title: Engineering the acceptor specificity of trehalose phosphorylase for the production of trehalose analogs.
abstract: Trehalose (α-D-glucopyranosyl-(1,1)-α-D-glucopyranoside) is widely used in the food industry, thanks to its protective effect against freezing and dehydration. Analogs of trehalose have the additional benefit that they are not digested and thus do not contribute to our caloric intake. Such trehalose analogs can be produced with the enzyme trehalose phosphorylase, when it is applied in the reverse, synthetic mode. Despite the enzyme's broad acceptor specificity, its catalytic efficiency for alternative monosaccharides is much lower than for glucose. For galactose, this difference is shown here to be caused by a lower K(m) whereas the k(cat) for both substrates is equal. Consequently, increasing the affinity was attempted by enzyme engineering of the trehalose phosphorylase from Thermoanaerobacter brockii, using both semirational and random mutagenesis. While a semirational approach proved unsuccessful, high-throughput screening of an error-prone PCR library resulted in the discovery of three beneficial mutations that lowered K(m) two- to three-fold. In addition, it was found that mutation of these positions also leads to an improved catalytic efficiency for mannose and fructose, suggesting their involvement in acceptor promiscuity. Combining the beneficial mutations did not further improve the affinity, and even resulted in a decreased catalytic activity and thermostability. Therefore, enzyme variant R448S is proposed as new biocatalyst for the industrial production of lactotrehalose (α-D-glucopyranosyl-(1,1)-α-D-galactopyranoside).
authors: Dept. of Biochemical and Microbial Technology, Center of Expertise for Industrial Biotechnology and Biocatalysis, Ghent University, Coupure Links 653, 9000 Ghent, Belgium.
authors: Jef
authors: J
authors: Van der Borght
authors: Wim
authors: W
authors: Soetaert
authors: Tom
authors: T
authors: Desmet
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D055162:Biocatalysis; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D015202:Protein Engineering; D013379:Substrate Specificity; D046968:Thermoanaerobacter / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D014199:Trehalose / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D014199:Trehalose; D005964:Glucosyltransferases; C023181:alpha,alpha-trehalose phosphorylase