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

Abstract

English:Lipase (EC 3.1.1.3) catalyses the hydrolysis of triacylglycerols and occur widely in nature. The' lipase reaction is reversible and a wide range of trans- and interesterification reactions can be catalysed. These enzymes could be used to manufacture products which could not be obtained by conventional chemical processes, and as the advantages of the use of lipases relative to traditional chemical processes are more and more recognised, lipases may be expected to gain even more importance in the enzyme market. For these purposes, new lipases with a wide range of novel characteristics will be needed. Several microbial isolates were screened for lipase production on agar plates containing different lipase inducers. The isolates (1 bacterial and 13 fungal) that showed lipase production with at least three inducers, were cultured in shake cultures containing olive oil as inducer of lipase production. The most promising lipase producer was identified as Aspergillus niger. Purification of lipase from Aspergillus niger was achieved using ion-exchange chrornatoqrapriy, iso-electric focussing, and size exclusion chromatography. It was shown that the iso-electric focussing was not essential for purification, if a more effective, gel filtration matrix with a narrower functional range was employed. The homogeneity of the lipase was confirmed on SOS-PAGE and iso-electric focussing gels. The purified lipase displayed a relative molecular weight of 43 600 Da in its glycosylated form and a pi of 6.1. The carbohydrate content of the lipase was estimated at 10 %. The pure lipase showed maximal activity at acidic pH values and a temperature range between 25 - 40 °C. The enzyme was stable over a wide pH range, and the presence of calcium increased the stability with the effect being most dramatic at high pH values. Even though the Aspergillus niger lipase is not considered as thermostable, the enzyme could be stabilised by calcium to such a degree that application even at higher temperatures seems feasible. Some heavy metal ions inhibited the enzyme's activity. The lipase activity was influenced by detergents forming ionic micelles, and the non-ionic and zwitterionic detergents had very little to no effect on the lipase activity. The functional analogy to serine proteases was also confirmed by modification of the "catalytic triad" residues. The positional and stereospecificity of the Aspergillus niger lipase was investigated with the monomolecular film technique. This technique is considered to be the most effective method for studying lipase kinetics. The lipase displayed a stereopreference for the sn-1 ester position and as expected, no marked hydrolysis of the ester in sn-2 position. As the surface pressure was increased the initial stereoselectivity can be altered to a preference for the sn-3 ester position; thus indicating that lipolysis is surface dependant. The regioselectivity of the lipase was also investigated, using this very sensitive technique, and these kinetic studies revealed that the lipase has a preference for adjacent ester groups at low surface pressures, but that the regioselectivity is less marked at higher surface pressures. The kinetic characterisation of Aspergillus niger lipase using the oil-drop tensiometer, showed very good lipolysis at the interface of the soybean oil drop. Even when compared to other known and well-characterised lipases, this lipase displayed exceptionally high activity. These studies show interfacial kinetics reported thus far can be misleading and special care must be taken when extracting kinetic parameters from a multiphase (emulsion) system. The Aspergillus niger lipase was also investigated at a molecular level; a successful cDNA library was constructed. Degenerate primers were designed according to amino acid sequence homology displayed between various fungal species, the peR product obtained with these primers were used to screen the library for the lipase gene. The partial nucleotide sequence of the Aspergillus niger lipase gene was obtained. The lipase from Aspergillus niger shows some unique aspects that should be investigated even more thoroughly to make this hyper producing fungus a prospect for biotechnological application.

Afrikaans:Lipase (EC 3.1.1.3), wat algemeen in die natuur voorkom, kataliseer die hidroliese van triasielgliserole. Die lipase-reaksie is omkeerbaar wat 'n wye reeks trans- en interesterifikasie reaksies moontlik maak. Die ensieme kan die vervaardiging van produkte wat andersins nie met konvensionele chemiese metodes gemaak kan word nie, moontlik maak. Na mate die voordele verbonde aan die toepassing van lipases erken word, kan daar verwag word dat lipases 'n groter wordende deel van die markaandeel sal begin inneem. Nuwe en verbeterde lipases sal daarom noodgedwonge gevind moet word. Verskeie mikrobiese isolate is getoets vir lipase-produksie op agar kwekingsplate met verskillende lipase-induseerders. Die isolate (een bakterie en dertien fungi) wat positief met ten minste drie induseerders getoets het, is in vloeibare skudkulture gekweek met olyfolie as ensiem-induseerder. Die mees belowende isolaat is as Aspergillus niger geïdentifiseer. Die ensiem is gesuiwer deur die toepassing van ioonuitruilingschromatografie, isoelektriese fokussering en grootte uitsluitingschromatografie. As 'n meer doeltreffende gelfiltrasiemedium met 'n enger fraksioneringswydte gebruik word, kan die iso-elektriese fokusseringstap uitgelaat word. Die homogeniteit van die ensiem is deur middel van "SOS-PAGE" en analitiese iso-elektriese fokussering bevestig. Die gesuiwerde ensiem het In relatiewe molekulêre massa van 43 000 Da in die geglikosileerde vorm en 'n pi van 6.1 getoon. Die koolhidraat-inhoud van die lipase was 10 %.Die gesuiwerde ensiem het maksimale aktiwiteit by lae pH-waardes en 'n by 'n temperatuurgebied van 25 - 45 oe getoon. Die ensiem is oor 'n wye pH-gebied stabiel en ea2+ het 'n dramatiese stabiliseringseffek, veral by hoë pH waardes. AI is die ensiem nie termostabiel nie, kon die ensiem in so 'n mate deur ea 2 + gestabiliseer word dat toepassing by hoër temperature moontlik is. Die lipase aktiwiteit is deur sommige swaarmetaalione geïnhibeer. Wasmiddels wat ioniese miselle vorm het ook die lipase aktiwiteit sterk beïnvloed, terwyl neutrale of zwitterioniese wasmiddels weinig effek gehad het. Die funksionele ooreenstemming met serien proteases is deur modifikasie van die katalitiese triade bevestig. Die posisionele- en stereoselektiwiteit van die lipase is deur middel van die monomolekulêre filmtegnieke bestudeer. Hierdie tegniek word as die mees betroubare hiervoor beskou. Die lipase het 'n besliste voorkeur vir die sn-1 esterbinding getoon en het, volgens verwagting, nie die sn-2 posisie teen 'n beduidende tempo gehidroliseer nie. Met 'n toename in oppervlakdruk is die aanvanklike stereovoorkeur verander na dié vir die sn-3 posisie. Hidroliese is dus oppervlakdruk afhanklik. Die regioselektiwiteit van die ensiem is ook deur middel van hierdie tegniek ondersoek. Kinetiese studie het daarop gedui dat naasliggende estergroepe by voorkeur by lae oppervlakdruk gehidroliseer word maar dat die regioselektiwiteit by hoë oppervlakdruk begin vervaag. Kinetiese karakterisering van die Aspergillus niger lipases deur van die oliedruppel tensiometer gebruik te maak het aangedui dat die ensiem 'n besonder hoë aktiwiteit besit by die interfase van die sojaboon oliedruppel. Hierdie studie dui op die gevaar dat kinetiese data bekom deur die toepassing van multifase (emulsie) sisteme, uiters misleidend kan wees. Die Aspergillus niger lipase is ook op molekulêre vlak ondersoek en 'n cONS biblioteek is voorberei. Degenererende voorlopers is ontwerp na aanleiding van aminosuurvolgorde homologie met ander fungusspesies. Die PKR-produk wat met' hierdie voorlopers verkry is, is gebruik om die cONS biblioteek te sif vir die lipasegeen. 'n Gedeeltelike nukleotiedvolgorde is vir die Aspergillus niger lipase-geen verkry. Die Aspergillus niger lipase het sommige unieke eienskappe wat verdere deeglike ondersoek na hierdie hiperproduserende fungus regverdig vir 'n biotegnologiese toepassing.