Lipolytic enzyme

In order to carry out PHA production from triglycerides, the cells need to hydrolyze the triglycerides into free fatty acids that can be transported into the cells. For this, the cells need to secrete an enzyme called lipase. A wide variety of organisms is known to secrete lipolytic enzymes, mainly for lipid metabolism and signal transduction. Lipolytic enzymes can be classified into different classes, including lipases, esterases, and phospholipases (Arpigny and Jaeger 1999). Esterases are enzymes that hydrolyze ester bonds of soluble or partially soluble molecules. 

For the oil-utilizing bacteria, extracellular lipolytic enzymes are secreted to the medium. As proteins, lipolytic enzymes need to be translocated across the membrane of bacteria. There are four pathways for lipolytic enzyme translocation in bacteria (Jaeger and Eggert 2002 Rosenau and Jaeger 2000) (Fig. 2.9). 

Basic Protocol 1 Titrimetric Determination of Lipase Activity Basic Protocol 2 Colorimetric Assay of Lipase Activity Using the Copper Soap Method 

Basic Protocol 3 Spectrophotometric Determination of Lipase Activity Using p-Nitrophenyl Laurate as Substrate Reagents and Solutions Commentary 

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There is quite a large body of literature on films of biological substances and related model compounds, much of it made possible by the sophisticated microscopic techniques discussed in Section IV-3E. There is considerable interest in biomembranes and how they can be modeled by lipid monolayers [35]. In this section we briefly discuss lipid monolayers, lipolytic enzyme reactions, and model systems for studies of biological recognition. The related subjects of membranes and vesicles are covered in the following section. 

Manufacture of Fatty Acids and Derivatives. Splitting of fats to produce fatty acids and glycerol (a valuable coproduct) has been practiced since before the 1890s. In early processes, concentrated alkaU reacted with fats to produce soaps followed by acidulation to produce the fatty acids. Acid-catalyzed hydrolysis, mostly with sulfuric and sulfonic acids, was also practiced. Pressurized equipment was introduced to accelerate the rate of the process, and finally continuous processes were developed to maximize completeness of the reaction (105). Lipolytic enzymes maybe utilized to spHt... 

Cutinase is a hydrolytic enzyme that degrades cutin, the cuticular polymer of higher plants [4], Unlike the oflier lipolytic enzymes, such lipases and esterases, cutinase does not require interfacial activation for substrate binding and activity. Cutinases have been largely exploited for esterification and transesterification in chemical synthesis [5] and have also been applied in laundry or dishwashing detergent [6]. 

The general rules that should therefore be observed include the use of a blanket of nitrogen whenever possible and evaporation of solvents at the lowest feasible temperatures, which must not exceed 50°C. The addition of an antioxidant such as butylated hydroxytoluene (2,6-di-/-butyl-4-methylphenol) to the extraction solvents (0.1 g 1 ) might be necessary to prevent deterioration of unsaturated lipids but it is essential for storage of lipid extracts at about 0.1% of the weight of lipid. Inactivation of lipolytic enzymes may usually be achieved by addition of an alcohol such as methanol or, in some cases, isopropanol. The latter is recommended for some more stable enzymes sometimes found in plant tissues. Alternatively the plant may be briefly immersed in boiling water. 

The best-known effect of APOE is the regulation of lipid metabolism (see Fig. 10.13). APOE is a constituent of TG-rich chylomicrons, VLDL particles and their remnants, and a subclass of HDL. In addition to its role in the transport of cholesterol and the metabolism of lipoprotein particles, APOE can be involved in many other physiological and pathological processes, including immunoregu-lation, nerve regeneration, activation of lipolytic enzymes (hepatic lipase, lipoprotein lipase, lecithin cholesterol acyltransferase), ligand for several cell receptors, neuronal homeostasis, and tissue repair (488,490). APOE is essential... 

Diacylglycerol is a better snbstrate than triacylglycerol for the lipolytic enzymes that are secreted by the pancreas. 

Selected entries from Methods in Enzymology [vol, page(s)] Activation of lipolytic enzymes by interfaces, 64, 341 model for lipase action on insoluble lipids, 64, 345 interfacial enzyme inactivation, 64, 347 reversibility of the adsorption step, 64, 347 monolayer substrates, 64, 349 kinetic models applicable to partly soluble amphiphilic lipids, 64, 353 surface dilution model, 64, 355 and 364 practical aspects, 64, 357. 

Triton X-100 has proved to be of great value in the surface dilution modeb for lipolytic enzyme action. In this experimental strategy, the surface concentration of phospholipid in mixed micelles is reduced by the addition of Triton as a neutral diluent, thereby increasing the average distance between phospholipids. This allows one to draw mechanistic inferences about the binding interactions of lipases and phospholipases with their lipid sub-stratesb... 

Fig. 3 Top row. Hydrolysis of PET with 7% crystallinity with a Humicola insolens cutinase for (a) 0, (b) 12 and (c) 48 h resulting in weight losses of 0, 18% and 54%, respectively. Bottom row. Hydrolysis of PET with (a) 34.8% crystallinity (blank), (b) a lipolytic enzyme (no weight loss) and (c) 1 M NaOH (modified from [14, 18])...

Hermetter, H., Paltauf, F., Zenzmaier, E., Schlacher, A., Schwab, H. and Faber, K. (1998) Fluorescense screening for lipolytic enzymes. In New frontiers in screening for microbial catalysis, edited by K.Kieslich,... 

Garton etal, (10) demonstrated that rumen microbial suspensions could hydrolyze triglycerides. It was later established that virtually any ester link between fatty acid and glycerol was subject to hydrolytic cleavage by rumen organisms (7 7). As aconsequence of the activity of the lipolytic enzymes, high levels of free fatty acids are produced in the rumen. The unsaturated fatty acids are substrates in biohydrogenation reactions. 

Different purified or partially purified enzymes were tested successfully, such as horse liver dehydrogenase [3], Sulfolobus solfataricus dehydrogenase [4], Pischia pastoris alcohol oxidase [5, 6], the baker s yeast alcohol dehydrogenase [7], and finally lipolytic enzymes, which probably constitute the major part of the work devoted to the use of enzymes working at the solid/gas interface, as summarized in a recent publication [8]. 

Deeth, H.C. and Fitz-Gerald, C.H. (1995) Lipolytic enzymes and hydrolytic rancidity in milk and milk products, in Advanced Dairy Chemistry, Vol. 2 Lipids, 2nd edn (ed. P.F. Fox), Chapman Hall, London, pp. 247-308. 

The second observation which does not support the unfolded protein model is that when phospholipase A (N. naja venom) was injected into the subphase under the lipid monolayer at equilibrium with globulin, lecithin was readily attacked, as indicated by the rapid fall of surface potential (4, 5, 6). If the penetrated protein were to cover entirely the polar groups of the lipid facing the aqueous subphase (as postulated in the unfolded protein model), the lipid molecules should not be accessible to the lipolytic enzyme. 

Parmesan or Grana, as it is known in Italy, is a group of very hard bacteria-ripened, granular-textured cheeses made from partially skimmed cow s milk. They originated in Parma, near Emilia, Italy, hence the name. Special lipolytic enzymes derived from animals are used, in addition to rennet, to produce the characteristic rancid flavor. 

Bacterial lipolytic enzymes (Not critical unless counts exceed 106-107/ml)... 

Downey, W. K. 1975. Identity of the major lipolytic enzyme activity of bovine milk in relation to spontaneous and induced lipolysis. Int. Dairy Fed. Doc. 86, 80-89. 

H. Brockerhoff and R. G. Jensen, Lipolytic Enzymes, Academic Press, New York, 1974. 

Since 1978. numerous works that show the ability of lipolytic enzymes to be active in organic solvents have been published2 4. Moreover, it appears that in certain conditions the stability of the enzyme is enhanced by the low content of water. In such conditions, as the organic substrates are soluble in the reactional media which is therefore homogeneous, continuous processes can be designed in a very classical way. 

G, Bell, R. Todd, J.A. Blain, J.D.E. Patterson and C.E.L. Shaw, Hydrolysis of triglycerides by solid phase lipolytic enzymes of Rhizopus arrhizus in continuous reactor systems. Biotech. Bioeng.,23,1981,1703-1719. 

Buchter D, Behre HM, Kliesch S, Chirazi A, Nieschlag E, Assmann G, von Eckardstein A. Effects of testosterone suppression in young men by the gonadotropin releasing hormone antagonist cetrorelix on plasma lipids, lipolytic enzymes, lipid transfer proteins, insulin, and leptin. Exp Clin Endocrinol Diabetes 1999 107(8) 522-9. 

Rassner, U. A., Crumrine, D. A., Nau, P., and Elias, P. M. 1997. Microwave incubation improves lipolytic enzyme preservation for ultrastructural cytochemistry. Histochem. J. 29 387-392. 

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