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Applied and Environmental Microbiology, July 2004, p. 4318-4325, Vol. 70, No. 7
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.7.4318-4325.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Restoration of a Defective Lactococcus lactis Xylose Isomerase

Joo-Heon Park{dagger} and Carl A. Batt*

Department of Food Science, Cornell University, Ithaca, New York 14853

Received 11 November 2003/ Accepted 8 March 2004

The genes (xylA) encoding xylose isomerase (XI) from two Lactococcus lactis subsp. lactis strains, 210 (Xyl–) and IO-1 (Xyl+), were cloned, and the activities of their expressed proteins in recombinant strains of Escherichia coli were investigated. The nucleotide and amino acid sequence homologies between the xylA genes were 98.4 and 98.6%, respectively, and only six amino acid residues differed between the two XIs. The purified IO-1 XI was soluble with Km and kcat being 2.25 mM and 184/s, respectively, while the 210 XI was insoluble and inactive. Site-directed mutagenesis on 210 xylA showed that a triple mutant possessing R202M/Y218D/V275A mutations regained XI activity and was soluble. The Km and kcat of this mutant were 4.15 mM and 141/s, respectively. One of the IO-1 XI mutants, S388T, was insoluble and showed negligible activity similar to that of 210 XI. The introduction of a K407E mutation to the IO-1 S388T XI mutant restored its activity and solubility. The dissolution of XI activity in L. lactis subsp. lactis involves a series of mutations that collectively eliminate enzyme activity by reducing the solubility of the enzyme.

* Corresponding author. Mailing address: Department of Food Science, Cornell University, Ithaca, NY 14853. Phone: (607) 254-5376. Fax: (607) 255-8741. E-mail: cab10{at}cornell.edu.

{dagger} Present address: Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853.

Applied and Environmental Microbiology, July 2004, p. 4318-4325, Vol. 70, No. 7
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.7.4318-4325.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.





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