Exploring the Mechanism Responsible for Cellulase Thermostability by Structure-Guided Recombination

• Published in: PloS one Vol. 11; no. 3; p. e0147485
• Main Authors: Chang, Chia-Jung, Lee, Cheng-Chung, Chan, Yueh-Te, Trudeau, Devin L, Wu, Mei-Huey, Tsai, Chih-Hsuan, Yu, Su-May, Ho, Tuan-Hua David, Wang, Andrew H-J, Hsiao, Chwan-Deng, Arnold, Frances H, Chao, Yu-Chan
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 17-03-2016; Public Library of Science (PLoS)
• Subjects: Agricultural biotechnology; Amino Acid Sequence; Amino acid sequencing; Analysis; Animal feed; Animals; Bacillus; Bacillus - chemistry; Bacillus - enzymology; Bacillus - genetics; Bacillus - metabolism; Bacillus subtilis; Bacteria; Biofuels; Biology and Life Sciences; Calcium; Cellulase; Cellulase - chemistry; Cellulase - genetics; Cellulase - metabolism; Cellulose; Chemical engineering; Chemistry; Chimeras; Cloning; Crown Ethers - chemistry; Crystal structure; E coli; Enzyme Stability; Enzymes; Feed industry; Geobacillus - chemistry; Geobacillus - enzymology; Geobacillus - genetics; Geobacillus - metabolism; Graphs; Heat treatment; High temperature; Homology; Hot Temperature; Medicine and Health Sciences; Models, Molecular; Molecular biology; Molecular Sequence Data; Mutagenesis; Physical Sciences; Protein Conformation; Proteins; Recombination; Research and Analysis Methods; Sequence Alignment; Thermal stability; Thermophiles; Thermophilic bacteria; United States > US; Taiwan; California
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0147485

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.