Molecular characterization of the Bambusa oldhamii BoPAL3–encoded phenylalanine ammonia-lyase

Abstract

• BoPAL3 gene was cloned from Bambusa oldhamii and heterologously expressed in Escherichia coli . • Recombinant BoPAL3 protein is an active enzyme, exhibiting obvious PAL activity and minuscule TAL activity. • BoPAL3 had similar biochemical properties with BoPAL2. Phenylalanine ammonia-lyase (PAL), which is ubiquitous in plants, catalyzes the formation of trans -cinnamic acid via the nonoxidative deamination of l -phenylalanine. Bambusa oldhamii contains four different forms of PAL proteins that differ in substrate specificity. Full-length BoPAL3 cDNA was cloned by a combination bamboo cDNA library screening and PCR-based cloning methods. Sequence alignment showed high homology between the deduced amino acid sequences of the BoPAL2 and BoPAL3 proteins (90%). Obvious PAL and tyrosine ammonia-lyase (TAL) activities were detected in Escherichia coli Top10 expressing recombinant BoPAL3 protein. Size-exclusion chromatography and denatured SDS–PAGE showed that the estimated molecular mass of recombinant BoPAL3 and the subunit form were approximately 330 kDa and 80 kDa, indicating that BoPAL3 presents as a tetrameric protein. The optimum temperature and pH for BoPAL3 activity were 50 °C and 8.5, respectively. The K m value of BoPAL3 for l -phenylalanine was 200 μM.