Engineering of pectinolytic enzymes for enhanced thermostability

Conversion of waste materials into valuable compounds is promising concerning transformation of byproduct streams such as sugar beet and potato pulp. In order to obtain those compounds with reduced energy consumption, carbohydrate active enzymes can be used as catalysts. Sugar beet and potato pulp consist of pectin that can be converted into beneficial polymeric and oligomeric carbohydrates requiring enzymes such as pectin lyases, rhamnogalacturonan I (RGI) lyases, polygalacturonases and galactanases. Enzymatic conversion of such pectinaceous biomasses at high temperatures is advantageous as it gives rise to lower substrate viscosity, easier mixing, higher substrate solubility and lowers the risk of contamination. The overall objective of this thesis was to discover enzymes for degradation of RGI structures in pectin and further engineer for enhanced thermostability. The hypotheses were that new enzymes could be selected through traditional screening methods and database searches, respectively. With the use of synthetic biology for recombinant gene expression, the amount of recombinant enzymes would be enhanced. The temperature profile could reveal whether the enzymes should be subjected to engineering in order to improve their thermostability. Development and implementation of a method for selection of candidate residues should reduce the number of residues subjected to mutagenesis and lower the cost and time for screening but still enhance the thermostability. A rhamnogalcturonan lyase from Bacillus licheniformis RGILY_BLI was selected through database searches for gene synthesis. Expression of RGILY_BLI in both Picha pastoris and Bacillus subtilis were successful. The optimum for RGILY_BLI was 61 ˚C, pH 8.1 with an activity of 17.8 U/mg and a half-life at 61 ˚C of 15 min. Based on a design of a small and smart library nine sites were selected for site-saturation mutagenesis. The mutant RGILY_BLI_Glu434Leu exhibited a half-life of 31 min, corresponding to a ~1.6-fold increase at 60 °C compared to the WT. Gly55Val was the second-best mutation with an increase of 35 % (27.0 min). The next best mutations were Glu434Trp, Glu434Phe, and Glu434Tyr with half-life of 27 min (33 %). Further increase in half-lives of RGILY_BLI was obtained in following variants: Glu434Leu/ Gly55Val (35.2±2.7 min), Glu434Leu/Gly326Glu (35.5±2.5 min), Glu434Leu /Gly55Val/Ala67Pro/Gly326Glu (35.7±3.4 min) and Glu434Leu/Gly55Val/ Gly326Glu/ (37.6±2.8 min). Two pectin lyases and two polygalacturonase where selected in a study for maximal release of prebiotic polysaccharides from potato pulp. The enzymes had different pH and temperature profiles where from different hypotheses were argued. In addition phosphate buffer was found to be chelating inducing the release of higher amount of dry matter than Tris-acetate buffer at pH 6. The optimal conditions for a high yield of polysaccharides from potato pulp were determined to be 1% (w/w) potato pulp treated with 1% (w/w) (E/S) for the pectin lyase from Emericella nidulans and the polygalacturonase from Aspergillus aculeatus at pH 6 and 60 °C for one minute. Talaromyces stipitatus was found to secrete endo-1,4-β- galactanase (TSGAL) on sugar beet pectin as sole carbon source. The encoding gene was codon optimized and expressed successfully in P. pastoris. TSGAL had an optimum at 46 °C at pH 4.5 with a half-life of 13 minutes at 55 °C. Nine single-site mutants were constructed exchanging the residue with specific amino acids. TSGAL_Lys31Pro exhibited a half-life of 20.6±3.7 min at 55 °C, which was 55 % better than the wild type enzyme and Thr172Pro gave an increase of half-life in 56%. The half-lives of Asn71Ala and Gly116Asp were increased with 17 % and 28 % respectively. The best mutant TSGAL_Gly305Ala gave a half-life of 114.4±8.1 min at 55 °C which corresponds to an 8.6 fold of increase.