Molecular and Biochemical Characteristics of ��-Propeller Phytase from Marine Pseudomonas sp. BS10-3 and Its Potential Application for Animal Feed Additives

Abstract

Phytate is an antinutritional factor that impacts the bioavailability of essential minerals such as Ca(2+), Mg(2+), Mn(2+), Zn(2+), and Fe(2+) by forming insoluble mineral-phytate salts. These insoluble mineral-phytate salts are hydrolyzed rarely by monogastric animals, because they lack the hydrolyzing phytases and thus excrete the majority of them. The β-propeller phytases (BPPs) hydrolyze these insoluble mineral-phytate salts efficiently. In this study, we cloned a novel BPP gene from a marine Pseudomonas sp. This Pseudomonas BPP gene (PsBPP) had low sequence identity with other known phytases and contained an extra internal repeat domain (residues 24-279) and a typical BPP domain (residues 280-634) at the C-terminus. Structurebased sequence alignment suggested that the N-terminal repeat domain did not possess the active-site residues, whereas the C-terminal BPP domain contained multiple calcium-binding sites, which provide a favorable electrostatic environment for substrate binding and catalytic activity. Thus, we overexpressed the BPP domain from Pseudomonas sp. to potentially hydrolyze insoluble mineral-phytate salts. Purified recombinant PsBPP required Ca(2+) or Fe(2+) for phytase activity, indicating that PsBPP hydrolyzes insoluble Fe(2+)-phytate or Ca2+-phytate salts. The optimal temperature and pH for the hydrolysis of Ca(2+)-phytate by PsBPP were 50°C and 6.0, respectively. Biochemical and kinetic studies clearly showed that PsBPP efficiently hydrolyzed Ca(2+)-phytate salts and yielded myo-inositol 2,4,6-trisphosphate and three phosphate groups as final products. Finally, we showed that PsBPP was highly effective for hydrolyzing rice bran with high phytate content. Taken together, our results suggest that PsBPP has great potential in the animal feed industry for reducing phytates.

In-depth characterization of phytase-producing plant growth promotion bacteria isolated in alpine grassland of Qinghai-Tibetan Plateau

“…In order to reveal the basic characteristics of enzymes and discover novel phytases with desirable properties, the enzyme must be purified and identified. In recent years, a considerable number of phytases have been isolated and cloned in microorganisms, which have achieved heterologous expression, and their enzymatic properties are tested ( Nam et al, 2014 ; Zhang et al, 2015 ; Rix et al, 2021 ). However, the phytases that possess high activity at low temperatures are still uncommon ( Jang et al, 2018 ), which play a role in P cycling and potentially be suitable for use in cold areas.…”

“…The optimal pH of PHY101 was 6.0, stable at pH 5.0–9.0, and the optimum temperature was 40°C, where the temperature is lower than most reported strains of Pseudomonas sp. (50°C) ( Nam et al, 2014 ) and Bacillus sp. (55–60°C) ( Borgi et al, 2014 ).…”

Cloning and characterization of the first actinomycete β‐propeller phytase from Streptomyces sp. US42

“…), value slightly higher than the theoretical molecular mass (45.8 kDa). This difference in size might be due to the high content in negatively charged amino acids (18%) (as calculated with ExPASy ProtParam tool, http://web.expasy.org/cgi-bin/protparam/protparam) as suggested by Nam et al and/or post‐translational modifications such as glycosylation that was shown to occur in Streptomyces .…”