Gene Cloning Of Phenylalanine Ammonia-lyase From Astragalus Membranaceus And Glydne Max And Secretory Expression In Pichia Pastoris

L-phenylalanine, as an essential amino acid for human nutrition, is mainly used as raw material of the rising food additives Aspartame. Recent years, as the global demand for Aspartame is increasing, the synthetic process of L-Phe has been a new research hotspot in biochemical industry. Utilizing reverse-catalytic property of phenylalanine ammonia-lyase to produce L-Phe is the main route. The emphasis is how to obtain PAL at a low cost and in large scale. In our study, we constructed the genetic engineering strains to achieve heterologous expression of gene PAL in Pichia pastoris GS115and in Escherichia coli BL21in order to realize industrialized production of PAL by microbial fermentation. The results are summarized as follows:1) The ORF of gene PAL was acquired from Astragalus membranaceus and Glycine max2) The cloning vector pUCm-T-PAL was constructed and transformed into E. coli DH5α3) The sequence analysis results of gene PAL by the software DNAStar and NCBI on line are as follows:For Astragalus membranaceus; it was predicted that ORF sequence of PAL gene was2157bp and PAL encoded a protein which was about78KD in molecular weight and6.04in pI, containing718amino acid residues. Amino acid sequence alignment revealed that PAL shared99%identity with PAL EF567076.1from Astragalus membranaceus published in the NCBI. For Glycine max, it was predicted that ORF sequence of PAL gene was about2142bp and PAL encoded a protein which was about77.6KD in molecular weight and6.31in pI, containing713amino acid residues. Amino acid sequence alignment revealed that PAL shared100%identity with PAL AK245610.1from Glycine max published in the NCBI.4) Expressing vectors pPIC9K-AmPAL, pPIC9K-GmPAL and pET32a-AmPAL were constructed by employing homologous recombination technique in vitro. 5) The genetic engineering strains GS115-pPIC9K-AmPAL, GS115-pPIC9K-GmPAL and BL21-pET32a-AmPAL were successfully constructed. After1hour being induced by1mmoL/L IPTG at25℃, SDS-PAGE of the strain BL21-pET32a-AmPAL demonstrated that a distinct96kD protein including some amino acids from pET32a which was equal to the predicted value was expressed in contrast to the parent BL21-pET32a. But the activity of PAL was just5U. After48hours being induced by1%methanol, SDS-PAGE of the strain GS115/pPIC9K-AmPAL showed that a78KD protein was expressed obviously which was coincident with the expectation. And the expression peak appeared at120h..By Q Sepharose Fast Flow chromatography, the protein was well purified. The concentration of purified protein was0.08mg/mL, accounting for11.54%of total proteins and the specific activity was up to4270U/mg.