In The Name Of God

1. In The Name Of God Lipase enzyme Dr. Bordbar By: Kamyar Khoshnevisan

2. Introduction • lipase or lipolytic enzyme is water-soluble enzyme that catalyze the hydrolysis of ester bonds in water-insoluble,lipid substrates. • Lipases are subclass of the esterases. • Lipases perform essential roles in the digestion, transport and processing of dietary lipids (e.g. triglycerides, fats, oils) in most- if not all- living organisms. Genes encoding lipases are even present in certain viruses.(Ec 3.1.1.34)

3. Triglyceride+water lipase glycerol+3fatty acids(figureI.12) Most lipases act at a specific position on the glycerol backbone of a lipid substrate. In the example of human pancreatic lipase (HPL), which is the main enzyme responsible for breaking down fats in the humandigestive system, a lipase acts to convert triglyceride substrates found in oils from food to monoglycerid and free fatty acids.

5. Lipases are very versatile enzymes that catalyze a large number of reactions . They catalyze the hydrolysis of AGs and a wide range of other esters(simple esters,phospholipi ds,acylglycosides,etc),including non-natural su bstrate. lipase are capable of performing the reverse reaction (ester synthesis).

6. Structure:While a diverse array of genetically distinct lipase enzymes are found in nature, and represent several types of protein folds and catalytic mechanisms, most are built on an alpha/beta hydrolase fold and employ a chymotrypsin-like hydrolysis mechanism involving a serinenucleophile, an acid residue (usually aspartic acid), and a histidine.

7. Schematic representation of the canonical α/β hydrolase fold.

9. Active site:The active site of the α/β hydrolase fold enzymes has three catalytic residues:a nucleophilic residue (serine, cysteine, or aspartate), a catalytic acid residue (aspartate or glutamate), and a histidine residue, always placed in this order in the amino acid sequence Nevertheless, this order is different from that observed in any of the other proteins that also contain catalytic amino acid triads.

10. Production&extraction: Bacteria produce different classes of lipolytic enzyme. Most of the lipases used in industry are microbial enzymes, of both fungal and bacterial origin. Pseudomonas are great classes for production lip- ase. The great versatility of fungal lipases (from genera such as Candida, Geotrichum, Rhizopus and thermomyces) .

11. Animal lipase: Normal New Zealand White rabbits and monkeys (Macaca mulatta) were fed standard chow diets or diets supplemented with cholesterol, coconut oil, and Menhaden fish oil until sacrifice. Pancreatic enzymes derived from cattle and pork are also available.

12. Lipase activity: • Influnce of carbon source on lipase production: • In GKM medium, glucose was replaced by other carbon sources.the best result was observed for sucrose present in the medium. • The activity of lipase synthesized under these conditions was of 118 U/ml.glucose and starch were also good carbon source for growth and production of lipase.

13. Effect of carbon source on lipase activity

14. Effect of nitrogen source on lipase production: • The effect of nitrogen source was tested in the GKM medium by removing urea and replacing it with selected nitrogen sources ( both organic and inorganic). • As shown in Fig. 3, the best result was observed for urea at a concentration of 0.4% (lipase production improved to 146 U/ml).

16. Optimization of lipase production and purification: • improving fermentation conditions such as carbon or nitrogen source supply, temperature, pH, or aeration. • using inductors for lipase expression and synthesis. • cloning and overexpresion of the lipase of interest in a host capable of producing and, if possible, secrete large amounts of enzyme, which sometimes requires the modification of the wild lipase-coding gene to add/change its signal peptide or certain codons to adapt them to those more suitable for the host.

17. improving the purification process

18. METHODS FOR LIPASE ACTIVITY DETERMINATION: • Plate assay • Spectrophotometric methods (colorimetric assays) • Spectrofluorimetric methods • Chromatographic assays • Titrimetric methods • Radiometric methods • Tensiometric methods

19. Properties of lipases useful in biotechnology: • Versatility: lipases are very versatile biocatalysts which can carry out many different reactions of hydrolysis and, in organic solvents, synthesis and acyl exchange, using a wide range of natural and non-natural compounds. • Specificity and selectivity: some of them show high substrate specificity, or high chemo-, regio-and stereoselectivity. • Absence of subproducts: most lipases do not perform lateral reactions.

20. Stability: lipases are active and stable in organic solvents, and in a wide range of pH and temperat-ures. • Knowledge: their structure and function is well known, and they can be modified to adapt them to novel uses. • Availability: lipases, mainly those which are secreted, are available in large amounts by fermentation processes of natural or recombinant strains. • No cofactors: most lipases do not require cofactors.

21. Low-cost and green: processes involving the use of lipases have a lower cost and are less polluting, because these enzymes act under mild conditions and with low energy and equipment requirements.

22. Biotechnological applications of lipases: • Food industry • Organic chemistry • Paper industry • Management of waste and toxic compounds

23. Kinetics: • The experimental data from the concentration variation experiments were fitted using the Michaelis-Menten model for enzyme reaction kinetics.

24. Km = 1480 mol TA/m3 and • Vmax = 1.2 x10-5 mol OH-/m2.s • Figure 3 shows that the studied hydrolysis reaction fully obeys Michaelis-Menten kinetics. • The experimental results obtained with the Andante membrane reactor are also plotted in Figure3. The Vmax for the Andante cellulose membrane reactor was nearly the same as for the Sepracor membrane reactor (1.1*10-5molOH-/ms). However,the Michaelis-Menten constant was smaller (approximately500 mol/m3).

28. thanks a lot for your attention