Abstract
For the removal of galactose inhibition, the predicted galactose binding residues, which were determined by sequence alignment, were replaced separately with Ala. The activities of the Ala-substituted mutant enzymes were assessed with the addition of galactose. As a consequence, amino acid at position 349 was correlated with the reduction in galactose inhibition. The F349S mutant exhibited the highest activity in the presence of galactose relative to the activity measured in the absence of galactose among the tested mutant enzymes at position 349. The K i of the F349S mutant (160 mM), which was 13-fold that of the wild-type enzyme, was the highest among the reported values of β-galactosidase. The wild-type enzyme hydrolyzed 62% of 100 g lactose/l with the addition of 30 g galactose/l, whereas the F349S mutant hydrolyzed more than 99%.
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Berger JL, Lee BH, Lacroix C (1997) Purification, properties and characterization of a high molecular-mass β-galactosidase isoenzyme from Thermus aquaticus. Biotechnol Appl Biochem 25:29–41
Chen W, Chen H, Xia Y, Zhao J, Tian F, Zhang H (2008) Production, purification, and characterization of a potential thermostable galactosidase for milk lactose hydrolysis from Bacillus stearothermophilus. J Dairy Sci 91:1751–1758
Coker JA, Sheridan PP, Loveland-Curtze J, Gutshall KR, Auman AJ, Brenchley JE (2003) Biochemical characterization of a β-galactosidase with a low temperature optimum obtained from an antarctic Arthrobacter isolate. J Bacteriol 185:5473–5482
Cornish-Bowden A (1974) A simple graphical method for determining the inhibition constants of mixed, uncompetitive and non-competitive inhibitors. Biochem J 137:143–144
de Alcantara PH, Martim L, Silva CO, Dietrich SM, Buckeridge MS (2006) Purification of a β-galactosidase from cotyledons of Hymenaea courbaril L. (Leguminosae). Enzyme properties and biological function. Plant Physiol Biochem 44:619–627
Di Lauro B, Strazzulli A, Perugino G, La Cara F, Bedini E, Corsaro MM, Rossi M, Moracci M (2008) Isolation and characterization of a new family 42 β-galactosidase from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius: identification of the active site residues. Biochim Biophys Acta 1784:292–301
Gutowski M, Skurski P, Simons J (2000) Dipole-bound anions of glycine based on the zwitterion and neutral structures. J Am Chem Soc 122:10159–10162
Hidaka M, Fushinobu S, Ohtsu N, Motoshima H, Matsuzawa H, Shoun H, Wakagi T (2002) Trimeric crystal structure of the glycoside hydrolase family 42 β-galactosidase from Thermus thermophilus A4 and the structure of its complex with galactose. J Mol Biol 322:79–91
Kim CS, Ji ES, Oh DK (2004) Characterization of a thermostable recombinant β-galactosidase from Thermotoga maritima. J Appl Microbiol 97:1006–1014
Ladero M, Perez MT, Santos A, Garcia-Ochoa F (2003) Hydrolysis of lactose by free and immobilized β-galactosidase from Thermus sp. strain T2. Biotechnol Bioeng 81:241–252
Mateo C, Monti R, Pessela BC, Fuentes M, Torres R, Guisan JM, Fernandez-Lafuente R (2004) Immobilization of lactase from Kluyveromyces lactis greatly reduces the inhibition promoted by glucose. Full hydrolysis of lactose in milk. Biotechnol Prog 20:1259–1262
Nguyen TH, Splechtna B, Steinbock M, Kneifel W, Lettner HP, Kulbe KD, Haltrich D (2006) Purification and characterization of two novel β-galactosidases from Lactobacillus reuteri. J Agric Food Chem 54:4989–4998
Park AR, Oh DK (2010a) Effects of galactose and glucose on the hydrolysis reaction of a thermostable β-galactosidase from Caldicellulosiruptor saccharolyticus. Appl Microbiol Biotechnol 85:1427–1435
Park AR, Oh DK (2010b) Galacto-oligosaccharide production using microbial β-galactosidase: current state and perspectives. Appl Microbiol Biotechnol 85:1279–1286
Pessela BCC, Mateo C, Fuentes M, Vian A, García J, Carrascosa AV, Guisán JM, Fernández-Lafuente R (2003) The immobilization of a thermophilic β-galactosidase on sepabeads supports decrease product inhibition complete hydrolysis of lactose in dairy products. Enzyme Microb Technol 33:199–205
Petzelbauer I, Nidetzky B, Haltrich D, Kulbe KD (1999) Development of an ultra-high-temperature process for the enzymatic hydrolysis of lactose. I. The properties of two thermostable β-glycosidases. Biotechnol Bioeng 64:322–332
Pisani FM, Rella R, Raia CA, Rozzo C, Nucci R, Gambacorta A, De Rosa M, Rossi M (1990) Thermostable β-galactosidase from the archaebacterium Sulfolobus solfataricus. Purification and properties. Eur J Biochem 187:321–328
Placier G, Watzlawick H, Rabiller C, Mattes R (2009) Evolved β-galactosidases from Geobacillus stearothermophilus with improved transgalactosylation yield for galacto-oligosaccharides production. Appl Environ Microbiol 75:6312–6321
Zhou QZ, Chen XD, Li X (2003) Kinetics of lactose hydrolysis by β-galactosidase of Kluyveromyces lactis immobilized on cotton fabric. Biotechnol Bioeng 81:127–133
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This study was conducted with the support of the 21C Frontier Project for Microbial Genomics, Ministry of Education, Science and Technology.
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Kim, YS., Yeom, SJ. & Oh, DK. Reduction of galactose inhibition via the mutation of β-galactosidase from Caldicellulosiruptor saccharolyticus for lactose hydrolysis. Biotechnol Lett 33, 353–358 (2011). https://doi.org/10.1007/s10529-010-0445-z
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DOI: https://doi.org/10.1007/s10529-010-0445-z