Modelling

Overview of Modelling

Laccases are versatile enzymes that catalyze oxidation reactions between phenolic and non-phenolic substrates. Laccase is one of the most well-studied enzymes, perhaps due to its wide presence in many fungi or plants. However, because of its wide presence, it is impossible to generalize the properties of different strains of Laccases. Our project specifically targeted the novel Lac1326 Laccase found in deep-sea bacteria.

We here built models to evaluate the potential improvements of a laccase we were considering using (a recently discovered laccase; Lac1326) and laccases introduced by other iGEM teams. Using data collected from various literature, we simulate and predict the enzyme activities and stabilities under varying conditions. We were able to build models that take parameters such as pH, temperature and EDC concentration into account.

Based on our prediction, we found that the laccase we wanted to pick (Lac1326) has better adaptability to the environment. Therefore, we decided to use this laccase (Lac1326) in our project. Moreover, after we got the real-life data (eg. water temperature and pH in wastewater treatment plant) from the local organizations, we also simulated how these laccases can degrade EDC in practice. This simulation further confirmed that our laccase K3021004(NSP4_Lac1326_HisTag) is expected to have better performance.

Background

The goal of our project is to find a solution for the degradation of EDC in wastewater, so it is of paramount importance that we understand what conditions are the laccases functional, in order to find the one that suits the job.

In terms of the normal pH range of wastewater, we consulted a Macau wastewater treatment plant that specifically deals with industrial wastewater. They suggested to us that we should look into a range from pH6-9 as that is what those are the pH values of which the industrial wastewater usually comes in.

In terms of temperature, it is advised that waste water treatment usually happens indoors with no temperature control. And we should be investigating in enzymes which performs best without any form of assistant in temperature, as it will save the cost of implementing such a system and make it more sustainable. Hence, we set our goals for an optimal temperature of 25°C.

It is also noted that wastewater tends to have the property of high salinity. A good laccase perform well in such situation.

pH	6-9
Temperature	25°C
Salinity	High

Through reading literature, we found a potential candidate of laccase K3021004（Lac1326）, which is claimed to have high adaptability https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6290062/, may be used in our project. Therefore, we decided to build models to compare this laccase K3021004（Lac1326） and laccase from previous iGEM teams.

Comparison between Laccases used by previous iGEM team and the K3021004（Lac1326）(we proposed to use).

There were previous teams who have constructed or tested laccases (from T.versicolour, check BBa_K863030 for more details) in waste water treatment (e.g. Biefield-Gemany 2012). While we are taking a different approach by augmenting living cells in our purposed method of treatment, there are previous standards we should reference and improve upon.

Our new construct should perform better in realistic wastewater conditions. The biggest decision we are making is to use a new strain of laccase (Lac1326), which is derived from deep-sea marine organisms, compare to the older K863000 construct, which is commonly found in higher fungi. It is believed that Lac1326 would outperform older constructs in terms of adaptability in a harsher environment.

Based on data from previous literatures (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6290062/; https://www.ncbi.nlm.nih.gov/pubmed/24152339), we here modeled the difference of laccase activity for both T. versicolour and K3021004（Lac1326） under different temperatures and pH.

As shown in the figures below:

As demonstrated in the graphs, both pH and Temperature has a profound influence on the activity of both laccases. However, the activity of fungal laccase (T. versicolour) is virtually ceased above pH 5, which is not suitable for the wastewater treatment according to the information we had. On the other hand, Lac1326 has retained 80% of its maximal activity in the range pH4-8. Also, the T. versicolour laccase showed a relatively bigger fall off as temperature decrease when compared to Lac1326, which maintained more than 50% of its maximal activity at 20-70°C. Therefore, we believe that Lac1326 could hypothetically perform better in wastewater, with its high adaptability characteristics.

We also model how Lac1326 is good with halotolerance. While the laccase activity of T. versicolour dropped below a quarter of its maximal activity at around NaCl concentration of 100mM, Lac1326 actually showed a significantly increased activity. This result shows the Lac1326 may have an advantage over the previous laccase introduced by other iGEM teams in terms of water treatment.

The regression model of Lac1326

As shown above, we built the model to predict activity of K3021004（Lac1326） based on pH and temperature. The regression is utilising a simple multilinear equation with two regressor variables:

Hence the formula will be

y	Enzyme activity
x₁	Temperature
x₂	pH

The regression has the R² value of 0.6977 and RSEM 3.521.

Under a typical waste water condition with a pH of 6, in 25°C, the prediction shows the proposed enzyme will retain 82.5% of its maximal activity. It goes to show that it is viable to investigate in using this new enzyme in the development of a new degradation system for waste water treatment.

Conclusion