Tweezing-Adsorptive Bubble Separation. Analytical Method for the Selective and High Enrichment of Metalloenzymes

摘要

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 (66.31percent recovery) and HPOX 17.8-fold (85.34percent) 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 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-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.