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.
Protocatechuate 3,4-dioxygenase belongs to the iron-dependent enzymes and catalyzes intradiol cleavage of aromatic compounds [
Calcium alginate gel is well known and the most widely used carrier in bioremediation process [
Protocatechuate 3,4-dioxygenase form
Cells were harvested in the late exponential growth phase (after 15 hours) and centrifuged at 4,500 g for 15 min at 4°C. Next, the cells were washed with 50 mM phosphate buffer, pH 7.0, and resuspended in the same buffer. Cells were sonicated 6x for 15 s and centrifuged at 9,000 g for 30 min at 4°C. The supernatant was used as crude extract for enzyme assays and immobilization procedures.
For immobilization of protocatechuate 3,4-dioxygenase in calcium alginate, 3 mL of enzyme solution were suspended in 7 mL of 3% (w/v) sodium alginate in 50 mM phosphate buffer solution (pH 7.0) and homogenized. After homogenization the mixture was dropped into 25 mL of 0.15 M
Immobilization of protocatechuate 3,4-dioxygenase on glyoxyl agarose was prepared as previously described [
9 mM 4-hydroxybenzoate was used as the inducer of protocatechuate 3,4-dioxygenase. Specific activity of free and immobilized protocatechuate 3,4-dioxygenase was assayed by measuring oxygen consumption [
One unit of enzyme activity was defined as the amount of enzyme required to generate 1
The effect of pH on the enzyme activity was determined by measuring the activity at 30°C over the pH range 2.2–10.0 using the following buffers: 0.05 M phosphate-citrate (pH 4.0 to 4.5), 0.05 M Sörensen (pH 5.0), 0.05 M phosphate (pH 5.7 to 8.0), 0.05 glycine (pH 10.0), 0.05 Britton-Robinson (pH 11.00 to 12.00), and 0.05 ammonia-sodium hydroxide (pH 13.00-14.00).
The optimum temperature was determined by assaying the enzyme activity at various temperatures (4 to 65°C) in 50 mM phosphate buffer solution (pH 7.4). The enzyme and the substrate solutions were preincubated, mixed, and followed by the enzymatic reaction at the same temperature.
Impact of various substituted derivatives of aromatic compounds on activity of free and immobilized enzyme was evaluated by incubating the enzyme with the respective aromatic compound for 3 min and assaying the activity. As a substrate, aromatic dihydroxy acids: 2,3-; 2,4-; 2,5-; 2,6-; 3,5-dihydroxybenzoate, caffeic acid, and 3,4-dihydroxyhydrocinnamic acid at 1 mM concentration were used.
Impact of various aliphatic alcohols and chelators on both free and immobilized enzyme was evaluated by incubating the enzyme with the respective inhibitor for 3 min and then assaying the residual activity. At regular time intervals (30 s), reaction progress was monitored by measuring oxygen consumption. Assay of catechol 2,3-dioxygenase was proceeding in the same way as in the case of noninhibited. Aliphatic alcohols studied were methanol, ethanol, propanol, and butanol at 100 mM, 200 mM, and 300 mM concentrations. For inhibition studies EDTA, 2,2′-dipyridyl, and phenanthroline at 1 mM, 2 mM, and 3 mM concentrations were used.
Protocatechuate 3,4-dioxygenase was immobilized in calcium alginate or on glyoxyl agarose. One of the most important parameters which should be considered during immobilization of enzyme is storage stability because of its practical application. 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. The entrapment of the protocatechuate 3,4-dioxygenase in calcium alginate matrix did not significantly influence the storage stability (Figure
Storage stabilities of protocatechuate 3,4-dioxygenase from
Environmental factors affecting enzymatic reactions include temperature and pH. Determination of the influence of protocatechuate 3,4-dioxygenase immobilization on pH optimum showed that entrapment of the enzyme in calcium alginate gel shifted its optimum pH towards high-alkaline pH (Figure
Effects of pH ((a), (b)) and temperature ((c), (d)) on protocatechuate 3,4-dioxygenase from
Many of intradiol dioxygenases show narrow substrate specificity and regioselectivity [
Protocatechuate 3,4-dioxygenase from KB2 strain showed activity against various dihydroxybenzoic acids; however, its activity towards these compounds was lower than against primary substrate (protocatechuic acid). Immobilization of enzyme in both carriers increased its activity against all examined substrates (Table
Substrate specificity of immobilized protocatechuate 3,4-dioxygenase from
Substrate | Relative activity of free enzyme, % | Relative activity of the enzyme immobilized in calcium alginate, % | Relative activity of the enzyme immobilized on glyoxyl agarose, % |
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Control-protocatechuate | 100.0 ± 0.00 | 100.0 ± 0.00 | 100.0 ± 0.00 |
2,3-dihydroxybenzoate | 23.60 ± 3.98 | 149.34 ± 22.94 | 119.83 ± 7.83 |
2,4-dihydroxybenzoate | 43.50 ± 5.73 | 96.52 ± 16.78 | 65.60 ± 13.61 |
2,5-dihydroxybenzoate | 33.46 ± 0.00 | 158.62 ± 11.71 | 72.11 ± 12.59 |
2,6-dihydroxybenzoate | 30.51 ± 2.05 | 90.40 ± 6.09 | 36.15 ± 7.42 |
3,5-dihydroxybenzoate | 44.33 ± 3.18 | 113.25 ± 2.81 | 42.86 ± 13.61 |
Caffeic acid | 34.21 ± 9.65 | 150.00 ± 16.39 | 97.08 ± 11.13 |
3,4-dihydroxyhydrocinnamic acid | 29.54 ± 5.42 | 95.03 ± 7.02 | 41.11 ± 15.26 |
Surprisingly, significant increase in activity of protocatechuate 3,4-dioxygenase immobilized in calcium alginate against 2,3-dihydroxybenzoate, 2,5-dihydroxybenzoate, and 3,5-dihydroxybenzoate (Table
Inhibitors interact with specific regions of the enzyme and inhibit enzymes. For that reason immobilization may reduce inhibition in two different ways based on the type of inhibition. After immobilization allosteric site of enzyme may be blocked which makes interaction between this site and allosteric inhibitor impossible. Immobilization may also slightly deform enzyme structure and as a consequence influence changes in affinity of inhibitor to the active site of enzyme [
Protocatechuate 3,4-dioxygenase possesses an iron ion in the active site and therefore it was interesting to check the influence of aliphatic alcohols on enzyme activity after its immobilization. As it is known, aliphatic alcohols may coordinate metal ions and have an influence on the reaction microenvironment [
Effect of aliphatic alcohols on the activity of immobilized protocatechuate 3,4-dioxygenase from
Compound | Concentration, ( |
Relative activity of free enzyme, % | Relative activity of the enzyme immobilized in calcium alginate, % | Relative activity of the enzyme immobilized on glyoxyl agarose, % |
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None | 100.0 ± 0.00 | 100.0 ± 0.00 | 100.0 ± 0.00 | |
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Methanol | 100 | 96.53 ± 0.54 | 110.70 ± 6.88 | 37.07 ± 17.47 |
200 | 91.53 ± 2.18 | 90.08 ± 2.48 | 45.17 ± 9.28 | |
300 | 101.54 ± 2.18 | 74.71 ± 2.2 | 100.00 ± 13.65 | |
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Ethanol | 100 | 144.61 ± 13.06 | 90.47 ± 3.58 | 67.18 ± 14.20 |
200 | 156.69 ± 3.81 | 116.54 ± 1.92 | 52.51 ± 5.46 | |
300 | 141.92 ± 1.63 | 155.65 ± 22.01 | 50.58 ± 28.94 | |
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Propanol | 100 | 160.78 ± 22.84 | 100.39 ± 4.40 | 93.82 ± 9.28 |
200 | 134.23 ± 5.98 | 101.75 ± 7.98 | 78.38 ± 8.19 | |
300 | 130.38 ± 1.63 | 101.75 ± 4.13 | 76.83 ± 11.47 | |
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Butanol | 100 | 112.69 ± 1.63 | 122.28 ± 0.83 | 108.49 ± 3.82 |
200 | 120.77 ± 2.18 | 118.10 ± 11.28 | 86.87 ± 30.03 | |
300 | 120.38 ± 2.72 | 109.15 ± 20.64 | 76.44 ± 0.0 |
Protective effect of immobilization in calcium alginate on enzyme activity was observed in the presence of
Effect of metals on the activity of immobilized protocatechuate 3,4-dioxygenase from
Compound | Concentration, (mM) | Relative activity of free enzyme, % [ |
Relative activity of the enzyme immobilized in calcium alginate, % | Relative activity of the enzyme immobilized on glyoxyl agarose, % |
---|---|---|---|---|
None | 100.0 ± 0.00 | 100.0 ± 0.00 | 100.0 ± 0.00 | |
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Co2+ | 1 | 74.34 ± 19.14 | 39.68 ± 8.98 | 61.65 ± 8.88 |
2 | 70.58 ± 8.45 | 70.63 ±7.86 | 67.27 ± 0.00 | |
3 | 51.55 ± 4.70 | 97.62 ± 3.37 | 86.11 ± 9.82 | |
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Cu2+ | 1 | 41.81 ±4.07 | 30.16 ± 0.00 | 85.12 ± 0.00 |
2 | 27.36 ± 2.61 | 28.57 ± 2.24 | 80.16 ± 5.61 | |
3 | 29.64 ± 0.63 | 22.22 ± 0.00 | 85.12 ± 0.00 | |
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Ni2+ | 1 | 78.65 ± 3.02 | 70.27 ± 0.00 | 69.58 ± 0.00 |
2 | 88.34 ± 2.29 | 84.24 ± 3.84 | 44.63 ± 10.05 | |
3 | 83.85 ± 0.31 | 63.51 ± 11.47 | 59.67 ± 0.93 | |
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Mn2+ | 1 | 108.06 ± 8.19 | 50.40 ± 1.13 | 70.74 ± 0.70 |
2 | 98.55 ± 5.08 | 20.00 ± 3.39 | 75.87 ± 6.76 | |
3 | 80.92 ± 5.43 | 29.60 ± 3.39 | 83.30 ± 2.10 | |
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Cd2+ | 1 | 69.33 ± 4.01 | 106.76 ± 7.64 | 84.70 ± 7.03 |
2 | 63.85 ± 2.13 | 115.54 ± 8.60 | 112.62 ± 6.49 | |
3 | 60.20 ± 12.64 | 62.16 ± 3.82 | 93.12 ± 8.11 | |
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Al3+ | 1 | 89.99 ± 5.43 | 46.62 ± 10.51 | 87.76 ± 4.33 |
2 | 68.39 ± 3.21 | 83.11 ± 0.96 | 78.97 ± 9.19 | |
3 | 33.50 ± 2.14 | 131.08 ± 11.47 | 91.20 ± 17.31 | |
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Zn2+ | 1 | 94.27 ± 7.21 | 27.20 ± 11.31 | 70.74 ± 0.70 |
2 | 71.79 ± 5.17 | 11.20 ± 6.79 | 82.97 ± 3.97 | |
3 | 50.95 ± 2.76 | 4.80 ± 0.00 | 67.76 ± 9.58 | |
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Fe3+ | 1 | 38.93 ± 6.17 | 180.16 ± 19.08 | 61.50 ± 0.00 |
2 | 31.17 ± 6.08 | 150.79 ± 9.00 | 56.03 ± 6.54 | |
3 | 22.76 ± 2.11 | 141.27 ± 6.73 | 54.38 ± 5.61 |
Immobilization of enzyme on glyoxyl agarose showed better protective effect than immobilization in calcium alginate (Table
It is known that enzymes possessing metal ions at their active sites are sensitive to chelators [
Effect of chelators on the activity of immobilized protocatechuate 3,4-dioxygenase from
Compound | Concentration, ( |
Relative activity of free enzyme, % | Relative activity of the enzyme immobilized in calcium alginate, % | Relative activity of the enzyme immobilized on glyoxyl agarose, % |
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None | 100.0 ± 0.00 | 100.0 ± 0.00 | 100.0 ± 0.00 | |
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EDTA | 100 | 135.07 ± 1.86 | 113.13 ± 7.11 | 46.72 ± 27.85 |
200 | 128.19 ± 23.46 | 97.54 ± 3.91 | 30.89 ± 6.55 | |
300 | 18.19 ± 0.12 | 116.92 ± 8.35 | 32.43 ± 3.28 | |
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2,2′-dipyridyl | 100 | 119.78 ± 0.87 | 123.81 ± 6.58 | 57.91 ± 0.00 |
200 | 99.30 ± 0.99 | 111.62 ± 3.55 | 30.12 ± 17.47 | |
300 | 87.62 ± 4.85 | 117.40 ±3.56 | 22.01 ± 12.56 | |
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Phenanthroline | 100 | 97.78 ± 7.33 | 117.53 ± 7.64 | 101.93 ± 1.09 |
150 | 81.46 ± 4.10 | 121.04 ± 5.87 | 60.62 ± 16.93 | |
200 | 69.69 ± 3.11 | 101.56 ± 6.76 | 21.62 ± 7.64 |
Immobilization of protocatechuate 3,4-dioxygenase from
All authors declare the absence of any conflict of interests including any financial, personal, or other relationships with other people or organizations that could inappropriately influence, or be perceived to influence, their work.
This work was supported by the Polish Ministry of Science and Higher Education (IP2010012170).