Studies On Purification And Enzyme Characterization Of Lipases From Antarctic Krill

Antarctic krill is a group of perennial crustacean zooplankton that widespread inthe Antarctic Ocean. They are characterized by large biological storage capacity, widedistribution, high nutritional value, important new marine resource of proteins, whichneed to be exploited and utilized. Antarctic krill lives in the special environment in itsparticularly active way of life，which has potential in producing novel bioactivesubstances．They are also the subject of a large fishery now．While a few producebioactive substances have been described and researched,the bioactive substancesderived from Antarctic krill are diverse，including enzymes，lipids，chitin，bioactivepeptides and the substances of UV-absorbing，and so on．Antarctic krill is known tohave a very efficient digestive system，which is based on a combination of enzymescontrolled by inhibitors. The efficiency of these enzymes is clearly visualized whenthe krill Post mortem is autolyzed.The high proteolytic activity has raised a lot ofinterest and a number of studies on the characteristics of these enzymes have beenperformed. Correspondingly, no attention has been paid on its lipases and theirfunctions. Lipase is a kind of important enzyme for industry application．It is widelyused in food，chemistry，paper and feed industry．Cold-adapted lipase is a kind ofhydrolase that mainly applied in fields such as food， medicine， environmentprotecting etc. Cold-adapted lipases have more advantages than theircounterparter-mesophilic and thermophilic lipases in applications, because they canreserve high activity even at very low temperatures. Because of their significantvaluable in theoretical research and applications, cold-adapted lipases have beenreceived more attentions recently and become one of research hot spots in the world.Accordingly, the purification of Lipases from Antarctic krill, the enzymaticcharacteristics study and the research on its biological activities have significanttheoretic and practical importance. In this paper, lipases were purified from Antarctic krill and characterized.This research is divided into two parts.Part1: Purification of Lipases from Antarctic krillThe esterolytic activity of these enzymes was routinely estimated by employingthe para-nitrophenyl palmitate (pNPP,C16) assay and para-nitrophenylbutyrate(pNPB,C4) assay.The separation and purification of lipase was conducted bycrude extract, DEAE-Sepharose Fast Flow ion-exchange chromatography, and then bySephacryl S100gel chromatography, which used the heads of Euphausia superba.The content of lipase that hydrolyze pNPP was too low. By solid ammoniumsulfate fractional precipitation with saturation between35%and80%,DEAEsepharose Fast Flow ion exchange chromatography, two activity peaks were obtained.The purification fold was6.59.The content of lipase that hydrolyze pNPB was high. By solid ammonium sulfatefractional precipitation with saturation between50%and80%,DEAE sepharose FastFlow ion exchange chromatography, sepharose S100gel chromatography, only oneactivity peak was obtained. The purification fold was5.41.This kind of lipase had amolecular weight of about42.81kDa by SDS-PAGE with12%separation gel.Part2: Enzyme Characterization of Lipase from Antarctic krillLipase which hydrolyze pNPB was obtained in part1.The study of temperature effects on enzyme stability showed that, the lipase wasstable in temperature range of5~25°C or lower, which was easy to be deactivatedwith temperature over35℃more than one hour，with optimum values at about35°Cat pH8.0. Antarctic krill lives in the cold environment,this kind of lipase looks like acold-adapted lipase.The purified lipase had above60%activity in pH range of6.0~10.0at4°C within12hours,and the optimum values was obtained at pH8.0.The study of effects of different metalions on reaction velocity of the enzymeshowed that, Mn2+(＜0.5mmol/L) and Cu2+(≥0.1mmol/L) showed certain degree ofinhibit effect to the purified lipase. Mn2+(＞0.5mmol/L) changed to promote theactivity.Mg2+(≤0.4mmol/L) and Ca2+(≤0.4mmol/L) slightly activated the lipase. K+ (0~0.3mmol/L) did not show significant effect on the activity,but it had slightpromotion with K+(≥0.4mmol/L). Of all these metal ions, Zn2+had the highest inhibiteffect on lipase activity.Generally, the purified lipase showed activity for esters of short-to medium-chain(C4) fatty acids(pNPB), and exhibited no activity for long-chain fatty acid esters likethat of palmitate(pNPP).