Part:BBa_K3711041
AOX1-α factor-Laccase-AOX1 Terminator
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1685
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1187
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 2102
- 1000COMPATIBLE WITH RFC[1000]
Description
This is a composite component for expressing Laccase outside the cell. Laccase is the key enzyme for degradation of indigo. It participates in the transformation from indigo to colorless substance. AOX1 promoter is a strong promoter induced by methanol. Under the condition of methanol induction, with the help of α factor, Laccase is translated and excreted from the cell.
Usage and Biology
Copper atom is usually contained in the laccase, which is one of the common characters of laccase and maintains its oxidizing/reducing reaction. As the key element of reaction center, copper atom is critical to the activity and specificity of laccase since it exists in the area that binds substrate and breaks & forms chemical bonds. According to crystal structure of laccase, most have 3 copper binding sites which bind 4 copper atoms[1]. Laccase has a wide variation of substrates, which includes phenol, aniline, carboxylic acid (and their derivatives), bio-pigments, lignans, organic metal compounds and other non-phenol compounds[2]. The optimum temperature of laccase is relatively low, while it has the maximum catalyzing efficient among environment of low pH. These characters give laccase a promising prospect in food industry, paper industry, textile industry and bio-repairing of soil. Coriolus versicolor from Aphyllophorales, Aphyllophorales, Hymenomycetes, Basidiomycota, is one of the ideal strains which is used for laccase production and have excellent degradation ability to lignans. Laccase in plants is involved in various botanic metabolism pathways, such as synthesis of lignans, synthesis and degradation of pigments and elongation of roots[2-4] .
Molecular cloning
Plasmid with target gene is transformed into E.coli. From them, we acquire large amount of target gene using as raw material for further operation.
The band of AOX1-α factor-Laccase-AOX1 Terminator from colony PCR is about 3000bp, identical to the theoretical length of 3416bp estimated by the designed primer location (promoter to terminator), which could demonstrate that this target plasmid had successfully transformed into E.coli
Using E.coli for amplification, we extract and digest them with Bgl I or Sal I to get linear plasmid, which could be integrated into yeast genome to avoid getting lost while being frozen. Then, concentration of linear plasmid is also applied to achieve higher copy number and higher expression level. Several rounds of electroporation later, we successfully get all the plasmid with AOX1 as promoter into yeast[5]
.
The bright bands are identical to the theoretical lengths, which could demonstrate that this target plasmid had successfully transformed into yeast.
SDS-PAGE
Because of the thin band of laccase from SDS-PAGE, we can't be entirely sure whether it's inactive or active but too low the concentration to be detected. So, purification through Nickel-affinity chromatography column is used to raise its concentration for further test of enzymatic activity.
Clear and thick band of about 75kDa is consistent to the result in the supernatant. Higher the eluent concentration, thicker the band. This indicates that the band of laccase in Fig3 is our target protein instead of other impurity with a high expression level
Enzyme activity determination
After target strips been detected, to measure the activity of Laccase, we mixed 1ml of 1mmol / L ABTS solution with 1ml of centrifuged supernatant and adjusted the final concentration of Cu2+ to 10mmol/L, and measured its absorbance at 420nm. On this basis, we carried out determination experiments to further explore the optimal conditions for inducing the highest activity of Laccase.
Since the active center of Laccase is copper ion, we first explored the copper ion concentration to be added into the culture media. Adjusted the concentration of supplementary CuSO4 solution to 9mmol / L and 10mmol / L respectively, and measured the enzyme activity of Laccase under the same conditions
We found that the enzyme activity reaches its zenith when the concentration of copper ion is 10mmol/L. It is probably because the concentration of Cu2+ can’t meet the need of Laccase active center when it is too low. When the concentration is too high, it will affect the binding of Laccase and substrate due to the existence of too much copper ions.
We also explored the effects of pH and temperature on activity of Laccase at the same time.
The optimum pH of Laccase was found to be acidic, therefore we prepared buffers with pH = 3, pH = 4.8 and pH = 6.6 respectively, with 100 mM citric acid and sodium citrate and added 1 ml buffer to control the pH of the reaction system while maintaining the final concentration of CuSO4 at 10 mmol / L
After comparison, we found that among the three groups of data measured, Laccase activity is highest at pH = 3.0, decreasing to nadir at pH = 4.8, and completely inactivated at pH = 6.6. As for the reason why the subsequent measured value was lower than that at the beginning at pH 6.6, we speculated that it was caused by undermixing of solution when started reading.
For the effect of temperature, we kept ABTS solution and supernatant at 10 ℃, 20 ℃ and 37 ℃ respectively in advance, and kept the final concentration of CuSO4 at 10mmol / L.
Through the analysis and comparison of the three groups of data, it is found that under testing conditions, the activity of Laccase is the highest when the temperature is 20 ℃, and the Laccase activity is inhibited by both low and high temperatures. As for the decrease of absorbance at the final phase of measuring enzyme activity at 10 ℃, it is suspected that the low temperature has a certain impact on ABTS cationic free radical, resulting in the decrease in absorbance
Reference
[1]ESWARAMOORTHY S, BONANNO J B, BURLEY S K, et al. Erratum: Mechanism of action of a flavincontaining monooxygenase (Proceedings of the National Academy of Sciences of the United States of America (2006) 103, (9832-9837) DOI: 10.1073/pnas.0602398103) [J]. Proceedings of the National Academy of Sciences - PNAS, 2007, 104(36): 14543.
[2]DITTMER N T, KANOST M R. Insect multicopper oxidases: Diversity, properties, and physiological roles [J]. Insect biochemistry and molecular biology, 2010, 40(3): 179-88.
[3]MATE D M, ALCALDE M. Laccase engineering: From rational design to directed evolution [J]. Biotechnology advances, 2015, 33(1): 25-40.
[4]SINGH G, BHALLA A, KAUR P, et al. Laccase from prokaryotes: a new source for an old enzyme [J]. Reviews in environmental science and biotechnology, 2011, 10(4): 309-26.
[5]STALEY C A, HUANG A, NATTESTAD M, et al. Analysis of the 5′ untranslated region (5′UTR) of the alcohol oxidase 1 (AOX1) gene in recombinant protein expression in Pichia pastoris [J]. Gene, 2012, 496(2): 118-27.
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