Increased Crystalline Cellulose Activity via Combinations of Amino Acid Changes in the Family 9 Catalytic Domain and Family 3c Cellulose Binding Module of Thermobifida fusca Cel9A

• Published in: Applied and Environmental Microbiology Vol. 76; no. 8; pp. 2582 - 2588
• Main Authors: Li, Yongchao, Irwin, Diana C, Wilson, David B
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.04.2010; American Society for Microbiology (ASM)
• Subjects: Actinomycetales - enzymology; Actinomycetales - genetics; Amino acid sequence; Amino Acid Substitution - genetics; Amino acids; Binding Sites; Biological and medical sciences; Carbohydrates; Catalytic Domain; Cellulase - genetics; Cellulase - isolation & purification; Cellulase - metabolism; Cellulose; Cellulose - metabolism; Enzymes; Enzymology and Protein Engineering; Fundamental and applied biological sciences. Psychology; Microbiology; Models, Molecular; Modifications; Protein Structure, Tertiary; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Aminoacid; Cellulose
• ISSN: 0099-2240; 1098-5336; 1098-6596
• DOI: 10.1128/AEM.02735-09

Amino acid modifications of the Thermobifida fusca Cel9A-68 catalytic domain or carbohydrate binding module 3c (CBM3c) were combined to create enzymes with changed amino acids in both domains. Bacterial crystalline cellulose (BC) and swollen cellulose (SWC) assays of the expressed and purified enzymes showed that three combinations resulted in 150% and 200% increased activity, respectively, and also increased synergistic activity with other cellulases. Several other combinations resulted in drastically lowered activity, giving insight into the need for a balance between the binding in the catalytic cleft on either side of the cleavage site, as well as coordination between binding affinity for the catalytic domain and CBM3c. The same combinations of amino acid variants in the whole enzyme, Cel9A-90, did not increase BC or SWC activity but did have higher filter paper (FP) activity at 12% digestion.