Purification And Characterization Of Protease And Lipase From Antarctic Krill

Antarctic krill?Euphausia Superba?is a crustacean zooplankton living in the Antarctic waters.It is the largest recognized single marine fishery resource that can be catched and utilized,and has the potential to build China's second offshore fishery.The development of Antarctic krill resources is of great strategic significance for promoting the development of China's polar fisheries and cultivating emerging industries of marine life.Antarctic krill contains protease,lipase,amylase,carboxypeptidase and cellulase and so on.Due to the low water temperature of the Antarctic waters,most Antarctic krill enzyme have more excellent low-temperature catalytic activities and good application prospects in the food,medicine and other fields.In this paper,the basic enzymatic properties of protease in the crude enzyme solution of Antarctic krill were studied,and a series of enzyme inhibitors were developed.A novel lipase was isolated and purified from Antarctic krill,and its enzymatic properties and catalytic ability were determined.This article is divided into three parts:?1?Study on the basic enzymatic properties and development inhibitors of protease in crude enzyme solution of Antarctic KrillThe effects of reaction temperature,pH,metal ions(Zn²⁺,Mg²⁺,Cu²⁺,Fe³⁺,Al³⁺and Ca²⁺)on the protease of crude enzyme of Antarctic krill were studied,and the inhibiting effects of PMSF,EDTA·2Na,iodoacetamide?IAM?and toluenesulfonyl-phenylalanine chloromethyl ketone?TPCK?on the protease were also investigated.The experimental results indicated that the optimum reaction temperature was 40?and the optimum pH value was 8.0.When the ultimate concentration of Zn²⁺was 0.5mmol/L,the inhibiting effect was the best,inhibition rate for 55.02%;Ca²⁺had a good activation effect on protease in crude enzyme solution of Antarctic krill;When the concentration of PMSF was 8.0 mmol/L,the inhibition rate was the highest,60%;The inhibiting effect of IAM was increasing with the ultimate concentration increasing from 0 mmol/L to 2.5 mmol/L,at 2.5 mmol/L,the inhibiting effect was the best and the inhibition rate was 66.67%;TPCK had a litter inhibiting effect inhibiting effect on enzyme activity,and even in the concentration range of 0-8.4 mmol/L.When the concentration increased to 15 mmol/L,the inhibitory effect was mild.EDTA·2Na had the best inhibiting effect when the concentration was 0.6 mmol/L,and the inhibition rate was 86.67%.With the increase of temperature in the range of 5°C to 30°C,the inhibiting effect of EDTA·2Na on enzyme activity improved gradually.At 30°C,the inhibition rate reached 85.02%.?2?Isolation and purification of an Antarctic krill lipaseWe constructed a purification technology to isolate a lipase from Antarctic Krill by comprehensive utilization of methods such as ammonium sulfate precipitation,dialysis,ion-exchange chromatography and molecular exclusion chromatography and so on,resulting in recovery rate of 5.2%and 22.4-fold purification,a single band of enzyme was detected in SDS-PAGE with molecular mass of approximately 71.27?kDa.?3?Studies on the enzymatic properties of Antarctic krill lipaseBy studying the effects of temperature,pH,metal ions,sugars,organic solvents and surfactants on the basic enzymatic properties of the new Antarctic krill lipase,we concluded that the lipase's optimal reaction temperature was 45°C,and the optimal pH was 8.0.The activity of lipase could be firstly enhanced with the addition of Ca²⁺and Mg²⁺in the concentration range of more than 0-0.5 mmol/L and more than 0-0.3mmol/L respectively,and the inhibiting effect of Mg²⁺improved with further increase of ionic concentration.Fe³⁺and Cu²⁺showed obvious inhibiting effect on the enzyme activity,and the inhibition rate were 71.79%and 53.33%respectively when the ionic concentration was 0.5 mmol/L.Sucrose at a final concentration of 6%had a protective effect on the Antarctic krill lipase;EDTA and SDS had a protective effect on the enzyme activity at the final concentration of 1 mmol/L,and the Tween 20 had the effect at the final concentration of 1%.Lipase had different hydrolytic activities for p-nitrophenyl esters with different side chain lengths?C4-C16?,among which the hydrolysis activity for p-nitrophenol octanoate was the best,and no hydrolysis on p-nitrophenol palmitate.The lipase's kinetic constants of the catalytic reaction using p-nitrophenol laurate as subtrate were studied,and the results showed that the Km and Vmax were 3.27 mM and 2.4 U/mg respectively.Through researching,we obtained the best inhibitor of protease in the crude enzyme solution of Antarctic krill was EDTA·2Na,and the purification route of the new lipase of Antarctic krill was established.The basic enzymatic properties and catalytic properties of the enzyme were studied.The study provides an important theoretical and data support for the development and diversification of Antarctic krill.