Bioconversion of Airborne Methylamine by Immobilized Recombinant Amine Oxidase from the Thermotolerant YeastHansenula polymorpha

Abstract

Aliphatic amines, including methylamine, are air-pollutants, due to their intensive use in industry and the natural degradation of proteins, amino acids, and other nitrogen-containing compounds in biological samples. It is necessary to develop systems for removal of methylamine from the air, since airborne methylamine has a negative effect on human health. The primary amine oxidase (primary amine : oxygen oxidoreductase (deaminating) or amine oxidase, AMO; EC 1.4.3.21), a copper-containing enzyme from the thermotolerant yeast Hansenula polymorpha which was overexpressed in baker's yeast Saccharomyces cerevisiae, was tested for its ability to oxidize airborne methylamine. A continuous fluidized bed bioreactor (CFBR) was designed to enable bioconversion of airborne methylamine by AMO immobilized in calcium alginate (CA) beads. The results demonstrated that the bioreactor with immobilized AMO eliminates nearly 97% of the airborne methylamine. However, the enzymatic activity of AMO causes formation of formaldehyde. A two-step bioconversion process was therefore proposed. In the first step, airborne methylamine was fed into a CFBR which contained immobilized AMO. In the second step, the gas flow was passed through another CFBR, with alcohol oxidase from the yeast H. polymorpha immobilized in CA, in order to decompose the formaldehyde formed in the first step. The proposed system provided almost total elimination of the airborne methylamine and the formaldehyde.

Methylamine-Sensitive Amperometric Biosensor Based on (His)6-TaggedHansenula polymorphaMethylamine Oxidase Immobilized on the Gold Nanoparticles

“…The value of K M app to MA derived from the calibration plot for the bienzyme sensor is 0.039 ± 0.003 mM, which is lower compared to K M app of free AMO in solution (0.22 mM) [11]. The sensitivity of the sensor for methylamine is 1450 ± 113  А · M −1 · m −2 (Figure 1(b)).…”

“…The maximal current at substrate saturation ( I max⁡ ) for the CP -AMO-HRP-ferrocene-modified carbon electrodes (area 7.3 mm 2 ) calculated from calibration graph is 1.23 ± 0.02 μ A ( Figure 1(c) ). The value of K M app to MA derived from the calibration plot for the bienzyme sensor is 0.039 ± 0.003 mM, which is lower compared to K M app of free AMO in solution (0.22 mM) [ 11 ]. The sensitivity of the sensor for methylamine is 1450 ± 113 А · M −1 · m −2 ( Figure 1(b) ).…”

“…The (His) 6 -tagged enzyme was purified from the cell-free extract of the recombinant strain by metal-affinity chromatography on Ni-NTA-agarose [11]. Purified AMO with specific activity 13.7 U · mg −1 was stored in 20 mM phosphate buffer, pH 7.5 (PB), at 4°C.…”

“…As a source of (His) 6 -tagged AMO, the constructed by us recombinant yeast strain Saccharomyces cerevisiae C13ABYS86 ( Mat a, leu2-3, ura3, his, pra1-1, prb1-1, prc1-1, cps1-3 ) able to overexpress the target enzyme was used. The (His) 6 -tagged enzyme was purified from the cell-free extract of the recombinant strain by metal-affinity chromatography on Ni-NTA-agarose [ 11 ]. Purified AMO with specific activity 13.7 U · mg −1 was stored in 20 mM phosphate buffer, pH 7.5 (PB), at 4°C.…”

“…A ferrocene layer was used as a final electron transfer mediator. The biorecognition membrane includes two enzymes: purified (His) 6 -tagged AMO from the recombinant yeast Saccharomyces cerevisiae [ 11 ] and commercial horseradish peroxidase (HRP). The enzymes were dropped on the ferrocene-modified graphite electrode surface and covered with a cathodic polymer GY 83-0270 0005.…”