Molecular cloning, expression and evaluation of phosphohydrolases for phytate-degrading activity.
Four acid phosphatase (phosphomonoesterase E.C.3.1.3.2) genes were cloned by polymerase chain reaction (PCR). These were pho3, pho5 and pho11 from Saccharomyces cerevisiae and the gene for a phosphate-respressible acid phosphatase from Aspergillus niger. The individual genes were subcloned into an A. oryzae expression vector downstream from a starch-inducible alpha-amylase promoter and the resulting expression constructs were transformed into a mutant strain of A. 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 fermentation media. 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, untransformed A. oryzae. Sufficient quantities of A. 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Moore
E
E
Alltech Research International, National University of Ireland, University College, Galway.
Helly
V R
VR
Conneely
O M
OM
Ward
P P
PP
Power
R F
RF
Headon
D R
DR
D000135:Acid Phosphatase / Q000096:biosynthesis / Q000235:genetics*; D000595:Amino Acid Sequence; D001230:Aspergillus / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D010833:Phytic Acid / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis
D011994:Recombinant Proteins; D010833:Phytic Acid; D000135:Acid Phosphatase