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Cloning, Expression, and Characterization of Rice α-Galactosidase

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Abstract

We have successfully cloned an α-galactosidase gene from a rice cDNA library and transformed it into Escherichia coli BL21. It was subsequently cloned to the pPIC9K vector and expressed in Pichia pastoris. A selected clone was found to result in high production yield of the galactosidase enzyme. The secreted enzyme was purified, and it revealed as a major protein band on an SDS-PAGE gel. The optimal pH value, enzyme stabilities, and substrate specificity were studied. The enzyme specificity toward the terminal α1→6, 1→4, and 1→3 linked galactosyl residue from various substrates was investigated. By determining the Michelis constant (Km) of the enzyme for melibiose, raffinose, and stachyose, our results showed that melibiose was hydrolyzed faster than raffinose, whereas the published data reported a reversed sequence, raffinose > melibiose. The enzyme also showed the ability of converting B red blood cells into O red cells. The objective of this work is to develop the Pichia system to produce a large quantity of enzyme for blood cell conversion for transfusion.

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Abbreviations

α-Gal:

α-galactosidase

M:

melibiose

pNP-α-Gal:

p-nitrophenyl-α-d-galactopyranoside

R:

raffinose

RBC:

red blood cell

S:

stachyose

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Acknowledgments

We would like to thank Dr. Horng-Jinh Chan, former president of Tamkang University, for partial support for this project. We also thank Dr. Horng-Ming Chen, Ye-Cherng Industrial Products Co., Ltd, Taiwan for helping us to clone pET23a-α-Gal/BL21(DE3)pLysS. We thank Mr. Chia-Cheng Liu, Strong Biotech Corp., Taiwan for his excellent assistance in yeast cloning and our best friend Dr. Catherine Dibello, English Department, Shippensburg University of Pennsylvania, USA for helping us to prepare this manuscript.

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Authors and Affiliations

  1. Department of Chemistry, Tamkang University, 151 Ying-Chuan Road, Tamsui, Taiwan, 25137

    Su-Fang Chien, Shi-Hui Chen & Ming-Yang Chien

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  1. Su-Fang Chien
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  2. Shi-Hui Chen
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Correspondence to Su-Fang Chien.

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Chien, SF., Chen, SH. & Chien, MY. Cloning, Expression, and Characterization of Rice α-Galactosidase. Plant Mol Biol Rep 26, 213–224 (2008). https://doi.org/10.1007/s11105-008-0035-6

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