Conversion of a cyclodextrin glucanotransferase into an alpha-amylase: assessment of directed evolution strategies.
ABSTRACT Glycoside hydrolase family 13 (GH13) members have evolved to possess various distinct reaction specificities despite the overall structural similarity. In this study we investigated the evolutionary input required to effeciently interchange these specificities and also compared the effectiveness of laboratory evolution techniques applied, i.e., error-prone PCR and saturation mutagenesis. Conversion of our model enzyme, cyclodextrin glucanotransferase (CGTase), into an alpha-amylase like hydrolytic enzyme by saturation mutagenesis close to the catalytic core yielded a triple mutant (A231V/F260W/F184Q) with the highest hydrolytic rate ever recorded for a CGTase, similar to that of a highly active alpha-amylase, while cyclodextrin production was virtually abolished. Screening of a much larger, error-prone PCR generated library yielded far less effective mutants. Our results demonstrate that it requires only three mutations to change CGTase reaction specificity into that of another GH13 enzyme. This suggests that GH13 members may have diversified by introduction of a limited number of mutations to the common ancestor, and that interconversion of reaction specificites may prove easier than previously thought.
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ABSTRACT: Directed evolution has become the preferred engineering approach to generate tailor-made enzymes. The method follows the design guidelines of nature: Darwinian selection of genetic variants. This review discusses the different stages of directed evolution experiments with the focus on developments in screening and selection procedures.International Union of Biochemistry and Molecular Biology Life 02/2009; 61(3):222-8. · 3.51 Impact Factor
Article: Engineering of cyclodextrin glucanotransferases and the impact for biotechnological applications.[show abstract] [hide abstract]
ABSTRACT: Cyclodextrin glucanotransferases (CGTases) are industrially important enzymes that produce cyclic alpha-(1,4)-linked oligosaccharides (cyclodextrins) from starch. Cyclodextrin glucanotransferases are also applied as catalysts in the synthesis of glycosylated molecules and can act as antistaling agents in the baking industry. To improve the performance of CGTases in these various applications, protein engineers are screening for CGTase variants with higher product yields, improved CD size specificity, etc. In this review, we focus on the strategies employed in obtaining CGTases with new or enhanced enzymatic capabilities by searching for new enzymes and improving existing enzymatic activities via protein engineering.Applied Microbiology and Biotechnology 09/2009; 85(4):823-35. · 3.42 Impact Factor
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ABSTRACT: Cyclodextrin glucanotransferases (CGTases) have attracted major interest from industry due to their unique capacity of forming large quantities of cyclic alpha-(1,4)-linked oligosaccharides (cyclodextrins) from starch. CGTases produce a mixture of cyclodextrins from starch consisting of 6 (alpha), 7 (beta) and 8 (gamma) glucose units. In an effort to identify the structural factors contributing to the evolutionary diversification of product specificity amongst this group of enzymes, we selected nine CGTases from both mesophilic, thermophilic and hyperthermophilic organisms for comparative product analysis. These enzymes displayed considerable variation regarding thermostability, initial rates, percentage of substrate conversion and ratio of alpha-, beta- and gamma-cyclodextrins formed from starch. Sequence comparison of these CGTases revealed that specific incorporation and/or substitution of amino acids at the substrate binding sites, during the evolutionary progression of these enzymes, resulted in diversification of cyclodextrin product specificity.Applied Microbiology and Biotechnology 05/2009; 84(1):119-33. · 3.42 Impact Factor