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Applied and Environmental Microbiology, December 2005, p. 8881-8887, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8881-8887.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Structural Characterization of Glucooligosaccharide Oxidase from Acremonium strictum

Meng-Hwan Lee,1,{dagger} Wen-Lin Lai,1,{dagger} Shuen-Fuh Lin,2 Cheng-Sheng Hsu,3 Shwu-Huey Liaw,4* and Ying-Chieh Tsai1*

Institute of BiochemistryFaculty of Life Science, National Yang-Ming University, Taipei,1 Department of Life Sciences, National University of Kaohsiung, Kaohsiung,2 Center of General Education, National Taipei College of Nursing, Taipei, Taiwan,3 Faculty of Life Science, National Yang-Ming University, Taipei4

Received 10 May 2005/ Accepted 22 August 2005

Glucooligosaccharide oxidase from Acremonium strictum was screened for potential applications in oligosaccharide acid production and carbohydrate detection. This protein is a unique covalent flavoenzyme which catalyzes the oxidation of a variety of carbohydrates with high selectivity for cello- and maltooligosaccharides. Kinetic measurements suggested that this enzyme possesses an open carbohydrate-binding groove, which is mainly composed of two glucosyl-binding subsites. The encoding gene was subsequently cloned, and one intron was detected in the genomic DNA. Large amounts of active enzymes were expressed in Pichia pastoris, with a yield of 300 mg per liter medium. The protein was predicted to share structural homology with plant cytokinin dehydrogenase and related flavoproteins that share a conserved flavin adenine dinucleotide (FAD)-binding domain. The closest sequence matches are those of plant berberine bridge enzyme-like proteins, particularly the characteristic flavinylation site. Unexpectedly, mutation of the putative FAD-attaching residue, H70, to alanine, serine, cysteine, and tyrosine did not abolish the covalent FAD linkage and had little effect on the Km. Instead, the variants displayed kcat values that were 50- to 600-fold lower, indicating that H70 is crucial for efficient redox catalysis, perhaps through modulation of the oxidative power of the flavin.

* Corresponding author. Mailing address: Institute of Biochemistry, National Yang-Ming University, 155, Sec. 2, Li-Nong St., Pei-Tou, Taipei 11221, Taiwan. Phone: (886) 2-2826-7125. Fax: (886) 2-2826-4843. E-mail for S.-H. Liaw: shliaw{at}ym.edu.tw. E-mail for Y.-C. Tsai: tsaiyc{at}ym.edu.tw.

{dagger} M.-H.L. and W.-L.L. contributed equally to this work.

Applied and Environmental Microbiology, December 2005, p. 8881-8887, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8881-8887.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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