Improvement of the Enzymatic Properties of Kojibiose Phosphorylase from Thermoanaerobacter brockii by Random Mutagenesis and Chimerization

Abstract

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...

Evolution of Phosphorylases from N-Acetylglucosaminide Hydrolases in Family GH3

“…Only a few dozen natural phosphorylase specificities have been reported to date, but enzyme engineers have steadily been developing modified phosphorylases that fulfill specific practical needs. Successful examples include the directed evolution of a cellobiose phosphorylase into a lactose phosphorylase, the (semi)-rational redesign of sucrose phosphorylase for the synthesis of rare sugars or glycosides, − and the modulation of chain sizes generated by disaccharide phosphorylases. , Unfortunately, the limited diversity of engineering templates is an important hurdle that narrows down the scope of functions that can be reached without making large and challenging leaps across the fitness landscape.…”

Evolution of Phosphorylase Activity in an Ancestral Glycosyltransferase

“…Further, our results suggest that transferases may be viable starting points for the development of novel phosphorylases that fulfill specific biotechnological needs. Over the years, various GPs with tailored substrate specificities or selectivities have already been obtained by directed evolution and (semi)­rational engineering efforts, each of them starting from one of the few dozen natural phosphorylases that have been discovered to date. ,− Unfortunately, given the small size of the pool of known GPs, it is not always possible to find an appropriate engineering template that can be adapted to recognize a substrate of interest without requiring extensive and challenging modifications to the active site. In those scenarios, it may be worth exploring the alternative strategy of scanning the enormous collection of characterized GTs for a template enzyme that already shows the desired specificity and subsequently evolving its glycosyl donor leaving group preference from nucleoside diphosphate to phosphate instead.…”

Enzymatic synthesis using glycoside phosphorylases

“…14). 28 The enzyme transferred d -glucose onto d - gluco -configured sugars, but tolerated either anomeric configuration and a range of anomeric substituents in the d -glucoside acceptor ( Fig. 6 ).…”

“…The ability to convert them into polymerising enzymes therefore represents an attractive prospect for polysaccharide synthesis. Mutagenesis studies have been used to enhance the flexibility of disaccharide phosphorylase enzymes: opening up the active site of CBP to allow glycosides of glucose to act as acceptor substrates 116 and increasing the length of koji-oligosaccharides produced by kojibiose phosphorylase 28 have both been reported.…”