Study On The Extraction Of Rongchang Pig's Lipase By Aqueous Two-phase System And Character Of Lipase

Aqueous two-phase extraction is a new bio-separation technique developed in recent years. Two hydrophilic polymer with different chemical constitution or hydrophilic polymer and inorganic salt do not dissolve mutually in suitable density. The object component could be separated by using aqueous two-phase extraction.The bio-separation technique has many advantages such as higher bio-compatibility, less separation procedures, milder operating condition, easier engineering amplification, less residue of organic solvents in the product and so on. It has been widely used to purify different enzymes, nucleic acids, hormone and bioactive component in plants.Lipase (EC 3.1.1.3) is a kind of biocatalysts to hydrolysis long-chain triglycerides to glycerol and long-chain fatty acids. Lipase is widely used in food, medicine, scour, leather and paper industry. The lipase holds an important status during the processing process. It has vital significance to meat product flavor regulation, craft improvement and production's industrialization.Aqueous two-phase system was used in this paper to extracting the lipase of Rongchang pig. The influencing factors of extracting and properties of lipase were studied.In this experiment the extraction effects of several factors on Rongchang pig's lipase were investigated. These factors included components of aqueous two-phase system and different operating conditions. The distribution coefficient and recovery rate are high when the density of (NH₄)₂SO₄ is 12%, PEG molecular weight is 2000 and density is 24%, pH value is 6.50, supplementary enzyme is 10%, counter-extract material Na₂HPO₄ density is 12%. The distribution coefficient and recovery rate are stable with enlarging this experimental result. And properties of the lipase from Rongchang pig are as follows.The lipase showed the highest activity at pH 6.5 and 37℃. It was stable under 37℃. The pH from 4.00 to 7.00 Rongchang pig's lipase has good acid-base stability. When ion density is 2mmol/L and 5mmol/L Ba²⁺ and Ca²⁺ stimulated the enzyme activity significantly while Fe²⁺,Mn²⁺,Zn²⁺,Sn²⁺had inhibitory effect. But K⁺,Na⁺,Mg²⁺had no effect on the activity of lipase. The density of surfactant is 0.05%. Cationic surfactant CATB, anionic surfactant SDS and non-ionic surfactant Trition-100 stimulated the enzyme activity significantly. Non-ionic surfactant Tween-80 had an inhibitory effect. Along with increasing density of surfactant the active effect will convert into inhibitory effect. The inhibitory effect will be enhanced at the same time. The Ca²⁺ reduced thermal stability of lipase. The activity of lipase was almost lost under 45℃treatment. The enzyme activity enhanced but the thermal stability reduced with adding Ca²⁺.The values of K_m and V_max were 18.17mmol/L and 588U/L in optimum reaction conditions.