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Characterization of Sulfolobus solfataricus β ‐galactosidase mutant F441Y expressed in Pichia pastoris
Author(s) -
Sun Xiaojun,
Duan Xuguo,
Wu Dan,
Chen Jian,
Wu Jing
Publication year - 2014
Publication title -
journal of the science of food and agriculture
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.782
H-Index - 142
eISSN - 1097-0010
pISSN - 0022-5142
DOI - 10.1002/jsfa.6419
Subject(s) - pichia pastoris , sulfolobus solfataricus , thermostability , chemistry , enzyme kinetics , pichia , enzyme , chromatography , lactose , biochemistry , recombinant dna , specific activity , yield (engineering) , active site , gene , materials science , archaea , metallurgy
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% . CONCLUSION The F441Y of β ‐galactosidase expressed in P. pastoris KM71 has potential application in the industrial preparation of GOS. © 2013 Society of Chemical Industry