Change of the Product Specificity of a Cyclodextrin Glucanotransferase by Semi-Rational Mutagenesis to Synthesize Large-Ring Cyclodextrins

Christian Sonnendecker,Wolfgang Zimmermann

doi:10.3390/catal9030242

Abstract

Cyclodextrin glucanotransferases (CGTases) convert starch to cyclodextrins (CD) of various sizes. To engineer a CGTase for the synthesis of large-ring CD composed of 9 to 12 glucose units, a loop structure of the protein involved in substrate binding was targeted for semi-rational mutagenesis. Based on multiple protein alignments and protein structure information, a mutagenic megaprimer was designed to encode a partial randomization of eight amino acid residues within the loop region. The library obtained encoding amino acid sequences occurring in wild type CGTases in combination with a screening procedure yielded sequences displaying a changed CD product specificity. As a result, variants of the CGTase from the alkaliphilic Bacillus sp. G825-6 synthesizing mainly CD9 to CD12 could be obtained. When the mutagenesis experiment was performed with the CGTase G825-6 variant Y183R, the same loop alterations that increased the total CD synthesis activity resulted in lower activities of the variant enzymes created. In the presence of the amino acid residue R183, the synthesis of CD8 was suppressed and larger CD were obtained as the main products. The alterations not only affected the product specificity, but also influenced the thermal stability of some of the CGTase variants indicating the importance of the loop structure for the stability of the CGTase.

Highlights

Evolutionary methods can be applied to change amino acid residues of proteins to alter their properties as desired [1]
When the mutagenesis experiment was performed with the Cyclodextrin glucanotransferases (CGTases) G825-6 variant Y183R, the same loop alterations that increased the total CD synthesis activity resulted in lower activities of the variant enzymes created
By selecting distinct amino acid residues instead of total randomization, which would lead to an incongruous number of combinations, a drastic decrease of the size of the variant library can be achieved allowing the alteration of further residues of the target protein without increasing its size [8]

Summary

Introduction

Evolutionary methods can be applied to change amino acid residues of proteins to alter their properties as desired [1]. Iterative rounds of diversification and selection thereby mimic an evolutionary process in vitro [2] Such directed evolution approaches on a molecular level are in in contrast to site-directed mutagenesis techniques, where rational considerations lead to specific and targeted alterations of the target protein [3]. Libraries with higher fitness can be constructed using mutagenic primers [5,6,7] Their design requires information obtained from multiple sequence alignments, consensus sequences, and from the structure and function of the protein [5,6,7]. By selecting distinct amino acid residues instead of total randomization, which would lead to an incongruous number of combinations, a drastic decrease of the size of the variant library can be achieved allowing the alteration of further residues of the target protein without increasing its size [8]

Objectives

Methods

Results

Discussion

Conclusion