Genetic engineering of xylose isomerase thermozymes for enhanced activity, stability, and utility

Molecular determinants responsible for high thermostability of the xylose isomerase from hyperthermophilic eubacterium Thermotoga neapolitana (TNXI) were identified by comparative thermostability and site-directed mutagenesis studies with the less thermostable counterpart enzyme− from thermophilic eubacterium Thermoanaerobacterium thermosulfurigenes (TTXI). Despite their highly similar structures and amino acid sequences (70.4% identity), no obvious differences in the enzyme structures can explain the differences in TNXI's stability compared to that of TTXI except for a few additional prolines and fewer Asn+Gln in TNXI. TNXI has 2 additional prolines in the Phe59 loop (Pro58 and Pro62). This loop helps forming another enzyme subunit's active site. When the 2 prolines in TNXI were substituted with the corresponding amino acids present in its less thermostable counterpart, TTXI, all mutant enzymes showed significant loss in thermostability compared to the wild-type TNXI. These data confirmed the hypothesis that prolines indeed play important roles in TNXI thermostability by reducing its entropy of unfolding.; TNXI's active site was engineered to improve its catalytic efficiency toward glucose. The TNXI V 185T mutant derivative was three times more efficient in glucose isomerization than the wild-type TNXI. Although this mutant derivative was highly thermostable and highly active at 97°C, it was less than 10% as active at 60°C and required neutral pH to work, To customize this TNXI mutant derivative, a directed evolution approach was applied to the TNXI V185T to improve its activity on glucose at low temperature and low pH. After two successive rounds of random mutagenesis and low temperature/low pH activity screening, a new mutant, TNXI 1F1, was obtained that exhibited dramatic improvement of glucose isomerase activity at low temperature and low pH as compared to TNXI V185T. TNXI 1F1 (V185T/L282P/F186S) with one mutation relatively distant (L282P) from the active site and the other (F186S) within the active site of the enzyme, was more active than TNXI V185T over all temperatures and pHs. TNXI 1F1 was also more stable than TNXI and TNXI V185T and this may have resulted from additional H-bond formation between Ser186's sidechain and the neighboring L229 residue's mainchain structure. This H-bond would strengthen local conformation and the affinity of E231 co-ordination with the structural metal.; Biochemical properties and fructose productivities of TNXI 1F1 and Gensweet™, a commercially available glucose isomerase were also compared. TNXI 1F1 displayed higher catalytic efficiencies on glucose at low or high temperature and pH ranges and had greater thermal stability than Gensweet™ despite having similar temperature optima of activity. This greater thermal stability together with the superior kinetic parameters on glucose render TNXI 1F1 an excellent candidate for the industrial glucose isomerization process based on the lifetime fructose productivity estimation.