Ecology, Environment and Conservation Paper

IDENTIFICATION OF L-ASPARAGINASE-PRODUCING BACTERIA AND CHARACTERIZATION OF THE EFFECT OF CHROMIUM ON THEIR GENE EXPRESSION AND PRODUCTION EFFICIENCY

Alawiah ALhebshi, Fada Mahja AL-Anazi and El-Hamshary, O. I. M.

Abstract

L-Asparaginase is a tetrameric protein produced from bacteria, having anti leukemic activity on human cells. Reactive oxygen species (ROS) production is the central mechanism for Chromium genotoxicity, which was demonstrated through its ability to generate superoxide radical anions as a type of ROS. Therefore, L asparaginase is a tetrameric protein produced from bacteria, having anti leukemic activity on human cells. Therefore, the present study is aimed to isolate, screen, identify L-asparaginase producing bacteria, study the resistance antibiotic and plasmid profile and examine effects of chromium CrO3 [Cr(VI)] on bacterial Lasparaginase activity and gene expression. Fifty isolates were obtained from the rhizospheres of four plants (Anethum graveolens, Portulaca oleracea, Eruca sativa and Phaseolus vulgaris), 5 isolates were obtained from the soil) collected from different plantations in Algamom, Makkah, Saudi Arabia. The fifty bacterial isolates were screened for potential L-asparaginase production using a rapid assay plate method on M9 medium containing L-asparagine with phenol red as an indicator. Four isolates showed potential L-asparaginase production. Based on morphological, biochemical characteristics and 16SrRNA gene sequencing data, the most potent isolates were selected and identified as three isolates of Acinetobacter and Tatumella ptyseos. The potent strains were designated Acinetobacter baumannii (HO6), Acinetobacter haemolyticus (FA3) and Acinetobacter lwoffii (FA1) and Tatumella ptyseos (FA2). The 16SrRNA sequences were submitted to the NCBI Gen Bank under accession numbers, KY313625 for A. lwoffii (FA1), KY313626 for T. ptyseos (FA2), KY313627 for A. haemolyticus (FA3) and KY313633 for A. baumannii (HO6) isolates. FA2 is the first T. ptyseos strain identified with high level of L-asparaginase enzyme production. Furthermore, these strains were used to examine the toxic effects of low concentrations of chromium (VI) on enzyme activity. Spectrophotometric analysis revealed that the three CrO3 [Cr(VI)] concentrations (0.05, 0.1 and 0.15 mM) reduced the enzyme activity of A.baumannii (HO6) and T. ptyseos (FA2) and enhanced the enzyme activity of A. haemolyticus (FA3). A. baumannii (HO6) was selected for to examine the toxic effects of low concentrations of chromium (VI) on L-asparaginase gene expression by quantitative real time PCR (qRT PCR to verify of L-asparaginase gene expression levels in A. baumannii (HO6) after treatment with three CrO3 [Cr(VI)] concentrations. Quantitative analysis of Asparaginase gene m RNA showed that addition of three CrO3 [Cr(VI)] concentrations (0.05, 0.1 and 0.15 mM) reduced the expression level to 9.402, 4.702 and 3.205 respectively after treatment compared with control. These strains were described for their antibiotic resistance profile, the results indicated that all strains were sensitive to six antibiotics, but resistant to ampicillin/sulbactam metronidazole was observed. In addition, the plasmid profile indicated that the three strains harbored one plasmid and A. lwoffii (FA1) harbored two plasmids. Therefore, the effect of different concentrations of mechachromium on the plasmid content of A.baumannii (HO6) was examined, and the results indicated plasmid curing with the three examined chromium concentrations. Therefore, four strains are good sources for Lasparagenase and is a new source of enzyme.