Optimizing the preparation conditions and characterization of cross-linked enzyme aggregates of a monoamine oxidase

Abstract

Monoamine oxidases are useful in determination of biogenic monoamines, particularly histamine and tyramine. In this study, cross-linked enzyme aggregates (CLEAs) technique was applied to improve the stability of a monoamine oxidase from (AMAO). Under the optimized condition (50% of saturated ammonium sulfate, 5 mM glutaraldehyde, 2.0 mg/mL AMAO, 4 h-cross-linking at 25°C, pH 8.0), CLEAs-AMAO was recovered with a yield of 82% based on the subjected total enzyme activity. Both pH activity and stability at alkaline pHs of CLEAs-AMAO were significantly improved compared to those of the free enzyme, resulting in the shift of optimum pH to pH 8.0 and a broader pH profile. The half-life of the CLEAs at 65°C was elongated by 1.7-fold compared to that of the free enzyme, suggesting the thermal stability of AMAO was also improved by the CLEAs formation.

Combined Cross-Linked Enzyme Aggregates of Monoamine Oxidase and Putrescine Oxidase as a Bifunctional Biocatalyst for Determination of Biogenic Amines in Foods

“…Through the optimization based on AMAO2 activity, the final condition for combi-CLEAs was determined as 1.25 mg/mL of each enzyme in 100 mM phosphate buffer (pH 8.0) containing 2 M AS and 40 mM glutaraldehyde (GA) for 12 h at 25 • C was determined. The relative residual tyramine-activity of the CLEAs based on the activities of the subjected enzymes (described as the yield of the CLEAs hereafter) was 71% (open circles in Figure 2), which was similar to the condition for CLEAs-AMAO2 (50 mM of GA and cross-linking for 17 h at pH 8.0 with 1 mg/mL of AMAO2) [23]. The optimum condition for APUO activity was same as that of AMAO2 (data not shown).…”

“…As ammonium sulfate (AS) has been previously selected as the precipitant for AMAO2 [23], AS was selected as the precipitant for CLEAs-MAPO and combi-CLEAs formation. As in the previous work, AMAO2 was completely precipitated at the concentrations of AS higher than 1.6 M, and fully recovered by the dilution of AS (Figure 1A).…”

“…The activities of the free and CLEAs amine oxidases were measured using the method described in the previous work [23]. The diluted enzyme solution was incubated in 100 mM potassium phosphate buffer (pH 7.0) containing tyramine or putrescine as the substrates for 10 min at 37 • C. After boiling the reaction mixture to terminate the reaction, the reaction mixture (75 µL) was mixed with 225 µL chromogen solution, consisting of 1 mM 4-aminoantipyrine, 10 mM hydroxyl benzoate, and 1 U/mL horseradish peroxidase (Sigma, St. Louis, MO, USA) in 100 mM potassium phosphate buffer (pH 7.0), followed by incubation at 37 • C for 30 min.…”

“…The effect of pH on the stabilities of the enzymes was investigated by measuring the residual activity after incubating at 4 • C for 24 h in a buffer. The thermostabilities of the enzymes were evaluated as previously described [23]. The first-order rate constant of irreversible thermal denaturation (k d ) was obtained from the slope of the plots of ln (residual activity/initial activity) versus time, and the half-lives were calculated as ln2/k d .…”

CLEAs, Combi-CLEAs and ‘Smart’ Magnetic CLEAs: Biocatalysis in a Bio-Based Economy

“…CLEAs have been prepared from a variety of oxidoreductases. These include oxidases such as glucose oxidase (GOX), galactose oxidase (GOase), laccase [89,90], monoamine oxidase (MAO) [91] peroxidases [55,[92][93][94][95] and dehydrogenases such as alcohol dehydrogenases/ketoreductases (KREDs), and formate dehydrogenase [95]. Laccase-CLEAs have been extensively studied because of the interest in using them for the removal of dyes, pharmaceutical residues and endocrine disruptors such as bisphenol A, from waste water [96][97][98].…”