Lipases purification

Baillargeon, M.W., McCarthy, S.G. 1991. Geotrichum candidum NRRL Y-553 lipase purification, characterization and fatty acid specificity. Lipids 26, 831-836. 

Hydrophobic-interaction chromatography (HIC) and affinity chromatography (AFC) [29-31] have been used in lipase purification. The ligands are very specific and each ligand is only applicable for the separation of the lipase from a certain source. Polypropylene glycol is reported to be a suitable ligand for the fractionation of Chromohacterium viscosum lipase [29]. 

This chapter examines in some detail the different techniques available to an investigator in the field of hpase purification. Lipases tend to follow very specific patterns of purification. The techniques used to purify the lipase need to be optimized to obtain maximum efficiency. Purification strategies need to be arranged in the proper order to maximize the level of lipase purification. Clearly, more detailed work is needed to estabhsh what factors enable the selection of one strategy over the other. Last, but not the least, theoretical models of continuous purifica-... 

Finally, hydrotrope solutions have been involved in natural product separation, e.g., in lipase purification and the evaluation of its thermal stability [46], as well as in the extraction of piperine from black pepper [47],... 

Most commercial applications, such as enzyme preparations for detergents, do not require pure lipases, but a certain degree of purity simplifies their successfiil usage as biocatalysts because reduces side-product formation and simplifies product downstream. Extensive lipase purification should be considered when structural studies are going to be performed or when it will be used as biocatalyst in a synthetic reaction for the pharmaceutical industry. The main drawbacks of traditional purification strategies are low yields and productivities. The extent of purification varies with the number and the order of purification steps (see section 2.2.3) the importance of designing optimal purification schemes has been highlighted in several comprehensive reviews on this topic (Taipa et al. 1992 Aires-Barros et al. 1994 Palekar et al. 2000 Saxena et al. 2003). 

One of the bottlenecks of enzyme technology is enzyme availability. When the biocatalyst is commercial, the price may be too high, but in most cases there is no commercial source available so that the enzyme must be produced by means of an overproducing strain and finally the enzyme should be purified. Enzyme purification (discussed in section 6.3.1) is a time consuming process and may represent up to 80% of the enzyme production cost. The usual procedures for lipase purification are sometimes troublesome, time consuming and result in low final yields (Gupta et al. 2004). Enzyme immobilization overcomes this handicap because it allows its reuse and can also enhance enzyme stability and activity (Sharma et al. 2001) furthermore, enzyme immobilization facilitates bioreactor design and final product downstream from reaction medium (see section 4.1). 

The most outstanding contributions to methods of lipase purification, however, have been made by Desnuelle and his colleagues in Marseilles. Their researches are published in a series of papers from 1957 (209, 216, 221-224). 

ZHO Zhou, L., Wan, J., and Cao, X, Synthesis of thermo-sensitive copolymer with affinity butyl ligand and its application in lipase purification, J. Chromatogr. B, 878, 1025, 2010. 

Chirazymes. These are commercially available enzymes e.g. lipases, esterases, that can be used for the preparation of a variety of optically active carboxylic acids, alcohols and amines. They can cause regio and stereospecific hydrolysis and do not require cofactors. Some can be used also for esterification or transesterification in neat organic solvents. The proteases, amidases and oxidases are obtained from bacteria or fungi, whereas esterases are from pig liver and thermophilic bacteria. For preparative work the enzymes are covalently bound to a carrier and do not therefore contaminate the reaction products. Chirazymes are available form Roche Molecular Biochemicals and are used without further purification. 

Ikeda, S., Nikaido, K., Araki, F K. et al. (2004) Production of recombinant human lysosomal acid lipase in Schizosaccharomyces pombe. development of a fed-batch fermentation and purification process. Journal of... 

The key-step of Mori s synthesis of 12 was pig pancreatic lipase (PPL)-cat-alyzed asymmetric hydrolysis of raeso-diacetate A to give B (Scheme 22) [32]. Purification of B (90.8% ee) afforded pure C, which was converted to 12. 

Hemiacetal 25 [(3 ,4S,l E)-3,4-bis(r-butenyl)tetrahydro-2-furanol] is the male pheromone of the spined citrus bug (Biprorulus bibax). Scheme 38 summarizes Mori s synthesis of 25 [61]. Claisen rearrangement (A B) and lipase-catalyzed asymmetric acetylation [meso-C >(5S,6R)-D] were the two key steps of the synthesis. Further purification of D was executed at the stage of its crystalline derivative E. In this particular case, the unnatural (3S,4R,l E)-25 was as active as the natural (3R,4S,VE)-25. Accordingly, a more efficient synthesis of ( )-25 was achieved by the rearrangement of F, avoiding the use of toxic HMPA [62]. 

J. G. N. De Jong, H. van den Bosch, D. Rijken, L. L. M. van Deenen, Studies on Ly-sophospholipases. III. The Complete Purification of Two Proteins with Lysophopho-lipase Activity from Beef Liver , Biochim. Biophys. Acta 1974, 369, 50-63. 

Selected entries from Methods in Enzymology [vol, page(s)] Detergent-resistant phospholipase Ai from Escherichia coll membranes, 197, 309 phospholipase Ai activity of guinea pig pancreatic lipase, 197, 316 purification of rat kidney lysosomal phospholipase Ai, 197, 325 purification and substrate specificity of rat hepatic lipase, 197, 331 human postheparin plasma lipoprotein lipase and hepatic triglyceride lipase, 197, 339 phospholipase activity of milk lipoprotein lipase, 197, 345. 

FI. Fielding, C. J., Human lipoprotein lipase. I. Purification and substrate specificity. Biochim. Biophys. Acta 206, 109-117 (1970). 

Rua, M L., Diaz-Maurino, T. Fernandez, V.M., Otero, C. and Ballesteros, A. (1993) Purification and partial characterization of two distinct lipases from Candida cylindracea. Biochim. Biophys. Acta., 1156, 181-189. 

Schemem 4.29 Lipase-catalyzed purification by removal of undesired stereoisomers from the product. Chirazyme L2 (CALB), vinyl acetate, n-heptane, mol. sieves , 25°C.

The binding of milk lipases to casein micelles apparently imparts some stability to the enzyme, for as purification progresses, the milk lipase becomes less stable, and more so as the concentration of casein decreases (Downey and Andrews 1966 Egelrud and Olivecrona 1972). 

Egelrud, T. and Olivecrona, T. 1972. The purification of a lipoprotein lipase from bovine skim milk. J. Biol. Chem. 247, 6212-6217. 

Lamberet, G. and Menassa, A. 1983. Purification and properties of an acid lipase from Penicillin roqueforti J. Dairy Res. 50, 459-468. 

The greatest advantage of the spectrophotometric method is that it is direct and rapid, requires no sample workup, and allows for continuous assays of lipase activity compared to the multiple fixed-time-point analyses incumbent within Basic Protocols 1 and 2. The spectrophotometric method can also be done using very small volumes (as small as 1 ml) and is suitable for following the course of purification (such as in chromatographic fractions) or adaptable to 96-well plates (and subject to automation, if available). Thus, it is the method of choice for screening several samples or preparations for lipase (esterase) activity. 

Yang, Y., and M.E. Lowe. 1998. Human pancreatic triglyceride lipase expressed in yeast cells Purification and characterization. Protein Expr Purif 13 36. 

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