title: Characterization of Sulfolobus solfataricus β-galactosidase mutant F441Y expressed in Pichia pastoris.
abstract: BACKGROUND
  β-Galactosidases are used to synthesize galactooligosaccharides (GOS). Since thermostable β-galactosidases have the advantages of high optimal temperature, thermostability as well as prevention of bacterial contamination in the production of GOS, they have received more attention in research. In this study a mutant of Sulfolobus solfataricus β-galactosidase, F441Y, was expressed in Pichia pastoris KM71.
  
  
  RESULTS
  The activity of the recombinant enzyme reached 204.9 U mL⁻¹ after induction by methanol in a 3 L bioreactor for 80 h. Purification using ion exchange and hydrophobic interaction chromatographies resulted in a substantially pure recombinant β-galactosidase that migrated with an apparent molecular weight of 56 kDa in an SDS-PAGE gel. The enzymatic properties were measured and the optimal pH was 5 and the optimal temperature was 85 °C. Using lactose as substrate, the K(m) and k(cat) values were found to be 26.3 mmol L⁻¹ and 160.1 min⁻¹ respectively. After optimizing the reaction conditions for GOS production, the yield of GOS produced by the recombinant enzyme at pH 6 and 75 °C with 700 g L⁻¹ lactose solution and an enzyme quantity of 9 U mL⁻¹ reached 61%.
  
  
  CONCLUSIONS
  The F441Y of β-galactosidase expressed in P. pastoris KM71 has potential application in the industrial preparation of GOS.
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China; School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China.
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Sun
authors.forename: Xuguo
authors.initials: X
authors.lastname: Duan
authors.forename: Dan
authors.initials: D
authors.lastname: Wu
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
authors.forename: Jing
authors.initials: J
authors.lastname: Wu
mesh_terms: D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019149:Bioreactors / Q000382:microbiology; D004790:Enzyme Induction / Q000187:drug effects; D004795:Enzyme Stability; D005690:Galactose / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D000432:Methanol / Q000494:pharmacology; D008970:Molecular Weight; D009154:Mutation; D009844:Oligosaccharides / Q000096:biosynthesis / Q000737:chemistry; D010843:Pichia / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012997:Solvents / Q000494:pharmacology; D048229:Sulfolobus solfataricus / Q000201:enzymology*; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D019843:Archaeal Proteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D012997:Solvents; D001616:beta-Galactosidase; D007785:Lactose; D005690:Galactose; D000432:Methanol
keywords: Pichia pastoris; characterization; expression; β-galactosidase