3752:
Enzymatic Properties of Yeast Expressed Tomato Beta-Galactosidase (TBG)1

Wednesday, August 4, 2010
Springs F & G
Megumi Ishimaru , Plant Biosciences, Osaka Prefecture University, Sakai, Japan
Hidemi Izumi , Bio-Oriented Sci & Tech, Kinki University, Kinokawa, Japan
Tatsuji Sakamoto , Biochemistry, Osaka Prefecture University, Sakai, Japan
Toshihisa Kotake , Biochemistry and Molecular Biology, Saitama University, Saitama, Japan
Yoichi Tsumuraya , Biochemistry and Molecular Biology, Saitama University, Saitama, Japan
Fruit softening occurs by several mechanisms, including modification of cell wall structure by cell wall degrading related enzymes. The most important change in tomato fruit, cell wall composition is the loss of galactosyl residues throughout development and ripening. In order to understand the role of galactosyl turnover in cell wall components, we successfully produced recombinant tomato β-galactosidase (TBG) fusion proteins in yeast. Previously, we reported the properties and substrate specificity of TBG1, 4 and 5. Here we assessed enzymatic properties and substrate specificity of TBG1 in detail using several linked galactooligosaccharides. Optimum pH of partially purified TBG1 was 5, and optimum temperature was 40°C.The Km for TBG1 was 0.45 mM, Vmax was 0.13 µM/s, and the IC50 by galactose was 0.24 M, using p-nitrophenyl -β-D-galactopyranoside as substrate. Using several galactooliogosaccharides has β-(13), β-(14) and β-(16) linkages. TBG1 released galactosyl residues from wide range of several galactooligosaccharides. When using β-(16)-galactohexaose as a substrate, the hydrolyzed pattern of TBG1 shows exo-β-(16)-galactosidase/ galactosyl transferase activities. Using tomato fruit cell wall materials, TBG1 released galactosyl residues from a variety of fruit stages and cell wall fractions. These results suggest that TBG1 which has strong β-(13)-galactosidase and exo-β-(16)-galactosidase/ galactosyl transferase activities, may hydrolyze to targeted arabinogalactan II in structure of wall pectic polysaccharides.