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

Abstract

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 Cu2+ and Fe3+. 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 Fe3+ 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.

High Enrichment of MMP-9 and Carboxypeptidase A by Tweezing Adsorptive Bubble Separation (TABS)

“…Gerken et al[16] made the same observation when applying TABS for the enrichment of laccase C and horseradish peroxidase. Both enzymes were enriched with highest efficiency values at the pH where complete inactivation was achieved.…”

“…This normally suggests the application of a surfactant in order to increase the foaming capacity of an enzyme [26]. Previous experience however revealed that, with using ADA alone or synthesized together with an octyl part, the enzyme's foaming capacity is enhanced due to a more unpolar characteristic [16]. Therefore, the ABS experiments were designed so that one process served as a standard for comparison, using a solution containing the enzyme only chelated with ADA (chelate #1), then three solutions using the enzyme in complex with ADA-C8 to observe the tweezing efficiency (chelates #2-#4), and another three solutions using a complex of zinc (II)-ADA-C8 in order to verify that indeed the catalytic center of MMP-9 is involved during the chelation (Fig.…”

“…According to classification, the pancreatic carboxypeptidase A (CPA) is [16]. The tweezer, chelating the metal ion in the metalloenzyme, consists of a seizing part covalently bound to an n-octyl part to enable the carriage into the foam phase.…”

Laccase treatment of recycled blue dyed paper: physical properties and fiber charge

“…Oxygen aeration had a negative effect on color removal for this recycled pulp. The results obtained from continuous bubbling may be attributed to greater laccase activity in the presence of oxygen bubbles [24].…”

Foaming of proteins: New prospects for enzyme purification processes

“…Foam inducing agents can be added to the media, if the genuine foaming capacity is not sufficient. The detergents used in the literature are nonionic, cationic, anionic and zwitterionic, such as Triton X-114 (Fernandes et al, 2002a), Tween 80 and Triton X-100 (Linke et al, 2005a(Linke et al, , 2009, saponin (Backleh et al, 2003b), sodium bis-(2-ethylhexyl)sulfosuccinate (Aerosol OT) (Jauregi and Varley, 1998), CTAB (Linke et al, 2007), betaine (Xu et al, 2009), SDS (Wang et al, 2009), and chelating tenside ADA-C8 (Gerken et al, 2005). Ionic surfactants form electrostatic forces between protein/enzyme and detergent molecule and develop an ion pair (Grieves and Bhattacharyya, 1966;Lemlich, 1968;Linke et al, 2007).…”