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A novelly developed tweezing-adsorptive bubble separation (ABS) method for the enrichment of metalloenzymes (laccase C and horseradish peroxidase) is introduced. The method is based on the chelation of the enzymes' active center and can also be applied for analysis. N.(2-Acetamido)iminodiacetic acid served as a chelator and was synthesized with an octyl unit to become ADA-C8. Laccase was enriched 13.3-fold (66.31% recovery) and HPOX 17.8-fold (85.34%) without a significant loss of enzymatic activity. To prove that the entire enzyme is tweezed at the active center, ABS trials were done using ADA-C8 already complexed with [Cu.sup.2+] and [Fe.sup.3+]. As only marginal enrichment occurred (ER laccase, 0.17; ER HPOX, 0.44), no chelating effect was concluded. It was determined how the chelation toward the active center was directed by applying other chelators such as EDTA, NTA, N,N-dimethylaminoglycine, oxalic acid, malonic acid, adipinic acid, and tripropylamine, which are similar in structure to ADA-C8. The results concluded that the chelation is 3-fold coordinated on the type 1 copper center of laccase, whereas that of HPOX only 1-fold at [Fe.sup.3+] and additionally at the cationic amino acid arginine, which is also located at the active center. Tweezing-ABS has been proven to selectively and effectively enrich metalloenzymes.

A combined system of a unique dye-decolorizing peroxidase (Ftr-DyP) and a laccase obtained from the basidiomycete Funalia trogii converted the precursor (+)-valencene completely to the high-value grapefruit flavour constituent (+)-nootkatone, reaching a concentration maximum of 1100 mg/L. In the presence of 1 mM Mn2+ and 2.5 mM p-coumaric acid, (+)-nootkatone was the predominating volatile product, and only traces of substrate and the nootkatols were detectable after 24 h. Hence, the two-enzyme-system reproduced the oxidizing activity observed before for the crude culture supernatant. The newly discovered Ftr-DyP was purified, sequenced and further characterized as a thermostable, non-glycosylated protein with a pH-optimum in the acidic range and a calculated mass of 52.3 kDa. Besides the typical activity of DyPs towards anthraquinone dyes, Ftr-DyP also oxidized Mn2+ and showed activity in the absence of hydrogen peroxide. Neither the DyP from Mycetinis scorodonius nor the manganese peroxidase from Nematoloma frowardii were able to replace Ftr-DyP in this reaction. A hypothetical reaction mechanism is presented. [ABSTRACT FROM AUTHOR]

The feruloyl esterase (FAE) gene EST1 from the basidiomycete Pleurotus sapidus was heterologously expressed in Escherichia coli and Pichia pastoris. Catalytically active recombinant Est1 was secreted using P. pastoris as a host. For expression in P. pastoris, the expression vector pPIC9K was applied. The EST1 gene was cloned with an N-terminal α-mating factor pre-pro sequence and expressed under the control of a methanol inducible alcohol oxidase 1 promotor. Est1 was purified to homogeneity using ion exchange and hydrophobic interaction chromatography. The recombinant Est1 showed optima at pH 5.0 and 50 °C, and released ferulic acid from saccharide esters and from the natural substrate destarched wheat bran. Substrate specificity profile and descriptor-based analysis demonstrated unique properties, showing that Est1 did not fit into the current FAE classification model. Transferuloylation synthesis of feruloyl-saccharide esters was proven for mono- and disaccharides. [ABSTRACT FROM AUTHOR]

Foam fractionation was used to concentrate and to purify extracellular alkaline lipases produced by Fusarium spec. in submerged cultures supplemented with Tween 80. Gas flow rate and pH-value proved to be crucial process parameters. Maximum recovery rates of lipolytic activity were obtained at 60 mL mm-1 N2 and pH 8.5. Under these conditions,> 90% of the total lipase activity was recovered in the foam with enrichment and purification factors of 8.8 and 5.9, respectively. The secreted lipases were characterized biochemically by means of IEF electrophoresis with activity staining and electrospray tandem mass spectrometry. [ABSTRACT FROM AUTHOR]

Abstract: An increasing demand of lignin-degrading enzymes makes an effective enrichment of laccase C from culture broth necessary. In this work, the continuous foam fractionation of laccase C was investigated as an alternative to other common applied methods, such as salting-out, dialyses or ultrafiltration, which are usually accompanied by losses of catalytic activity or low operational capacity. After optimization of different process parameters (feed position, gas flow rate and surfactant concentration) an enrichment ratio of 11.7 was achieved for laccase C along with 70.2% recovery. Feed position at the top of the foaming column increased the recovery. Increasing the gas flow rate led to a lower enrichment but higher recovery, and an increased outlet flow rate had only negative effect on the recovery. With increasing concentration of cetyl trimethyl ammonium bromide (CTAB), which was applied as a surfactant to enable foaming, the enrichment ratio decreased (from 11.7 to 1.8) and recovery increased (from 70.2 up to 80.2%). A for the enrichment ratio and recovery optimum CTAB-concentration of 0.4mg/ml was found. The method has been proven to be effective for the enrichment of laccase C. [Copyright &y& Elsevier]