Paper Sample on Aromatic Amino Acids

Introduction

An aromatic amino acid is a type of amino acid that majorly includes an aromatic ring in its structural formulae. For the 20 amino acids largely studied, there are three prominent examples of aromatic amino acids, including phenylalanine, tryptophan, and tyrosine. (Knott,S et al. 2020. Pp. 25476).

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Tyrosine

The polymerization of ethylene terephthalate results in the formation of polyethylene terephthalate as the end product. It may exist as amorphous or transparent or as a crystalline polymer depending on its processing, and thermal history. It is one of the most common thermoplastic polymer resins of a polyester family (Pellis et al., 2016, p.316). The polymerization process is majorly utilized in containers for liquids and foods, and in the manufacturing of apparel products. (Gigante, et al.2016. p.g.1857)

Polyethylene Terephthalate

Major companies in the world that are tasked in beverage production, cloth making, carpeting and packaging all employ crystalline poly- (ethylene terephthalate) which is resistant to biological de-polymerization and catalysis due to limited penetration and accessibility to its ester linkages. The bacteria Ideonella sakaensis has been discovered to use poly (ethylene terephthalate) as its carbon source for growth and energy. I. sakaiensis enzyme dubbed PETase-(Poly (ethylene terephthalate)-digesting enzymes) converts Poly (ethylene terephthalate) to mono(2-hydroxyethyl) terephthalic acid, including minimal traces of TPA (Terephthalic Acid). (Han, et al.2017 p.g 1)

Secondly, an enzyme referred to as the MHETase is responsible for the conversion MHET into dual monomers: EG (ethylene glycol); and TPA. The two enzymes are produced by I. Sakaiensis. Even though polyethylene terephthalate in humans uses a wide scope of polyesters majorly distinguished by both aromatic and aliphatic content, polyethylene terephthalate remains a semi-aromatic polyester generally. A small number of aliphatic polyesters like PBS (Polybutylene Succinate), and PLA (Polylactic Acid) generated from energy renewable sources as indicated by (Gigante et al. (2019, p.1857). These aliphatic polyesters are often promoted as biodegradable plastic materials due to their minimal glass transition temperatures, and crystallinity. These factors consequently offer a direct enzymatic association with ester bonds (Sharon et al, 2017, p.257). Aromatic, including semi-aromatic polyesters, exhibit enhanced material, as well as thermal elements and, hence an implication to have reached a remarkable higher market volume but are only that they are not as biodegradable as their aliphatic ones. An emerging, bio-based auxiliary involves polyethylene-2,5-furandicarboxylate and PEF grounded on sugar-derived FDCA (2,5-furandicarboxylic acid). The PE has got advanced gas barrier features over polyethylene terephthalate. which makes it essential for 9968377156447industrial research. Even though PEF is a bio-based semi-aromatic polyester predicted to offset greenhouse gas emissions compared to polyethylene terephthalate, the process takes a long period in the environment.

Enhanced Studies

Polyethylene terephthalate affinity analysis through surface affinity essays reveals an enhanced studies that entails how it binds and interacts with proteins which is achieved through quantitative close analysis obtained through a detailed experiment undertaken in the laboratory. All this process involve the selection of carbohydrate binding module of a planar architecture involving aromatic acid residues. Three of the samples are subjected to molecular dynamics simulations to detect and identify their mode of action with an aim of making comparison from the experimental essay with the calculated models known. Now through the known simulations and most preferably tryptophan quenching practical and experiments, the model describing the mode of interaction between carbohydrates binding module and polyethylene terephthalate as a model basis of understanding other major interactions that majorly exist. (Han, et al.2017 pg. 1)

Construction of plasmids encoding carbohydrate binding module for better understanding of the basis of interaction that exist between polyethylene terephthalate and peptides with even other polymers. The first step involved the employment of E.coli codon optimized DNA sequences named as follows; TrCBM1, PpCBM1, BaCBM2, BsCBM2, BaCBM5, ucCBM10, and PaCBM10 with the existence of a fluorescence marker named as Bs2.Affinity tag, strep tag II. Every sequence that encoded for one of the carbohydrate binding module peptides C would terminally fuse to Bs2 and strep tag II which then gets inserted to a vector hence lacking the leader sequence the followed by cloning through standard techniques.

The plasmids are then transformed through heat shock formula onto a competent cells of E.coli named as XL1 blue cells and BL21 cells. All the above described the precise protocol that is carried to enhance the expression and identification of expected and detailed mode of interaction that exist.

Secondly, enzyme, MHETase (MHET-digesting enzyme), further converts MHET to two monomers, TPA with ethylene glycol (EG). The two enzymes are produced by I. sakaiensis and majorly act synergistically to depolymerize PET. Sequence studies and recent structural research studies of PETase list the similarities to α/β-hydrolase enzymes, including the cutinase and lipase families, which catalyze hydrolysis of cutin and fatty acids, respectively. This provides clues towards describing the origin of PETase.

Purification and Expression

Purification and expression as per the analysis. Proteins, recombinant proteins expressed in E.coli were from B21 cells through the method of auto induction method cell disruption are done by sonication using the method of sonoplus sonication homogenisator adjusted 3*5min ,with 30%power and cycle 5. Centrifugation was meant to isolate and distinguish the soluble fragments with revolutions per min of 11000 revolutions per min carried out for 30 mins on a standard temperature of 4 degrees.

Carbohydrate binding module proteins are purified by affinity tag and also through the process of ion exchange chromatography. Affinity tag method the fraction sample is loaded onto a strep tactin super flow plus which is 1ml cartridge.(Knott, et al . 2020. Pp. 25476)

The biotechnological modification of poly (ethylene terephthalate) is majorly achieved through the hydrolyzing cutinases. The hydrophobicity of poly (ethylene terephthalate) signified as a crucial issue, which should be understood, especially when the procedure is applied in an industrial process. The enzymatic treatment of polyethylene terephthalate includes one of the challenging features associated with poor affinity of various enzymes to polyethylene terephthalate substrates. Due to this effect, the challenge of enzymatic modification prompts the application of polyethylene terephthalate hydrolases fused to binding modules, and hydrophobins to initiate binding of an enzyme to the polyethylene terephthalate surfaces.(Knott, et al . 2020. Pp. 25476)

Through the application of the carbohydrate-binding module, we use the context with the aim of ensuring the concentration of enzymes on the surface of a substrate. Due to the similarity in the sequence of amino acids, the carbohydrate-binding modules are classified into varied families, which are then identified by their structural and individual functional features giving rise to three types of carbohydrate-binding modules denoted as A, B and C.

Type A modules are majorly surface binding elements, and consist of members belonging to carbohydrate-binding module families of 1, 2a, 3, 5, and 10. A major feature of this type includes its high binding capability to crystalline cellulose and chitin, which is promoted by their characteristic platform consisting of aromatic amino acid residues (Kasyanov et al., 2016, p.309). Aromatic amino acids exposed on the surface of carbohydrate plays an important role in the interaction of carbohydrate-binding module with its respective synthetic substrate polyethylene terephthalate. (Khazanov, et al.2016 pg. 309)

Conclusion

MD simulations of the carbohydrate binding module–poly (ethylene terephthalate) interface revealed π-stacking interactions of exposed aromatic residues of the peptide with the phenyl rings of poly (ethylene terephthalate) and hydrogen bonding of polar residues (Weber et al., 2019). Therefore, balanced point involving the contact area of hydrophobic with polar residues can positively impacts the strength of the carbohydrate binding module–poly (ethylene terephthalate) interaction. (Han, et al.2017 pg. 1)

References

Gigante, V., Coltelli, M.B., Vannozzi, A., Panariello, L., Fusco, A., Trombi, L., Donnarumma, G., Danti, S. and Lazzeri, A., 2019. Flat die extruded biocompatible poly (lactic acid)(PLA)/poly (butylene succinate)(PBS) Based Films. Polymers, 11(11), p.1857.
https://doi.org/10.3390/polym11111857

Han, X., Liu, W., Huang, J.W., Ma, J., Zheng, Y., Ko, T.P., Xu, L., Cheng, Y.S., Chen, C.C. and Guo, R.T., 2017. Structural insight into catalytic mechanism of PET hydrolase. Nature communications, 8(1), pp.1-6. https://doi.org/10.1038/s41467-017-02255-z.