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Biotechnology and Applied Biochemistry (2001) 34, (99–102) (Printed in Great Britain)

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Improvement of tagatose conversion rate by genetic evolution of thermostable galactose isomerase

Pil Kim*¹, Sang Hyun Yoon*, Myung Ji Seo*, Deok Kun Oh† and Jin Hwan Choi*

*R & D Center, Tongyang Confectionery Co., 30-10 Munbai-dong, Yongsan-ku, Seoul 140-715, Korea, and †Department of Bioscience and Biotechnology, Sejong University, Seoul 143-747, Korea

Key words: activity enhancement, error-prone PCR, isomerization.

Abbreviations used: gali, Gali; gene and protein product of thermostable galactose (arabinose) isomerase; LB, Luria–Bertani.

¹Present address and address for correspondence: Metabolic Engineering Laboratory, Department of Bioscience and Biotechnology, Sejong University, 98 Kunja-dong, Kwangjin-ku, Seoul 143-747, Korea (e-mail kpil1718@hanmail.net).

To enhance the isomerization rate of galactose into tagatose, a thermostable galactose isomerase, which was isolated from bacteria growing in a hot spring, was genetically improved using the error-prone PCR method. From 500 mutant clones, a clone showing improved conversion activity was selected. The sequence of the selected clone had five amino acid changes: His²²⁸ Asp, Gly³⁸⁴ Asp, Ser³⁹³ Thr, Lys⁴²⁸ Asn and Asp⁴⁷⁵ Lys. The improved galactose isomerase had an 11-fold higher reaction rate than the original.

Received 4 May 2001; accepted 23 July 2001

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