Phosphorylases that have been reported to date are classified into fourteen kinds by the substrates phosphorolyzed.1) All of them catalyze an exowise phosphorolysis at the nonreducing end of the glycosyl linkage and, more specifically, most phosphorolyze glucosyl linkages. Kojibiose phosphorylase (KP; EC 2.4.1.230) from Thermoanaerobacter brockii ATCC35047 catalyzes the reversible phosphorolysis of the α 1,2 glucosyl bond of kojibiose (α D glucopyranosyl (1 2) D glucopyranose). T. brockii also produces trehalose phosphorylase (TP; EC 2.4.1.64) that reversibly phosphorolyzes the α,α 1,1 glucosyl bond of trehalose (α D glucopyranosyl (1 1) α D glucopyranoside). KP and TP produce β D glucose 1 phosphate (β G1P) and D glucose by the phosphorolysis of each substrate. Functionally, KP and TP catalyze the phosphorolysis of the α glucosyl bond with an inversion of the anomeric configuration. Structurally, they are classified into glycoside hydrolase family 65 (GH65) based on the amino acid sequence (http: afmb.cnrs mrs.fr CAZY GH.html). We have already purified KP and TP,2,3) and cloned these phosphorylase genes from T. brockii. 4,5) Furthermore, various oligosaccharides were synthesizing by using KP or TP. Protein engineering techniques have often been employed to improve the characteristics of targeted enzymes. For industrial purposes, the enhancement of thermostability of targeted enzymes is generally favorable, and the change in specificities of enzymes could increase the yield of the main products, thereby reducing unfavorable by products, or would facilitate the synthesis of novel products. Random mutagenesis has been employed to improve the characteristics of various enzymes.10 12) Chimerization is also used to improvement on the enzymatic properties. Since it is possible to exchange or insert regions in the target enzymes, chimerization could result in remarkable improvements in the characteristics of enzymes.13)The regiospecificities of phosphorylases are very strict and they phosphorolyze only their specific type of glycosyl linkage. These strict specificities are important in the exploitation of these enzymes in the synthesis of oligosaccharides by their synthetic reaction. Namely, oligosaccharides with specific glycosyl linkages can be synthesized using phosphorylases. Therefore, improvement of enzymatic properties of phosphorylases would open an opportunity for production of useful oligosaccharides.In this review, we introduce our recent studies about the random mutagenesis of KP, and the construction and characterization of chimeric enzymes from KP and TP. J. Appl. Glycosci., 53, 123 129 (2006) ! C 2006 The Japanese Society of Applied Glycoscience Proceedings of the Symposium on Amylases and Related Enzymes, 2005 Improvement of the Enzymatic Properties of Kojibiose Phosphorylase fromThermoanaerobacter brockii by Random Mutagenesis and Chimerization (Received December 20, 2005) Takuo Yamamoto, Abstract: Random mutagenesis by error-prone PCR was introduced to kojibiose phosphorylase (KP; EC 2.4.1.230) fro...

Cyclic nigerosyl-(+̮0)-nigerose (CNN) has a unique structure consisting of four glucose residues connected by alternate a-(+̮-)and a-(+̮0)-linkages. We investigated the crystal properties of CNN and the stability of powdered functional oils using CNN. As a result of thermal analysis, we found that CNN takes crystal forms, /H,O, +H,O and anhydrous crystals. The alcohol retention capacity of variously cyclic saccharides, such as a-, band g-cyclodextrin (CD), was also examined. Ethanol and propanol retention capacities were observed in the order b-CD̮CNN̮b-CD̮g-CD. We then examined the stability of powdered fat-soluble vitamins, vitamin D-, vitamin E and eicosapentaenoic acid, by using anhydrous CNN ; CNNfunctional oil powders showed the highest residual values. These results indicate that CNN is useful for powdering easily oxidized fat-soluble substances.