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Characterization of a mutated Geobacillus stearothermophilus l ‐arabinose isomerase that increases the production rate of d ‐tagatose
Author(s) -
Kim H.J.,
Kim J.H.,
Oh H.J.,
Oh D.K.
Publication year - 2006
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/j.1365-2672.2006.02975.x
Subject(s) - isomerase , geobacillus stearothermophilus , arabinose , biochemistry , enzyme kinetics , enzyme , galactose , escherichia coli , chemistry , biology , gene , active site , thermophile , xylose , fermentation
Aims: Characterization of a mutated Geobacillus stearothermophilus l ‐arabinose isomerase used to increase the production rate of d ‐tagatose. Methods and Results: A mutated gene was obtained by an error‐prone polymerase chain reaction using l ‐arabinose isomerase gene from G. stearothermophilus as a template and the gene was expressed in Escherichia coli. The expressed mutated l ‐arabinose isomerase exhibited the change of three amino acids (Met 322 →Val, Ser 393 →Thr, and Val 408 →Ala), compared with the wild‐type enzyme and was then purified to homogeneity. The mutated enzyme had a maximum galactose isomerization activity at pH 8·0, 65°C, and 1·0 mM Co 2+ , while the wild‐type enzyme had a maximum activity at pH 8·0, 60°C, and 1·0‐mM Mn 2+ . The mutated l ‐arabinose isomerase exhibited increases in d ‐galactose isomerization activity, optimum temperature, catalytic efficiency ( k cat / K m ) for d ‐galactose, and the production rate of d ‐tagatose from d ‐galactose. Conclusions: The mutated l ‐arabinose isomerase from G. stearothermophilus is valuable for the commercial production of d ‐tagatose. Significance and Impact of the Study: This work contributes knowledge on the characterization of a mutated l ‐arabinose isomerase, and allows an increased production rate for d ‐tagatose from d ‐galactose using the mutated enzyme.