Directed evolution of 1,4-beta-D-glucan glucohydrolase from Thermotoga neapolitana: Tracking improvements in catalytic efficiency by thermostable coupled enzyme assay derived from Thermotoga maritima

A continuous, thermostable coupled enzyme assay was developed to screen for increased catalytic efficiency in the hydrolysis of cellobiose by mutant Thermotoga neapolitana 1,4-β-D-glucan glucohydrolase (GghA), produced by error-prone PCR. The assay, linking the production of glucose to the reduction of NADP (by monitoring the formation of NADPH at 340 nm), uses Thermotoga maritima enzymes: glucokinase and glucose 6-phosphate dehydrogenase. Glucokinase (EC 2.7.1.2) had a specific activity of 166 U/mg and a monomeric MW of 33.8 kDa, while that of glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate: NADP oxidoreductase, EC 1.1.1.4.9) was 76 U/mg and 57.5 kDa, respectively. Glucokinase had a K _m of 0.038 mM for glucose and 0.189 mM for ATP. Glucose 6-phosphate dehydrogenase had a K_m of 0.019 mM for glucose 6-phosphate and 0.08 mM for NADP. The temperature range of the coupled enzyme assay was 70°–95°C, and its pH range was 6.8 to 8.5.; A first generation mutant GghA library of 1500 clones was produced by the directed evolution technique of error-prone PCR. The library, expressed in Escherichia coli, was picked into 96-well plates and screened at 85°C for increased hydrolysis of cellobiose using the coupled enzyme assay. DNA sequencing of two positive clones and two null clones identified 11 single base shifts. The nucleotide transition of one of the positive clones, IE8A9, caused an Ile → Thr amino acid substitution at position 170 in the primary amino acid sequence. The nucleotide substitution in the null clone, IE1A5 caused the conserved, catalytically important Asn at position 163 to be replaced by an Ile. Comparative kinetic behavior of the pure proteins was determined using the continuous, thermostable coupled enzyme assay. The V_max for the mutant (107.4 U mg−1) was 31% higher than that of the WT (74.1 U mg−1) while the K_m for both proteins was nearly the same. The new enzyme's k_cat increased by 31% and its catalytic efficiency (k_cat/K _m) of for cellobiose rose by 27% as compared to the parent. Structural models were made for IE8A9 and WT proteins, based on sequence homology with 1BGA (PDB) from Paenibacillus polymyxa. Comparative analysis of the 3-D structures indicated that the I;e170Thr substitution had repositioned a key conserved catalytic residue Asn 163 and reconfigured the entry to the active site, providing cellobiose with greater access to the catalytic mechanism of the enzyme.