Combinatorial approach for screening and assessment of multiple therapeutic enzymes from marine isolate Pseudomonas aeruginosa AR01 Yogeswaran Jagadeesan, a Shanmugapriya Meenakshisundaram, a Lokha Ranjani Alagar Boopathy, b Vijay Pradhap Singh Mookandi c and Anandaraj Balaiah * a 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 IC 50 value of 12 mg 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. 1. Introduction The world, with a population of 7.63 billion people, is highly susceptible to more than 30 000 diseases, which have been clinically characterized. Of these, less than one-third can be treated and only a few can be cured. 1 Emptiness and ineffec- tiveness in the arsenal of therapeutic agents led to a medical massacre with a huge death toll. The concept of the therapeutic enzyme has been around for at least 40 years. 2 Screening of therapeutic enzymes is principally preferred from microbes, as they are cheaper, easy to cultivate and their enzymes are more predictable and controllable, unlike plant and animal enzymes. Therapeutic enzymes derived from existing native and engi- neered bacterial origins have become less effective. Hence, there is an urgent need for new effective therapeutic compounds that are potent, safe and cheaper for curing threatening diseases. Nature has been a provider of therapeutic compounds for millennia with many novel drugs isolated from microorganisms. 3 Sometimes terrestrial microbes are consid- ered when there is a need for novel therapeutics. 2 Owing to the alarming emergence of dreadful diseases, there has been a great and growing market for therapeutic enzymes in the past decade. As a result, more biopharmaceuticals have been entering drug discovery and development pipelines in recent times. However, due to extensive research, exploration of ground-based natural bioactive compounds has become increasingly challenging. Instead, water-based natural compounds have become more promising alternatives, not only as pharmacological leads but also in industrial and commercial applications. 4 It is believed that life on Earth began from the great ocean – the origin of life. The ocean covers 71% of the Earth's surface and contains 1.4  10 21 litres of water. In the a Department of Biotechnology, University College of Engineering, BIT Campus, Anna University, Room no. 124, New Academic Block-B, Tiruchirappalli, Tamilnadu, 620 024, India. E-mail: drananadaraj74@gmail.com; yogi.affable@gmail.com; priyamsundaram94@gmail.com; Fax: +91-431-2407333; Tel: +91-431-2407947; +91- 9790915036; +91-9843707586; +91-9840669723 b Department of Biomedical Sciences, University of Illinois College of Medicine, Rockford, Illinois, 61107, USA. E-mail: lr.au.bit@gmail.com c Department of Biotechnology, Vivekananda College of Engineering for Women, Namakkal, Elaiyampalayam, Tiruchengode, Tamil Nadu, 637 205, India. E-mail: vijaypradhapsingh@gmail.com; Tel: +91-8668157693 Cite this: RSC Adv. , 2019, 9, 16989 Received 4th April 2019 Accepted 14th May 2019 DOI: 10.1039/c9ra02555c rsc.li/rsc-advances This journal is © The Royal Society of Chemistry 2019 RSC Adv., 2019, 9, 16989–17001 | 16989 RSC Advances PAPER Open Access Article. Published on 31 May 2019. Downloaded on 10/31/2020 3:11:30 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online View Journal | View Issue