Improved Activity of a Thermophilic Cellulase, Cel5A, from Thermotoga maritima on Ionic Liquid Pretreated Switchgrass

Chen, Zhiwei; Pereira, Jose H.; Liu, Hanbin; Tran, Huu M.; Hsu, Nathan S.  Y.; Dibble, Dean; Singh, Seema; Adams, Paul D.; Sapra, Rajat; Hadi, Masood Z.; Simmons, Blake A.; Sale, Kenneth L.

doi:10.1371/journal.pone.0079725

Title: Improved Activity of a Thermophilic Cellulase, Cel5A, from Thermotoga maritima on Ionic Liquid Pretreated Switchgrass

Journal Article · Thu Nov 14 00:00:00 EST 2013 · PLoS ONE

DOI:https://doi.org/10.1371/journal.pone.0079725· OSTI ID:1511377

Chen, Zhiwei ^[1]; Pereira, Jose H. ^[1]; Liu, Hanbin ^[2]; Tran, Huu M. ^[2]; Hsu, Nathan S. Y. ^[3]; Dibble, Dean ^[2]; Singh, Seema ^[2]; Adams, Paul D. ^[4]; Sapra, Rajat ^[2]; Hadi, Masood Z. ^[2]; Simmons, Blake A. ^[2]; Sale, Kenneth L. ^[2]

Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Sandia National Lab. (SNL-CA), Livermore, CA (United States)
Univ. of California, Los Angeles, CA (United States)
Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States)

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.

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Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23), Biological Systems Science Division (SC-23.2 )

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1511377

Journal Information:: PLoS ONE, Vol. 8, Issue 11; ISSN 1932-6203

Publisher:: Public Library of ScienceCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 20 works

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References (53)

Automated saccharification assay for determination of digestibility in plant materials Gomez, Leonardo D.; Whitehead, Caragh; Barakate, Abdellah Biotechnology for Biofuels, Vol. 3, Issue 1 https://doi.org/10.1186/1754-6834-3-23	journal	January 2010
Microplate-based carboxymethylcellulose assay for endoglucanase activity Xiao, Zhizhuang; Storms, Reginald; Tsang, Adrian Analytical Biochemistry, Vol. 342, Issue 1 https://doi.org/10.1016/j.ab.2005.01.052	journal	July 2005
Why High-error-rate Random Mutagenesis Libraries are Enriched in Functional and Improved Proteins Drummond, D. Allan; Iverson, Brent L.; Georgiou, George Journal of Molecular Biology, Vol. 350, Issue 4 https://doi.org/10.1016/j.jmb.2005.05.023	journal	July 2005
Techno-economic analysis of a lignocellulosic ethanol biorefinery with ionic liquid pre-treatment Klein-Marcuschamer, Daniel; Simmons, Blake A.; Blanch, Harvey W. Biofuels, Bioproducts and Biorefining, Vol. 5, Issue 5 https://doi.org/10.1002/bbb.303	journal	June 2011
Influence of physico-chemical changes on enzymatic digestibility of ionic liquid and AFEX pretreated corn stover Li, Chenlin; Cheng, Gang; Balan, Venkatesh Bioresource Technology, Vol. 102, Issue 13 https://doi.org/10.1016/j.biortech.2011.04.005	journal	July 2011
Introduction of glycine and proline residues onto protein surface increases the thermostability of endoglucanase CelA from Clostridium thermocellum Yi, Zhuo-Lin; Pei, Xiao-Qiong; Wu, Zhong-Liu Bioresource Technology, Vol. 102, Issue 3 https://doi.org/10.1016/j.biortech.2010.11.043	journal	February 2011
Ionic liquid tolerant hyperthermophilic cellulases for biomass pretreatment and hydrolysis Datta, Supratim; Holmes, Bradley; Park, Joshua I. Green Chemistry, Vol. 12, Issue 2, p. 338-345 https://doi.org/10.1039/b916564a	journal	January 2010
Climate change and evolutionary adaptation Hoffmann, Ary A.; Sgrò, Carla M. Nature, Vol. 470, Issue 7335 https://doi.org/10.1038/nature09670	journal	February 2011
Biomass Recalcitrance: Engineering Plants and Enzymes for Biofuels Production Himmel, M. E.; Ding, S.-Y.; Johnson, D. K. Science, Vol. 315, Issue 5813, p. 804-807 https://doi.org/10.1126/science.1137016	journal	February 2007
The Path Forward for Biofuels and Biomaterials Ragauskas, Arthur J.; Williams, Charlotte K.; Davison, Brian H. Science, Vol. 311, Issue 5760, p. 484-489 https://doi.org/10.1126/science.1114736	journal	January 2006
A hierarchy of timescales in protein dynamics is linked to enzyme catalysis Henzler-Wildman, Katherine A.; Lei, Ming; Thai, Vu Nature, Vol. 450, Issue 7171 https://doi.org/10.1038/nature06407	journal	November 2007
Protein engineering in designing tailored enzymes and microorganisms for biofuels production Wen, Fei; Nair, Nikhil U.; Zhao, Huimin Current Opinion in Biotechnology, Vol. 20, Issue 4, p. 412-419 https://doi.org/10.1016/j.copbio.2009.07.001	journal	August 2009
Deconstruction of Lignocellulosic Biomass to Fuels and Chemicals Chundawat, Shishir P. S.; Beckham, Gregg T.; Himmel, Michael E. Annual Review of Chemical and Biomolecular Engineering, Vol. 2, Issue 1, p. 121-145 https://doi.org/10.1146/annurev-chembioeng-061010-114205	journal	July 2011
Improved Catalytic Efficiency and Active Site Modification of 1,4-β-d-Glucan Glucohydrolase A from Thermotoga neapolitana by Directed Evolution McCarthy, James K.; Uzelac, Aleksandra; Davis, Diane F. Journal of Biological Chemistry, Vol. 279, Issue 12 https://doi.org/10.1074/jbc.M305642200	journal	December 2003
Molecular simulations provide new insights into the role of the accessory immunoglobulin-like domain of Cel9A Liu, Hanbin; Pereira, Jose Henrique; Adams, Paul D. FEBS Letters, Vol. 584, Issue 15, p. 3431-3435 https://doi.org/10.1016/j.febslet.2010.06.041	journal	July 2010
Dynamical Contributions to Enzyme Catalysis: Critical Tests of A Popular Hypothesis Olsson, Mats H. M.; Parson, William W.; Warshel, Arieh Chemical Reviews, Vol. 106, Issue 5 https://doi.org/10.1021/cr040427e	journal	May 2006
Compositional changes during storage of large round switchgrass bales Wiselogel, A. E.; Agblevor, F. A.; Johnson, D. K. Bioresource Technology, Vol. 56, Issue 1 https://doi.org/10.1016/0960-8524(95)00171-9	journal	April 1996
Directed evolution of a thermophilic endoglucanase (Cel5A) into highly active Cel5A variants with an expanded temperature profile Liang, Chaoning; Fioroni, Marco; Rodríguez-Ropero, Francisco Journal of Biotechnology, Vol. 154, Issue 1 https://doi.org/10.1016/j.jbiotec.2011.03.025	journal	June 2011
Visualization of biomass solubilization and cellulose regeneration during ionic liquid pretreatment of switchgrass Singh, Seema; Simmons, Blake A.; Vogel, Kenneth P. Biotechnology and Bioengineering, Vol. 104, Issue 1, p. 68-75 https://doi.org/10.1002/bit.22386	journal	September 2009
Plants to power: bioenergy to fuel the future Yuan, Joshua S.; Tiller, Kelly H.; Al-Ahmad, Hani Trends in Plant Science, Vol. 13, Issue 8 https://doi.org/10.1016/j.tplants.2008.06.001	journal	August 2008
Intrinsic motions along an enzymatic reaction trajectory Henzler-Wildman, Katherine A.; Thai, Vu; Lei, Ming Nature, Vol. 450, Issue 7171 https://doi.org/10.1038/nature06410	journal	November 2007
Production and extraction of sugars from switchgrass hydrolyzed in ionic liquids Sun, Ning; Liu, Hanbin; Sathitsuksanoh, Noppadon Biotechnology for Biofuels, Vol. 6, Issue 1 https://doi.org/10.1186/1754-6834-6-39	journal	January 2013
Microfluidic Glycosyl Hydrolase Screening for Biomass-to-Biofuel Conversion Bharadwaj, Rajiv; Chen, Zhiwei; Datta, Supratim Analytical Chemistry, Vol. 82, Issue 22 https://doi.org/10.1021/ac102243f	journal	November 2010
Biofuels: Biomolecular Engineering Fundamentals and Advances Li, Han; Cann, Anthony F.; Liao, James C. Annual Review of Chemical and Biomolecular Engineering, Vol. 1, Issue 1 https://doi.org/10.1146/annurev-chembioeng-073009-100938	journal	June 2010
Protein frustratometer: a tool to localize energetic frustration in protein molecules Jenik, M.; Parra, R. G.; Radusky, L. G. Nucleic Acids Research, Vol. 40, Issue W1 https://doi.org/10.1093/nar/gks447	journal	May 2012
Coot model-building tools for molecular graphics Emsley, Paul; Cowtan, Kevin Acta Crystallographica Section D Biological Crystallography, Vol. 60, Issue 12, p. 2126-2132 https://doi.org/10.1107/S0907444904019158	journal	November 2004
Protein aggregation in recombinant bacteria: biological role of inclusion bodies Villaverde, Antonio; Mar Carrió, M. Biotechnology Letters, Vol. 25, Issue 17, p. 1385-1395 https://doi.org/10.1023/A:1025024104862	journal	September 2003
Automated assay for screening the enzymatic release of reducing sugars from micronized biomass Navarro, David; Couturier, Marie; da Silva, Gabriela Microbial Cell Factories, Vol. 9, Issue 1 https://doi.org/10.1186/1475-2859-9-58	journal	January 2010
Protein production by auto-induction in high-density shaking cultures Studier, F. William Protein Expression and Purification, Vol. 41, Issue 1, p. 207-234 https://doi.org/10.1016/j.pep.2005.01.016	journal	May 2005
Critical evaluation of random mutagenesis by error-prone polymerase chain reaction protocols, Escherichia coli mutator strain, and hydroxylamine treatment Rasila, Tiina S.; Pajunen, Maria I.; Savilahti, Harri Analytical Biochemistry, Vol. 388, Issue 1 https://doi.org/10.1016/j.ab.2009.02.008	journal	May 2009
Microbial production of fatty-acid-derived fuels and chemicals from plant biomass Steen, Eric J.; Kang, Yisheng; Bokinsky, Gregory Nature, Vol. 463, Issue 7280, p. 559-562 https://doi.org/10.1038/nature08721	journal	January 2010
Thermostabilization of cellulosomal endoglucanase EngB from Clostridium cellulovorans by in vitro DNA recombination with non-cellulosomal endoglucanase EngD: Thermostabilization of endoglucanase EngB from C. cellulovorans Murashima, Koichiro; Kosugi, Akihiko; Doi, Roy H. Molecular Microbiology, Vol. 45, Issue 3 https://doi.org/10.1046/j.1365-2958.2002.03049.x	journal	July 2002
Biochemical characterization and crystal structure of endoglucanase Cel5A from the hyperthermophilic Thermotoga maritima Pereira, Jose H.; Chen, Zhiwei; McAndrew, Ryan P. Journal of Structural Biology, Vol. 172, Issue 3, p. 372-379 https://doi.org/10.1016/j.jsb.2010.06.018	journal	December 2010
Engineering for biofuels: exploiting innate microbial capacity or importing biosynthetic potential? Alper, Hal; Stephanopoulos, Gregory Nature Reviews Microbiology, Vol. 7, Issue 10 https://doi.org/10.1038/nrmicro2186	journal	October 2009
Site-directed mutagenesis and CBM engineering of Cel5A (Thermotoga maritima) Arumugam Mahadevan, Shobana; Gon Wi, Seung; Lee, Dae-Seok FEMS Microbiology Letters, Vol. 287, Issue 2, p. 205-211 https://doi.org/10.1111/j.1574-6968.2008.01324.x	journal	October 2008
Effect of hemicellulose and lignin removal on enzymatic hydrolysis of steam pretreated corn stover Ohgren, K.; Bura, R.; Saddler, J. Bioresource Technology, Vol. 98, Issue 13 https://doi.org/10.1016/j.biortech.2006.09.003	journal	September 2007
Outlook for cellulase improvement: Screening and selection strategies Percival Zhang, Y.-H.; Himmel, Michael E.; Mielenz, Jonathan R. Biotechnology Advances, Vol. 24, Issue 5, p. 452-481 https://doi.org/10.1016/j.biotechadv.2006.03.003	journal	September 2006
Optimization of pH controlled liquid hot water pretreatment of corn stover Mosier, Nathan; Hendrickson, Richard; Ho, Nancy Bioresource Technology, Vol. 96, Issue 18, p. 1986-1993 https://doi.org/10.1016/j.biortech.2005.01.013	journal	December 2005
Correlation between thermostability and stability of glycosidases in ionic liquid Ferdjani, Salim; Ionita, Marina; Roy, Bimalendu Biotechnology Letters, Vol. 33, Issue 6 https://doi.org/10.1007/s10529-011-0560-5	journal	February 2011
Directed evolution of a para-nitrobenzyl esterase for aqueous-organic solvents Moore, Jeffrey C.; Arnold, Frances H. Nature Biotechnology, Vol. 14, Issue 4 https://doi.org/10.1038/nbt0496-458	journal	April 1996
THEORY OF PROTEIN FOLDING: The Energy Landscape Perspective Onuchic, José Nelson; Luthey-Schulten, Zaida; Wolynes, Peter G. Annual Review of Physical Chemistry, Vol. 48, Issue 1 https://doi.org/10.1146/annurev.physchem.48.1.545	journal	October 1997
Combined sugar yields for dilute sulfuric acid pretreatment of corn stover followed by enzymatic hydrolysis of the remaining solids Lloyd, Todd A.; Wyman, Charles E. Bioresource Technology, Vol. 96, Issue 18, p. 1967-1977 https://doi.org/10.1016/j.biortech.2005.01.011	journal	December 2005
Thermodynamic prediction of protein neutrality Bloom, J. D.; Silberg, J. J.; Wilke, C. O. Proceedings of the National Academy of Sciences, Vol. 102, Issue 3 https://doi.org/10.1073/pnas.0406744102	journal	January 2005
Fermentable sugars by chemical hydrolysis of biomass Binder, J. B.; Raines, R. T. Proceedings of the National Academy of Sciences, Vol. 107, Issue 10, p. 4516-4521 https://doi.org/10.1073/pnas.0912073107	journal	March 2010
Optimization of the ammonia fiber explosion (AFEX) treatment parameters for enzymatic hydrolysis of corn stover Teymouri, Farzaneh; Laureano-Perez, Lizbeth; Alizadeh, Hasan Bioresource Technology, Vol. 96, Issue 18, p. 2014-2018 https://doi.org/10.1016/j.biortech.2005.01.016	journal	December 2005
Deep-Sea Oil Plume Enriches Indigenous Oil-Degrading Bacteria Hazen, Terry C.; Dubinsky, Eric A.; DeSantis, Todd Z. Science, Vol. 330, Issue 6001 https://doi.org/10.1126/science.1195979	journal	August 2010
Dynamical Contributions to Enzyme Catalysis: Critical Tests of a Popular Hypothesis Olsson, Mats H. M.; Parson, William W.; Warshel, Arieh ChemInform, Vol. 37, Issue 31 https://doi.org/10.1002/chin.200631289	journal	August 2006
Tracing Determinants of Dual Substrate Specificity in Glycoside Hydrolase Family 5 Chen, Zhiwei; Friedland, Gregory D.; Pereira, Jose H. Journal of Biological Chemistry, Vol. 287, Issue 30 https://doi.org/10.1074/jbc.m112.362640	journal	May 2012
Carbohydrate-induced Differential Gene Expression Patterns in the Hyperthermophilic Bacterium Thermotoga maritima Chhabra, Swapnil R.; Shockley, Keith R.; Conners, Shannon B. Journal of Biological Chemistry, Vol. 278, Issue 9 https://doi.org/10.1074/jbc.m211748200	journal	December 2002
Engineering of Clostridium phytofermentans Endoglucanase Cel5A for Improved Thermostability Liu, W.; Zhang, X. -Z.; Zhang, Z. Applied and Environmental Microbiology, Vol. 76, Issue 14 https://doi.org/10.1128/aem.00958-10	journal	May 2010
Bacterial Cell Surface Display of an Enzyme Library for Selective Screening of Improved Cellulase Variants Kim, Y. -S.; Jung, H. -C.; Pan, J. -G. Applied and Environmental Microbiology, Vol. 66, Issue 2 https://doi.org/10.1128/aem.66.2.788-793.2000	journal	February 2000
Regulation of Endo-Acting Glycosyl Hydrolases in the Hyperthermophilic Bacterium Thermotoga maritima Grown on Glucan- and Mannan-Based Polysaccharides Chhabra, S. R.; Shockley, K. R.; Ward, D. E. Applied and Environmental Microbiology, Vol. 68, Issue 2, p. 545-554 https://doi.org/10.1128/aem.68.2.545-554.2002	journal	February 2002
Thermodynamic Prediction of Protein Neutrality Bloom, Jesse D.; Silberg, Jonathan J.; Wilke, Claus O. arXiv https://doi.org/10.48550/arxiv.q-bio/0409013	text	January 2004

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