Bacterial laccase from Bacillus sp. - codon optimized

Description

This is the codon-optimized coding sequence of laccase (cotA) from Bacillus sp. HR03. The enzyme has been expressed in E. coli BL21(DE3), and its properties have been discussed by Mohammadian, Fathi-Roudsari, Mollania, Badoei-Dalfard, and Khajeh (2010). It was shown to have the highest activity when expressed at 18℃ and put at 70℃ (Mohammadian et al., 2010).

As an oxidase, laccase has been used to catalyze dye synthesis from plant-derived pigment precursors (Jeon et al., 2010). In the study, 15 phenols and 120 combinations were studied. Three combinations, gallic acid and syringic acid, catechin and catechol, and ferulic acid and syringic acid, were further studied as they were oxidized into brown, black, and red materials, respectively (Jeon et al., 2010). Inspired by the principle of synthetic dyes, it was hypothesized that such combinations could be used as an alternative to direct dying with pigments.

The complete coding sequence for laccase (cotA) from Bacillus sp. HR03 has been included in the GenBank database (reference code: FJ663050.1). iGEM20_Shanghai_SFLS_SPBS optimized the codons based on that sequence, constructed the plasmid pET28b-T7-laccase, and tested the expression and activity of laccase.

Experiments & Results

Successful production in E.coli BL21(DE3)

The plasmid was transformed into E. coli BL21(DE3). 0.1 mM IPTG induction was added when the culture reached an OD600 of 1.0. Then, the shake flask was cultured at 25℃ for 20 h (cultured at 25℃ instead of 18℃ because no more shakers were available in the laboratory). Then, the bacterial solution was centrifuged, the cell pellet was resuspended in PBS (pH=7.2-7.4), and the resuspension was sonicated.

SDS-PAGE

The resuspension after sonication was loaded into an SDS-PAGE. The experiment showed an expected result, with a very evident strap at ~58.4 kDa.

Figure 1. SDS-PAGE results of laccase expressed at 25℃ for 20 h. After sonication and heating at 95℃ for 10 min, the bacterial solution was loaded in Lane 12. The marker is loaded in Lane 15. It is evident that a high level of laccase was expressed.

Enzyme Activity Assay

EL-ABTS Chromogenic Reagent kit from Sangon Biotech was used to test laccase activity. ABTS can be oxidized by laccase and result in absorbance peaks at 405 nm and 650 nm. The experiment was carried out according to the procedure given by the kit. Enzyme activity of 10% bacterial solution and 2% bacterial solution was compared to the negative control, pure water. The pigment precursor was oxidized, and absorbance increased.

Figure 2. Enzyme Activity Assay results.

Sequence & Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 885
Illegal AgeI site found at 1192
Illegal AgeI site found at 1348
1000
COMPATIBLE WITH RFC[1000]

References

1. Shraddha, R. Shekher, S. Sehgal, M. Kamthania and A. Kumar, "Laccase: Microbial Sources, Production, Purification, and Potential Biotechnological Applications", Enzyme Research, vol. 2011, pp. 1-11, 2011. Available: 10.4061/2011/217861.

2. J. Jeon et al., "Laccase-catalysed polymeric dye synthesis from plant-derived phenols for potential application in hair dyeing: Enzymatic colourations driven by homo- or hetero-polymer synthesis", Microbial Biotechnology, vol. 3, no. 3, pp. 324-335, 2010. Available: 10.1111/j.1751-7915.2009.00153.x.

3. D. Kumar, A. Kumar, S. Sondhi, P. Sharma and N. Gupta, "An alkaline bacterial laccase for polymerization of natural precursors for hair dye synthesis", 3 Biotech, vol. 8, no. 3, 2018. Available: 10.1007/s13205-018-1181-7.

4. M. Mohammadian, M. Fathi-Roudsari, N. Mollania, A. Badoei-Dalfard and K. Khajeh, "Enhanced expression of a recombinant bacterial laccase at low temperature and microaerobic conditions: purification and biochemical characterization", Journal of Industrial Microbiology & Biotechnology, vol. 37, no. 8, pp. 863-869, 2010. Available: 10.1007/s10295-010-0734-5.

Team: SMS_Shenzhen 2021

In order to determine the optimal temperature for modified Laccase CotA, we conducted a temperature gradient experiment. We found that 60℃ is the optimal temperature of the original Laccase CotA, which is close to the normal temperature of the ground in semitropic and tropic zones, suggesting that Laccase CotA can degrade gum base that stuck to the ground effectively.

<img src=""> Fig.1 Temperature gradient for CotA

We then utilized two approaches to improve the enzyme activity of Laccase CotA. One is rational design serving to modify the substrate affinity between Laccase CotA and ABTS, and the other one is directed evolution with the purpose of improving the thermal stability of Laccase CotA at ground temperature.

We carried out homology modeling to identify the probable 3D-structure of laccase CotA, molecular docking to predict the binding sites on laccase CotA, and multiple sequence alignment to verify the validity of binding sites. Then, recommended mutations, which can improve substrate affinity, were compared by molecular docking software. Laccase CotA with mutation of HIS496>ASP, LEU386>GLU, and LEU386>GLU& HIS496>ASP, which show the highest mutation energy, was determined via rational design.

We also made a plan of conducting directed evolution experiments. Randomly mutated sequences coding Laccase CotA would be yielded from Error Prone-PCR(EP-PCR). Then, single colonies would be cultured to express mutated Laccase CotA. We implemented library screening for beneficial variations by testing activity of crude enzymes. Finally, two beneficial variations were obtained, CotA-2-12 and CotA-2-8.

We then characterized those mutations separately. As for the rational design mutations, CotA-386 shows approximately none activity. The enzymatic activities of CotA-497 and CotA-386-497 increased compared to the original one. Surprisingly, enzymatic activities for mutation from directed evolution, CotA-2-12 and CotA-2-8, shows a higher increase.

<img src=""> Fig.2 Enzymatic Activities of original and mutaional CotA

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