Synthesis of carotenoid-containing polyesters and protein engineering to improve synthetic efficiency of Pseudomonas fluorescens esterase.

• Main Author: Jiang, Yun
• Format: Thesis
• Language: English
• Published: 2009
• Subjects: Acyltransferase; Biocatalysis; Protein Engineering; Biochemistry; Molecular Bio; and Biophysics; Romas; Antarc*; Antarctica
• Online Access: http://purl.umn.edu/57161

University of Minnesota Ph.D. dissertation. October 2009. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: Romas Kazlauskas. 1 computer file (PDF); ix, 139 pages. Ill. (some col.) The conjugated double bonds in carotenoids central chain make these compounds potential building blocks for conductive polymers. Bixin and crocetin's capability of forming carboxylester bonds at end are used to synthesis polyesters. Among 20 esterases and lipases, Candida antarctica Lipase B (CalB) is the only enzyme that catalyzes bixin esterification reaction with linear alcohols. None of these enzyme catalyzes crocetin esterification reaction. Candida antarctica Lipase B added alcohols, such as, n-propanol, polyethylene glycol 400 and 1, 10- decanediol to bixin to make bixin diesters, which worked as initiators and were added into "-caprolactone to synthesis polyesters. The corresponding polymers had the MW 10,600, 12,900 and 11,100. Esterases/lipases and some acyltransferases share a Ser-His-Asp catalytic triad and similar mechanisms. Comparison of the x-ray structures of these structurally related esterases/lipases with acyltransferases reveals a different conformation of the oxyanion loop. In esterases/lipases this loop adopts a type II ! turn conformation with C=O of the main chain facing the active site. In acyltransferases this loop adopts a type I ! turn with the N–H of the main chain facing the active site. The x-ray crystal structure of Pseudomonas fluorescens esterase containing a sulfonate transition state analog shows the C=O facing active site activate via a bridging water molecule to the attacking water molecule. While in acyltransferases an opposing interaction with the N– H may deactivate the attacking water molecule. Oxyanion turn GWLL of Pseudomonas fluorescens esterase (PFE) was engineered in order to switch to a type I # turn and favor acyltransfer reaction. Replacing GWLL with acyltransferases oxyanion turn peptides yielded several mutants with only PFE-GLRA soluble. However PFE-GLRA doesn't show activity. Saturation mutagenesis at position W28 and L29, yielded mutants L29I, L29T, L29V, L29W, with acyltransfer/hydrolysis (A/H) ratios 2.2, 2.5, 2.5, 4 fold of that of the wild type. Some of these mutants might contain type I # turns.