Biomass Depolymerization projects

Practical Synthesis of Thermoresponsive Cellulase-Polymer Bioconjugates

This team is generating cellulase enzymes with enhanced physical properties by attaching polymer stands with low critical solution temperature (LCST) values to specific locations on the cellulase surfaces. Raising the temperature above the LCST threshold value causes the temporary precipitation of the attached cellulases, enabling them to be recovered and added to fresh biomass samples (at temperatures below the LCST) and resume their catalytic activity. This "thermorecycling" concept will improve the practicality and decrease the cost of using these enzymes in cellulose depolymerization, and provide a useful design concept that could be applied to other enzymatic transformations required for feedstock processing.

project Highlights

2014 Highlights

Our research seeks to increase the performance of cellulases through the use of synthetic polymer additives. As one way to do this, we have attached the enzymes to polymers that display thermally-switchable solubility properties. This allows the enzymes to be precipitated and recovered through subtle increases in the solution temperature. Using this strategy, we have shown that cellulases can be carried through three or more rounds of biomass depolymerization. We have also found that amphiphilic polymers can substantially enhance the lifetime of cellulases, producing 2- to 7-fold increases in the amount of overall activity that each enzyme can achieve.

2013 Highlights

Our research seeks to increase the performance of cellulases through the use of synthetic polymer additives. As one way to do this, we have attached the enzymes to polymers that display thermally switchable solubility properties. This allows the enzymes to be precipitated and recovered through subtle increases in the solution temperature. Using this strategy, we have shown that cellulases can be carried through three or more rounds of biomass depolymerization. We have also found that amphiphilic polymers can substantially enhance the lifetime of cellulases, producing 2- to 7-fold increases in the amount of overall activity that each enzyme can achieve.

2012 Highlights

The main goal of our research is the development of new strategies to allow cellulases to be carried through multiple rounds of biomass depolymerization. To do this, we have used site-selective bioconjugation chemistry to attach the enzymes to polymers displaying lower critical solution temperatures. The resulting enzyme conjugates can be precipitated from solution and recovered simply by heating them above a specific transition temperature. Upon cooling, the conjugates can be dissolved in a fresh reaction solution and used for an additional round of cellulose processing. In a forthcoming publication, we have demonstrated that these conjugates can be carried through three or more reaction cycles, affording substantially more glucose equivalents than would be obtained using non-recyclable enzymes. We have also developed a convenient synthetic method to tune the temperatures at which the polymers exhibit this solubility change. This allows the approach to be extended to a variety of mesophilic and thermophilic enzymes.

2011 Highlights

The group developed chemical methods to attach special polymers to mixtures of cellulases from T. reesei. They successfully recovered the polymers and attached cellulases after heating the solution above the LCST threshold. Upon cooling, the cellulases returned to solution and could be used for another round of cellulose depolymerization.  The enzymes retained their activity through many cycles of reuse. Francis’ group also expressed a thermostable cellulase from Pyrococcus horikoshii with yields exceeding 80 mg/L in E. coli and modified the enzyme to possess a chemical modification handle at the N-terminus for site-selective polymer attachment. They achieved a high yield of this modified cellulase under mild conditions, with the N-terminal selectivity being confirmed using mass spectrometry. Francis’s group now has a library of polymers with different LCST properties that can be attached to various positions on virtually any enzyme of interest to the EBI.

 

Publications

Published in 2014

Effects Of Nipam Polymer Additives On The Enzymatic Hydrolysis Of Avicel And Pretreated Miscanthus, K. J. Mackenzie, M. B. Francis, Biotechnology and Bioengineering, V. 111, pp. 1792-1800.

Published in 2013

Recyclable Thermoresponsive Polymer-Cellulase Bioconjugates for Biomass Depolymerization, K. J. Mackenzie, M. B. Francis, Journal of the American Chemical Society, 135, pp. 293-300, doi:10.1021/Ja309277v, January 9, 2013

 

Site-Specific Protein Transamination Using N-Methylpyridinium-4-Carboxaldehyde, L. S. Witus, C. Netirojjanakul, K. S. Palla, E. M. Muehl, C. H. Weng, A. T. Lavarone, M. B. Francis, Journal of the American Chemical Society, doi: 10.1021/Ja408868a.


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