We introduced mutations into prxA3a, a peroxidase gene of hybrid aspen, Populus kitakamiensis, to substitute the amino acid residues at the surface of the protein, and analyzed substrate specificities. PrxA3a and mutated enzymes heterogeneously gene expressed in Saccharomyces cerevisiae were purified by Ni affinity chromatography, hydrolysis of sugar chain (Endoglycosidase H f ) and gel filtration. The substrate specificities were altered by substituted amino acid residues. PrxA3a F77Y A165W acquired the substrate specificity to m-chlorophenol. PrxA3a F77Y and PrxA3a F77YA165W could polymerize sinapyl alcohol. In addition, PrxA3a A165W, F77Y, and F77YA165W improved cytochrome c oxidizing activity. These substituted amino acid residues should function as a catalytic site outside of the heme pocket.
β-fructofuranosidase (FFase) of Aspergillus niger ATCC 20611 can transfer fructosyl residues from one sucrose to another for the synthesis of glucose and fructooligosaccharides composed of 1-kestose (GF 2 ), nystose (GF 3 ), and β-fructofuranosylnystose (GF 4 ). The FFase gene, under the control of the sporamin gene promoter from sweet potato, was introduced into tobacco plants. Sporamin promoter activity is induced by sugar and exhibits preferential expression in stem and root tissues. Thin-layer and high performance liquid chromatographic analyses showed that soluble extracts from the transgenic plants contained considerable amounts of fructooligosaccharides such as GF 2 and GF 3 . The conversion of sucrose into fructooligosaccharides did not affect plant growth or development. Our results indicate that the transgenic plants could be utilized as bioreactors, and this opens up the possibility for efficient production of fructooligosaccharides in sucroseproducing plants such as sugar beet and sugarcane.
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