Cloning And Expression Of Gene Encoding The Phytase From Aspergillus Niger WP1 In Pichia Pastoris

Phytate and phytic acid are the major phosphorus storage form contained in food or feeds of plant. Phytases are myo-inositol hexakisphosphate phosphohydrolase that catalyze the stepwise removal of inorganic orthophosphate from phytate. Addition of phytase into feed can result in the following advantages: increasing the absorptive efficiency of phosphate in animal body, decreasing the phosphate pollution in environment, eliminating the chelate reaction between phytate and metal ion. So that phytase can improve the utilization efficiency of the feed. Hence the study on phytase, especially phytase produced by microorganism, has been paid much attention by scientists in China and abroad. A broad range of microorganisms, including bacteria, filamentous fungi and yeasts can produce phytases. However, due to the low production and the weak thermostability of the phytase products, only a limited amount of commercial microbial phytases are used in feedstuff. Thus economical and thermostable phytases are badly required in the feedstuff industry. It could not only increase the production of phytase in wide range with technology of genetic engineering, but also rebuild the characteristics of protein to obtain improved phytase according to the demand.In this study, the fragment of phyA about 1.4 kb and phyB about 1.5 kb were amplified by PCR with primers designed according to published sequences, they were also cloned and sequenced. The results showed that the sequence of phyA₁ was 1347bp in size, and encoded a peptide of 448 amino acid residues with eleven putative N-glycosylation sites in the sequence. It was found that active-site sequence, RHGXRYP and HD. Comparison of this sequence with the nature A. niger NRRL3135 (GenBank Accession: M94550), the amino acid homology came up to 97.77%. The sequence of phyA₁ of A. niger WP1 strain in this paper had been accessed by GenBank (Accession:DQ815852). The other results showed that the coding region of phyB₁ was 1560bp in size, including 3 introns, and encoded a peptide of 460 amino acid residues. Comparison of this sequence with the aph of the nature A. niger ALKO243(GenBank Accession:L02420), showed that the amino acid homology was 99.13%, and the nucleotide homology came up to 96.79%. The sequence of phyB₁ of A. niger WP1 strain in this paper had been accessed by GenBank (Accession: DQ836360).The recombinant plasmid pPIC9K/phyA₁ was constructed by connecting phyA₁ and plasmid together and then was transformed into Pichia pastoris GS115. Transformants with inserted target fragment were screened using MM, MD, G418 and phytin culture midium. Induced by methanol, the phyA₁ gene was expressed and the phytase activity was reached 64510 U/mL after inducing 144 hours, which was more than 1535 times of that produced from the original strain Aspergillus niger WP1(42 U/mL). The molecular mass of PHYA₁ was determined to be 66 kD by SDS-PAGE analysis. The analysis of enzymatic properties showed that its optimal temperature was 50℃, and its optimal pH was 5.5. Under the condition of 50℃and 60℃for 20 min, the PHYA₁ remained 67.8ï¼…and 57.2ï¼…of the initial activity arid it still remained more than 40ï¼…after 40min at 50℃; it retained 40ï¼…of its activity after denaturation at 70℃for 5 min, and there was nearly no activity retained after 80 min. The metal-ions had different effect on the activity of phytase.There was not much research on phyB₁ in our country. It has not been reported before that two kinds of phytase genes were cloned from one stain. The successful cloning of phyB₁ has enriched the resource of phytase gene in our country and provided experimental material for in-depth research of phytase gene. We successfully constructed the genetic engineering strains: GA-22. It provided the fine strains for making the bio-reactor quantity and high effective producing the phytase into realization. At the same time, the Pichia pastoris/phyA₁ expression was the base of studying and developing new multi-functional ecological agent with phytase, and supplied some message to realize the green engineering of livestock farming.