Mining of Ruminant Microbial Phytase (RPHY1) from Metagenomic Data of Mehsani Buffalo Breed: Identification, Gene Cloning, and Characterization

Abstract

Phytases have been widely used as animal feed supplements to increase the availability of digestible phosphorus, especially in monogastric animals fed cereal grains. The present study describes the identification of a full-length phytase gene of Prevotella species present in Mehsani buffalo rumen. The gene, designated as RPHY1, consists of 1,251 bp and is expressed into protein with 417 amino acids. A homology search of the deduced amino acid sequence of the RPHY1 phytase gene in a nonredundant protein database showed that it shares 92% similarity with the histidine acid phosphatase domain. Subsequently, the RPHY1 gene was expressed using a pET32a expression vector in Escherichia coli BL21 and purified using a His60 Ni-NTA gravity column. The mass of the purified RPHY1 was estimated to be approximately 63 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal RPHY1 enzyme activity was observed at 55°C (pH 5) and exhibited good stability at 5°C and within the acidic pH range. Significant inhibition of RPHY1 activity was observed for Mg²⁺ and K⁺ metal ions, while Ca²⁺, Mn²⁺, and Na⁺ slightly inhibited enzyme activity. The RPHY1 phytase was susceptible to SDS, and it was highly stimulated in the presence of EDTA. Overall, the observed comparatively high enzyme activity levels and characteristics of the RPHY1 gene mined from rumen prove its promising candidature as a feed supplement enzyme in animal farming.

Evolution of E. coli Phytase for Increased Thermostability Guided by Rational Parameters

“…Numerous nonrational, mostly labor-intensive protein engineering methods such as eqPCR [8], DNA shuffling [9] and mutagenesis [10,11] were also applied. At the same time, strategies such as metagenome analysis [12] and screening of thermophilic bacteria [13,14] were also utilized to develop novel heatresistant phytases. Phytases from well-known microorganisms such as Aspergillus niger [15], Escherichia coli [16] and Bacillus subtilis [1] have been reengineered by directed evolution employing random mutagenesis and multigene DNA shuffling.…”