Production of enzymes for food industry via solid state fermentation and optimization of process conditions

Enzymes are biocatalysts, accelerating biochemical reactions in vivo or in vitro, with a wide range of applications in different industries like food, feed, detergent, paper, and pharmaceuticals. Aspartic protease, an acid protease, and Transglutaminase are important enzymes for the food industry, like milk, meat, brewery, and wine industries. Limited GRAS native strain and low extracellularly produced APase and TGase, makes the research for recombinant alternatives imperative. Pichia pastoris a well-known yeast host for heterologous protein production is a good alternative for recombinant expression of these enzymes. Once the production host is selected, the determination of fermentation steps is the key in enzyme production. Solid-state fermentation (SSF), which is performed in the nearabsence of free water is considered to be a cheap and simple process with high protein titers. Despite being a promising process option, SSF presents several challenges, primarily because of its heterologous nature, e.g., maintaining fermentation temperature and pH constant, enhancing oxygen consumption, and homogeneous nutrient distribution. Addressing these challenges constitutes the main theme of this thesis. This thesis covers molecular work, optimization of production via SSF of the two target enzymes, and application of these in various food processes. The molecular work covers the design, de novo synthesis and cloning, and construction of methanol inducible system with P. pastoris as host. Once the host is obtained, a series of submerged (SmF) and solid-state fermentation (SSF) were conducted to comparatively analyze growth and production yields as well as oxygen consumption and methanol evaporation rates. To find optimum conditions, SSF in a modified reactor with forced aeration and forced stirring were performed. The produced enzyme is tested in the different food products. SSF process is verified to be promising conditions for recombinant enzyme production, during which high cell density are reached. A higher oxygen uptake rate was proved by results compared with SmF. Both recombinant enzymes were successfully expressed when the complex medium was used. While, when the defined medium was used the production was in good titer only in the SSF process.