Abstract
Microbial intradiol dioxygenases have been shown to have a great potential for bioremediation; however, their structure is sensitive to various environmental and chemical agents. Immobilization techniques allow for the improvement of enzyme properties. This is the first report on use of glyoxyl agarose and calcium alginate as matrixes for the immobilization of protocatechuate 3,4-dioxygenase. Multipoint attachment of the enzyme to the carrier caused maintenance of its initial activity during the 21 days. Immobilization of dioxygenase in calcium alginate or on glyoxyl agarose resulted in decrease in the optimum temperature by 5°C and 10°C, respectively. Entrapment of the enzyme in alginate gel shifted its optimum pH towards high-alkaline pH while immobilization of the enzyme on glyoxyl agarose did not influence pH profile of the enzyme. Protocatechuate 3,4-dioygenase immobilized in calcium alginate showed increased activity towards 2,5-dihydroxybenzoate, caffeic acid, 2,3-dihydroxybenzoate, and 3,5-dihydroxybenzoate. Slightly lower activity of the enzyme was observed after its immobilization on glyoxyl agarose. Entrapment of the enzyme in alginate gel protected it against chelators and aliphatic alcohols while its immobilization on glyoxyl agarose enhanced enzyme resistance to inactivation by metal ions.
Highlights
Protocatechuate 3,4-dioxygenase belongs to the irondependent enzymes and catalyzes intradiol cleavage of aromatic compounds [1]
The stability of free and the immobilized protocatechuate 3,4-dioxygenase was determined after storage of preparations in the phosphate buffers (50 mM, pH 7.0, for free enzyme or enzyme immobilized in calcium alginate and 40 mM, pH 8.0, for protocatechuate 3,4-dioxygenase immobilized on glyoxyl agarose) at 4∘C for predetermined period
Relative activity of the enzyme immobilized on glyoxyl agarose, % 100.0 ± 0.00 61.65 ± 8.88 67.27 ± 0.00 86.11 ± 9.82 85.12 ± 0.00 80.16 ± 5.61 85.12 ± 0.00 69.58 ± 0.00 44.63 ± 10.05 59.67 ± 0.93 70.74 ± 0.70 75.87 ± 6.76 83.30 ± 2.10 84.70 ± 7.03 112.62 ± 6.49 93.12 ± 8.11 87.76 ± 4.33 78.97 ± 9.19 91.20 ± 17.31 70.74 ± 0.70 82.97 ± 3.97 67.76 ± 9.58 61.50 ± 0.00 56.03 ± 6.54 54.38 ± 5.61 ion for the active site
Summary
Protocatechuate 3,4-dioxygenase belongs to the irondependent enzymes and catalyzes intradiol cleavage of aromatic compounds [1]. Interactions between substrate and atom of iron(III) cause activation of the substrate for an electrophilic attack by molecular oxygen. It leads to the peroxobridge formation between the iron and C4 of substrate. Criegee rearrangement of this structure occurs, leading to the cyclic anhydride formation [1]. These enzymes take part in degradation of various aromatic compounds but they can be inhibited by different agents such as substrate analogues, chelators, and metal ions [3,4,5,6,7]. One of them is immobilization which has been used as a tool to improve many of enzymes’ properties such as operational stability, inhibitor resistance, and performance in organic solvents [8,9,10]
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