Recombinant production of pectinolytic enzymes in pichia pastoris and process optimization for food industry

Pectinolytic and ligninolytic enzymes degrade pectin and lignin respectively, main constituents of plant-based biomass, used in several industries ranging from food, feed and energy, wastewater treatment, textile, oil extraction, retting of plant fibers and paper making industries. Pectinolytic enzymes dominate 10% of total enzyme market and 25% of the worldwide food enzyme market. A key bottleneck in these domestic industries is the supply of affordable enzymes to local markets. Heterologous enzyme production is a key technology that uses the powerful tools of molecular biology, allowing to manufacture tailor-made products for specific applications, e.g. desired enzyme blends. Pichia pastoris is a methylotrophic yeast and a promising host for industrial biotechnology, as it can be used for high level of protein production. Additionally, suitability for high cell density cultivation, capacity to secrete produced proteins, rendering in turn downstream processes cheaper, availability of genome sequence, ease in genetic manipulations and capability of post translational modifications makes P. pastoris an interesting host for heterologous protein production. In this thesis, five pectinolytic and ligninolytic enzymes namely, polygalacturonase (EC 3.2.1.15), pectin methyl esterase (EC 3.1.1.11), pectin lyase (EC 4.2.2.10), laccase (EC 1.10.3.2) and lignin peroxidase (EC 1.11.1.14) were heterologously produced in P. pastoris. The recombinant enzymes are produced in lab scale (both in Erlenmeyer flasks as well as in controlled bioreactor conditions), under the methanol-inducible AOX1 promoter and the activities of enzymes from the crude extracts (analyzed separately by SDS-PAGE) are measured based on released products. Subsequently, five different Pichia pastoris strains are engineered via recombinant DNA technology to produce target enzymes.