title: Rational design to improve thermostability and specific activity of the truncated Fibrobacter succinogenes 1,3-1,4-β-D-glucanase.
abstract: 1,3-1,4-β-D-Glucanase has been widely used as a feed additive to help non-ruminant animals digest plant fibers, with potential in increasing nutrition turnover rate and reducing sanitary problems. Engineering of enzymes for better thermostability is of great importance because it not only can broaden their industrial applications, but also facilitate exploring the mechanism of enzyme stability from structural point of view. To obtain enzyme with higher thermostability and specific activity, structure-based rational design was carried out in this study. Eleven mutants of Fibrobacter succinogenes 1,3-1,4-β-D-glucanase were constructed in attempt to improve the enzyme properties. In particular, the crude proteins expressed in Pichia pastoris were examined firstly to ensure that the protein productions meet the need for industrial fermentation. The crude protein of V18Y mutant showed a 2 °C increment of Tm and W203Y showed ∼30% increment of the specific activity. To further investigate the structure-function relationship, some mutants were expressed and purified from P. pastoris and Escherichia coli. Notably, the specific activity of purified W203Y which was expressed in E. coli was 63% higher than the wild-type protein. The double mutant V18Y/W203Y showed the same increments of Tm and specific activity as the single mutants did. When expressed and purified from E. coli, V18Y/W203Y showed similar pattern of thermostability increment and 75% higher specific activity. Furthermore, the apo-form and substrate complex structures of V18Y/W203Y were solved by X-ray crystallography. Analyzing protein structure of V18Y/W203Y helps elucidate how the mutations could enhance the protein stability and enzyme activity.
authors.affiliation: Genozyme Biotechnology Inc., Taipei 106, Taiwan.
authors.forename: Jian-Wen
authors.initials: JW
authors.lastname: Huang
authors.forename: Ya-Shan
authors.initials: YS
authors.lastname: Cheng
authors.forename: Tzu-Ping
authors.initials: TP
authors.lastname: Ko
authors.forename: Cheng-Yen
authors.initials: CY
authors.lastname: Lin
authors.forename: Hui-Lin
authors.initials: HL
authors.lastname: Lai
authors.forename: Chun-Chi
authors.initials: CC
authors.lastname: Chen
authors.forename: Yanhe
authors.initials: Y
authors.lastname: Ma
authors.forename: Yingying
authors.initials: Y
authors.lastname: Zheng
authors.forename: Chun-Hsiang
authors.initials: CH
authors.lastname: Huang
authors.forename: Peijian
authors.initials: P
authors.lastname: Zou
authors.forename: Je-Ruei
authors.initials: JR
authors.lastname: Liu
authors.forename: Rey-Ting
authors.initials: RT
authors.lastname: Guo
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D044644:Endo-1,3(4)-beta-Glucanase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D045848:Fibrobacter / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering
chemical_list: D001426:Bacterial Proteins; D044644:Endo-1,3(4)-beta-Glucanase