Enhanced thermostability of mesophilic endoglucanase Z with a high catalytic activity at active temperatures

This is the first study for therrmostable mutants of mesophilic endoglucanase EngZ from Clostridium cellulovorans using by site-directed mutagenesis. K94R, S365P and their double mutant K94R/S365P had a wide range of active temperatures (30-60 °C). In addition, the optimal temperature of K94R/S365P was increased by 7.5 °C. K94R/S365P retained 78.3% relative activity at 70 °C, while the wild type retained only 5.8%. Especially, K94R/S365P remained 45.1-fold higher activity than the wild type at 70 °C. In addition, K94R/S365P was 3.1-fold higher activity than the wild type at 42.5 °C, which is the optimal temperature of the wild type. K94R/S365P showed also stimulated in 2.5-fold lower concentration of CaCl₂ and delayed aggregation temperature in the presence of CaCl₂ compared to the wild type. In pH stability, K94R/S365P was not influenced, but the optimum pH was transferred from pH 7 to pH 6. In long-term hydrolysis, K94R/S365P reduced the newly released reducing sugar yields after 12 h reaction; however, the yields consistently increased until 72 h. Finally, the total reducing sugar of K94R/S365P was 5.0-fold higher than the wild type at 50 °C, pH6. EngZ (K94R/S365P) can support information to develop thermostability of GH9 endoglucanase with a high catalytic efficiency as the potential industrial bioprocess candidate.