ABSTRACT
Phenylalanine ammonia-lyase (PAL, EC 4.3.1.24) is common in plants and catalyzes the formation of trans-cinnamic acid and ammonia via phenylalanine deamination. Recombinant Bambusa oldhamii BoPAL3 protein expressed in Escherichia coli was immobilized on an electrospun nanofibrous membrane using dextran polyaldehyde as a crosslinker. The immobilized BoPAL3 protein exhibited comparable kinetic properties with the free BoPAL3 protein and could be recycled for six consecutive cycles compared with the free BoPAL3 protein. The residual activity of the immobilized BoPAL3 protein was 84% after 30 days of storage at 4 °C, whereas the free BoPAL3 protein retained 56% residual activity in the same storage conditions. Furthermore, the resistance of the immobilized BoPAL3 protein to chemical denaturants was greatly increased. Therefore, the BoPAL3 protein can be immobilized using the natural dextran polyaldehyde crosslinker in place of the conventional chemical crosslinker. Nanofibrous membranes made from polyvinyl alcohol (PVA), nylon 6, and chitosan (CS) are incredibly stable and useful for future industrial applications.
ABSTRACT
Cytokinin oxidase/dehydrogenase (CKX) catalyzes the irreversible breakdown of active cytokinins, which are a class of plant hormones that regulate cell division. According to conserved sequences of CKX genes from monocotyledons, PCR primers were designed to synthesize a probe for screening a bamboo genomic library. Cloned results of three genes encoding cytokinin oxidase were named as follows: BoCKX1, BoCKX2, and BoCKX3. In comparing the exon-intron structures among the above three genes, there are three exons and two introns in BoCKX1 and BoCKX3 genes, whereas BoCKX2 contains four exons and three introns. The amino acid sequence of BoCKX2 protein shares 78% and 79% identity with BoCKX1 and BoCKX3 proteins, respectively. BoCKX1 and BoCKX3 genes are particularly closely related given that the amino acid and nucleotide sequence identities are more than 90%. These three BoCKX proteins carried putative signal peptide sequences typical of secretion pathway, and a GHS-motif was found at N-terminal flavin adenine dinucleotide (FAD) binding domain, suggesting that BoCKX proteins might covalently conjugate with an FAD cofactor through a predicted histidine residue.
ABSTRACT
Malate dehydrogenase (MDH), which is ubiquitously occurred in nature, catalyzes the interconversion of malate and oxaloacetate. Higher plants contain multiple forms of MDH that differ in coenzyme specificity, subcellular localization and physiological function. A putative Bambusa oldhamii BoMDH cDNA was screened with the specific probe from the bamboo cDNA library. Sequence alignment shows that there's a high homology between the deduced amino acid sequence of BoMDH and MDH protein in Oryza sativa glyoxysome (92%). A 57 kDa fusion protein was expressed by IPTG induction in Escherichia coli BL21 (DE3), and an obvious MDH activity was detected in the recombinant protein. The molecular mass of recombinant BoMDH was estimated to be 120 kDa, and the subunit form was 57 kDa by denatured SDS-PAGE, indicating that BoMDH presents as a homodimer. The optimum temperature and pH for BoMDH activity were 40 °C and 9.5, respectively. The Km values of BoMDH for malate and NAD+ were 5.2 mM and 0.52 mM. The kcat/Km values of BoMDH for malate and NAD+ were 163 min-1 mM-1 and 3060 min-1 mM-1.
