Name: Amna Mukhtar

Roll No: 0314

Session: M Phil Morning (2020-2022)

Semester: 2nd

Subject: Bioremediation

Date: 11th August, 2021

Q-2

Enzyme Engineering

Enzyme engineering, also termed as protein engineering, is the technique of changing the amino
acid sequence in order to construct proteins or enzymes via the process of recombinant DNA
mutation. This procedure is required to change the catalytic activity of individual enzymes in
order to generate novel metabolites and to facilitate the development of a new route for carrying
out activities.

Laccase Modification

Laccase

Laccase is a copper-dependent enzyme. Laccase's molecular structure consist of three major

domains coated with additional four copper atoms, that comprises of further three sub-types for
producing the biocatalyst’s active sites. However, each Type 1 and Type 2 possesses one copper
atom whereas Type 3 has two copper atoms.

Laccases basically catalyzes the oxidative process of several phenols and aromatic substances
found in soil and water.

Why to select Laccase?

Laccase is a multi – gene encoded family of enzymes that is present in plants, microorganism
(fungi and bacteria) as well as insects. I would like to modify laccase because it is essential and
capable of oxidizing both hazardous and harmless substances. And it is primarily used in the
textile, food, and wood manufacturing sectors, as well as the pharmaceutical and chemical
industries.

Lignin engineering by modification of laccase

Biofuel has attracted much interest in past few years as a renewable, environmentally beneficial,
and cost-effective resource. Following the “first generation” biofuel was generated from sugar
cane, while in “second generation”, biofuel is made from starch and raw sugar-based products
including sugarcane and maize etc. However, the use of lignocellulose biomass for production
processes is becoming more common as an emerging new business (Chang, 2007).

The plant cell wall, a heterogeneous structure primarily composed of compounds like cellulose,
hemicellulose, and lignin, one of the most essential elements of lignocellulosic biomass. Because
it has tendency to attract cellulolytic enzymes and limit the release of cellulose, as well as
decomposition of byproducts, can also restrict cellulolytic enzyme activity, lignin has been
identified as the major resistive element in the saccharification process (Van and Zeng, 2014).

Lignin is a natural phenolic monomer possessing large molecular mass, with unique
composition, and structure as well as one of the primary components of cell walls. In plants,
growth, tissue formation, resistance and sensitivity to a number of biological and chemical
stresses are all regulated by lignin production (Turlapati et al., 2011).

Laccase Catalytic Cycle

The above figure is representing a diagram of the laccase catalytic cycle. Cu1 is oxidizing the
laccase substrate, causing an electron to be lost while on the other hand production of a free
radical, which can then be further, oxidized and may undergo polymerization. However, the
catalytic cycle is showing the loss of Cu1 is responsible for limiting reaction rate. The derived
electron is then transported to the tri-nuclear copper complex via the highly conserved His-Cys-
His pattern, in which water is formed by molecular oxygen. The oxidization process of four
molecules of substrate occurs by the reduction of single oxygen molecule to double water
molecules (Baldrian, 2006).

Enzymology of Laccase

Laccases in plants are generally glycoproteins having huge amount of carbon as 20% to 45% and
in fungus about 10 % to 25%, which is mainly involved in retention of copper, activity and
stability of enzymes, amino acid ranging from 500-600. Mostly, they are secreted proteins
having certain exceptions, but active sites usually share same molecular structure as well as
reaction process in case of fungal laccases. They have less oxidation-reduction potential with
varying pH values. The less oxidation-reduction is mainly because of the presence of methionine
at T1 site instead of phenylalanine in plant laccases. The optimum pH range is different between
laccases of plant or fungus. For plant laccases, optimum pH is approximately 7-10 whereas,
fungus origin represents relatively lower acidic range under optimum conditions.

What will be Benefits?

For effective utilization of biomass of plants and decrease in the cost of pretreatment for the
production of bioethanol, it is important to engineer the cell wall matrix of polysaccharides and
to manage by mass production. Lignin is one of the important substances that are responsible for
biomass recalcitrance. However, decreased lignin content will surely improve the absorbance of
biomass and recalcitrance. But improving lignin content by using laccase modification will be
beneficial in increasing the density of energy and biomass as well as large amount of chemicals
will be incorporated in lignin monomer.

Enhancing lignin produce high energy Feedstock

Lignin instead of all others cell well polymers possess high energy density with great amount of
heat. This biomass with high lignin content can be used for the manufacturing of high energy
fuels such as bio energy, bioelectricity or may be jet fuels by using thermochemical mechanism.
Because of the cost required for the manufacturing of bioethanol, the current interest has been
transformed to the production of hydrocarbon bio fuels by the process of thermal deconstruction
like the process of pyrolysis. Hence, improving the lignin content will surely enhance the
feedstock suitability for bio oil and bio electricity production.

Changing the components of cell wall (lignin, cellulose) usually follows enhancement in
polymers of cell walls to maintain and cell wall strength against pressure of cells in plants.

Application in Bio-fuels and Bio-products

Other than energy production, lignin is antimicrobial, antioxidant and one of the biodegradable
substances. It can also neutralize CO2, can be converted to liquid fuel, and commercially used
chemical additives. Lignin possess distinguished group that has potential to be used as reagent
for emulsification, absorption and dispersion in cement blending and can cat as chelating agent
(Doherty at al,. 2011).

Conclusion

Laccase enzyme is a subclass of oxidoreductase enzyme that catalyzes the oxidation of various
aromatic compounds and it can be able to degrade amino groups containing lignin or phenols.
Laccases can reduce to molecules of oxygen into water by eliminating the electrons from the
substrate. Laccase modification into lignin engineering is involving the process oxidation by
converting single molecules of oxygen into two water molecules. However, enhancement in
lignin content will increase the biomass production, as well as used as antioxidant. It has
property to enhance the energy feedstock.

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