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Applied and Environmental Microbiology, November 1998, p. 4489-4494, Vol. 64, No. 11
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Cloning and High-Level Expression of alpha -Galactosidase cDNA from Penicillium purpurogenum

Hajime Shibuya,1 Hiroaki Nagasaki,1 Satoshi Kaneko,2 Shigeki Yoshida,1 Gwi Gun Park,3 Isao Kusakabe,1 and Hideyuki Kobayashi2,*

Institute of Applied Biochemistry, University of Tsukuba,1 and National Food Research Institute, Ministry of Agriculture, Forestry, and Fisheries,2 Tsukuba, Ibaraki 305, Japan, and Department of Food Processing and Technology, Kyungwon University, Kyunggi-do 461-701, Korea3

Received 20 April 1998/Accepted 28 August 1998

The cDNA coding for Penicillium purpurogenum alpha -galactosidase (alpha Gal) was cloned and sequenced. The deduced amino acid sequence of the alpha -Gal cDNA showed that the mature enzyme consisted of 419 amino acid residues with a molecular mass of 46,334 Da. The derived amino acid sequence of the enzyme showed similarity to eukaryotic alpha Gals from plants, animals, yeasts, and filamentous fungi. The highest similarity observed (57% identity) was to Trichoderma reesei AGLI. The cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast GAL10 promoter. Almost all of the enzyme produced was secreted into the culture medium, and the expression level reached was approximately 0.2 g/liter. The recombinant enzyme purified to homogeneity was highly glycosylated, showed slightly higher specific activity, and exhibited properties almost identical to those of the native enzyme from P. purpurogenum in terms of the N-terminal amino acid sequence, thermoactivity, pH profile, and mode of action on galacto-oligosaccharides.

* Corresponding author. Mailing address: National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki 305, Japan. Phone: 81-298-38-8063. Fax: 81-298-38-7996. E-mail: hkobayas{at}nfri.affrc.go.jp.

Applied and Environmental Microbiology, November 1998, p. 4489-4494, Vol. 64, No. 11
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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