br Conclusion br Acknowledgments The authors extend

Conclusion
Acknowledgments The authors extend their appreciation to Kayyali Chair for Pharmaceutical Industry, Department of Pharmaceutics, College of Pharmacy, King Saud University for funding this work through the research project Number (G-2016-2).
Introduction L-ornithine is a natural but non-essential amino colorimetric assay in mammals. This amino acid is an effective reagent in healing wounds, protecting the liver, relieving stress and improving sleep and fatigue symptoms [1]. Hence L-ornithine could be used as a nutritional supplement for bodybuilders and applied to the pharmaceutical industry. Owing to its multiple useful applications, a convenient strategy for L-ornithine production is needed. Arginase (L-arginine amidinohydrolase, EC 3.5.3.1) is a binuclear metalloenzyme that hydrolyzes L-arginine to L-ornithine and urea. This reaction is accomplished by cleaving the guanidine group from L-arginine. Arginase is considered to be a key enzyme responsible for the urea cycle for the organisms which contain arginase are able to carry out the complete urea cycle. Arginase is widely distributed throughout the biosphere and has been found in a diverse set of organisms, including animals, plants, fungus and bacteria [2]. In animals, arginase depletes arginine levels by hydrolyzing arginine to inhibit the growth of cancer cells which are auxotrophic for arginine. It has been reported that site-directed pegylated human arginase I could be used as anti-cancer and anti-viral agents [3], [4]. Moreover, arginase could be a useful bioanalytical tool, allowing for the monitoring of arginine levels in blood [5] and pharmaceuticals [6]. In plants, the major functions of arginase is nitrogen fixation, which is a crucial step during fruit development [7]. In bacteria, arginases in Bacillus caldovelox, Helicobacter pylori, Bacillus thuringiensis and Bacillus subtilis have been well characterized [8]. Studies revealed that most of arginases are homo-oligomers including trimer, tetramer and hexamer. Furthermore, arginases have been proved to be metal ion activated enzymes, particular activated by divalent cations. There are three methods for the industrial production of L-ornithine [9]. Compared to chemical synthesis and fermentative production, the method using arginase is advantageous owning to its specificity for the selected reactions, its use of simple apparatuses and procedures, and its tolerance to the co-solvents used to solubilize substrates with low water solubility [10]. Currently, arginase applied in L-ornithine biosynthesis is mainly extracted from animal livers, however this approach would be high cost and viral contamination [8]. It seems that genetic technology might be an efficient method to overcome these problems. With the overexpression of human arginase I and B. caldovelox arginase gene in Pichia pastoris and Escherichia coli, the production of 149 and 112.3g/L L-ornithine were obtained [11], [12]. Nevertheless, low enzyme activity and poor stability of these arginases could restrict the further application of these enzymes. Therefore, a high enzyme activity and thermostable arginase is needed to be found. Previously, Rummeliibacillus pycnus SK31.001 was isolated from watermelon planting soil samples by our laboratory. Before this study, the only genomic information for R. pycnus was its 16S rRNA sequence. It is the first report regarding the nucleotide and amino acid sequence of R. pycnus arginase. In this study, arginase encoding gene from R. pycnus SK31.001 was overexpressed in E.coli. This enzyme displayed considerable activity and significant thermostability. Kinetic parameters of the enzyme were also determined and compared to the other arginases. Additionally, high productivity revealed that this arginase could be a good candidate for L-ornithine biosynthesis.
Materials and methods
Results
Discussion Before this study, the only genomic information for R. pycnus was its 16S rRNA sequence. This is the first report regarding the nucleotide and amino acid sequence of R. pycnus arginase. Several primers for the R. pycnus arginase were designed based on sequences from homologous species, but these primers all failed to amplify the correct gene. Nevertheless, BLAST analysis of nucleotide sequences from Bacillus genera revealed that arginases possess highly conserved motifs [23]. Primers for degenerate PCR were therefore designed from these conserved motifs. Sequences upstream and downstream of the R. pycnus arginase gene were obtained by inverse PCR as described by Kataoka et al. [28]. Compared with conventional methods [16], this method is simple and reliable. The strategy for amplifying R. pycnus arginase DNA uses degenerate PCR and inverse PCR, generalizing the technology used for screening of unknown genes.