Abstract
As oil reserves decline, the search for alternative sources of energy is paramount. There is increasing interest in biofuels derived from organic wastes as a potential source of energy. Around the world, the seafood industry contributes millions of tons crustacean shell waste from crabs, lobsters, and shrimp, most of which ends up in landfills or back into the ocean (Yan and Chen, 2015). The chemical composition of crustacean shell waste consists of protein, calcium carbonate, and chitin. Chitin is of primary interest due to its abundance as an amino-polysaccharide and its unique properties which can be used in the pharmaceutical, agricultural, and biomedical industries. In a previous study, Rhodococcus opacus PD630 was able to utilize N-acetyl-glucosamine (NAG), a monomer of chitin, the sole source of carbon to produce triacylglycerols (TAGs), a precursor to biodiesels (Palmer and Brigham, 2016). However, R. opacus lacks the chitinolytic mechanisms to degrade chitin into NAG. The implementation of a cell-free protein synthesis system (TXTL), which utilizes Escherichia coli's transcription and translation mechanisms (Garamella et al., 2016), to produce recombinant chitinases, derived from Serratia marcescens db11, to enzymatically degrade chitin into NAG. Three chitinases were of interest: ChiA, ChiB, and ChiC. The genes: chiA, chiB, and chiC, were isolated from S. marcescens db11 and inserted into plasmids (p70a) obtained from TXTL to produce 3 recombinant chitinase plasmids: p70a-chiA, p70a-chiB, and p70a-chiC. The recombinant plasmids were used to successfully transform E. coli which expressed chitinolytic activity on colloidal chitin plates. The recombinant plasmids were used in TXTL reactions. When the proteins extracted were analyzed through SDS-PAGE, there was no indication of chitinase from the TXTL reactions; however, the TXTL chitinases demonstrated some degradation of chitin in the colloidal chitin plates when cultured with R. opacus which enabled the growth of R. opacus on chitin.