Optimization of heterologous expression of the phytase (PPHY) of Pichia anomala in P-pastoris and its applicability in fractionating allergenic glycinin from soy protein

摘要

The phytase (PPHY) of Pichia anomala has the requisite properties of thermostability and acidstability, broad substrate spectrum, and protease insensitivity, which make it a suitable candidate as a feed and food additive. The 1,389-bp PPHY gene was amplified from P. anomala genomic DNA, cloned in pPICZ alpha A, and expressed extracellularly in P. pastoris X33. Three copies of PPHY have been detected integrated into the chromosomal DNA of the recombinant P. pastoris. The size exclusion chromatography followed by electrophoresis of the pure rPPHY confirmed that this is a homohexameric glycoprotein of similar to 420 kDa with a 24.3 % portion as N-linked glycans. The temperature and pH optima of rPPHY are 60 A degrees C and 4.0, similar to the endogenous enzyme. The kinetic characteristics K (m), V (max), K (cat), and K (cat)/K (m) of rPPHY are 0.2 +/- A 0.03 mM, 78.2 +/- A 1.43 nmol mg(-1) s(-1), 65,655 +/- A 10.92 s(-1), and 328.3 +/- A 3.12 mu M-1 s(-1), respectively. The optimization of medium components led to a 21.8-fold improvement in rPPHY production over the endogenous yeast. The rPPHY titer attained in shake flasks could also be sustained in the laboratory fermenter. The rPPHY accounts for 57.1 % of the total secreted protein into the medium. The enzyme has been found useful in fractionating allergenic protein glycinin from soya protein besides dephytinization.