Transformation of AFLATOXIN B1 and ZEARALENONE using an evolved HIGH-REDOX POTENTIAL LACCASE
Jog Raj*1, Hunor Farkaš1, Miguel Alcalde2, Bernardo J. Gomez-Fernandez2, Jasna Bošnjak-Neumüller1 and Marko Vasiljević1 PATENT CO, DOO., Mišićevo, Serbia CSIC- Department of Biocatalysis - Institute of Catalysis, CSIC, Madrid, Spain *Corresponding and presenting author: jog.raj@patent-co.com 1 2
The World Mycotoxins Forum (WMF) meets IUPAC, Belfast, Oct 14-16, 2019
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OBJECTIVE Mycotoxins are the biggest challenge for animal feed producers and, therefore, regular monitoring and remediation is necessary. Mycotoxins can be remediated by various methods, but enzymes and microbes for deactivating mycotoxins are becoming popular these days.
MATERIALS & METHODS
1. INCUBATION Deoxynivalenol (DON), Aflatoxin B1 (AFB1), Zearalenone (ZEN), T-2 toxin, Ochratoxin A (OTA) and Fumonisin B1 (FB1) (2 ppm) were incubated with 500 Οl of DooKu enzyme (Laccases) at 37°C; with syringaldehyde (10mM) as redox mediator for 1 hour.
Among enzymes involved in mycotoxin degradation, fungal high-redox potential laccases are promising biocatalysts due to their minor requirements and broad substrate range.
2. QUANTIFICATION After incubation, the samples were centrifuged and analysed by a LC-MS/ MS based multi-mycotoxin method for quantification of all the mycotoxins (Aflatoxin B1, Ochratoxin A, Zearalenone, Deoxynivalenol, FB1 and T-2 toxin).
We have recently engineered a high-redox potential laccase by consensus design and DNA recombination to enhance its thermostability and substrate scope. In the present study, we have tested the transformation of mycotoxins using the evolved laccase, DooKu variant.
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RESULTS
Different variants of Laccases were screened and Laccase 240 was among the best enzymes for transformation of AFB1 and ZEN as shown in Table 1. Syringaldehyde proved or be a better cofactor as compared to TEMPO.
Table 1. Screening different variants of laccases using different cofactors at pH 6.5.
Enzyme Variant name
240 (MA) Chu-B (MA) OB-1 (MA) KyLo (MA) 9C1 (MA) Vader (MA)
Cofactor
Transformation (%)
DON
AFB1
FB1
T-2
ZEN
OTA
TEMPO, 10mM
14.1
7.4
1.7
16.2
100.3
11.5
Syringaldehyde, 10mM
7.9
73.7
0.2
2.0
100.5
3.5
TEMPO, 10mM
10.1
5.1
3.3
2.1
92.4
4.4
Syringaldehyde, 10mM
6.3
9.8
4.1
0.9
24.6
2.5
TEMPO, 10mM
6.4
1.3
2.3
4.2
100.0
1.8
Syringaldehyde, 10mM
4,0
15.2
1.1
3.3
65.4
8.0
TEMPO, 10mM
1.3
0.8
6.8
1.6
5.1
0.5
Syringaldehyde, 10mM
8.8
12.9
2.9
3.0
17.3
1.2
TEMPO, 10mM
3.3
5.8
2.1
4.3
74.5
5.4
Syringaldehyde, 10mM
4.6
14.5
11.3
8.0
20.4
8.6
TEMPO, 10mM
1.0
8.4
4.1
5.2
58.0
6.3
Syringaldehyde, 10mM
6.8
3.7
6.6
3.6
3.1
0.1
Table 2. Effect of different pH on the activity of Laccase 240.
Transformation (%)
A240G (PM1 laccase variant)
pH
DON
AFB1
FB1
T-2
ZEN
OTA
3
-0.3
7.2
-5.3
2.0
44.2
-0.3
4
9.0
6.9
-5.8
2.8
88.7
2.5
5
6.7
7.4
-7.6
2.2
99.0
3.7
6.5
13.2
73.2
-3.8
-1.2
70.4
1.2
3
The effect of pH was studied by carrying out assays at pH 3.0, 4.0, 5.0 and 6.5 pH. Laccase 240 showed 73% transformation for AFB1 at pH 6.5 and 99% transformation of ZEN at pH 5.0 as shown in Table 2.
Figure 1. Transformation of AFB1 and ZEN using Laccase 240 over a period of time .
120 % Transformation
100 80 60
% AFB1 transformation at pH 6.5
40
% ZEN transformation at pH 5.0
20 0 0
200
400
600
800
1000
1200
1400
1600
Time in minutes
Effect of incubation time was also studied as shown in Figure 1. ZEN was transformed within 2 hours, whereas AFB1 was transformed in 3 hours.
Experiments related to metabolites derived from AFB1 and ZEN are in progress and results will be updated soon. This laccase enzyme can be used as a solution for mycotoxins remediation in animal feeds.
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