Expression of Phytase in Microalgae with Potential Application as A Feed Supplement

Abstract

Phytate, a prevalent anti-nutrient in plant-derived animal feed, impairs the bioavailability of essential minerals such as phosphorus, calcium, and iron in monogastric animals. The enzymatic hydrolysis of phytate by phytase has emerged as an effective strategy to enhance mineral absorption and mitigate phosphate pollution. Although fungal and yeast-derived phytases are extensively utilized, bacterial phytases demonstrate superior biochemical properties that hold potential for optimized feed applications. This study focuses on the isolation, characterization, and heterologous expression of a phytase- encoding gene from food-grade probiotic bacteria, culminating in its expression in Schizochytrium sp., a eukaryotic host renowned for docosahexaenoic acid (DHA) production. newlineProbiotic-derived microbial isolates were obtained with an enumerated density of 1.32 × 10and#8309; CFU/mL and identified as Gram-positive rods via biochemical assays. Among the strains screened, Bacillus sp. PS4 exhibited a hydrolytic zone of 6.2 mm on Phytase Screening Medium (PSM) agar, indicative of substantial phytase activity. Subsequent 16S rRNA gene sequencing identified the strain as Bacillus sp. BAB 3372 (Accession No. KF952780.1), with measured phytase activity of 1.15 U/mL. Genomic DNA was isolated with a concentration of 19.773 ng/and#956;L and an A260/A280 ratio of 1.8. PCR amplification yielded a phytase gene fragment of 1152 bp, which was cloned into Schizochytrium sp. ATCC 20888. Recombinant strains were selected on G418-supplemented plates (300 1000 and#956;g/mL), and the expressed phytase was purified using Ni-NTA affinity chromatography newline