Strain-screening For Producing Phenylalanine Ammonia Lyase, And Construction Of Its Recombinant Strains And Research On Fermentation Process

Phenylalanine ammonia lyase(PAL, EC4.3.1.5) is an endocellular inducible enzyme,existing in all plants and a portion of microbes, which has industrial application because of itsreversible catalysis of trans-cinnamic to L-phenylalanine. With in-depth study of it, thepotential value of PAL is discovered for application in industrial and pharmaceutical fields.A Rhodosporidium toruloides Y01with the ability of producing PAL was screened, thenits pal gene was cloned by RT-PCR and expressed in Escherichia coli and Picha pastoris,respectively. Fermentation experiments were performed by the recombinant E.coli. The mainconclusions were summarized as follows:1) The PAL producing strain was obtained using L-Tyrosine, analogue of its substrate,and identified as Rhodosporidium toruloides based on the results of morphologiccharacteristics and26S rDNA. After cultivating it in induction medium for20h, the highestPAL activity could reach10.670.49U/L.2) The RNA of R. toruloides was extracted by the method of hot phenol after comparingit with methods of Trizol and glass beads mechanical disruption. Then the pal gene wascloned by RT-PCR and PCR. Nucleotide sequencing identified the gene of99%similaritywith reported pal gene.3) Recombinant plasmids pET28a(+)-pal and pPIC9K-pal were constructed andtransformed into E.coli BL21(DE3) and Picha pastoris GS115respectively. It was shown thatthere was a specific protein band about76kDa as reported by SDS-PAGE ofcell lysate supernatant from the recombinant E.coli, which was detected PAL activity. A76kDa band from Picha pastoris, but didn’t detected PAL activity form recombinant strain.4) The optimization condition was investigated on the basis of LB medium. After singlefactor experiments, the optimal condition:0.1mmol/L IPTG was added at3rd hour andinduced at25℃for16h. The highest PAL activity reached45.810.17U/L, which was morethan4.3times as original strain. The specific activity of PAL was22.8%higher thanrecombinant E.coli which has been constructed before.5) By investigating the lactose induction condition in TB medium.It shows that the bestinitial concentrations of glycerol and lactose added amount were2g/L and1.5g/Lrespectively. The optimal temperature was25℃. After the process, the PAL activity was6103.97U/L, enhancing about5.3times comparing with former batch fermentation in15L and57.2times over the original strain (R. toruloides)production.6) The optimum transformation conditions were pH10.5, temperature30℃,12g/Ltrans-cinnamic acid,40%ammonium hydroxide(v/v). The conversion ratio of cinnamic acidto phenylalanine was62.371.33%, and the concentration of L-phenylalanine was2.25g/L.