Part:BBa_K5386000
co_E.coli laccase ORF
Currently, multicopper oxidase (MCO), represented by laccase, is the most widely used class of enzymes to degrade biogenic amines in fermented foods. Biogenic amines are a class of nitrogenous small molecule organic compounds widely present in fermented foods, and controlling the content of biogenic amines in fermented foods is necessary to ensure food safety. The principle of amine degradation by laccase and other biogenic amine-degrading enzymes is to oxidize amines into aldehydes, and at the same time to generate non-toxic and easily degradable products. The gene encoding laccase was cloned from Rhizobium, and heterologously expressed in E. coli Rosetta (DE3) to form recombinant laccase. The recombinant laccase was studied for its enzymatic properties and its tolerance to ethanol. The enzyme has good heat resistance, is active and stable only under acidic conditions, and has good tolerance to low concentrations of ethanol, which is suitable for the degradation of biogenic amines in acidic alcoholic beverages containing low concentrations of ethanol; the recombinant laccase was studied for the degradation of eight biogenic amines, and could almost completely degrade tyramine and tryptamine, and degrade spermidine. The degradation of eight biogenic amines by the recombinant laccase was investigated, and it could almost completely degrade tyramine and tryptamine, with a degradation rate of 49.60% for spermidine and 25.27% and 21.80% for phenylethylamine and histamine, respectively.
Usage and Biology
Laccase is a kind of multi-copper oxidase (MCO), belonging to the family of copper-containing oxidases. It catalyzes the oxidation of substrates through the catalytic reaction mechanism involving molecular oxygen and free radicals, with water as the product. In recent years, as the demand for the degradation of organic pollutants has grown, laccase featuring high catalytic activity and wide substrate applicability has drawn extensive attention. Laccase demonstrates significant value in fields such as wastewater treatment, bioremediation, and anti-cancer. In our project, laccase is employed to degrade sex hormones in the marine environment, thereby reducing their inhibition on the root development of seagrass and facilitating the recovery of seagrass populations, with the aim of reconstructing marine ecosystems.
Construction of plasmid
The construction and validation of the plasmid were conducted by VectorBuilder Inc. As illustrated in Figures 1 and 2, the DNA fragments digested by restriction enzymes exhibited distinct bands following agarose gel electrophoresis. Figure 3 demonstrates that the sequencing results were fully aligned with the reference sequence, confirming successful plasmid construction.
Culture of engineered bacteria and determination of laccase properties
Culture of recombinant BL21 Escherichia coli
A single colony on the medium was picked into a liquid LB medium (containing 100 μg/mL ampicillin) and cultured overnight at 37℃ and 150 rpm. The next day, IPTG with a final concentration of 0.5 mM and Cu2+ with a final concentration of 0.5 mM, which had been filtered through a 0.45 μm filter membrane, were added, and the culture was continued at 30℃ for 16 h to obtain the best culture effect.
Detection of laccase enzyme activity
In accordance with the ratio of bacterial quantity (108 per mL) to the volume of the extract (mL) (20:1), the cells were subjected to ultrasound in an ice bath (power 300 W, sonication for 3 seconds, interval of 7 seconds, total duration 3 minutes); subsequently, centrifugation was performed at 10,000 g at 4℃ for 10 minutes, and the supernatant was placed on ice. In the glass cuvette, 150 μL of distilled water + 850 μL of ABTS working solution and 150 μL of sample + 850 μL of ABTS working solution were added respectively as the sample and the control. The absorbance value A1 at 10 seconds was determined at 420 nm. The cuvette was rapidly placed in a 45℃ water bath for 3 minutes, then taken out, rapidly dried, and the absorbance value A2 at 190 seconds was measured. The ΔA of the measurement tube = A2 - A1 of the measurement tube; the ΔA of the blank tube = A2 - A1 of the blank tube, and the ΔA of the measurement tube = ΔA - ΔA of the blank tube.
Investigation of the effect of estradiol concentration on the removal effect
A laccase with a starting amount of 7.3U/mL was set, and the starting concentrations of estradiol were 0.0005, 0.001, 0.005, 0.01, 0.05 mmol/L respectively. A Britton - Robinson buffer solution with a pH of 5.5 was used as the reaction system, and the total volume of the reaction mixture was 200 μL. The reaction system was placed in a 35℃ constant temperature water bath for 3 h, then a sample was taken, and the estradiol concentration was measured with an estradiol Elisa kit to calculate the removal rate. The specific detection steps are as follows:
(1) The microporous plate strip to be used was taken down from the plate frame, the remaining plate strips were put back into the aluminum foil bag containing a desiccant, and then resealed for storage.
(2) 350 μL of 1x washing buffer was added to each hole, and the liquid was discarded after standing for 40 seconds. This step was washed 3 times in total.
(3) A biotinylated antigen (100x) working solution was prepared 15 minutes before use.
(4) 50 μL of standard/sample diluent (R1) was added to the blank hole, and different concentrations of standard products and samples to be tested were added to the other holes. Then 50 μL of biotinylated antigen was immediately added to each hole and a new sealing film was covered, and the incubation was carried out at 37℃ for 1.5 hours.
(5) A streptavidin - HRP (100x) working solution was prepared 15 minutes before use.
(6) The liquid in the hole was discarded, and the washing step in step 2 was repeated.
(7) Streptavidin - HRP working solution (100 μL/hole) was added to each hole, and a new sealing film was covered, and the incubation was carried out at 37℃ for 30 minutes.
(8) The enzyme marker was preheated.
(9) The liquid in the hole was discarded, and each hole was washed with 350 μL of washing liquid, soaked for 1 - 2 minutes, and the plate was washed 5 times in total.
(10) TMB substrate (90 μL/hole) was added to the hole. Incubation was carried out at 37℃ in the dark for 15 - 20 minutes.
(11) A termination liquid (50 μL/hole) was added, and it was immediately put into the enzyme marker, and the OD value at 450 nm of each hole was measured within 5 minutes. A correction wavelength was selected and set to 630 nm. And the reading at 450 nm was subtracted from the reading at 630 nm. This way can correct and remove the OD value of non - coloring substances to obtain more accurate detection results.
The experimental results demonstrate that the MCO exhibits the ability to degrade estradiol under various concentrations. At a concentration of 0.0100 mmol/L, the MCO reaches its peak degradation capacity for the substrate (40%). However, it is surprising to note that the overall trend of MCO's degradation capability shows an initial decrease followed by an increase with the elevation of estradiol concentration. This suggests that in similar verification experiments assessing degradation capacity, we must also consider other factors, such as the photodegradation of estradiol and temperature effects.
Investigating the yield of laccase produced by recombinant BL21 E. coli on PE film
A single colony on the medium was picked into a liquid LB medium (containing 100 μg/mL ampicillin) and cultured overnight at 37℃ and 150 rpm. The next day, IPTG with a final concentration of 0.5 mM and Cu2+ with a final concentration of 0.5 mM were added, and the culture was transferred to a culture dish containing a PE film. A group without a PE film was used as a control group, and the culture was continued at 30℃ for 16 h. The culture medium was taken respectively to detect the laccase activity. At the same time, the PE film group was placed on ice, an ultraviolet lamp was turned on for 1 h, then a laccase extraction liquid was added. One part was detected for laccase activity according to the method in experiment 3.2, and one part was placed in an estradiol solution with a final concentration of 136.19 ng/mL, and after a reaction for 3 h at 35℃, the estradiol was detected according to the method in experiment 3.3.
Investigating the effect of seawater on the survival rate of recombinant BL21 E. coli
(1) Recombinant BL21 Escherichia coli was taken into a liquid LB medium (pH = 7.0 ±0.2) (containing 100 μg/mL ampicillin) and cultured overnight at 37℃ and 150 rpm. The next day, IPTG with a final concentration of 0.5 mM and Cu2+ with a final concentration of 0.5 mM were added, and the culture was continued at 30℃ for 16 h.
(2) The cultured recombinant BL21 Escherichia coli was centrifuged at 2500 rpm for 3 minutes, the supernatant was removed, and it was washed 3 times with an LB liquid medium (containing 100 μg/mL ampicillin). A bacterial suspension was prepared with an LB liquid medium; then, the bacterial suspension was counted with a hemocytometer to make the number of bacteria 1×108 cfu/mL and set aside.
(3) The experiment was divided into 3 groups, namely the experimental group, the positive control group, and the negative control group. Experimental group: The bacterial liquid was added to an LB culture medium containing a final concentration of 30 g/L sea salt; positive control group: Normal LB culture medium plus bacterial liquid; negative control group: Liquid medium; The above experiment was repeated 3 times. After covering the lid and gently mixing, the culture was carried out for 1 h, and the colony count was carried out by the spread plate method to calculate the sterilization rate.
Reference
[1]CAO, Y., WANG, S., LIU, Y., GAO, M., DU, L., MA, L., ZHANG, H., TIAN, X., & YANG, W. (2023). Enzymatic Characteristics of Laccase from Lactobacillus plantarum and Its Effect on Degradation of Biogenic Amines. Food Science, 44(12), 157-163. doi:10.7506/spkx1002-6630-20221025-258
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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