19.04.2022 lysozyme essay PDF

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Egg white lysozyme: how has it been cloned and how would you purify it? Lysozymes are a class of hydrolytic glycosidases that specifically target peptidoglycan. Cleavage occurs at 1,4-beta linkages between the polymer’s primary sugar components, N-acetylmuramic acid and N-acetyl-D-glucosamine (Cao et al, 2015). Given the importance of peptidoglycan as a structural element in bacterial cell walls, this digestion increases susceptibility to osmotic pressures, resulting in cell lysis. Patterns of lysozyme sensitivity observed between grampositive and gram-negative bacterial species are reflective of this substrate specificity (Farkye and Bansal, 2011). Animals exploit this functional property, employing lysozymes as an offensive weapon in their innate immune system (Hirsch et al, 2017). The potential pharmacological, therapeutic, and commercial applications conferred by this highlight lysozyme as a high-yield target for further study and molecular manipulation (Silvetti et al, 2017). Indeed, even standard bacterial protein extraction methods in the laboratory rely on lysozyme activity. This essay focuses on hen egg white lysozymes (HEWL) and aims to (i) outline a method for cloning its gene into a host and (ii) propose a pathway for its extraction and purification.

The first stage of the cloning process is obtaining the relevant DNA fragment, in this case HWEL DNA. Traditional methods rely on the generation of a DNA library followed by screening of respective clonal colonies using hybridisation methods such as Southern Blotting. DNA libraries can either be in the genomic or cDNA form. Given the high abundance and localisation of lysozyme in the outer layer of the egg’s vitelline membrane, utilisation of lysozyme RNA transcripts as a template for reverse transcriptase is preferable. The process of creating a cDNA library, as described above, is extensive and time consuming. It is possible to

reduce the number of steps by extracting only the relevant lysozyme mRNA transcript. This is can be achieved through the utilisation of primer design software. As opposed to using random hexamers or oligo dT primers, this software works backwards from regions of minimal degeneracy within lysozyme’s amino acid sequence to create specific primer pairs (Aozhong, 2015). Amplification of this selected cDNA transcript is then achieved through the polymerase chain reaction.

To choose an appropriate cloning vector, it is important to consider what system it will be expressed in. Given the eukaryotic origins of the enzyme, expression in bacterial systems, such as Escherichia coli, would result in the formation of insoluble protein aggregates. Yeast, on the other hand, is capable of implementing eukaryotic modifications whilst withstanding the intrinsic degradative power of the enzyme itself, enabling high production yields to be reached. A fungal species which fits this criterion is Aspergillus Niger (Archer et al., 1990). Constructed plasmids compatible with this expression system include pAN7-1 and pAN52-1. These contain a BamHI cloning site that is cut by the restriction endonuclease BamHI to generate sticky ends, complementary to that of lysozyme’s digested cDNA. The fungal host is cultured in a complete medium, supplemented with buffer, and induced to uptake vectors upon application of an electrical current. Gene insertion occurs adjacent to the vector’s inducible GAM promoter region supporting control of expression in the host. Furthermore, the hydromycin antibiotic selection marker on these plasmids allows screening of successful recombinants via insertional inactivation.

Cytoplasmic contents of these transformed cells can be released mechanically or chemically and then separated in a centrifuge to yield a protein mixture. Given

lysozyme’s various distinct properties, multiple potential purification routes exist to separate it from its supernatant. For example, its enzyme substrate-binding property can be applied in the context of affinity column chromatography through the use chitin based or N-acetylglucosamine coated (Wolman et al, 2013). Alternatively, a higher purification fold of up to 98% can be reached using polyacrylamide-based Cation-exchange Cryogels (ion-exchange chromatography) (Yan et al, 2011). The latter relies on lysozyme’s sulfo groups, which give it a characteristically high isoelectric point of 10.6. A possible set up includes a negatively charged column, e.g. the CM-Sepharose fast column, equilibrated with buffer at a pH below 10.6 (Archer et al, 1990). Passing the filtered medium through the column binds the protein, which can be eluted sequentially with various concentrations of NaCl to maximise final purity and yield values (Yan et al, 2011).

The concentration of the purified lysozyme can be determined using the Lowry assay, and its functionality can be tested by quantifying lysis of the bacterial species Microccus Lysodeikticus as a function of time (Archer et al, 1990). Evaluation of protein purity can be done through visualisation using SDS-PAGE. Further confirmation can be provided using 1H-NMR spectroscopy. Identical fingerprint regions between the recombinant and naturally-sourced HWEL is indicative of correct processing and folding.

In summary, molecular cloning and purification of HEWL lysozyme first requires extraction of the relevant gene sequence using carefully designed primers in the presence of reverse transcriptase. Integration of this gene into a suitable plasmid relies on two enzyme families – ligases and restriction endonucleases, such as BamHI. Purification and analysis of the protein from the recombinant host,

Aspergillus Niger, exploits multiple intrinsic properties of the enzyme, including its charge at certain pHs, its substrate binding and hydrolytic properties, and its molecular mass. Heterologous lysozyme expression through host transformation creates an accessible and abundant source for scientists to better characterise the enzyme’s properties and behaviour.

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Hirsch, D., Baieli, M., Urtasun, N., Lázaro- Martínez, J., Glisoni, R., Miranda, M., Cascone, O. and Wolman, F., 2017. Sulfanilic acid-modified chitosan mini-spheres and their application for lysozyme purification from egg white. Biotechnology Progress, 34(2), pp.387-396.

Silvetti, T., Morandi, S., Hintersteiner, M. and Brasca, M., 2017. Use of Hen Egg White Lysozyme in the Food Industry. Egg Innovations and Strategies for Improvements, pp.233-242.

YAN, L., SHEN, S., YUN, J. and YAO, K., 2011. Isolation of Lysozyme from Chicken Egg White Using Polyacrylamide-based Cation-exchange Cryogel. Chinese Journal of Chemical Engineering, 19(5), pp.876-880.

Archer, D., Jeenes, D., MacKenzie, D., Brightwell, G., Lambert, N., Lowe, G., Radford, S. and Dobson, C., 1990. Hen Egg White Lysozyme Expressed in and Secreted from, Aspergillus niger is Correctly Processed and Folded. Nature Biotechnology, 8(8), pp.741-745.].

Aozhong, J., 2015. Cloning of egg white lysozyme gene (RJM) and yeast expression method. Patentimages.storage.googleapis.com.

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Cao, D., Wu, H., Li, Q., Sun, Y., Liu, T., Fei, J., Zhao, Y., Wu, S., Hu, X. and Li, N., 2015. Expression of Recombinant Human Lysozyme in Egg Whites of Transgenic Hens. PLOS ONE, 10(2), p.e0118626....