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.

The objective of this study was to isolate and characterize a strain of Aspergillus capable of producing xylanase. According to the morphology and comparison of ITS (Internal Transcribed Spacer) rDNA gene sequence, the strain Aspergillus sp. ZH-26 was identified as a strain of Aspergillus awamori. Statistically based experimental designs were applied for the optimization of xylanase production from A. awamori ZH-26. The considered medium components included 17 components as follows: yeast extract, tryptone, urea, NH(4)Cl, (NH(4))(2)SO(4), NaNO(3), KH(2)PO(4), K(2)HPO(4), NH(4)NO(3), MgSO(4), CaCl(2), CuSO(4), ZnCl(2), FeSO(4), MnSO(4), vitamin B(1), and EDTA. Yeast extract, tryptone, (NH(4))(2)SO(4), KH(2)PO(4), and CaCl(2) were identified to have significant effects on xylanase production using the Plackett-Burman experimental design. These 5 major components were subsequently optimized using the Box-Behnken experimental design. By response surface methodology and canonical analysis, the optimal concentrations for higher production of xylanase were yeast extract 5.95 g/L, tryptone 6.79 g/L, (NH(4))(2)SO(4) 13.37 g/L, KH(2)PO(4) 1.14 g/L, CaCl(2) 0.81 g/L. Under optimal conditions, the xylanase activity from A. awamori ZH-26 reached 47.3 U/mL. A small-scale mashing was carried out to evaluate the performance of the xylanase on degradation of arabinoxylans in mashing. Results showed that polymeric arabinoxylan content and wort viscosity in mashing with grist containing wheat malt sharply decreased to the basal level (from 470 to 185 mg/L) with the addition of xylanase.