ABSTRACT
A bread-baking process was developed using a potential novel enzyme, cyclodextrin glucanotransferase[3-18] (CGTase[3-18]), that had previously been engineered to have enhanced hydrolyzing activity with little cyclodextrin (CD) formation activity toward starch. CGTase[3-18] was primarily manipulated to be displayed on the cell surface of Saccharomyces cerevisiae. S. cerevisiae carrying pdeltaCGT integrated into the chromosome exhibited starch-hydrolyzing activity at the same optimal pH and temperature as the free enzyme. Volumes of the bread loaves and rice cakes prepared using S. cerevisiae/pdeltaCGT increased by 20% and 45%, respectively, with no detectable CD. Retrogradation rates of the bread and rice cakes decreased significantly during storage. In comparison to the wild type, S. cerevisiae/pdeltaCGT showed improved viability during four freeze-thaw cycles. The results indicated that CGTase[3-18] displayed on the surface of yeast hydrolyzed starch to glucose and maltose that can be used more efficiently for yeast fermentation. Therefore, display of an antistaling enzyme on the cell surface of yeast has potential for enhancing the baking process.
Subject(s)
Bread , Glucosyltransferases/genetics , Yeasts/genetics , Cell Membrane/enzymology , Cloning, Molecular , Cooking , Enzyme Stability , Gene Transfer Techniques , Genetic Engineering , Glucosyltransferases/metabolism , Oryza , Plasmids , Polymerase Chain Reaction , Recombinant Proteins/metabolismABSTRACT
The enzymatic modification of genistin to enhance its water solubility was studied using two glycosyltransferases, cyclodextrin glucanotransferase from alkalophilic Bacillus sp. I-5 and 4-alpha-glucanotransferase from Thermus scotoductus. Two different catalytic reactions, the transglycosylation and cyclization activities, were observed when the reaction was performed with soluble starch as a donor and genistin as an acceptor. The reaction products were isolated and identified as [Glc(alpha1-4)](1-22)-Glc(beta1-7)-genisteins and cycloamylose with DP 8-12 by HPLC and MALDI-TOF MS. A beta-amylase treatment revealed inclusion complexes composed of Glc(alpha1-4)-Glc(beta1-7)-genistein/Glc(alpha1-4)-Glc(alpha1-4)-Glc(beta1-7)-genistein and cycloamylose with DP 8-12. The results indicated that the cycloamylose formed by the cyclization reaction of the enzyme included Glc(alpha1-4)-Glc(beta1-7)-genistein/Glc(alpha1-4)-Glc(alpha1-4)-Glc(beta1-7)-genistein. The presence of cycloamylopectin, in which the Glc(alpha1-4)-Glc(beta1-7)-genistein/Glc(alpha1-4)-Glc(alpha1-4)-Glc(beta1-7)-genistein was enclosed, was also observed with HPLC, HPSEC-MALLS, and MALDI-TOF MS analyses. The solubility of genistin was highly improved, and the solution containing glycosylated genistin and the inclusion complex demonstrated excellent properties of transparency and stability during storage at 4 degrees C.
