Development of a New Hydrogel Anion Exchange Membrane for Swine Wastewater Treatment.

We developed a proprietary anion exchange membrane (AEM) for wastewater treatment as an alternative to commercial products. Following the successful development of a hydrogel cation exchange membrane on a porous ceramic support, we used the same approach to fabricate an AEM. Different positively charged monomers and conditions were tested, and all AEMs were tested for nitrate and phosphate anion removal from buffers by electrodialysis. The best AEM was tested further with real swine wastewater for phosphate removal by electrodialysis and nitrate removal in a bioelectrochemical denitrification system (BEDS). Our new AEM showed better phosphate removal compared with a commercial membrane; however, due to its low permselectivity, the migration of cations was detected while operating a two-chambered biocathode BEDS in which the membrane was utilized as a separator. After improving the permselectivity of the membrane, the performance of our proprietary AEM was comparable to that of a commercial membrane. Because of the usage of a porous ceramic support, our AEM is self-supporting, sturdy, and easy to attach to various frames, which makes the membrane better suited for harsh and corrosive environments, such as swine and other animal farms and domestic wastewater.

Effect of the amino acid substitution in the DNA-binding domain of the Fur regulator on production of pyoverdine.

The ferric uptake regulator gene (fur), its promoter region and Fur box of pvdS gene involved in siderophore-mediated iron uptake system were sequenced in the parent strain Pseudomonas aeruginosa PAO1 and in the fur mutant FPA121 derived from the strain PAO1. We identified the gene fur 179 bearing a novel, single-point mutation that changed the amino acid residue Gln60Pro in the DNA-binding domain of the Fur protein. The synthesis of pyoverdine was studied in cultures of the strains PAO1 and FPA121 grown in iron-deplete and iron-replete (60 µmol/L FeIII) medium. The amino acid replacement in the regulatory Fur protein is responsible for the overproduction of pyoverdine in iron-deplete and iron-replete medium. No mutation was identified in the Fur box of the gene pvdS.

Whole-cell oxidation of omeprazole sulfide to enantiopure esomeprazole with Lysinibacillus sp. B71.

Production of enantiopure esomeprazole by biocatalysis is of great demand by pharmaceutical industry. A Gram-positive bacterium oxidizing omeprazole sulfide 1a (5-methoxy-2-[((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)thio]-1H-benzoimidazole) to (S)-sulfoxide esomeprazole 2a (S)-5-methoxy-2-[(4-methoxy-3,5-dimethylpyridin-2-yl) methylsulfinyl]-3H-benzoimidazole was isolated from soil polluted with elemental sulfur. The strain exhibited the highest identity with the genus Lysinibacillus and catalyzed oxidation of 1a into enantiopure esomeprazole with conversion of 77% in a stirred bioreactor, fed-batch culture. No consecutive oxidation of (S)-sulfoxide to sulfone was observed during whole-cell catalysis. The unique characteristics of the catalyst provide a solid basis for further improvement and development of sustainable green bioprocess.

Five-substrate cocktail as a sensor array for measuring enzyme activity fingerprints of lipases and esterases.

Substrate arrays for measuring enzyme activity fingerprints can be conveniently formulated as cocktails designed such that the reaction products can be separated and quantified by analytical high-performance liquid chromatography (HPLC). Fingerprinting of lipases and esterases, an important class of microbial enzymes, is reported with a cocktail of only five substrates as a practical fingerprinting reagent. An unusually strong C4-esterase activity was thus revealed in a recently discovered microbial esterase.

Screening systems.

Enzyme screening technology has undergone massive developments in recent years, particularly in the area of high-throughput screening and microarray methods. Screening consists of testing each sample of a sample library individually for the targeted reaction. This requires enzyme assays that accurately test relevant parameters of the reaction, such as catalytic turnover with a given substrate and selectivity parameters such as enantio- and regioselectivity. Enzyme assays also play an important role outside of enzyme screening, in particular for drug screening, medical diagnostics, and in the area of cellular and tissue imaging. In the 1990s, methods for high-throughput screening of enzyme activities were perceived as a critical bottleneck. As illustrated partly in this chapter, a large repertoire of efficient screening strategies are available today that allow testing of almost any reaction with high-throughput.

Low background FRET-substrates for lipases and esterases suitable for high-throughput screening under basic (pH 11) conditions.

FRET-based fluorogenic substrates for lipases and esterases were prepared in four steps from commercially available building blocks. The substrates are pyrenebutyric acid monoesters of aliphatic 1,2-diols bearing a dinitrophenylamino group as a quencher. The most enzyme-reactive substrate is ester 2a. The substrates do not show any measurable background reaction in the absence of enzyme even at pH 11, but react fast and specifically with lipases and esterases. These substrates offer an unprecedented and practical solution to the long-standing problem of a simple yet efficient high-throughput screening tool for lipase activities under basic conditions.

A high-throughput, low-volume enzyme assay on solid support.

A high-throughput enzyme assay is described that uses 1 microL or less of enzyme solution for each test. Enzyme solutions are deposited by robotic handling in a throughput of over 1000 tests/h on the surface of silica gel plates that have been preimpregnated with fluorogenic substrates. The reaction is quantitated by fluorescence. The method is compatible with water-insoluble substrates (lipases), water-soluble substrates (glycosidases), whole-protein substrates (proteases), and enzyme inhibition measurements. Hydrolytically labile umbelliferyl esters can be used to assay lipases in this format without background hydrolysis. High throughput and reproducibility were tested by fingerprint analysis of lipases and esterases against 37 different fluorogenic ester substrates. A set of eight fluorogenic unbelliferyl esters was selected for optimal activity screening of lipases and esterases on silica gel plates.