Biology:Lysozyme

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Short description: Antimicrobial enzyme produced by animals
Lysozyme
Identifiers
EC number3.2.1.17
CAS number9001-63-2
Databases
IntEnzIntEnz view
BRENDABRENDA entry
ExPASyNiceZyme view
KEGGKEGG entry
MetaCycmetabolic pathway
PRIAMprofile
PDB structuresRCSB PDB PDBe PDBsum
Gene OntologyAmiGO / QuickGO
Glycoside hydrolase, family 22, lysozyme
Lysozymecrystals1.png
Lysozyme crystals stained with methylene blue.
Identifiers
Symbol?
InterProIPR000974

Lysozyme (EC 3.2.1.17, muramidase, N-acetylmuramide glycanhydrolase; systematic name peptidoglycan N-acetylmuramoylhydrolase) is an antimicrobial enzyme produced by animals that forms part of the innate immune system. It is a glycoside hydrolase that catalyzes the following process:

Hydrolysis of (1→4)-β-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan and between N-acetyl-D-glucosamine residues in chitodextrins

Peptidoglycan is the major component of gram-positive bacterial cell wall.[1] This hydrolysis in turn compromises the integrity of bacterial cell walls causing lysis of the bacteria.

Lysozyme is abundant in secretions including tears, saliva, human milk, and mucus. It is also present in cytoplasmic granules of the macrophages and the polymorphonuclear neutrophils (PMNs). Large amounts of lysozyme can be found in egg white. C-type lysozymes are closely related to α-lactalbumin in sequence and structure, making them part of the same glycoside hydrolase family 22.[2] In humans, the C-type lysozyme enzyme is encoded by the LYZ gene.[3][4]

Hen egg white lysozyme is thermally stable, with a melting point reaching up to 72 °C at pH 5.0.[5] However, lysozyme in human milk loses activity very quickly at that temperature.[6] Hen egg white lysozyme maintains its activity in a large range of pH (6–9).[7] Its isoelectric point is 11.35.[8] The isoelectric point of human milk lysozyme is 10.5–11.[9]

Function and mechanism

The enzyme functions by hydrolyzing glycosidic bonds in peptidoglycans. The enzyme can also break glycosidic bonds in chitin, although not as effectively as true chitinases.[10]

Overview of the reaction catalysed by lysozyme

Lysozyme's active site binds the peptidoglycan molecule in the prominent cleft between its two domains. It attacks peptidoglycans (found in the cell walls of bacteria, especially Gram-positive bacteria), its natural substrate, between N-acetylmuramic acid (NAM) and the fourth carbon atom of N-acetylglucosamine (NAG).

Shorter saccharides like tetrasaccharide have also shown to be viable substrates but via an intermediate with a longer chain.[11] Chitin has also been shown to be a viable lysozyme substrate. Artificial substrates have also been developed and used in lysozyme.[12]

Mechanism

Phillips

The Phillips mechanism proposed that the enzyme's catalytic power came from both steric strain on the bound substrate and electrostatic stabilization of an oxo-carbenium intermediate. From X-ray crystallographic data, Phillips proposed the active site of the enzyme, where a hexasaccharide binds. The lysozyme distorts the fourth sugar (in the D or -1 subsite) in the hexasaccharide into a half-chair conformation. In this stressed state, the glycosidic bond is more easily broken.[13] An ionic intermediate containing an oxo-carbenium is created as a result of the glycosidic bond breaking.[14] Thus distortion causing the substrate molecule to adopt a strained conformation similar to that of the transition state will lower the energy barrier of the reaction.[15]

The proposed oxo-carbonium intermediate was speculated to be electrostatically stabilized by aspartate and glutamate residues in the active site by Arieh Warshel in 1978. The electrostatic stabilization argument was based on comparison to bulk water, the reorientation of water dipoles can cancel out the stabilizing energy of charge interaction. In Warshel's model, the enzyme acts as a super-solvent, which fixes the orientation of ion pairs and provides super-solvation (very good stabilization of ion pairs), and especially lower the energy when two ions are close to each other.[16]

The rate-determining step (RDS) in this mechanism is related to formation of the oxo-carbenium intermediate. There were some contradictory results to indicate the exact RDS. By tracing the formation of product (p-nitrophenol), it was discovered that the RDS can change over different temperatures, which was a reason for those contradictory results. At a higher temperature the RDS is formation of glycosyl enzyme intermediate and at a lower temperature the breakdown of that intermediate.[17]

Covalent intermediate of lysozyme enzyme, with covalent bond in black and experimental evidence as blue mesh.[18]

Covalent mechanism

Substrates in Vocadlo's experiment

In an early debate in 1969, Dahlquist proposed a covalent mechanism for lysozyme based on kinetic isotope effect,[14] but for a long time the ionic mechanism was more accepted. In 2001, a revised mechanism was proposed by Vocadlo via a covalent but not ionic intermediate. Evidence from ESI-MS analysis indicated a covalent intermediate. A 2-fluoro substituted substrate was used to lower the reaction rate and accumulate an intermediate for characterization.[19] The amino acid side-chains glutamic acid 35 (Glu35) and aspartate 52 (Asp52) have been found to be critical to the activity of this enzyme. Glu35 acts as a proton donor to the glycosidic bond, cleaving the C-O bond in the substrate, whereas Asp52 acts as a nucleophile to generate a glycosyl enzyme intermediate. The Glu35 reacts with water to form hydroxyl ion, a stronger nucleophile than water, which then attacks the glycosyl enzyme intermediate, to give the product of hydrolysis and leaving the enzyme unchanged.[20] This type of covalent mechanism for enzyme catalysis was first proposed by Koshland.[21]

More recently, quantum mechanics/ molecular mechanics (QM/MM) molecular dynamics simulations have been using the crystal of HEWL and predict the existence of a covalent intermediate.[22] Evidence for the ESI-MS and X-ray structures indicate the existence of covalent intermediate, but primarily rely on using a less active mutant or non-native substrate. Thus, QM/MM molecular dynamics provides the unique ability to directly investigate the mechanism of wild-type HEWL and native substrate. The calculations revealed that the covalent intermediate from the covalent mechanism is ~30 kcal/mol more stable than the ionic intermediate from the Phillips mechanism.[22] These calculations demonstrate that the ionic intermediate is extremely energetically unfavorable and the covalent intermediates observed from experiments using less active mutant or non-native substrates provide useful insight into the mechanism of wild-type HEWL.

Two Possible Mechanisms of Lysozyme

Inhibition

Imidazole derivatives can form a charge-transfer complex with some residues (in or outside active center) to achieve a competitive inhibition of lysozyme.[23] In Gram-negative bacteria, the lipopolysaccharide acts as a non-competitive inhibitor by highly favored binding with lysozyme.[24]

Further information: Glycoside hydrolase

Non-enzymatic action

Despite that the muramidase activity of lysozyme has been supposed to play the key role for its antibacterial properties, evidence of its non-enzymatic action was also reported. For example, blocking the catalytic activity of lysozyme by mutation of critical amino acid in the active site (52-Asp -> 52-Ser) does not eliminate its antimicrobial activity.[25] The lectin-like ability of lysozyme to recognize bacterial carbohydrate antigen without lytic activity was reported for tetrasaccharide related to lipopolysaccharide of Klebsiella pneumoniae.[26] Also, lysozyme interacts with antibodies and T-cell receptors.[27]

Enzyme conformation changes

Lysozyme exhibits two conformations: an open active state and a closed inactive state. The catalytic relevance was examined with single walled carbon nanotubes (SWCN) field effect transistors (FETs), where a singular lysozyme was bound to the SWCN FET.[28] Electronically monitoring the lysozyme showed two conformations, an open active site and a closed inactive site. In its active state lysozyme is able to processively hydrolyze its substrate, breaking on average 100 bonds at a rate of 15 per second. In order to bind a new substrate and move from the closed inactive state to the open active state requires two conformation step changes, while inactivation requires one step.

Superfamily

The conventional C-type lysozyme is part of a larger group of structurally and mechanistically related enzymes termed the lysozyme superfamily. This family unites GH22 C-type ("chicken") lysozymes with plant chitinase GH19, G-type ("goose") lysozyme GH23, V-type ("viral") lysozyme GH24 and the chitosanase GH46 families. The lysozyme-type nomenclature only reflects the source a type is originally isolated from and does not fully reflect the taxonomic distribution.[29] For example, humans and many other mammals have two G-type lysozyme genes, LYG1 and LYG2.[30]

Role in disease and therapy

Lysozyme is part of the innate immune system. Reduced lysozyme levels have been associated with bronchopulmonary dysplasia in newborns.[31] Piglets fed with human lysozyme milk can recover from diarrheal disease caused by E. coli faster. The concentration of lysozyme in human milk is 1,600 to 3,000 times greater than the concentration in livestock milk. Human lysozyme is more active than hen egg white lysozyme. A transgenic line of goats (with a founder named "Artemis") were developed to produce milk with human lysozyme to protect children from diarrhea if they can't get the benefits of human breastfeeding.[32][33]

Since lysozyme is a natural form of protection from Gram-positive pathogens like Bacillus and Streptococcus,[34] it plays an important role in immunology of infants in human milk feeding.[35] Whereas the skin is a protective barrier due to its dryness and acidity, the conjunctiva (membrane covering the eye) is, instead, protected by secreted enzymes, mainly lysozyme and defensin. However, when these protective barriers fail, conjunctivitis results.

In certain cancers (especially myelomonocytic leukemia) excessive production of lysozyme by cancer cells can lead to toxic levels of lysozyme in the blood. High lysozyme blood levels can lead to kidney failure and low blood potassium, conditions that may improve or resolve with treatment of the primary malignancy.

Serum lysozyme is much less specific for diagnosis of sarcoidosis than serum angiotensin converting enzyme; however, since it is more sensitive, it is used as a marker of sarcoidosis disease activity and is suitable for disease monitoring in proven cases.[36]

Chemical synthesis

The first chemical synthesis of a lysozyme protein was attempted by Prof. George W. Kenner and his group at the University of Liverpool in England.[37] This was finally achieved in 2007 by Thomas Durek in Steve Kent's lab at the University of Chicago who made a synthetic functional lysozyme molecule.[38]

Other applications

Lysozyme crystals have been used to grow other functional materials for catalysis and biomedical applications.[39][40][41] Lysozyme is a commonly used enzyme for lysing gram positive bacteria.[42] Due to the unique function of lysozyme in which it can digest the cell wall and causes osmotic shock (burst the cell by suddenly changing solute concentration around the cell and thus the osmotic pressure), lysozyme is commonly used in lab setting to release proteins from bacterium periplasm while the inner membrane remains sealed as vesicles called the spheroplast.[43][44]

For example, E. coli can be lysed using lysozyme to free the contents of the periplasmic space. It is especially useful in lab setting for trying to collect the contents of the periplasm.[1] Lysozyme treatment is optimal at particular temperatures, pH ranges, and salt concentrations. Lysozyme activity increases with increasing temperatures, up to 60 degrees Celsius, with a pH range of 6.0-7.0. The salts present also affect lysozyme treatment, where some assert inhibitory effects, and others promote lysis via lysozyme treatment. Sodium chloride induces lysis, but at high concentrations, it is an active inhibitor of lysis. Similar observations have been seen with the use of potassium salts. Slight variations are present due to differences in bacterial strains.[45] A consequence of the use of lysozyme in extracting recombinant proteins for protein crystallization is that the crystal may be contaminated with units of lysozyme, producing a physiologically irrelevant combination. In fact, some proteins simply cannot crystalize without such contamination.[46][47]

Furthermore, lysozyme can serve as a tool in the expression of toxic recombinant proteins. Expressing recombinant proteins in BL21(DE3) strains is typically accomplished by the T7-RNA-polymerase. Via IPTG induction, the UV-5 repressor is inhibited, leading to the transcription of the T7-RNA-polymerase and thereby of the protein of interest. Nonetheless, a basal level of the T7-RNA-polymerase is observable even without induction. T7 lysozyme acts as an inhibitor of the T7-RNA-polymerase. Newly invented strains, containing a helper plasmid (pLysS), constitutively co-express low levels of T7 lysozyme, providing high stringency and consistent expression of the toxic recombinant protein.[48]

History

The antibacterial property of hen egg white, due to the lysozyme it contains, was first observed by Laschtschenko in 1909.[49] The bacteria-killing activity of nasal mucus was demonstrated in 1922 by Alexander Fleming, the discoverer of penicillin, who coined the term "lysozyme".[50] He is reported as saying: "As this substance has properties akin to those of ferments I have called it a 'Lysozyme'."[51] Fleming went on to show that an enzymic substance was present in a wide variety of secretions and was capable of rapidly lysing (i.e. dissolving) different bacteria, particularly a yellow "coccus" that he studied".[52]

Lysozyme was first crystallised by Edward Abraham in 1937, enabling the three-dimensional structure of hen egg white lysozyme to be described by David Chilton Phillips in 1965, when he obtained the first 2-ångström (200 pm) resolution model via X-ray crystallography.[53][54] The structure was publicly presented at a Royal Institution lecture in 1965.[55] Lysozyme was the second protein structure and the first enzyme structure to be solved via X-ray diffraction methods, and the first enzyme to be fully sequenced that contains all twenty common amino acids.[56] As a result of Phillips' elucidation of the structure of lysozyme, it was also the first enzyme to have a detailed, specific mechanism suggested for its method of catalytic action.[57][58][59] This work led Phillips to provide an explanation for how enzymes speed up a chemical reaction in terms of its physical structures. The original mechanism proposed by Phillips was more recently revised.[19]

See also

  • Egg allergy

References

  1. ↑ 1.0 1.1 "Lysozyme". Handbook of Detection of Enzymes on Electrophoretic Gels. Boca Raton, Fla.: CRC Press. 1994. p. 223. ISBN 978-0-8493-8935-1. https://www.google.com/search?q=Hydrolysis+of+%281-%3E4%29-beta-linkages+between+N-acetylmuramic+acid+and+N-acetyl-D-glucosamine+residues+in+a+peptidoglycan+and+between+N-acetyl-D-glucosamine+residues+in+chitodextrins#tbs=cdr:1%2Ccd_min:1900%2Ccd_max:2006&tbm=bks&q=Hydrolysis+linkages+between+N-acetylmuramic+acid+N-acetyl-D-glucosamine+residues+in+a+peptidoglycan+and+between+N-acetyl-D-glucosamine+residues+in+chitodextrins&*. 
  2. ↑ "Glycoside hydrolase family 22". https://www.cazypedia.org/index.php/Glycoside_Hydrolase_Family_22. 
  3. ↑ "Human lysozyme: sequencing of a cDNA, and expression and secretion by Saccharomyces cerevisiae". Biochemical and Biophysical Research Communications 150 (2): 794–801. January 1988. doi:10.1016/0006-291X(88)90461-5. PMID 2829884. 
  4. ↑ "The human lysozyme gene. Sequence organization and chromosomal localization". European Journal of Biochemistry 182 (3): 507–516. July 1989. doi:10.1111/j.1432-1033.1989.tb14857.x. PMID 2546758. 
  5. ↑ "Thermal stability of high concentration lysozyme across varying pH: A Fourier Transform Infrared study". Journal of Pharmacy & Bioallied Sciences 5 (2): 148–153. April 2013. doi:10.4103/0975-7406.111821. PMID 23833521. 
  6. ↑ "Lysozyme Content of Human Milk". Nature 204 (4953): 76–77. October 1964. doi:10.1038/204076a0. PMID 14240122. Bibcode: 1964Natur.204...76C. 
  7. ↑ "Lysozyme, Product information". Sigma-Aldrich. https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Datasheet/7/l7651dat.pdf. 
  8. ↑ "Lysozyme, Product information". Sigma-Aldrich. https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Datasheet/7/l7651dat.pdf. 
  9. ↑ "Isolation and characterization of human milk lysozyme". Archives of Biochemistry and Biophysics 130 (1): 59–65. March 1969. doi:10.1016/0003-9861(69)90009-5. PMID 5778672. 
  10. ↑ "Adsorption and reactions of chitinase and lysozyme on chitin". Molecular and Cellular Biochemistry 2 (2): 221–228. December 1973. doi:10.1007/BF01795475. PMID 4359167. 
  11. ↑ "The chemical structure of lysozyme substrates and their cleavage by the enzyme". Proceedings of the Royal Society of London. Series B, Biological Sciences 167 (1009): 402–415. April 1967. doi:10.1098/rspb.1967.0037. PMID 4382803. Bibcode: 1967RSPSB.167..402S. 
  12. ↑ "Lysozyme Substrates". Lysozymes: Model Enzymes in Biochemistry and Biology. Experientia Supplementum. 75. 1996-01-01. pp. 105–110. doi:10.1007/978-3-0348-9225-4_7. ISBN 978-3-0348-9952-9. 
  13. ↑ "Crystallographic studies of the activity of hen egg-white lysozyme". Proceedings of the Royal Society of London. Series B, Biological Sciences 167 (1009): 378–388. April 1967. doi:10.1098/rspb.1967.0035. PMID 4382801. Bibcode: 1967RSPSB.167..378B. 
  14. ↑ 14.0 14.1 "Application of secondary α-deuterium kinetic isotope effects to studies of enzyme catalysis. Glycoside hydrolysis by lysozyme and β-glucosidase". Biochemistry 8 (10): 4214–4221. October 1969. doi:10.1021/bi00838a045. PMID 5388150. 
  15. ↑ "Lysozyme and α-lactalbumin: structure, function, and interrelationships". Advances in Protein Chemistry 41: 173–315. 1991. doi:10.1016/s0065-3233(08)60198-9. ISBN 9780120342419. PMID 2069076. 
  16. ↑ "Energetics of enzyme catalysis". Proceedings of the National Academy of Sciences of the United States of America 75 (11): 5250–5254. November 1978. doi:10.1073/pnas.75.11.5250. PMID 281676. Bibcode: 1978PNAS...75.5250W. 
  17. ↑ "Temperature-dependent change in the rate-limiting step of β-glucosidase catalysis". The Journal of Biological Chemistry 255 (19): 9030–9032. October 1980. doi:10.1016/S0021-9258(19)70521-3. PMID 6773958. 
  18. ↑ "Hen Egg-White (HEW) Lysozyme - Proteopedia, life in 3D". http://proteopedia.org/wiki/index.php/Lysozyme#Covalent_intermediate_and_product_complex. 
  19. ↑ 19.0 19.1 "Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate". Nature 412 (6849): 835–838. August 2001. doi:10.1038/35090602. PMID 11518970. Bibcode: 2001Natur.412..835V. https://eprints.whiterose.ac.uk/131/1/daviesgj1.pdf. 
  20. ↑ "Chapter 14: Mechanism of enzyme action". Biochemistry. Australia: Thomson Brooks/Cole. 2007. pp. 467–9. ISBN 978-0-495-11912-8. https://books.google.com/books?id=W4o_5YGqfYsC&q=lysozyme%20mechanism%20of%20action%20glu-35%20asp-52&pg=PA468. 
  21. ↑ "Stereochemistry and the Mechanism of Enzymatic Reactions". Biological Reviews 28 (4): 416–436. November 1953. doi:10.1111/j.1469-185X.1953.tb01386.x. https://digital.library.unt.edu/ark:/67531/metadc1255185/. 
  22. ↑ 22.0 22.1 "QM/MM simulations predict a covalent intermediate in the hen egg white lysozyme reaction with its natural substrate". Chemical Communications (37): 4425–4427. October 2008. doi:10.1039/b810099c. PMID 18802578. 
  23. ↑ "The inhibition of hen egg-white lysozyme by imidazole and indole derivatives". Journal of Molecular Biology 65 (1): 59–62. March 1972. doi:10.1016/0022-2836(72)90491-3. PMID 5063023. 
  24. ↑ "Lipopolysaccharide interaction with lysozyme. Binding of lipopolysaccharide to lysozyme and inhibition of lysozyme enzymatic activity". The Journal of Biological Chemistry 264 (8): 4434–4441. March 1989. doi:10.1016/S0021-9258(18)83761-9. PMID 2647736. 
  25. ↑ "Genetic evidence that antibacterial activity of lysozyme is independent of its catalytic function". FEBS Letters 506 (1): 27–32. September 2001. doi:10.1016/S0014-5793(01)02872-1. PMID 11591365. 
  26. ↑ "Lysozyme's lectin-like characteristics facilitates its immune defense function". Quarterly Reviews of Biophysics 50: e9. January 2017. doi:10.1017/S0033583517000075. PMID 29233221. 
  27. ↑ Lysozyme: Antigenic structure as defined by antibody and T cell responses. CRC Press. 1996. pp. 91–144. ISBN 978-0-8493-9225-2. 
  28. ↑ "Single-molecule lysozyme dynamics monitored by an electronic circuit". Science 335 (6066): 319–324. January 2012. doi:10.1126/science.1214824. PMID 22267809. Bibcode: 2012Sci...335..319C. 
  29. ↑ Wohlkönig, Alexandre; Huet, Joëlle; Looze, Yvan; Wintjens, René (9 November 2010). "Structural Relationships in the Lysozyme Superfamily: Significant Evidence for Glycoside Hydrolase Signature Motifs". PLOS ONE 5 (11): e15388. doi:10.1371/journal.pone.0015388. PMID 21085702. Bibcode: 2010PLoSO...515388W. 
  30. ↑ Irwin, David M (December 2014). "Evolution of the vertebrate goose-type lysozyme gene family". BMC Evolutionary Biology 14 (1): 188. doi:10.1186/s12862-014-0188-x. PMID 25167808. 
  31. ↑ "Lactoferrin and lysozyme deficiency in airway secretions: association with the development of bronchopulmonary dysplasia". The Journal of Pediatrics 121 (2): 262–270. August 1992. doi:10.1016/S0022-3476(05)81201-6. PMID 1640295. https://zenodo.org/record/1259643. 
  32. ↑ "Consuming transgenic goats' milk containing the antimicrobial protein lysozyme helps resolve diarrhea in young pigs". PLOS ONE 8 (3): e58409. 2013. doi:10.1371/journal.pone.0058409. PMID 23516474. Bibcode: 2013PLoSO...858409C. 
  33. ↑ "Spilled Milk". Case Studies: News Features. Undark: Truth, Beauty, Science. 30 June 2016. http://undark.org/article/gmo-goats-lysozyme-uc-davis-diarrhea/. 
  34. ↑ Microbiology: A Human Perspective (5th ed.). Boston, Mass.: McGraw-Hill Higher Education. 2007. ISBN 978-0-07-110706-8. 
  35. ↑ "Immunological aspects of human milk". Nutrition Reviews 36 (9): 265–272. September 1978. doi:10.1111/j.1753-4887.1978.tb07393.x. PMID 362248. 
  36. ↑ "Serum lysozyme levels and clinical features of sarcoidosis". Lung 177 (3): 161–167. 1999. doi:10.1007/pl00007637. PMID 10192763. 
  37. ↑ "The Bakerian lecture. Towards synthesis of proteins". Proceedings of the Royal Society of London. Series B, Biological Sciences 197 (1128): 237–253. June 1977. doi:10.1098/rspb.1977.0068. PMID 19745. Bibcode: 1977RSPSB.197..237K. 
  38. ↑ "Convergent chemical synthesis and high-resolution x-ray structure of human lysozyme". Proceedings of the National Academy of Sciences of the United States of America 104 (12): 4846–4851. March 2007. doi:10.1073/pnas.0610630104. PMID 17360367. Bibcode: 2007PNAS..104.4846D. 
  39. ↑ "Time-dependent, protein-directed growth of gold nanoparticles within a single crystal of lysozyme". Nature Nanotechnology 6 (2): 93–97. February 2011. doi:10.1038/nnano.2010.280. PMID 21278750. Bibcode: 2011NatNa...6...93W. 
  40. ↑ "Expanding coordination chemistry from protein to protein assembly". Chemical Communications 49 (39): 4114–4126. May 2013. doi:10.1039/C2CC36935D. PMID 23211931. 
  41. ↑ "Porous protein crystals as reaction vessels". Chemistry: A European Journal 19 (28): 9096–9102. July 2013. doi:10.1002/chem.201300250. PMID 23813903. 
  42. ↑ "Lysis of gram-negative bacteria by lysozyme". Biochimica et Biophysica Acta 22 (1): 189–191. October 1956. doi:10.1016/0006-3002(56)90240-2. PMID 13373865. 
  43. ↑ Protein Condensation : Kinetic Pathways to Crystallization and Disease. Cambridge: Cambridge University Press. 2007. pp. 156–158. ISBN 9780511535321. https://archive.org/details/proteincondensat00gunt. 
  44. ↑ Fundamental Laboratory Approaches for Biochemistry and Biotechnology. John Wiley. 2010. ISBN 978-0470087664. 
  45. ↑ "The properties of lysozyme and its action on microorganisms". Bacteriological Reviews 21 (2): 82–100. June 1957. doi:10.1128/MMBR.21.2.82-100.1957. PMID 13436356. 
  46. ↑ "Lysozyme contamination facilitates crystallization of a heterotrimeric cortactin-Arg-lysozyme complex". Acta Crystallographica. Section F, Structural Biology and Crystallization Communications 68 (Pt 2): 154–158. February 2012. doi:10.1107/S1744309111056132. PMID 22297987. 
  47. ↑ "Biochemical and structural characterization of an aromatic ring-hydroxylating dioxygenase for terephthalic acid catabolism". Proceedings of the National Academy of Sciences of the United States of America 119 (13): e2121426119. March 2022. doi:10.1073/pnas.2121426119. PMID 35312352. Bibcode: 2022PNAS..11921426K. 
  48. ↑ "Reduced background expression and improved plasmid stability with pET vectors in BL21 (DE3)". BioTechniques 29 (6): 1234–1238. December 2000. doi:10.2144/00296st03. PMID 11126126. 
  49. ↑ "Über die keimtötende und entwicklungshemmende Wirkung Hühnereiweiß" (in de). Zeitschrift für Hygiene und Infektionskrankheiten 64: 419–427. 1909. doi:10.1007/BF02216170. 
  50. ↑ "Ernest Duchesne and the concept of fungal antibiotic therapy". Lancet 354 (9195): 2068–2071. December 1999. doi:10.1016/S0140-6736(99)03162-1. PMID 10636385. 
  51. ↑ "On a remarkable bacteriolytic element found in tissues and secretions". Proceedings of the Royal Society B 93 (653): 306–317. May 1922. doi:10.1098/rspb.1922.0023. Bibcode: 1922RSPSB..93..306F. 
  52. ↑ Advances in Protein Chemistry. Academic Press. 13 June 1991. pp. 176–. ISBN 978-0-08-058214-6. https://books.google.com/books?id=U1P3a5hjbSAC&pg=PA176. 
  53. ↑ "Structure of hen egg-white lysozyme. A three-dimensional Fourier synthesis at 2 Angstrom resolution". Nature 206 (4986): 757–761. May 1965. doi:10.1038/206757a0. PMID 5891407. 
  54. ↑ "Structure of some crystalline lysozyme-inhibitor complexes determined by X-ray analysis at 6 Angstrom resolution". Nature 206 (4986): 761–763. May 1965. doi:10.1038/206761a0. PMID 5840126. 
  55. ↑ "The early history of lysozyme". Nature Structural Biology 5 (11): 942–944. November 1998. doi:10.1038/2917. PMID 9808036. 
  56. ↑ "The Amino Acid Sequence of Egg White Lysozyme". The Journal of Biological Chemistry 238 (8): 2698–2707. August 1963. doi:10.1016/S0021-9258(18)67888-3. PMID 14063294. 
  57. ↑ "The mechanisms of hydrolysis of glycosides and their revelance [sic] to enzyme-catalysed reactions". Proceedings of the Royal Society of London. Series B, Biological Sciences 167 (1009): 389–401. April 1967. doi:10.1098/rspb.1967.0036. PMID 4382802. Bibcode: 1967RSPSB.167..389V. 
  58. ↑ "The binding and cleavage by lysozyme of N-acetylglucosamine oligosaccharides". Proceedings of the Royal Society of London. Series B, Biological Sciences 167 (1009): 416–428. April 1967. doi:10.1098/rspb.1967.0038. PMID 4382804. Bibcode: 1967RSPSB.167..416R. 
  59. ↑ "The chemical structure of lysozyme substrates and their cleavage by the enzyme". Proceedings of the Royal Society of London. Series B, Biological Sciences 167 (1009): 402–415. April 1967. doi:10.1098/rspb.1967.0037. PMID 4382803. Bibcode: 1967RSPSB.167..402S. 

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