Starch-based bioethanol process innovation

Starch liquefaction and saccharification are key processing steps in the bioethanol industry. The rate-limiting α-amylase plays an important role due to its endo-glycosidic activity. Work carried out in this thesis on barley α-amylase focused on ER retention to boost recombinant protein expression in planta and purification of the protein to facilitate a cascade refinery approach allowing other high value proteins to be co-produced together with starch for bioethanol production. Results obtained generated evidence for context-dependence of the ER retention motif HDEL, the existence of an HDEL-independent ER retention mechanism and quantitative data showing toxicity or detrimental effects of HDEL overdose. Results also revealed an effect of peptide tags on N-linked glycosylation as well as evidence that expression levels and systems can strongly affect glycosylation of proteins in the secretory pathway. Furthermore, α-amylase endo-glycosidic action on long glucan chains was shown not to be rate limiting in starch saccharification but the presence of short oligomers and their susceptibility to hydrolysis by fungal amyloglucosidase must be considered next. Interestingly, starch saccharification using acid hydrolysis was more efficient compared to enzyme catalysed hydrolysis. In order to optimise the saccharification process further, the research centred on exploring enzymes with raw-starch digesting properties at low temperature and increased specificity for low molecular weight oligosaccharides. Therefore, an alpha amylase-like gene was identified in ripening plantain (Musa acuminata × balbisiana) using degenerate primers. The gene encodes a putative protein product with close homology to chloroplast α-amylases from Ricinus communis, and Arabidopsis thaliana (AtAMY3).