The R pycnus arginase was identical to the published

The R. pycnus arginase was 65% identical to the published B. caldovelox arginase sequence (AAB06939). Furthermore, the R. pycnus arginase gene was 62%, 45%, 36% and 22% identical to the published arginase sequences from Bacillus thuringiensis (AJI37018), Thermus thermophiles (WP_038039155), Human arginase I (2AEB) and Helicobacter pylori (ABO15436), respectively [25]. The major differences between eukaryotic and bacterial arginases occur in extensions at the C and N termini; the active sites of eukaryotic and bacterial arginases are largely similar. A 3D homology model of the R. pycnus arginase was built based on the published B. caldovelox arginase [17]. The active site is created by two long loops of residues 125–142 and 230–248, with the catalytic manganese ions (Mn22+) are at the bottom of the active site cavity. Compared with the Mn2+ bound to H100, the Mn2+ bound to H125 is closer to the surface and more exposed to the external solvent. It has been reported that second-shell residues could affect the geometry of the active site with respect to positioning of the manganese ligands [24]. Changes in geometry may affect water coordination and metal release [17]. Arginases are homo-oligomers consisting of typical 32–34kDa subunits. In general, eukaryotic arginases are trimeric, while bacterial arginases are hexameric [2]. The molecular mass of the full R. pycnus arginase complex indicated that the protein is a hexamer. The size of the R. pycnus arginase was similar to the sizes of arginases from Bacillus thuringiensis and B. caldovelox [9,24]. The optimal pH of the R. pycnus arginase is similar to most arginases, exception of the arginase from H. pylori and arginase I with Co2+, which both exhibited optimal activities at alkaline conditions with a wide range pH spectrum from 7.0 to 10.5 [23], [29]. In this study, an arginase from Bacillus JTP74057 receptor was found that could maintain a high enzyme activity with Ni2+ at low pH other than at the optimal pH 9.5 with Mn2+. Though Bacillus anthracis arginase could remain active at pH 6.3 with Ni2+, the activity of is quite low [27]. Previously, Abhishek et al. reported that the low optimal pH for H. pylori might due to its possession of a unique 13-residue sequence motif [23]. The pH shift in R. pycnus arginase is similar to Human arginase I with Co2+ but different from H. pylori arginase. The metal ligand bonds are asymmetrical, furthermore, the metal ligand lengths would change accordingly when the pH changed. This will lead to modification in metal selectivity while remain enzyme active at different pH [29]. Currently, the main strategy for L-ornithine production via enzyme hydrolysis uses beef liver L-arginase. The optimal temperature of beef liver L-arginase is 37°C, and its t1/2 is less than 100min at its optimal temperature [20]. A thermostable arginase, such as the arginase produced by the extreme thermophile B. caldovelox, has a t1/2=107min at 60°C without any additives (bovine serum albumin) [24]. Compared with the B. caldovelox arginase and the beef liver L-arginase, the R. pycnus arginase had a higher optimal temperature and significantly higher thermal stability. Generally, industrial applications for enzymatic L-ornithine production require a thermophilic arginase with relatively high optimum temperature. L-arginine hydrolysis performed at higher temperature may improve the reaction rate and prevent the reaction mixture from being biologically contaminated. The optimal temperature of R. pycnus arginase was determined to be 80°C. This significant high optimal temperature could obviously certify its application in L-ornithine biosynthesis at the range of 40–70°C. Moreover, such high optimal temperature suggested that this arginase had a high structural stability. More than 85% and 60% enzyme activity was remained after incubating at 40–50 and 60°C for 24 and 15h, respectively. This enzyme revealed promising thermal stability which could maintain high enzyme efficiencies for production.