| Extracellular phytase (phyA) from Aspergillus niger NRRL 3135, aglycoprotein which catalyzes the hydrolysis of phosphate moieties from phyticacid, improves the bioavailability of phytate phosphorus in plant foods tohumans and animals and reduces phosphorus pollution of animal waste. It is asubfamily of the high-molecular-weight histidine acid phosphatases. A.nigerphytase (phyA) is a acid stable enzyme with high specific activity as thecommercial Natuphos from A.niger NRRL 3135. However, the thermostabilityconstraint and the cost of the enzyme limit its use in animal production.At present, the phytase from A.fumigatus refolded efficiently after heatdenaturation at 90℃and consensus phytase retained maximal activity at 71℃.But the activity of wild-type A.niger phytase with phytic acid as the substrate at37°C is obviously more than that of consensus phytase and A.fumigatus phytase.The Thermomyces phytase retained activity at assay temperatures up to 75°Cand demonstrated superior catalytic efficiency to any known fungal phytase at65°C. Thermo-acido-tolerant phytase production from a PH01 bacteriumretained Less than 20% fluctuation in activity when the reactions were carriedout at temperatures ranging from 37 to 75℃. However, further application ofthese phytases is also constrained in animal feed production due to the high costand the constraint of hight activity or acid resistance of the enzyme product.Some researchers express A.niger phytase (phyA) in different eukaryotichosts. Probably because of heavy glycosylation, these expressed phyA seem tobe more thermostable than wild-type phyA in these hosts. Han et al successfullyexpressed A.niger phytase (phyA) as an active, extracellular phytase inSaccharomyces cerevisiae. The recombinant phyA retained 75% of its activityafter being heated 80°C for 15 min, whereas the commercial phytase retained51% of its activity. Wild-type phytase retained only 40% of its activity after itwas heated at 68°C for 10 mins. However, purification means of phyAexpressed in Saccharomyces cerevisiae are too complicated to rapidly harvestthe enzyme. Han also successfully expressed A.niger phytase (phyA) inP.pastoris X33 and KM71. The expressed phytase was almost the only proteinin the supernatant for both X33 and KM71 transformants and enzyme expressedin P.pastoris X33 retained 56% of its activity after being heated 80°C for 15mins. P.pastoris GS115 has been widely used as a host to express heterologousproteins, and the expressed proteins are generally hyperglycosylated. Ourobjectives were to study the effects of glycosylation on the thermostability ofA.niger phytase (phyA) expressed in P.pastoris GS115 transformed withpPICZaA-phyA.A 1.4-kb DNA fragment containing the coding region of the phyA genefrom Aspergillus niger NRRL 3135 was inserted into the expression vectorpPICZaA and was expressed in Pichia pastoris GS115 under the induction ofmethanol. Clones expressing phytase were selected from 37 transformants. Twoimportant results were found by determining property of purified phytase. Atfirst, the expressed phytase retained approximately 48% of its activity afterbeing subjected to 85℃ for 10 minutes. At the second, almost the only proteinwas expressed in Pichia pastoris GS115 transformed with pPICZaA-phyA as anactive, extracellular phytase. The expressed phytase had two pH optima (2.5 and5.5) and a temperature optimum between 55 and 60°C. Due to the heavyglycosylation, the expressed phytase had a molecular size of approximately 95kDa with a specific activity of approximately 100units/mg of protein. Inconclusion, the phyA gene was expressed as an active, extracellular phytase inPichia pastoris GS115 and its thermostability was improved.
|
PDF Full Text Request