In Silico modification and In Vitro validation of novel functional histidine acid phosphatase from Saccharomyces cerevisiae for in-feed poultry enzyme

Phytic acid (myo-inositol, 1,2,3,4,5,6-hexakisphosphate) is the primary storage and indigestible organic form of phosphorous, accounting for up to 85% of total phosphorous in plant seeds, legumes, and cereals. Excreted phytic acid from monogastric animals can cause an adverse effect on the environment through eutrophication. Positively charged minerals such as iron, zinc, and calcium are chelated by negatively charged phytic acid in the digestive tract of monogastric animals. It is essential to use an exogenous phytase enzyme to mitigate the adverse effects of phytic acid as an anti-nutritional factor in monogastric animals. In the current investigation, firstly, phytase-producing microorganisms were isolated through screening on PSM (phytase-specific medium) agar medium. Afterward, the whole genome of the phytase-producing yeast (SPA) was extracted and subjected to PCR for amplification of the histidine acid phosphatase (phytase) gene through degenerate primers. The amplified gene was codon optimized and expressed in the E. coil expression system. The functional phytase was obtained through the co-expression of the phytase gene along with the molecular chaperones of dnaK, dnaJ, and GroESL that shifted the phytase enzyme from insoluble (inclusion bodies) to soluble (bioactive) form. The co-expressed phytase was evaluated through biochemical and biophysical characterization to check the specific activity, pH and temperature profiles, and thermostability. Afterward, the phytase was subjected to protein engineering based on genetic engineering through site-directed mutagenesis to improve its thermostability and activity. Two mutants, E61A and D205E, were designed based on the hypothesis and Gibbs' free energy. The molecular dynamics (MD) simulation was also performed to confirm the mutants through in silico study. The mutations were performed by designing the mutagenic primers. SPA isolate attributed to S. cerevisiae illustrated the lowest extracellular phytase (HAP) activity of 194 mU/mL compared to other phytase-producing isolates. However, histidine acid phosphatases in the SPA strain isolate were detected through the in silico analyses, which made it appropriate for animal feed application. The BlastN illustrated that the amplified HAP gene (PhySc) shared 99.57% similarity with the PHO5 gene. The translation of the PhySc gene sequence to protein sequence and multiple sequence alignment demonstrated two natural mutations at the N- and C-terminal of the PhySc. It was demonstrated that hydrophobic-aromatic Phe2 was naturally substituted to the hydrophobic Leu2 at the N-terminus region. Meanwhile, the positively charged Arg466 residue was naturally mutated to the positively charged Lys466 residue at the C-terminus region. The confirmation of his-tagged protein and activity was carried out using a Histag ELISA kit and zymogram, at which both results were positive and showed that PhySc at 110 kDa is his-tag protein and bioactive. The expressed PhySc showed the optimum specific activity of 28.75 ± 0.39 U/mg at pH 5.5 and 40°C. The biochemical characterization of thermostability demonstrated a half-life of 55.44 sec at 60 ̊C and 2.75 min at 55 ̊C. The secondary structure prediction using circular dichroism (CD) demonstrated that random coil with 30.5% is the dominant content emphasized by structural model prediction. The E61A and D205E showed the optimum specific activities of 32.43 ± 0.45 and 26.715 ± 0.32 U/mg at pH 5.5 and 40 ˚C. The mutant E61A demonstrated half-lives of 5.44 min, 2.12 min, and 48 sec at 55, 60, and 65 ˚C temperatures, respectively. However, the mutant of D205E illustrated the half-lives of 4.38 and 2 min at temperatures of 55 and 60 ˚C, respectively. The E61A and D205E showed the km values of 1.4825 ± 0.07 and 1.9685 ± 0.065 mM, respectively. Furthermore, the Kcat/Km values of E61A and D205E were 461.09 ± 4.65 and 347.095 ± 4.15 (S-1.mM-1). The measured Tm recorded for mutants E61A and D205E were 57.7 and 58.65 ˚C. In conclusion, a new variant of the HAP gene was amplified from phytaseproducing S. cerevisiae SPA isolate and was functionally expressed through coexpression with molecular chaperones. A comprehensive biochemical, biophysical, and in silico characterization demonstrated that the features of this phytase are compatible with the poultry body for application in the animal feed industry. However, it suffers from low thermostability for a pelleting process. Its thermostability was also improved by implementing a site-directed mutagenesis approach, making it one more step closer to industrial application in poultry feed.

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