Description

Inspiration

On March 21 this year, a disastrous explosion occurred in a chemical plant in Xiangshui, China, killing 47 people and hurting 566 people. This explosion was due to the burning of an environmentally unfriendly pollutant--m-Phenylenediamine.

m-Phenylenediamine will not only cause health hazards like allergy, but also cause environmental hazards such as water pollution. Instead of using traditional degradation methods, we resorted to microbial biodegradation, the breakdown of organic matter by microorganisms such as bacteria and fungi. We aimed to applicate three methods: 1) Adaptive evolution of E. coli; 2) Engineered Laccase Expressing E.coli; 3) Adaptive evolution of Bacteria from Aniline-Polluted Activated Sludge.

Harm of Aniline Compound

Aniline is toxic by inhalation of the vapour, ingestion, or percutaneous absorption. The early manufacture of aniline resulted in increased incidents of bladder cancer, but these effects are now attributed to naphthylamines, not anilines. [1] Apart from this, aniline compound has been implicated as one possible cause of forest dieback. [2] Aniline and its derivatives can cause damage to human blood circulation system by forming methemoglobin, which can directly cause toxic damage. After entering the body, these compounds can easily pass through the blood barrier and react with a large number of lipid-like nervous systems, resulting in nervous system damage. In addition, aniline compounds do have carcinogenic and mutagenic effects.

Traditional Degradation & Biodegradation

Generally speaking, the traditional degradation of m-phenylenediamine is roughly divided into physical and chemical degradation. The physical method mainly uses resin drilling to adsorb m-phenylenediamine. However, the shortcoming is that it can not completely absorb m-phenylenediamine in water. For the chemical method, as m-phenylenediamine is a special kind of pollution, a large number of chemicals need to be added to ensure that the chemical reaction is carried out in water, and the presence of such chemicals might confuse the effect of degradation. Even worse, it can lead to the secondary pollution of water. For example, the Fenton reaction is now widely used in chemical industry to degrade m-phenylenediamine. During the reaction, transition-metalions such as iron and copper can donate or accept free electrons via intracellular reactions and so contribute to the formation of free radicals. In oxygen respirating organisms, most of the intracellular iron is in the ferric (Fe3+) state and must therefore be reduced to the ferrous (Fe2+) form to take part in the Fenton reaction. Superoxideions and transition metals act in a synergistic way in the appearance of free radical damages. [3] In this reaction, acidic substances will be produced, and +3 valence iron hydroxide will cause secondary pollution to the water.
In the case of microbial degradation, it will not affect pH value of the water, and m-phenylenediamine will also be completely mineralized to the form of organic matter, thus acting as the energy source of microorganisms and providing carbon dioxide and water. Biodegradable material is capable of decomposing anaerobically into carbon dioxide, water, and biomass. Different from composting, an accelerated biodegradation process due to optimized circumstances, [4] biodegradation can occur in different time frames under different circumstances, but is meant to occur naturally without human intervention.

Adaptive Laboratory Evolution (ALE)

Adaptive laboratory evolution (ALE) strategies simulate and accelerate natural evolution, which allow for the metabolic engineering of microorganisms by combining genetic variation with the selection of beneficial mutations in an unbiased fashion. ALE can be performed in the laboratory by sequential serial passages in shake flasks, where nutrients or additives are in certain parameters. This technology is wildly used, ranging from biofuel production, commodity chemical synthesis, production of industrial and biopharmaceutical proteins, through to the utilization of new substrates. [5-7]

Laccase

Laccase was first discovered from the juice of Rhus venicifera. It belongs to multinuclear copper-containing oxidases. This enzyme catalyzes the reduction of molecular oxygen by various organic compounds straightforwardly to water without the step of hydrogen peroxide formation.[8-9] In the past few years, laccase has received much attention because laccases are capable of oxidizing phenolic and non-phenolic lignin-related compounds as well as highly persistent environmental pollutants. This makes them very useful for applications involving a variety of biotechnological processes. This includes detoxification of industrial wastewater, such as paper, pulp, textile and petrochemical industries. Laccase is also valuable as a medical diagnostic tool and as a tool for removing biocides from herbicides, pesticides and certain explosives in the soil. Besides, these enzymes can also be used as catalysts for the manufacture of anticancer drugs, and even as ingredients in cosmetics. Their ability to remove heterogeneous biomass and produce polymer products makes them useful tools for bioremediation purposes. [8-9]

Signal peptide

A signal peptide (sometimes referred to as signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide) is a short(5-30 amino acids long) peptide present at the N-terminus of the majority of newly synthesized proteins that are destined towards the secretory pathway. SP is in most cases a transient extension of the end of the protein amino, and once its target function is performed, it is called a small class of enzyme serotas ease removal. Many complete membrane proteins are anchored to the membrane through uncut SPs or through a series of SP-like "start-up transfer" and "termination transfer" sequences. [10]

Reference:

1.Thomas Kahl, Kai-Wilfrid Schröder, F. R. Lawrence, W. J. Marshall, Hartmut Höke, Rudolf Jäckh "Aniline" in Ullmann's Encyclopedia of Industrial Chemistry, 2007. John Wiley & Sons: New York.doi:10.1002/14356007.a02_303
2.Krahl-Urban, B., Papke, H.E., Peters, K. (1988) Forest Decline: Cause-Effect Research in the United States of North America and Federal Republic of Germany. Germany: Assessment Group for Biology, Ecology and Energy of the Julich Nuclear Research Center.
3.Robbins & Cotran (2008). Pathologic basis of disease (7th ed.). Elsevier. p. 16. ISBN 9780808923022.
4.Magdoff F (November 1993). "Building Soils for Better Crops". Soil Science. 156 (5): 371. doi:10.1097/00010694-199311000-00014.
5.LaCroix R A, Sandberg T E, O'Brien E J, et al. Use of adaptive laboratory evolution to discover key mutations enabling rapid growth of Escherichia coli K-12 MG1655 on glucose minimal medium[J]. Appl. Environ. Microbiol., 2015, 81(1): 17-30.
6.Guzmán G I, Sandberg T E, LaCroix R A, et al. Enzyme promiscuity shapes adaptation to novel growth substrates[J]. Molecular systems biology, 2019, 15(4).
7.Dragosits M, Mattanovich D. Adaptive laboratory evolution–principles and applications for biotechnology[J]. Microbial cell factories, 2013, 12(1): 64.
8.Yaropolov A I, Skorobogat’Ko O V, Vartanov S S, et al. Laccase[J]. Applied Biochemistry and Biotechnology, 1994, 49(3): 257-280.
9.Couto S R, Herrera J L T. Industrial and biotechnological applications of laccases: a review[J]. Biotechnology advances, 2006, 24(5): 500-513.
10.Choi J H, Lee S Y. Secretory and extracellular production of recombinant proteins using Escherichia coli[J]. Applied microbiology and biotechnology, 2004, 64(5): 625-635.

Team:Xiamen City/Description