Improved activity of a thermophilic cellulase, Cel5A, from Thermotoga maritima on ionic liquid pretreated switchgrass.
Ionic liquid pretreatment of biomass has been shown to greatly reduce the recalcitrance of lignocellulosic biomass, resulting in improved sugar yields after enzymatic saccharification. However, even under these improved saccharification conditions the cost of enzymes still represents a significant proportion of the total cost of producing sugars and ultimately fuels from lignocellulosic biomass. Much of the high cost of enzymes is due to the low catalytic efficiency and stability of lignocellulolytic enzymes, especially cellulases, under conditions that include high temperatures and the presence of residual pretreatment chemicals, such as acids, organic solvents, bases, or ionic liquids. Improving the efficiency of the saccharification process on ionic liquid pretreated biomass will facilitate reduced enzyme loading and cost. Thermophilic cellulases have been shown to be stable and active in ionic liquids but their activity is typically at lower levels. Cel5A_Tma, a thermophilic endoglucanase from Thermotoga maritima, is highly active on cellulosic substrates and is stable in ionic liquid environments. Here, our motivation was to engineer mutants of Cel5A_Tma with higher activity on 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) pretreated biomass. We developed a robotic platform to screen a random mutagenesis library of Cel5A_Tma. Twelve mutants with 25-42% improvement in specific activity on carboxymethyl cellulose and up to 30% improvement on ionic-liquid pretreated switchgrass were successfully isolated and characterized from a library of twenty thousand variants. Interestingly, most of the mutations in the improved variants are located distally to the active site on the protein surface and are not directly involved with substrate binding.
Deconstruction Division, Joint BioEnergy Institute, Emeryville, California, United States of America ; Biomass Science and Conversion Technology, Sandia National Laboratories, Livermore, California, United States of America.
Zhiwei
Z
Chen
Jose H
JH
Pereira
Hanbin
H
Liu
Huu M
HM
Tran
Nathan S Y
NS
Hsu
Dean
D
Dibble
Seema
S
Singh
Paul D
PD
Adams
Rajat
R
Sapra
Masood Z
MZ
Hadi
Blake A
BA
Simmons
Kenneth L
KL
Sale
D018533:Biomass; D002480:Cellulase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D057166:High-Throughput Screening Assays; D052578:Ionic Liquids; D008958:Models, Molecular; D016296:Mutagenesis; D006109:Poaceae; D011487:Protein Conformation; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics
D052578:Ionic Liquids; C407024:endoglucanase Cel5A; D002480:Cellulase