Abstract
Most carbohydrate degrading enzymes have a two-domain structure consisting of a catalytic domain and a carbohydrate binding module (CBM) domain. CBMs mediate the binding of enzyme to the carbohydrate substrate. Family 21 CBMs contain ~100 amino acid residues, and some members have starch-binding functions or glycogen-binding activities. CBMs have shown to be functional even when it is independent from the enzyme. Glucoamylases containing starch-binding domains (SBDs) are used in a variety of scientific and technological applications. In this dissertation a protein engineering strategy – circular permutation – was employed, which yielded variants with greatly enhanced catalytic performance and modified selectivity. A circularly permutated RoCBM21 (CP90) with improved affinity and selectivity toward longer-chain carbohydrates were synthesized, suggesting a new starch-binding protein may be developed for specific scientific and industrial applications. Many studies using random mutagenesis and DNA shuffling could induce only a minor impact to improve the affinity of SBDs while the circular permutation on RoCBM21 could significantly alter the selectivity and affinity. The thermodynamic and chemical stability of circular permutants were determined as well as the functional characteristics were analyzed. Four of the circular permutants predicted could fold with native-like β-barrel fold. The pH, thermodynamics and chemical stability of the circular permutants were investigated. However significant thermal-denaturation differences were observed in two permutants. The circular permutants expressed diverse rate of binding affinity towards carbohydrates. Circular permutation on the 90th position of amino acid (CP90) could generate a highly efficient candidate with higher binding affinity and higher selectivity towards long chain carbohydrates. Qualitative and quantitative experiments were carried out to substantiate the increased affinity and altered selectivity. Further, we used a standard soluble ligand (amylose EX-I) to characterize the detailed functional and structural aspects of CP90. Site-directed mutagenesis along with the crystal structure reveals an altered binding path, which could be the deciding factor to improve affinity and alter the selectivity of this newly created starch-binding domain. The circularly permuted RoCBM21 (CP90) postulates a novel and potential starch binding domain for efficient industrial applications.