Dataset Information

Improved activity of a thermophilic cellulase, Cel5A, from Thermotoga maritima on ionic liquid pretreated switchgrass.

ABSTRACT: 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.

SUBMITTER: Chen Z

PROVIDER: S-EPMC3828181 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Improved activity of a thermophilic cellulase, Cel5A, from Thermotoga maritima on ionic liquid pretreated switchgrass.

Chen Zhiwei Z Pereira Jose H JH Liu Hanbin H Tran Huu M HM Hsu Nathan S Y NS Dibble Dean D Singh Seema S Adams Paul D PD Sapra Rajat R Hadi Masood Z MZ Simmons Blake A BA Sale Kenneth L KL

PloS one 20131114 11

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 lignocellulo ...[more]

PMID: 24244549

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Project description:BACKGROUND:The development of advanced biofuels from lignocellulosic biomass will require the use of both efficient pretreatment methods and new biomass-deconstructing enzyme cocktails to generate sugars from lignocellulosic substrates. Certain ionic liquids (ILs) have emerged as a promising class of compounds for biomass pretreatment and have been demonstrated to reduce the recalcitrance of biomass for enzymatic hydrolysis. However, current commercial cellulase cocktails are strongly inhibited by most of the ILs that are effective biomass pretreatment solvents. Fortunately, recent research has shown that IL-tolerant cocktails can be formulated and are functional on lignocellulosic biomass. This study sought to expand the list of known IL-tolerant cellulases to further enable IL-tolerant cocktail development by developing a combined in vitro/in vivo screening pipeline for metagenome-derived genes. RESULTS:Thirty-seven predicted cellulases derived from a thermophilic switchgrass-adapted microbial community were screened in this study. Eighteen of the twenty-one enzymes that expressed well in E. coli were active in the presence of the IL 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) concentrations of at least 10% (v/v), with several retaining activity in the presence of 40% (v/v), which is currently the highest reported tolerance to [C2mim][OAc] for any cellulase. In addition, the optimum temperatures of the enzymes ranged from 45 to 95°C and the pH optimum ranged from 5.5 to 7.5, indicating these enzymes can be used to construct cellulase cocktails that function under a broad range of temperature, pH and IL concentrations. CONCLUSIONS:This study characterized in detail twenty-one cellulose-degrading enzymes derived from a thermophilic microbial community and found that 70% of them were [C2mim][OAc]-tolerant. A comparison of optimum temperature and [C2mim][OAc]-tolerance demonstrates that a positive correlation exists between these properties for those enzymes with a optimum temperature >70°C, further strengthening the link between thermotolerance and IL-tolerance for lignocelluolytic glycoside hydrolases.

Dataset Information

Improved activity of a thermophilic cellulase, Cel5A, from Thermotoga maritima on ionic liquid pretreated switchgrass.

Publications

Improved activity of a thermophilic cellulase, Cel5A, from Thermotoga maritima on ionic liquid pretreated switchgrass.

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