Combinatorial approach for screening and assessment of multiple therapeutic enzymes from marine isolate Pseudomonas aeruginosa AR01

Abstract

Industrialization and modernization have led to humans being more susceptible to diseases. Therapeutic enzymes from traditional earthbound bacterial origin result have less therapeutic value. Hence, the hunt for a novel source of enzymes is indispensable. Twenty different marine bacterial strains were isolated from mangrove soil around S. P. Pattinum, Tamilnadu, India. From repeated qualitative and quantitative experiments, the study results were that, out of twenty bacterial isolates, only one Gram-negative bacterium was positive for multiple therapeutic enzymes such as asparaginase, glutaminase, uricase and collagenase. Based on its 99% 16S rRNA sequence similarity with Pseudomonas aeruginosa, the isolate was designated as Pseudomonas aeruginosa AR01. Modified minimal medium amended with asparagine results in a simple and cost-effective, one-pot production medium for enhanced production and easy purification of all therapeutic enzymes. The biochemical studies imply that the therapeutic enzymes from P. aeruginosa AR01 may find a significant role in medical applications. The in vitro cytotoxic study reveals that the anticancer enzyme from P. aeruginosa is considerably effective with an IC₅₀ value of 12 μg mL^-1 against K-562 cell line. Colony PCR was performed for the detection of specific therapeutic enzyme-coding genes in the genome of P. aeruginosa AR01. PCR results confirm that P. aeruginosa AR01 possesses nucleotide regions for corresponding therapeutic enzymes in its gene cluster. BLASTN and BLASTX analyses of the partial nucleotide sequences of therapeutic enzymes were deposited in GenBank. The results appear so promising that Pseudomonas aeruginosa AR01 may be a potent candidate for medical biotechnology.

Fig. 1. Growth of P. aeruginosa AR01 and therapeutic enzyme production patterns in different production media. (a) Growth patterns in different production media; (b) asparaginase and glutaminase activity; (c) uricase activity; (d) collagenase activity. MB, marine broth; MM, modified minimal medium; MM-ASP, modified minimal medium amended with 2.5% (w/v) asparagine; MM-GLU, modified minimal medium amended with 2.5% (w/v) glutamine; MM-GEL, modified minimal medium amended with 2.5% (w/v) gelatin; MM-UA, minimal medium amended with 0.3% (w/v) uric acid.

Fig. 2. Effect of protease inhibitors on collagenase activity. (a) Residual collagenase activity of CFCS against protease inhibitors. ***The significance of the test sample against control (untreated). (b) Zymography analysis of culture filtrate treated with different protease inhibitors: (1) control, untreated culture filtrate; (2) PMSF-treated culture filtrate; (3) PHNT-treated culture filtrate.

Fig. 3. Effect of temperature, pH and metal ions on therapeutic enzymes. (a) Effect of various temperatures, (b) effect of various pH and (c and d) effect of various monovalent and divalent metal ions on anticancer enzymes (asparaginase, glutaminase, uricase and collagenase).

Fig. 4. In vitro cytotoxic activity of P. aeruginosa AR01 CFCS against K-562 cell line. (A) Control, untreated cells; (B) camptothecin, 25 μM; C–H, CFCS at 3, 6, 12, 24, 48 and 96 μg mL⁻¹. The IC₅₀ value was found to be 12 μg mL⁻¹. ***The significance of the test sample against control, untreated; ^###the significance of the test sample against the commercial drug camptothecin.

Fig. 5. Detection of therapeutic genes from P. aeruginosa AR01 by colony PCR. Agarose gel images of purified PCR products (therapeutic genes). Lane M: 100-bp DNA Ladder (GeneRuler 100 bp Plus DNA Ladder, Thermo Scientific SM0323); lanes 1, 3, 5, 7 and 9 correspond to elastase (1497 bp), uricase (1485 bp), asparaginase (987 bp), glutaminase (909 bp) and rhodanese (816 bp) respectively; lanes 2, 4, 6, 8 and 10 correspond to negative controls for respective genes.

Fig. 6. Phylogenetic tree relationships of partial 16S rRNA and therapeutic enzyme sequences of P. aeruginosa AR01 with its closely related P. aeruginosa strains. (a) 16S rRNA sequence. Bootstrap values are indicated. The tree illustrates the maximum likelihood of P. aeruginosa AR01 strain among other P. aeruginosa strains. Evolutionary analysis by maximum likelihood method and Kimura 2-parameter model. The tree with the highest log likelihood is shown. The evolutionary analysis was conducted in MEGA X. (b) Asparaginase, (c) glutaminase, (d) elastase, (e) rhodanese, and (f) uricase phylogenetic tree relations between the protein sequences of therapeutic enzymes obtained from P. aeruginosa AR01 compared with protein sequences of closely related P. aeruginosa. The evolutionary history was inferred using the UPGMA method. Evolutionary analyses were conducted in MEGA X.