Optimization of ethanol production from starch by an amylolytic nuclear petite Saccharomyces cerevisiae strain

Ethanol fermentation characteristics of the 100% respiratory-deficient nuclear petite amylolytic Saccharomyces cerevisiae NPB-G strain was investigated in both shake-flask and controlled bioreactor cultivation conditions, and comparison with the earlier reported results revealed 54.6% increase in ethanol yield. Efforts to improve the starch utilization rate by increasing the selection pressure or supplying the fermentation medium with glucose did not prevent the observed decrease in time-dependent amylolytic activity. Response surface methodology (RSM) was then used as a statistical tool to optimize the initial yeast extract and starch contents of the medium, which resulted in a substantial increase in the stability of the expression plasmid in both the respiratory-deficient NPB-G and the parental respiratory-sufficient WTPB-G strains, with concomitant improvement in their amylolytic potentials. High ethanol yields on substrate values of the bioreactor cultures, which were very close to the theoretical yield, indicated that the amylolytic respiratory-deficient NPB-G strain was effective in the direct fermentation of starch into ethanol. Copyright (c) 2006 John Wiley & Sons, Ltd

Levansucrase from Halomonas smyrnensis AAD6(T): first halophilic GH-J clan enzyme recombinantly expressed, purified, and characterized

Fructans, homopolymers of fructose produced by fructosyltransferases (FTs), are emerging as intriguing components in halophiles since they are thought to be associated with osmotic stress tolerance and overall fitness of microorganisms and plants under high-salinity conditions. Here, we report on the full characterization of the first halophilic FT, a levansucrase from Halomonas smyrnensis AAD6T (HsLsc; EC 2.4.1.10). The encoding gene (lsc) was cloned into a vector with a 6xHis Tag at its C-terminus, then expressed in Escherichia coli. The purified recombinant enzyme (47.3 kDa) produces levan and a wide variety of fructooligosaccharides from sucrose, but only in the presence of high salt concentrations (> 1.5 M NaCl). HsLsc showed Hill kinetics and pH and temperature optima of 5.9 and 37 °C, respectively. Interestingly, HsLsc was still very active at salt concentrations close to saturation (4.5 M NaCl) and was selectively inhibited by divalent cations. The enzyme showed high potential in producing novel saccharides derived from raffinose as both fructosyl donor and acceptor and cellobiose, lactose, galactose, and ʟ-arabinose as fructosyl acceptors. With its unique biochemical characteristics, HsLsc is an important enzyme for future research and potential industrial applications in a world faced with drought and diminishing freshwater supplies.status: publishe