title: Expression of a functional cold active β-galactosidase from Planococcus sp-L4 in Pichia pastoris.
abstract: Lactase deficiency problem discourages many adults from consuming milk as a major source of micro- and macronutrients. Enzymatic hydrolysis of lactose is an ideal solution for this problem but such processing adds significant costs. In this study, a cold active β-galactosidase from Planococcus sp-L4 (bgal) was optimized for expression of recombinant "BGalP" in Pichia pastoris. As a result of codon optimization, the codon adaptation index was improved from 0.58 to 0.85 after replacing rare codons. After transformation of two P. pastoris strains (KM71H and GS115), the activity of BGalP enzyme was measured in the culture supernatants using ortho-Nitrophenyl-β-galactoside (ONPG). Maximal activity was recorded as 3.7U/ml on day 11 in KM71H clone #2 which was 20% higher than the best GS115 clone. Activity measurements under different conditions indicated optimal activity at pH 6.5. It was active at temperatures ranging from 0 to 55°C with deactivation occurring at or above 60°C. Protein analysis of the crude ultra-filtrate showed the enzyme was ∼75kDa and was the major constituent (85%) of the sample. This enzyme have the potential to find utility for the breakdown of lactose in chilled milk and subsequently can be deactivated by pasteurization. The use of BGalP would minimize energy consumption thus decreasing cost and also help to preserve the nutritional elements of the milk.
authors.affiliation: Biochemistry of Nutrition Research Center, Department of Medical Biochemistry, School of Medicine, Mashhad University of Medical University, Mashhad, Iran.
authors.forename: Seyed Mohammad Amin
authors.initials: SM
authors.lastname: Mahdian
authors.affiliation: Biochemistry of Nutrition Research Center, Department of Medical Biochemistry, School of Medicine, Mashhad University of Medical University, Mashhad, Iran.
authors.forename: Ehsan
authors.initials: E
authors.lastname: Karimi
authors.affiliation: Biochemistry of Nutrition Research Center, Department of Medical Biochemistry, School of Medicine, Mashhad University of Medical University, Mashhad, Iran.
authors.forename: Mohammad Hossein
authors.initials: MH
authors.lastname: Tanipour
authors.affiliation: Biochemistry of Nutrition Research Center, Department of Medical Biochemistry, School of Medicine, Mashhad University of Medical University, Mashhad, Iran.
authors.forename: Seyed Mohammad Reza
authors.initials: SM
authors.lastname: Parizadeh
authors.affiliation: Biochemistry of Nutrition Research Center, Department of Medical Biochemistry, School of Medicine, Mashhad University of Medical University, Mashhad, Iran.
authors.forename: Majid
authors.initials: M
authors.lastname: Ghayour-Mobarhan
authors.affiliation: Department of Community Medicine, School of Medicine, Mashhad University of Medical University, Mashhad, Iran.
authors.forename: Mojtaba Mousavi
authors.initials: MM
authors.lastname: Bazaz
authors.affiliation: Biochemistry of Nutrition Research Center, Department of Medical Biochemistry, School of Medicine, Mashhad University of Medical University, Mashhad, Iran. Electronic address: mashkaniba@mums.ac.ir.
authors.forename: Baratali
authors.initials: B
authors.lastname: Mashkani
mesh_terms: D000818:Animals; D003062:Codon; D003080:Cold Temperature; D004789:Enzyme Activation; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007785:Lactose / Q000378:metabolism*; D008892:Milk; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D058642:Planococcus Bacteria / Q000201:enzymology* / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014170:Transformation, Genetic; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D003062:Codon; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase; D007785:Lactose
keywords: Codon optimization; Cold-active; ONPG; Pichia pastoris; β-Galactosidase