A kinetic and molecular study of the purified lipase from Aspergillus niger

[摘要] English:Lipase (EC 3.1.1.3) catalyses the hydrolysis of triacylglycerols and occur widely innature. The' lipase reaction is reversible and a wide range of trans- andinteresterification reactions can be catalysed. These enzymes could be used tomanufacture products which could not be obtained by conventional chemicalprocesses, and as the advantages of the use of lipases relative to traditionalchemical processes are more and more recognised, lipases may be expected togain even more importance in the enzyme market. For these purposes, newlipases with a wide range of novel characteristics will be needed. Several microbial isolates were screened for lipase production on agar platescontaining different lipase inducers. The isolates (1 bacterial and 13 fungal) thatshowed lipase production with at least three inducers, were cultured in shakecultures containing olive oil as inducer of lipase production. The most promisinglipase producer was identified as Aspergillus niger. Purification of lipase from Aspergillus niger was achieved using ion-exchangechrornatoqrapriy, iso-electric focussing, and size exclusion chromatography. It wasshown that the iso-electric focussing was not essential for purification, if a moreeffective, gel filtration matrix with a narrower functional range was employed. Thehomogeneity of the lipase was confirmed on SOS-PAGE and iso-electric focussinggels. The purified lipase displayed a relative molecular weight of 43 600 Da in itsglycosylated form and a pi of 6.1. The carbohydrate content of the lipase wasestimated at 10 %.The pure lipase showed maximal activity at acidic pH values and a temperaturerange between 25 - 40 °C. The enzyme was stable over a wide pH range, and thepresence of calcium increased the stability with the effect being most dramatic at high pH values. Even though the Aspergillus niger lipase is not considered asthermostable, the enzyme could be stabilised by calcium to such a degree thatapplication even at higher temperatures seems feasible. Some heavy metal ionsinhibited the enzyme's activity. The lipase activity was influenced by detergentsforming ionic micelles, and the non-ionic and zwitterionic detergents had very littleto no effect on the lipase activity. The functional analogy to serine proteases wasalso confirmed by modification of the catalytic triad residues. The positional and stereospecificity of the Aspergillus niger lipase was investigatedwith the monomolecular film technique. This technique is considered to be themost effective method for studying lipase kinetics. The lipase displayed astereopreference for the sn-1 ester position and as expected, no marked hydrolysisof the ester in sn-2 position. As the surface pressure was increased the initialstereoselectivity can be altered to a preference for the sn-3 ester position; thusindicating that lipolysis is surface dependant. The regioselectivity of the lipase wasalso investigated, using this very sensitive technique, and these kinetic studiesrevealed that the lipase has a preference for adjacent ester groups at low surfacepressures, but that the regioselectivity is less marked at higher surface pressures.The kinetic characterisation of Aspergillus niger lipase using the oil-droptensiometer, showed very good lipolysis at the interface of the soybean oil drop.Even when compared to other known and well-characterised lipases, this lipasedisplayed exceptionally high activity. These studies show interfacial kineticsreported thus far can be misleading and special care must be taken whenextracting kinetic parameters from a multiphase (emulsion) system. The Aspergillus niger lipase was also investigated at a molecular level; asuccessful cDNA library was constructed. Degenerate primers were designedaccording to amino acid sequence homology displayed between various fungalspecies, the peR product obtained with these primers were used to screen thelibrary for the lipase gene. The partial nucleotide sequence of the Aspergillus nigerlipase gene was obtained. The lipase from Aspergillus niger shows some uniqueaspects that should be investigated even more thoroughly to make this hyperproducing fungus a prospect for biotechnological application.

[发布机构] University of the Free State