A Novel Phytase appA from Citrobacter amalonaticus CGMCC 1696: Gene Cloning and Overexpression in Pichia pastoris

Abstract

A novel phytase gene appA, with upstream and downstream sequences from Citrobacter amalonaticus CGMCC 1696, was cloned by degenerate polymerase chain reaction (PCR), and thermal asymmetric interlaced (TAIL) PCR and was overexpressed in Pichia pastoris. Sequence analysis revealed one open reading frame that consisted of 1311 bp encoding a 436-amino-acid protein, which had a deduced molecular mass of 46.3 kDa. The phytase appA belongs to the histidine acid phosphatase family and exhibits the highest identity (70.1%) with C. braakii phytase. The gene was overexpressed in P. pastoris. The secretion yield of recombinant appA protein was accumulated to approximately 4.2 mg.mL(-1), and the enzyme activity level reached 15,000 U x mL(-1), which is higher than any previous reports. r-appA was glycosylated, as shown by Endo H treatment. r-appA was purified and characterized. The specific activity of r-appA for sodium phytate was 3548 U.mg(-1). The optimum pH and temperature for enzyme activity were 4.5 and 55 degrees C, respectively. r-appA was highly resistant to pepsin or trypsin treatment. This enzyme could be an economic and efficient alternative to the phytases currently used in the feed industry.