Wood is a potential source for biofuels such as ethanol if it can be digested into sugars and fermented by yeast. Biomass derived from wood is a challenging substrate for ethanol production since it is made of lignin and cellulose which cannot be broken down easily into fermentable sugars. Some insects, and termites in particular, are specialized at using enzymes in their guts to digest wood into sugars. If termite gut enzymes could be made abundantly by a recombinant protein expression vector system, they could be applied to an industrial process to make biofuels from wood. In this study, a large cDNA library of relevant termite genes was made using termites fed a normal diet, or a diet with added lignin. A subtracted library yielded genes that were overexpressed in the presence of lignin. Termite gut enzyme genes were identified and cloned into recombinant insect viruses called baculoviruses. Using our PERLXpress system for protein expression, these termite gene recombinant baculoviruses were prepared and used to infect insect larvae, which then expressed abundant recombinant termite enzymes. Many of these expressed enzymes were prepared to very high purity, and the activities were studied in conjunction with collaborators at Purdue University. Recombinant termite enzymes expressed in caterpillars were shown to be able to release sugars from wood. Mixing different combinations of these enzymes increased the amount of sugars released from a model woody biomass substrate. The most economical, fastest and energy conserving way to prepare termite enzymes expressed by recombinant baculoviruses in caterpillars was by making crude liquid homogenates. Making enzymes stable in homogenates therefore was a priority. During the course of these studies, improvements were made to the recombinant baculovirus expression platform so that caterpillar-derived homogenates containing expressed termite enzymes would be more stable. These improvements in the baculoviruses included significantly reducing proteases and preventing blackening immune reactions that occur when caterpillars are homogenized. Proteases may degrade enzymes and immune reaction blackening may inactivate enzymes thus compromising the ability of these crude recombinant expressed termite enzyme preparations to release sugars. Commercial preparations of fungal enzymes currently are used to digest wood for ethanol production. We demonstrated in this study that termite enzymes could improve the efficiency of fungal enzyme cocktails. Although the economic feasibility of using caterpillar expressed termite enzymes alone to treat wood was not proven, this work points to the potential to combine C-PERLXpressed insect enzymes with industrial enzyme cocktails to boost their efficiency at treating wood for biofuels.
1. 09 biomass fuels
2. 59 basic biological sciences
3. 60 applied life sciences
4. Termite, cabbage looper, caterpillar,beetle, insect, baculovirus, cellulase, xylanase, lignase, ligninase, superoxide dismutase, glutathione peroxidase, aldo-keto reductase, glucosidase, catalase, laccase