Recombinant lactase enzyme production in pichia pastoris

Lactase enzyme is a biocatalyst that breaks down lactose, commonly known as milk sugar, through catabolic reactions into its submonomers glucose and galactose. Also known as β-galactosidase, the lactase enzyme is especially utilized in the food industry to produce lactose-free milk and dairy products. The widespread prevalence of lactose intolerance among the population has significantly increased the demand for lactose-free products. This sensitivity, which affects approximately 70% of the global population, can lead to gastrointestinal problems such as bloating, intestinal pain, vomiting, and diarrhea in adults, starting from infancy. Due to its considerable impact on quality of life, the enzyme is also used as a nutritional supplement in daily diets for sensitive individuals. Today, lactase (β-galactosidase) is predominantly produced on an industrial scale using microbial fermentation and recombinant DNA techniques. These production methods offer advantages such as high yield, low cost, and sustainability. In this study, the enzyme was recombinantly produced in Pichia pastoris (P. pastoris) yeast under the control of an ethanol-inducible mutant ADH2 promoter. As part of the study, a synthetic codon-optimized lactase gene from Aspergillus oryzae (A. oryzae) was cloned under a synthetic SNT5 promoter, and the resulting pSNT5α-Lac expression vector was transformed into a P. pastoris MK115 (PDI) strain. Following transformation, P. pastoris MK115 (PDI) #1 and #2 clones were used for production in baffled flasks, during which temperature (20–24–28°C) and pH (3.0–7.0) optimization studies were conducted. The optimum conditions for lactase enzyme production were determined to be pH 6 and 28°C. Using these parameters, shake-flask scale production with the selected P. pastoris MK115 (PDI) #1 clone led to extracellular secretion of the enzyme, achieving a maximum enzyme activity of 6,800,000 U/mL and a maximum total protein concentration of 512 mg/L. Subsequently, enzyme production was scaled up to a bioreactor under the same optimized temperature and pH conditions using FM22 medium. After approximately 95 hours of fermentation, the enzyme activity and total protein concentration in the supernatant samples were determined as 18,400,000 U/mL and 3177 mg/L, respectively. As a result of the study, A. oryzae lactase enzyme was successfully produced in the P. pastoris MK115 (PDI) strain under the control of the ethanol-inducible SNT5 promoter, and bioprocess data were obtained for industrial-scale lactase production.