Maurer Hein (hockeyseal38)

In this work, the expression of an α-amylase from Bacillus megaterium on the cell surface of Escherichia coli strains WDHA (Δ hycA and Δ ldhA) and WDHFP (Δ hycA, Δ frdD and Δ pta) by the autodisplay adhesin involved in diffuse adherence (AIDA) system was carried out with the purpose to confer the ability to E. coli strains to degrade starch and thus produce hydrogen, ethanol and succinic acid. For the characterization of the biocatalyst, the effect of temperature (30-70 °C), pH (3-6) and CaCl2 concentration (0-25 mM), as well as the thermostability of the biocatalyst (55-80 °C) at several time intervals (15-60 min) were evaluated. The results showed that the biocatalyst had a maximum activity at 55 °C and pH 4.5. Calcium was required for the activity as well for the thermal stability of the biocatalyst. The calculated Vmax and Km values were 0.24 U/cm3 and 5.8 mg/cm3, respectively. Furthermore, a set of anaerobic batch fermentations was carried out using 10 g/dm3 of starch and 1 g/dm3 of glucose as carbon sources in 120 cm3 serological bottles, using WDHA and WDHFP strains harboring the pAIDA-amyA plasmid. The hydrogen production for WDHA was 1056.06 cm3/dm3 and the succinic acid yield was 0.68 g/gstarch, whereas WDHFP strain produced 1689.68 cm3/dm3 of hydrogen and an ethanol yield of 0.28 g/gstarch. This work represents a promising strategy to improve the exploitation of starchy biomass for the production of biofuels (hydrogen and ethanol) or succinate without the need of a pre-saccharification process. Soybean is a most promising sustainable protein source for feed and food to help meet the protein demand of the rapidly rising global population. To enrich soy protein, the environment-friendly enzymatic processing requires multiple carbohydrases including cellulase, xylanase, pectinase, α-galactosidase and sucrase. Besides enriched protein, the processing adds value by generating monosaccharides that are ready feedstock for biofuel/bioproducts. Aspergillus could produce the required carbohydrases, but with deficient pectinase and α-galactosidase. Here we address this critical technological gap by focused evaluation of the suboptimal productivity of pectinase and α-galactosidase. A carbohydrases-productive strain A. niger (NRRL 322) was used with soybean hull as inducing substrate. Temperatures at 20 °C, 25 °C and 30 °C were found to affect cell growth on sucrose with an Arrhenius-law activation energy of 28.7 kcal/mol. LY3009120 The 30 °C promoted the fastest cell growth (doubling time = 2.1 h) and earliest enzyme production, but it gave lower final enzyme yield due to earlier carbon-source exhaustion. The 25 °C gave the highest enzyme yield. pH conditions also strongly affected enzyme production. Fermentations made with initial pH of 6 or 7 were most productive, e.g., giving 1.9- to 2.3-fold higher pectinase and 2.2- to 2.3-fold higher α-galactosidase after 72 h, compared to the fermentation with a constant pH 4. Further, pH must be kept above 2.6 to avoid limitation in pectinase production and, in the later substrate-limiting stage, kept below 5.5 to avoid pectinase degradation. α-Galactosidase production always followed the pectinase production with a 16-24 h lag; presumably, the former relied on pectin hydrolysis for inducers generation. Optimal enzyme production requires controlling the transient availability of inducers. Quorum sensing is a population density-dependent gene expression regulation mechanism in bacteria. The substrate specificity of RhlI, an enzyme in the RhlI-RhlR quorum sensing system of Pseudomonas aeruginosa, was explored by directed evolution to gain insight into the molecular mechanisms of quorum sensing. RhlI catalyzes S-adenosyl methionine and butanoyl or hexanoyl acyl carrier protein to form N-butanoyl homoserine lactone (BHL) and or N-hexanoyl homoserine lactone (HHL), respectively, none of which contain 3-oxo groups. We developed high-throughput genetic screening and selection methods to id