Aims: To achieve high phytase yield with improved enzymatic activity in Pichia pastoris.
Methods and Results:The 1347-bp phytase gene of Aspergillus niger SK-57 was synthesized using a successive polymerase chain reaction and was altered by deleting intronic sequences, optimizing codon usage and replacing its original signal sequence with a synthetic signal peptide (designated MF4I) that is a codon-modified Saccharomyces cerevisiae mating factor a-prepro-leader sequence. The gene constructs containing wild type or modified phytase gene coding sequences under the control of the highly-inducible alcohol oxidase gene promoter with the MF4I-or wild type a-signal sequence were used to transform Pichia pastoris. The P. pastoris strain that expressed the modified phytase gene (phyA-sh) with MF4I sequence produced 6AE1 g purified phytase per litre of culture fluid, with the phytase activity of 865 U ml )1 . The expressed phytase varied in size (64, 67, 87, 110 and 120 kDa), but could be deglycosylated to produce a homogeneous 64 kDa protein. The recombinant phytase had two pH optima (pH 2AE5 and pH 5AE5) and an optimum temperature of 60°C. Conclusions: The P. pastoris strain with the genetically engineered phytase gene produced 6AE1 g l )1 of phytase or 865 U ml )1 phytase activity, a 14AE5-fold increase compared with the P. pastoris strain with the wild type phytase gene. Significance and Impact of the Study: The P. pastoris strain expressing the modified phytase gene with the MF4I signal peptide showed great potential as a commercial phytase production system.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.