The first place that I looked for information about my enzyme is the protein data base molecule of the month page. Here I learned that Lysozyme is an enzyme that attacks bacteria. It does so by breaking the carbohydrate chains that are produced in the bacteria as a cell wall. The cell wall will be destroyed and the cell will rupture due to its turgor pressure. Lysozyme was discovered as an accidental antibiotic because of its characteristics. It is also because of its characteristics that it is located in plants, eyes, blood and other areas that are susceptible to potential bacteria growth. This information was provided by the molecule of the month page on the protein data bank.
1) http://www.scientificsocieties.org/jib/papers/2010/G-2010-0330-1033.pdf
There are two things that college students will remember when looking back at their college experience. The first is their experience in biochem and the second is beer. My first journal about lysozyme brings the two together. The authors of this paper use the enzyme lysozyme to counteract the bacteria that cause beer spoilage. Bacteria go through lactic acid fermentation for metabolism. The resulting lactic acid spoils beer by raising the acidic acid and giving an undesirable flavor. This causes the brewing industry financial burden. Both gram-positive and negative bacteria cause beer spoilage, so the authors want to find a cost effective solution to this problem. Lysozyme is an antimicrobial enzyme that cleaves the β(1→4) glycosidic bond in the cell walls of the bacteria. This is also cost effective because lysozyme can be produced in an industrial level from egg whites. It is also a natural solution which is appealing to customers. The study used four breweries and 37 different unpasteurized beers. Each beer was tested to show a high ability for bacterial spoilage. Duplicate studies were done to the beers during the bottling stage of production. Each of the beers was analyzed with and without lysozyme addition. The study also performed “taste tests” on the beer with lysozyme added to make sure that the added protein did not affect taste at all. The results of the lab showed that the addition of lysozyme is able to prolong the shelf life of unpasteurized beer. There was also noted an increase in flavor and texture when the lysozyme was added. Further studies are being carried out to understand these phenomena and to determine how the quality of beer can be improved. Who wouldn’t think that lysozyme is the greatest protein if it can give us better longer lasting beer!
2) http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0016788
The second article that I found was a little more important than improving beer sales. It has to do with the human immune system. It is known that lysozyme is a key part in the human immune system to fight off bacterial infections. However, bacteria are able to mutate and become resistant to lysozyme through inhibition. Some serious infections occur due to lysozymes functional limitations to overcome the resistance. Cystic fibrosis and pneumonia are due to resistant bacterial infections. Both of these infections are in the lungs. Medical research has attemeted to administer lysozyme through inhalation technology, however the protein has a dense positive charge that be easily be deactivated by the negative bacteria. The authors if this lab have engineered a lysozyme with enhanced anti-bacterial activity by mutating two of its amino acids.
They did this by taking the human lysozyme as a starting template, over 150,000 mutants were screened for anti-bacterial activity and stability. A double mutated lysozyme Arg101→Asp and Arg115→His was found to have the best anti-bacterial activity in presence of the bacteria’s inhibitors. Studies showed that the mutation did not diminish the enzymes Km or Vmax in vitro, and actually proved to be kinetically faster than the wild type.
Through structural and sequence studies it was shown that the mutation had little effect on the active site because the mutations were on opposite sides of the “substrate binding groove.” This means the mutations do not noticeably change the function of the enzyme, but only prevent the inhibitory effect of resistant bacteria. Through stability analysis the authors were able to determine that the stability of the enzyme when subject to aggregation and proteases was no worse than wild type lysozyme. These authors have engineered the first lysozyme that can kill both gram negative and positive bacteria under inhibitory conditions. This enzyme can help to specifically combat cystic fibrosis bacteria, and also provide insight to effectively combating other resistant bacteria.
3) http://www.jstor.org.lib-proxy.calvin.edu/stable/34573?&Search=yes&searchText=lysozyme&list=hide&searchUri=%2Faction%2FdoAdvancedSearch%3Fq0%3Dlysozyme%2B%26f0%3Dall%26c1%3DAND%26q1%3D%26f1%3Dall%26acc%3Don%26wc%3Don%26Search%3DSearch%26sd%3D%26ed%3D%26la%3D%26jo%3D&prevSearch=&item=3&ttl=7664&returnArticleService=showFullText
This journal article explains that the main goal to protein engineering is stability. And disulfide bonds add a significant amount to tertiary structure stability, adding to the over all stability when altering proteins. However the addition of disulfide bonds does not always increase stability. So, the authors of this paper tried to identify the mechanism to which additional disulfide bonds increase stability. First the authors had to determine spots to introduce disulfide bonds. The positions of CYS residues were determined. Then all potential pairs were scanned into the computer to determine pairs that satisfied three requirements. Separated by 20 residues, must have Υ atoms, and they must be 6 A away. Next they tried to determine the strain due to the addition of each disulfide bond. And finally they analyzed the stabilizing interactions like hydrogen bonding and vanderwalls that contribute to protein stability. The authors then engineered four mutants by sight mutations. The results of the study showed several points relating disulfide bonds and stability. First introduction of CYS residues minimize the disruption that stabilize the native structure. Second, the size of the loop formed by the crosslink should be as large as possible. Next, the strain energy should be kept as low as possible, because the disulfide bonds with the lowest calculated strain had the lowest redox potential resulting in a more stable enzyme.
This journal displays another great attribute about lysozyeme. It gives us a template enzyme that we can easily study to determine the characteristics of more complex enzymes. Lysozyme allows us to learn how to develop and stabilize more complex proteins, this can have major medical implications.
