Expression in yeast of secreted lignin peroxidase with improved 2,4-dichlorophenol degradability by DNA shuffling.

Lignin peroxidase (LiP) from Phanerochaete chrysosporium was shown to mineralize a variety of recalcitrant aromatic compounds and oxidize a number of polycyclic aromatic and phenolic compounds. The major problem of the wild type LiP is that it can be inactivated by excess H(2)O(2) and high concentrations of aromatic compounds. We applied a directed evolution technique coupled with a rapid colorimetric screening method to obtain mutant genes with improved H(2)O(2) stability and polychlorinated phenol degradability, and they were successfully expressed as the secretive LiPs in recombinant Saccharomyces cerevisiae. The resulting variants showed approximately 1.6-fold improved 2,4-dichlorophenol (2,4-DCP) degradation activity and stability against H(2)O(2) compared with the parent strain. The kinetic properties of the variants toward 2,4-DCP and H(2)O(2) were also increased compared with the wild type for all three mutants studied. Amino acid sequence analysis indicated that the greatest number of amino acid substitutions was located near the surface or Ca(2+) binding sites of the enzyme.

Functionality improvement of fungal lignin peroxidase by DNA shuffling for 2,4-dichlorophenol degradability and H2O2 stability.

One of the major problems of wild-type lignin peroxidase (LiP) is its inactivity at the presence of excess H(2)O(2) and high concentration of aromatic compounds. Little is known about the substrate-binding site of LiP, and functionality improvement of LiP was not actively tried by genetic engineering and directed evolution. In order to improve LiPs functionality, we performed directed evolution with a colorimetric screening method. Finally, three types of LiP mutants were screened. The catalytic efficiency of the variants toward 2,4-dichlorophenol (DCP) degradation activity and the stability against H(2)O(2) was increased over the wild type. The K(m) value of the variants toward H(2)O(2) was increased, but K(m) value toward 2,4-DCP degradation was reduced. Overall, The K(cat)/K(m) values of the mutants toward 2,4-DCP was increased ca. 4-fold, and that toward H(2)O(2) was increased ca. 89-fold. Amino acid sequence analysis indicated that the most of the mutations were located on the enzyme surface. We expect that these results coupled with recombining mutation can be successfully applied to the molecular evolution cycles for screening of LiPs and other oxidative enzymes with improved functionality and stability.

Improving immunobinding using oriented immobilization of an oxidized antibody.

Recent technical advances in biorecognition engineering and microparticle fabrication enabled us to develop a single-step purification process using magnetic particles (MPs). The process is simple, efficacious, easy to automate, and economical. The method immobilizes the ligand molecule in a particular orientation on commercial MPs that have surface carboxyl groups. Mouse IgG and anti-mouse IgG antibody were the model capture and ligand molecules for this study. The immunobinding efficacy of anti-mouse IgG antibody using "oriented immobilization" was compared with the efficacy of a conventional amine-coupling system that results in random orientation and of another standard method, the biotin-streptavidin system. The oriented immobilization was accomplished by oxidizing the sugar moiety in the CH(2) domain of the antibody's Fc and covalently conjugating the moiety to the hydrazine-coated MP. The specific binding affinity of the oriented immobilization process was about 2.5 times that of the amine-coupling system, and selectivity from a binary mixture was about 2 times greater for the oriented immobilization method. Results were nearly identical for the biotin-streptavidin system and the oriented immobilization system, matching the calculated binding stoichiometry between mouse IgG and anti-mouse IgG antibody. The binding improvement over the amine-coupling system shown by assay was confirmed by a separate surface plasmon resonance experiment. In summary, the oriented immobilization method was as effective as the streptavidin-biotin system, yet simpler and cost-effective.