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

Autor: Yu-Chan Chao, Chwan-Deng Hsiao, Su-May Yu, Andrew H.-J. Wang, Chia-Jung Chang, Yueh-Te Chan, Mei-Huey Wu, Tuan-Hua David Ho, Chih-Hsuan Tsai, Devin L. Trudeau, Frances H. Arnold, Cheng-Chung Lee
Rok vydání: 2015
Předmět:
0301 basic medicine
Models
Molecular
Protein Structure Comparison
Hot Temperature
Protein Conformation
lcsh:Medicine
Bacillus
Bacillus subtilis
Pathology and Laboratory Medicine
Biochemistry
Protein structure
Crown Ethers
Enzyme Stability
Medicine and Health Sciences
Macromolecular Structure Analysis
Cellulases
lcsh:Science
Homologous Recombination
Thermostability
Multidisciplinary
Crystallography
Organic Compounds
Physics
Condensed Matter Physics
Recombinant Proteins
Enzymes
Bacterial Pathogens
Nucleic acids
Chemistry
Bacillus Subtilis
Medical Microbiology
Physical Sciences
Crystal Structure
Prokaryotic Models
Pathogens
Sequence Analysis
Research Article
Ethers
Protein Structure
DNA recombination
Molecular Sequence Data
Sequence alignment
Cellulase
Biology
Research and Analysis Methods
Microbiology
03 medical and health sciences
Model Organisms
Genetics
Animals
Solid State Physics
Amino Acid Sequence
Molecular Biology Techniques
Sequencing Techniques
Molecular Biology
Microbial Pathogens
Bacteria
Thermophile
lcsh:R
Organic Chemistry
Chemical Compounds
Organisms
Geobacillus
Biology and Life Sciences
Proteins
DNA
biology.organism_classification
Fusion protein
030104 developmental biology
Mutagenesis
biology.protein
Enzymology
lcsh:Q
Homologous recombination
Sequence Alignment
Zdroj: PLoS ONE
PLoS ONE, Vol 11, Iss 3, p e0147485 (2016)
ISSN: 1932-6203
Popis: 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 3_(10) 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.
Databáze: OpenAIRE
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