Molecular cloning, expression and evaluation of phosphohydrolases for phytate-degrading activity

Abstract

Four acid phosphatase (phosphomonoesterase E.C.3.1.3.2) genes, werecloned by polymerase chain reaction (PCR). These were pho3, pho5 and pho11 fromSaccharomyces cerevisiae and the gene for a phosphate-respressible acid phosphatase fromAspergillus niger. The individual genes were subcloned into anA. oryzae expression vector downstream from a starch-inducible α-amylase promoter and the resulting expression constructs were transformed into a mutant strain ofA. oryzae, AO7. Southern hybridization analysis confirmed that the acid phosphatase genes had been integrated into the host genome with estimates of integrated copy numbers ranging from 2 to 20 for individual transformants. Northern hybridization analysis of total RNA from individual transformants revealed the presence of a single transcript of the expected size of 1.8 kb. Production of recombinant protein was induced by the addition of 30 g L⁻¹ of soluble starch in the fermentationmedia. Active acid phosphatases, not present in control cultures, were detected in the supernatant fractions of transformant cultures by acid phosphatase activity staining of non-denaturing polyacrylamide gels. The ability of the recombinant acid phosphatases to hydrolyze phytate was assessed by referenced phytase (myoinositol hexakisphosphate phosphohydrolase E.C. 3.1.3.8) activity assay procedures. A two- to six-fold increase in phytase activity was measured in transformants compared to control, untransformedA. oryzae. Sufficient quantities ofA. niger and pho5 recombinant acid phosphatases were generated from large-scale fermentations to assess the efficacy of these enzymes as phytate-degrading enzymes when included in poultry diets. Data indicated an increase in available phosphorus of 1 g kg⁻¹ obtained with yeast acid phosphatase andA. niger acid phosphatase representing 40% utilization of unavailable dietary P compared to 48% utilization for commercial phytase.