5-88: Directed Evolution of Hyperthemophilic Endoglucanase, Cel5A, from Thermotoga maritma MSB8

Biomass conversion to biofuels is a four-step process – 1) biomass diminution and physicochemical pretreatment; 2) enzymatic hydrolysis of (hemi-)cellulosic biomass to sugars; 3) conversion of sugars to fuels and 4) finally, recovery and blending of the fuels. Enzymatic hydrolysis of cellulose to glucose is carried out by the action of enzymes known as cellulases, which include endoglucanases (EC 3.1.2.4), exoglucanases (EC 3.1.2.91) and beta-glucosidases (EC 3.1.2.21).  Enzymes from extremophiles, organisms that live under extreme conditions of pH, salt, temperature etc., are adapted to work under the conditions for chemical and physical conditions in the pretreatment steps and are thus chosen as targets for improvement of enzymatic hydrolysis.
Hyperthermophilic endo-β-1,4-glucanase, Cel5A from Thermotoga maritime MSB8, was  chosen as our initial target for directed evolution to improve the hydrolysis efficiency under high temperature. Codon-optimized cel5A gene was cloned into pCDF2 Ek/LIC vector (Novagen) and highly soluble expressed in E. coli strain, BL21 (DE3). It has an optimal pH and temperature at 4.8 (citric acid buffer) and 81°C, respectively. Error-prone PCR was used to generate a low error-rate library. Thousands of colonies were screened for activity improvement by DNS assay compared with wild type cel5A. About 10 hits with 30-50% improvements were further investigated. After expression and purification by Ni-NTA columns, these mutants were assayed for specific activity (U/mg protein). 5 mutant cel5A genes with 20-30% improvement were confirmed by DNA sequencing.