Wednesday, July 29, 2009 - 10:00 AM
S100
Molecular evolution for biomass-degrading enzymes
Chie Imamura, Akinori Ikeuchi, and Haruo Takahashi. Biotechnology Lab., Toyota Central R&D Labs., Inc., Nagakute,, Aichi, 4801192, Japan
To increase the efficiency of converting cellulosic biomass to fermentable sugars, we attempted to improve the catalytic activity of biomass-degrading enzymes by evolutionary protein engineering approaches. Recently, we have developed a new molecular evolution method called SIMPLEX (single-molecule-PCR-linked in vitro expression) for the purpose of changing protein function and stability. In addition, in order to solve protein folding problems, a functional expression system was developed using an E.coli in vitro coupled transcription/translation system by addition of chaperones and protein disulfide isomerase. Using this system, disulfide-containing cellobiohydrolases (CBH), endoglucanases, homo-dimeric β-glucosidases (BGL) and lignin-degrading heme proteins were successfully synthesized in an active form, representing a success rate of approximately 90 percent. Our functional expression system has made it possible to screen a large number of mutant proteins in a high-throughput manner.
On the other hand, in our recent work, L-lactic acid, which is a raw material for a plastic, was produced by metabolically engineered yeast. A BGL was expressed on the surface of a lactic-acid-producing yeast strain to enable lactic acid fermentation with cellobiose, due to the enzymatic saccharification. In such case, BGL convert cellobiose into glucose in the acidic culture condition. We aimed to improve the Thermotoga maritima BGL activity under both acidic pH and low temperature using SIMPLEX method. Furthermore, we have succeeded in increasing the catalytic activity of a fungus CBH, which is a key enzyme in cellulose degradation. Our novel system can be widely applied for various kind of screening of proteins with desired properties.
On the other hand, in our recent work, L-lactic acid, which is a raw material for a plastic, was produced by metabolically engineered yeast. A BGL was expressed on the surface of a lactic-acid-producing yeast strain to enable lactic acid fermentation with cellobiose, due to the enzymatic saccharification. In such case, BGL convert cellobiose into glucose in the acidic culture condition. We aimed to improve the Thermotoga maritima BGL activity under both acidic pH and low temperature using SIMPLEX method. Furthermore, we have succeeded in increasing the catalytic activity of a fungus CBH, which is a key enzyme in cellulose degradation. Our novel system can be widely applied for various kind of screening of proteins with desired properties.
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See more of Invited Oral Papers
See more of The Annual Meeting and Exhibition 2009 (July 26 - 30, 2009)