title: Enhanced thermostability of mesophilic endoglucanase Z with a high catalytic activity at active temperatures.
abstract: 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 12h reaction; however, the yields consistently increased until 72h. 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.
authors.affiliation: Department of Biotechnology, Korea University, Seoul 02841, Republic of Korea.
authors.forename: Su Jung
authors.initials: SJ
authors.lastname: Kim
authors.affiliation: Department of Biotechnology, Korea University, Seoul 02841, Republic of Korea.
authors.forename: Ji Eun
authors.initials: JE
authors.lastname: Joo
authors.affiliation: Department of Biotechnology, Korea University, Seoul 02841, Republic of Korea.
authors.forename: Sang Duck
authors.initials: SD
authors.lastname: Jeon
authors.affiliation: Department of Biotechnology, Korea University, Seoul 02841, Republic of Korea.
authors.forename: Jeong Eun
authors.initials: JE
authors.lastname: Hyeon
authors.affiliation: Department of Chemical and Biological Engineering, Korea University, Seoul 02841, Republic of Korea.
authors.forename: Seung Wook
authors.initials: SW
authors.lastname: Kim
authors.affiliation: Clean Energy Research Center, Korea Institute of science of Technology (KIST), Seoul 02792, Republic of Korea.
authors.forename: Young Soon
authors.initials: YS
authors.lastname: Um
authors.affiliation: Department of Biotechnology, Korea University, Seoul 02841, Republic of Korea. Electronic address: samhan@korea.ac.kr.
authors.forename: Sung Ok
authors.initials: SO
authors.lastname: Han
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D055162:Biocatalysis; D002118:Calcium / Q000494:pharmacology; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D046974:Clostridium cellulovorans / Q000201:enzymology; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D009154:Mutation; D066329:Protein Aggregates; D013696:Temperature
chemical_list: D066329:Protein Aggregates; C080692:endoglucanase Z; D002480:Cellulase; D002118:Calcium
keywords: Cellulolytic efficiency; EngZ; Family 9 endoglucanase; Site-directed mutagenesis; Thermostability