Cloning and expression of a phenylalanine ammonia-lyase gene ( BoPAL2) from Bambusa oldhamii in Escherichia coli and Pichia pastoris

• Published in: Protein expression and purification 2010-06, Vol.71 (2), p.224-230
• Main Authors: Hsieh, Lu-Sheng, Yeh, Chuan-Shan, Pan, Hung-Chi, Cheng, Chieh-Yang, Yang, Chien-Chih, Lee, Ping-Du
• Format: Article
• Language: English
• Subjects: Ammonia-Lyases - chemistry; Ammonia-Lyases - genetics; Ammonia-Lyases - metabolism; Bambusa; Bambusa - genetics; Bambusa - metabolism; Bambusa oldhamii; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Gene expression; Gene Library; Molecular cloning; Open Reading Frames; Phenylalanine ammonia-lyase (PAL); Phenylalanine Ammonia-Lyase - chemistry; Phenylalanine Ammonia-Lyase - genetics; Phenylalanine Ammonia-Lyase - metabolism; Pichia - genetics; Pichia - metabolism; Pichia pastoris; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Tyrosine ammonia-lyase (TAL)
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2010.01.009

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is the first committed enzyme of phenylpropanoid pathway. A PAL gene, designated as BoPAL2, was cloned from a Bambusa oldhamii cDNA library. The open reading frame of BoPAL2 was 2142 bp in size encoding a 713-amino acid polypeptide. BoPAL2 was heterologous expressed in Escherichia coli and Pichia pastoris. The recombinant proteins were exhibited PAL and tyrosine ammonia-lyase activities. The recombinant BoPAL2 had a subunit mass of 80 kDa and existed as a homotetramer. The optimum temperature and pH of BoPAL2 were 50–60 °C and 8.5–9.0, respectively. The K m and k cat values of BoPAL2 expressed in E. coli were 250 μM and 10.12 s −1. The K m and k cat values of BoPAL2 expressed in P. pastoris were 331 μM and 16.04 s −1. The recombinant proteins had similar biochemical properties and kinetic parameters with PALs reported in other plants.