Tweezing-Adsorptive Bubble Separation. Analytical Method for the Selective and High Enrichment of Metalloenzymes Birte M. Gerken, † Carsten Wattenbach, † Diana Linke, ‡ Holger Zorn, ‡ Ralf G. Berger, ‡ and Harun Parlar* ,† Department of Chemical-Technical Analysis and Chemical Food Technology, Research Center Weihenstephan for Brewing and Food Quality, Technical University of Munich, Weihenstephaner Steig 23, D-85354 Freising-Weihenstephan, Germany, and Institute of Food Chemistry, University of Hannover, Wunstorfer Strasse 14, D-30453 Hannover, Germany A novelly developed tweezing-adsorptive bubble separa- tion (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-Acet- amido)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 2+ and Fe 3+ . As only marginal enrich- ment 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 apply- ing other chelators such as EDTA, NTA, N,N-dimethyl- aminoglycine, 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 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. The production of enzymes for technological applications usually implies their separation from the biological source. Frequently, applied are salting-out, dialysis, or ultrafiltration, which are often followed by chromatographic purification processes. Each of these steps, however, is bound to losses of enzymatic activity. 1-3 To overcome this problem, an alternative method was investigated, the so-called adsorptive bubble separation (ABS). 4 ABS can well be applied for trace analysis and the elimination of undesired byproducts at common analysis. 5 In this respect, a variety of applications have been reported for mineral ores and hazardous metal ions, proteins, and surfactants. 6-10 ABS has become important for the removal of trace metals such as cadmium, chromium, and copper. 11 In principle, soluble and surface-active substances separate from aqueous solutions at a gas-liquid interface layer, which is generated by the inflow of gas. 12,13 When gas (mostly air or nitrogen) is led through the liquid placed in a column via a porous glass frit, foam molds by starting first with spheric (lower column) and then polyhedral bubbles (upper column). 14 A polyhedral foam is necessary for the purpose of concentrating substances. During transition, surface-active molecules concentrate at the interface gaseous bubble-liquid either due to drainage of the laminar liquid or because of collapsing lamellas, leading as well to a reflux. The foam thereafter flows into a beaker, where it disintegrates back to liquid, the so-called “foamate”. Common varied process param- eters are as follows: gas flow rate, initial substance concentration, addition of surface-active substances, start volume of the matrix, column geometry, pH value, and foaming time. Surface-active substances such as carnosic acid, flavokavins, and solanidine alkaloids from respective plant materials could already be suc- cessfully enriched or eliminated. 15-17 * Corresponding author. E-mail: parlar@wzw.tum.de. Phone: +49 (0)8161 71-3283. Fax: +49 (0)8161 71-4418. † Technical University of Munich. ‡ University of Hannover. (1) De Souza, C. G. M.; Peralta, R. M. J. Basic Microb. 2003, 43(4), 278-286. (2) Shin, K. S.; Lee, Y. J. Arch. Biochem. 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