Journal of Pure and Applied MicrobiologyVol. 7 No. 1

Cloning, Expression and Characterization of Three Novel Histidine acid Phytases from Aspergillus niger, Aspergillus oryzae and Penicillium sp

Junjun Liu1, Honglei Liu1, Qianqian Zhao, Liping Jiang, Xiao zhang and Yuzhen Zhang*

State Key Laboratory of Microbial Technology, Shandong University, Jinan - 250 100, China.

Received on 03 November 2012 and accepted on 24 December 2012

 

ABSTRACT

In the present study, a strategy for cloning three novel phytase genes (phyA) from different filamentous fungi strains by using combined utilization of Consensus-Degenerate Hybrid Oligonucleotide Primer (CODEHOP) PCR and Thermal Asymmetric Interlaced PCR (TAIL-PCR) was developed. Based on the conserved regions recognized by ClustalW online, CODEHOP primers were designed to amplify a partial sequence of the phytase gene. TAIL-PCR was then employed to clone the 5? and 3? flanking sequences. Using this strategy, three novel phytase genes of Aspergillus niger, Aspergillus oryzae and Penicillium sp. were cloned. After expression in Pichia pastoris, the properties of the three recombinant phytase were characterized. Sequence analysis and characterization of the recombinant enzymes revealed that they belonged to the family of histidine acid phosphatases. The results showed that combination of CODEHOP PCR and TAIL-PCR was an efficient strategy for cloning unknown genes from potential species.

Keywords : Phytase, CODEHOP, TAIL-PCR, Aspergillus niger, A. oryzae, Penicillium sp.