title: Exploring the Mechanism Responsible for Cellulase Thermostability by Structure-Guided Recombination.
abstract: Cellulases from Bacillus and Geobacillus bacteria are potentially useful in the biofuel and animal feed industries. One of the unique characteristics of these enzymes is that they are usually quite thermostable. We previously identified a cellulase, GsCelA, from thermophilic Geobacillus sp. 70PC53, which is much more thermostable than its Bacillus homolog, BsCel5A. Thus, these two cellulases provide a pair of structures ideal for investigating the mechanism regarding how these cellulases can retain activity at high temperature. In the present study, we applied the SCHEMA non-contiguous recombination algorithm as a novel tool, which assigns protein sequences into blocks for domain swapping in a way that lessens structural disruption, to generate a set of chimeric proteins derived from the recombination of GsCelA and BsCel5A. Analyzing the activity and thermostability of this designed library set, which requires only a limited number of chimeras by SCHEMA calculations, revealed that one of the blocks may contribute to the higher thermostability of GsCelA. When tested against swollen Avicel, the highly thermostable chimeric cellulase C10 containing this block showed significantly higher activity (22%-43%) and higher thermostability compared to the parental enzymes. With further structural determinations and mutagenesis analyses, a 310 helix was identified as being responsible for the improved thermostability of this block. Furthermore, in the presence of ionic calcium and crown ether (CR), the chimeric C10 was found to retain 40% residual activity even after heat treatment at 90°C. Combining crystal structure determinations and structure-guided SCHEMA recombination, we have determined the mechanism responsible for the high thermostability of GsCelA, and generated a novel recombinant enzyme with significantly higher activity.
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, ROC.
authors.forename: Chia-Jung
authors.initials: CJ
authors.lastname: Chang
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, ROC.|Core Facilities for Protein Structural Analysis, Academia Sinica, Taipei, Taiwan, ROC.
authors.forename: Cheng-Chung
authors.initials: CC
authors.lastname: Lee
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, ROC.|Core Facilities for Protein Structural Analysis, Academia Sinica, Taipei, Taiwan, ROC.
authors.forename: Yueh-Te
authors.initials: YT
authors.lastname: Chan
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, United States of America.
authors.forename: Devin L
authors.initials: DL
authors.lastname: Trudeau
authors.affiliation: Institute of Biotechnology, National Cheng Kung University, Tainan, Taiwan, ROC.|Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan, ROC.
authors.forename: Mei-Huey
authors.initials: MH
authors.lastname: Wu
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, ROC.
authors.forename: Chih-Hsuan
authors.initials: CH
authors.lastname: Tsai
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, ROC.|Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan, ROC.
authors.forename: Su-May
authors.initials: SM
authors.lastname: Yu
authors.affiliation: Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan, ROC.|Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan, ROC.
authors.forename: Tuan-Hua David
authors.initials: TH
authors.lastname: Ho
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, ROC.|Core Facilities for Protein Structural Analysis, Academia Sinica, Taipei, Taiwan, ROC.
authors.forename: Andrew H-J
authors.initials: AH
authors.lastname: Wang
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, ROC.
authors.forename: Chwan-Deng
authors.initials: CD
authors.lastname: Hsiao
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, United States of America.
authors.forename: Frances H
authors.initials: FH
authors.lastname: Arnold
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, ROC.|Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan, ROC.
authors.forename: Yu-Chan
authors.initials: YC
authors.lastname: Chao
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001407:Bacillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D043844:Crown Ethers / Q000737:chemistry; D004795:Enzyme Stability; D056506:Geobacillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011487:Protein Conformation; D016415:Sequence Alignment
chemical_list: D043844:Crown Ethers; D002480:Cellulase