Advanced Recombinant Manganese Peroxidase for Biosynthesis of Lignin Bioproducts, Phase I Final Report, STTR Grant #: DE-SC0007503.

Beatty, Christopher ; Kitner, Joshua ; Lajoie, Curtis ; McClain, Sean ; Potochnik, Steve

关键词: lignin;    manganese peroxidase;    pichia pastoris;    UV-Visible spectroscopy;    heterologous protein expression;   

DOI  :  10.2172/1057291
RP-ID  :  DOE/SC0007503.1
PID  :  OSTI ID: 1057291

学科分类：燃料技术

美国|英语

来源: SciTech Connect

【 摘 要 】

The core purpose of this Phase I STTR was to evaluate the feasibility of a new method of producing a recombinant version of manganese peroxidase (MnP) enzyme. MnP is a potentially valuable enzyme for producing high value lignin products and also for industrial de-coloring operations such as biobleaching of pulp and color removal from textile dye effluents. This lignin-modifying enzyme is produced in small amounts by the native host, a white rot fungus. Previous work by Oregon State University developed a secreted recombinant version of the enzyme in the yeast Pichia pastoris. Unfortunately, the expression is barely moderate and the enzyme is heavily glycosylated, which inhibits purification. In this work, the gene for the enzyme is given a tag which targets production of the enzyme to the peroxisome. This is a promising approach since this location is also where heme and hydrogen peroxide are sequestered, which are both necessary cofactors for MnP. More than ten recombinant strains were constructed, verified, and expressed in the Pichia system. Constitutive (GAP) and methanol-induced promoters (AOX) were tried for peroxisomal targeted, cytosolic, and secreted versions of MnP. Only the secreted strains showed activity. The amount of expression was not significantly changed. The degree of glycosylation was lessened using the AOX (methanol) promotoer, but the resulting enzyme was still not able to be purified using immobilized metal affinity chromatography. Additional work beyond the scope of the defined Phase I project was undertaken to construct, verify, and express Pichia strains that mutated the MnP glycosylation sites to inhibit this process. These strains did not show significant activity. The cause is not known, but it is possible that these sites are important to the structure of the enzyme. Also beyond the scope proposed for our Phase I STTR, the team collaborated with AbSci, a startup with a new E. coli based expression system focused on the production of antibodies and enzymes containing disulfide bonds and requiring folding/post-translational modification. With only limited time remaining in the Phase I schedule, a single construct was made to produce MnP with this system. The enzyme was produced in the soluble fraction of the cell lysate, but no activity was measured. MnP from the existing recombinant source was used to act on lignin. The lignin was from a Kraft process and had a molecular weight of about 10,000 Da. Using 1000 Da dialysis membranes and UV-visible spectroscopy, no modification of either lignin was evident in the dialysate or the retentate. Assays using 2,6 dimethoxy phenol (DMP) as a substrate showed consistent activity throughout the project. In summary, these results fell far short of our expectations. A Phase II proposal was not submitted. Possible reasons for the failure of peroxisomal targeting include destruction by native hydrogen peroxide, native proteases, or unforeseen causes. The AbSci system was only lighted tested and further work may yield a strain with active enzyme. The lack of evidence for lignin modification may be due to the techniques employed. NMR or GC-MS studies may reveal evidence of modification.