloning, Expression in E. coli and Characterization.

L-asparaginases (L-ASNases; EC 3.5.1.1) are aminohydrolases that catalyze the hydrolysis of L-asparagine (L-Asn) to L-aspartic acid and ammonia, resulting in the death of acute lymphoblastic leukemic cells and other blood cancer cells. In this study, Bacillus sonorensis (accession number MK523484) uncharacterized L-ASNase gene (accession number MN562875) was isolated by polymerase chain reaction (PCR), cloned into pET28a (+) vector, and expressed in Escherichia coli as a cytosolic protein. The recombinant enzyme was purified by affinity chromatography at 23.79-fold and 49.37% recovery. Denaturing polyacrylamide gel (10%) analysis of the purified enzyme resulted in a single protein band at 36 kDa that immunoreacted strongly with 6His-tag monoclonal antibody. The purified enzyme exhibited optimal activity at 45 °C and pH 7.0 and retained 92% and 85% of its initial activity after incubation for 60 min at 37 °C and 45 °C, respectively. The purified enzyme exhibited substrate specificity toward L-asparagine and low glutaminase activity (15.72%) toward L-glutamine at a concentration of 10 mM. The Km and Vmax values were 2.004 mM and 3723 µmol min1-, respectively.

Microbial control of the cotton leafworm Spodoptera littoralis (Boisd.) by Egyptian Bacillus thuringiensis isolates.

Four local Bacillus thuringiensis (Bt) isolates that had been serologically identified as Bt var. kurstaki (Btk2, Btk3, and Btk66) and Bt var. mexicanensis (Btm27), in addition to two reference strains (4D20 and 4AC1), were laboratory assayed as microbial control agents against the Egyptian cotton leafworm Spodoptera littoralis (Boisd.). Polymerase chain reaction (PCR) amplification analysis revealed that each of the six experimental strains carries, at least, a cry1 type gene which expresses a protein toxin active against lepidopterous insects. Additionally, PCR amplification results demonstrated that 4D20 and Btk66 contain the Lepidoptera- and Diptera-active cry2 type gene and that Btk66 contains Coleoptera-active cry7 and cry8 genes. Among the six strains, Btk66 and Btm27 were the most promising microbial control agents against S. littoralis. The present findings were the first to report that Btm27 (classified as B. thuringiensis var. mexicanensis) is a very potent microbial control agent against S. littoralis-tested larvae. For more characterization of these two isolates, the sspO gene was investigated as a molecular chronometer. The DNA sequencing results proved that Btk66 and Btm27 carry sspO open reading frames with identical nucleotide sequences, suggesting a strong phylogenetic relationship between the two strains.