Beiercassidy8853

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. 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. RK 24466 order We developed high-throughput genetic screening and selection methods to identify RhlI mutants via four rounds of directed evolution and identified RhlI-4M1 as the mutant that generated new catalytic activity and synthesized 3-oxo-hexanoyl homoserine lactone (OHHL) containing the 3-oxo group in Escherichia coli. Additionally, the synthesizing activities of BHL and HHL were improved by 3.98- and 3.01-fold, respectively. RhlI-4M1 contains five amino acid substitutions (A15D, K31R, T92S, Y129N, and L184Q) and one stop codon (Q193*) mutations. The deletion of nine amino acids in the C-terminus was crucial for OHHL production by RhlI mutants. This work demonstrates that the genetic screen/selection should be useful in the development of applications involving the manipulation of bacterial quorum sensing. The new catalytic activity of these RhlI mutants will prove beneficial in elucidating the mechanistic understanding of bacterial quorum sensing and similarly, may prove beneficial in the development of new drugs including antimicrobial compounds. Transglutaminases (TGases) are a class of transferases widely used in the food and biotechnology industries. In this work, we describe the production of recombinant Bacillus amyloliquefaciens TGase in Escherichia coli, obtaining the protein in its soluble and active form. In order to reduce TGase activity inside host cells and consequently its toxicity, we constructed a bicistronic plasmid containing the B. amyloliquefaciens TGase gene fused to the inhibitory Streptomyces caniferus prodomain. To make the enzyme active and avoid the need of prodomain removal in vitro, we also cloned the 3C protease gene into the same plasmid. After a fast single-step purification protocol, we obtained a partially purified recombinant TGase with 37 mU/mg protein activity, that crosslinked bovine serum albumin (BSA). This is the first report on the expression of B. amyloliquefaciens TGase in E. coli in its mature and active form. 2,5-Bis(hydroxymethyl)furan (BHMF) is a versatile building block in the synthesis of polymers, fuels, and macrocycle polyethers. In this work, alcohol dehydrogenases (ADHs) were identified from Meyerozyma guilliermondii SC1103 and were heterologously expressed in Saccharomyces cerevisiae for the synthesis of BHMF from 5-hydroxymethylfurfural (HMF). Of recombinant strains constructed, S. cerevisiae expressing an aryl ADH (MgAAD1669) was observed to be the best catalyst. Upon process optimization, BHMF was afforded with a 99% selectivity and a 94% yield within 24 h at the substrate concentration of 250 mM. The space-time yield up to 3.4 g/L h was achieved in the fed-batch synthesis of BHMF. Inexpensive corncob hydrolysate proved to be a promising alternative to glucose as co-substrate for biocatalytic synthesis of BHMF, thus resulting in the significantly reduced production cost.