HistoryEven reaction 107 times than without enzyme.In

HistoryEven that the first use of the term “Enzyme” was less than 150 years ago, using enzymes in our daily life was centuries before that.Eating vegetables rich in enzymes, baking (which depends on yeast enzymes), and using leaves to promote healing of wounds, are all examples of enzyme usage throughout the ages.One of the earliest discoveries of enzymes starts with a debate between the French biologist and chemist Louis Pasteur and the German physiologist Wilhelm Kühne. They debate about the fermentation of sugar into alcohol, Pasteur thinks that the process depends on the yeast viability and cannot occur without the presence of a living organism. Kühne, on the other hand, thinks it’s the result of a chemical process and the yeast viability has nothing to do with the process.It wasn’t until the death of both scientists when the German chemist Eduard Buchner studied the yeast extracts and found that the fermentation happened even there were no viable yeast cells. He published his paper twenty years after Kühne first used the term Enzyme.Enzymology backgroundEnzymes are macromolecules biological catalysts that show a high degree of specificity to their substrates. Their function is to speed up biochemical reactions taking place in biological systems. Without enzymes, reactions take place at a very slow rate.For example, The hydrolysis of carbon dioxide.This reaction is catalyzed by an enzyme called carbonic anhydrase. The presence of enzyme speeds up the reaction 107 times than without enzyme.In fact, carbonic anhydrase is one of the fastest enzymes known. Each enzyme molecule can hydrate 106 molecules of CO2 per second.All enzymes are proteins except catalytic RNA molecules. As it’s known protein functions are based on their 3D structure (conformation). That means the primary, secondary, tertiary and quaternary structures play an important role in enzymes catalytic activity.Many enzymes depend on the presence of prosthetic group to do their job. A prosthetic group may be a cofactor (inorganic ions such as Mg2+, Mn2+ or Zn2+) or a coenzyme (small organic or metalloorganic molecules such as pyridoxal phosphate).    An enzyme without its cofactor is referred to as an apoenzyme while the complete, catalytically active enzyme is called a holoenzyme.Some enzymes require both a coenzyme and one or more cofactor for activity. Others do not need any external assistance.How enzymes workTemperature, pH and aqueous environment of the cell is not the most favoured conditions for many reactions to take place. The active site of enzyme takes the shape of a pocket, this conformation provides a proper environment for the reaction by shielding the substrate from the solvent. Creating suitable conditions by this shielding or bringing two substrates together in the right orientation lowers its activation energy. The portion of the substrate undergoing chemical changes meets a specific site containing amino acid residues that catalyze the chemical transformation. Specificity of enzymes is attributed to the amino acid sequence in the active site. Biofilms and Antibiotic resistanceAlexander Fleming, the Nobel prize winner for the discovery of Ampicillin, the world’s first antibiotic, once said: “There is probably no chemotherapeutic drug to which in suitable circumstances the bacteria cannot react by in some way acquiring resistance”.Most bacteria tend to live in groups, they form what is known as biofilms. The formation of biofilms starts with the bacteria being adsorbed and attached on a solid material, and then they start to grow and secrete polysaccharides and other molecules. On on those molecules is quorum sensing molecule, which helps the bacteria sensing and communicating with other bacteria.Biofilm is the perfect structure for the microbes, helping them to overcome drugs and antibiotics by allowing the transfer of genes (including drug-resistance genes) between bacterial cells.This figure shows a Scanning electron micrograph of biofilm contaminating the inner surface of a medical device. And as we can tell from Fleming’s comment that was mentioned earlier, The bacterial resistance is not a new thing, yet, it keeps getting harder and harder for us to combat. The current rate of new antibiotics development isn’t enough to keep us ahead in the battle with bacteria.A new strategy to combat microbes is via bio-based materials. And one of those is antimicrobial enzymes.Antimicrobial enzymesAntimicrobial enzymes are found naturally in many organisms to protect themselves against bacteria. Scientists exploit this advantage to replace antibiotics. Anti-microbial enzymes can destroy microorganism by destroying the biofilm or catalyzing reactions that produce antimicrobial compounds. One enzyme or more is used to produce this formulation, It may also combine with other antimicrobial or anti-biofilm agents.1. Proteolytic enzymes    Proteases are enzymes that hydrolyze proteins. Proteases may be exopeptidase or endopeptidase depending on whether the cleavage is terminal or not.  Subtilisins :    Several surface proteins (adhesions) have been shown to be essential in the adhesion of many bacteria species to surfaces and solid support. If bacteria failed to attach to surfaces, biofilm formation stops. Subtilisins are non-specific proteases produced by Bacillus sp. that can hydrolyze adhesions.Treatment of bacteria with subtilisins was tested and it reduced effectively the ability of bacteria to form biofilm colonies. Scientists are working now on subtilisin-like proteases produced by hyperthermophiles to remove biofilms in heat exchangers. 2. Polysaccharide-degrading enzymes Bacterial cell wall and membrane are associated with a variety of glycoconjugates and polysaccharides which aids in structural formation as well as performing various functions in the bacterial cell.    Alginate lyase is antimicrobial enzyme that, which cleaves ?-glycosidic bonds of bacteria alginate polymer (structural co-polymer Alginate made up of alpha-L-guluronate and beta-D-mannuronate assist in building the cell walls) Mechanism of the alginate lyase:    (i) removal of the negative charge on the carboxylate anion(ii) abstraction of the proton on C5(iii) ?-elimination of the 4-O-glycosidic bond (lyase)Figure 4. Alginate lyase catalyzes the hydrolysis of alginate, a copolymer of a-L-guluronate (G) and C5 epimer-D-mannuronate (M), through ?-elimination.P. aeruginosa ( Gram-negative pathogenic bacteria) is colonizing the respiratory tracts of patients with cystic fibrosis. Treating patients with a combination of alginate lyase and gentamicin is an effective treatment. A genetically engineered A1-III alginate lyase immobilized on PEG removed more than 90% of bacteria adherent polymers of P. aeruginosa (wound pathogen).     DNA from dead microorganisms play an important role in crosslinking of biofilms due to its electronegativity. Deoxyribonuclease is used to hydrolyze this DNA in cystic fibrosis mucus. Combining DNAses with alginate lyase leads to better results. Using a mixture of a protein-degrading enzyme (protease) and polysaccharide-degrading enzymes (alpha-amylase, ?-glucuronidase, glucose oxidase, dextranase and pectinase) achieves more effective results when targeting biofilms.3. Anti-quorum sensing enzymes Other developing groups of antimicrobial enzymes include the so-called “anti-quorum sensing” enzymes. Bacteria use quorum sensing to regulate various physiological activities, including virulence, competence, conjugation, antibiotic and bacteriocin production, motility, and spore and biofilm formation. Among the quorum-sensing molecules proposed as targets are the acyl homoserine lactones (AHLs), which are implicated in the regulation of bacterial virulence in greater than 50 bacterial species. Scientists have also discovered quorum-quenching enzymes such as the lactonases which hydrolyze the ester bond of the homoserine lactone ring of acylated homoserine lactones, in that way preventing AHLs from binding to their target transcriptional regulators. AHL acylase hydrolyses the amide linkage between the acyl chain and the homoserine moiety of AHL molecules. By suppressing the virulence of pathogenic microorganisms, these enzymes are likely to play major biotechnological role in agriculture and health industries.Challenges in application of enzymes as antimicrobial and anti-biofilm agentsThe high cost of enzyme production is a very challenging obstacle, especially for biomedical uses because pure enzymes are required. A synthetic biology may provide the solution, by integrating the genes coding for different antimicrobial enzymes into a single host. Some microorganisms gain resistance against anti-microbial enzymes. This has been attributed to mutations in their genes.Example: femA gene which encodes the factor responsible for the addition of the second and third glycines residues to the pentaglycine bridge of the cell wall. This addition results in the replacement of glycine residues with serine residues, disrupting the binding of lysostaphin onto the pentaglycine cross-bridge and subsequently blocking the influence of lysostaphin.References:Oxford Companion to the History of Modern SciencePrinciples of Biochemistry (6th ed.)Lubert Stryer Biochemistry (5th ed.)Biofilms: Microbial Life on Surfaces: Rodney M. Donlan (ncbi.nlm.nih.gov/pmc/articles/PMC2732559)Antimicrobial enzymes: An emerging strategy to fight microbes and microbial biofilms (Thallinger, Barbara, et al. Biotechnology journal. 2013 Jan 1;8(1):97-109.)


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