Enzyme Chemistry and Application of Carbohydrate-Relating Enzymes

Abstract

This paper from the 1995 award address of the Society is composed of the following three research topics that were carried out at the Laboratories of Biological Chemistry, and Molecular Enzymology and Protein Engineering, Faculty of Agriculture, Tokyo University of Agriculture and Technology, from 1983 to 1995. A summary of these is given below. 1) Subsite structure and substrate specificity. The subsite structures of Thermoactinomyces vulgaris α-amyalse (TVA I), and native porcine pancreatic α-amyalse II (PPA II) and PPA II modified with DTNB were determined by the SUGANUMA-HIROMI method. It was obvious that the characteristics of the TVA I subsite structure caused its hydrolysis of (α1→6) glucosidic linkages of isopanose and 62-α-maltosylmaltose as well as (α1→4) glucosidic linkage of maltotriose (G3) on the same active site. The difference between the subsite structures of native and modified PPA Its reflected the change of their substrate specificities; the rate of hydrolysis of G³ by PPA II was lower than that of the modified PPA II and the cleavage point of G³ was changed from its reducing end to its nonreducing end by the DTNB modification. The thermal stability of Bacillus acidopullulyticus pullulanase (BAPase), increased by the addition of sugar alcohol, was dependent on its molecular weight; the sugar alcohols larger than maltotriitol (0.2 M) stabiliyzed BAPase completely for 7 hr at 60t. 2) Cloning of TVA genes and the structure and fucntion of TVAs. Genes of TVA (tva I, tva II) were cloned in Escherichia coli MV1184 using two different methods, the probe DNA method and the shotgun cloning method. TVA I was the same enzyme as described previously, and TVA II was a different gene product from TVA I, whereas these enzymes have the α-amylase and pullulan-hydrolyz-ing (panose-forming) acivities. Their primary structure, amylase family conserved region, subsrtrate specificity and kinetic parameters for low and high molecular weight substrates were compared. The modification of TVA II was tried using site-directed mutagenesis, and enzyme activities of most of mutant enzymes decreased, except for the activity of one mutant (H2O2N). Crystals of TVA II were obtained by the sitting drop vapor diffusion technique and diffraction data to 2.9 A resolution were collected using the R-AXIS llc system (Rigaku). The crystal belongs to an orthorhombic system with cell dimensions of a119.5 Å, b=120.6 Å, and c=114.6 Å and a space group P2¹2¹2¹. 3) Oligosaccharide syntheses by pullulanase, TVA II and isomalto-dextranase. Branched cyclodextrins were synthesized from maltooligosaccharides and cyclodextrins (CDs) using the reverse reaction of BAPase. Under the conditions of 70-75% of substrates and 100-200 units of BAPase/g of CD at 60-70t, the yield of branched CDs was higher than 40%. TVA II transfered panosyl residues from pullulan to the C-4 and C-6 positions of glucose (acceptor). Also isomaltodextranase transferred isomaltosyl residues from dextran to the C-6 and C-4 positions of glucose (acceptor) as well as to sucrose, trehalose and primary alcohols.