Studies On Purification, Lyophillization And Application Of Marine Microorganism Lysozyme

Many antibacterial and antiviral substances possessed special function could be obtained from marine microorganism.Our research group had gotten a marine bacteria strain S-12-86 from the sea mud sample collected from the East Sea of China which producing lysozyme.Based on the research,the project studies mainly focused on marine microorganism purification,lyophillization and fresh-keeping effect on the refrigerant pork.The results showed as:A novel bacteriolytic enzyme was purified from the crude culture of Marine Microorganism Lysozyme by centrifugation of culture broth,ultrafiltration using a hollow fiber（molecular weight cut of,50kD） and concentration using a hollow fiber（molecular weight cut of,10kD）,extracting with ethanol,superdex 75 chromatography and reversed phase HPLC.The lysozyme was purified 41.98 fold to electrophoretic homogeneity with a recovery of 21.7%.The molecular weight was 17.1 kD by SD—PAGE electrophoresis.Amino acid analysis of this Marine Microorganism Lysozyme showed that it was mainly composed of Leu、Thr、Gly、Glu、Ser、Ala、Phe.The optimal pH and temperature was 8.0 and 35℃respectively.Zn²⁺ and Cu²⁺ could stimulate the activity,whereas Mn²⁺ and Ag⁺ inhibited the lytic activity.Ba²⁺, Li⁺,Ca²⁺,Na⁺,K⁺ and Fe³⁺ had no effects on the enzyme activity.Marine Microorganism Lysozyme was stable in some chemicals and thickening agents. Containing 10mg/ml surfactant,the relative enzyme activity was improved for 18%, 11%,5%by tween80,APG and cAPG respectively,decreased 15%by SDS,and had no change in Tween 20 solution.Native marine microorganism lysozyme preparations were formulated with different additives for freezing,lyophillization and storage stabilization.The additives studied were:trehalose,sucrose,maltose and trehalose/tween 80.All the additives showed good protecting effect and trehalose/tween 80 showed the best performance for freeze-drying stabilization.Comparing with the native lysozyme in absence of protective agents,the activity of the freeze-dried lysozyme（50 mg/ml） treated by trehalose（0.5 M） and tween 80（20 mg/l） as protective agents,increased by 28%after lyophillization.After one-month storage,it could retain more than 92%of activity. Therefore the freeze-drying process for marine microorganism lysozyme was successfully established.The activity of liquid enzyme had more loss at room temperature than that at low temperature.Freeze-dried powder of the enzyme at room temperature or low temperature had no significant differences at the enzyme activity. The results showed that freeze-dried power was fitter to be saved than that in liquid for Marine Microorganism Lysozyme.Marine Microorganism Lysozyme-had a broad antimicrobial spectrum against standard strains including G⁺,G^- and fungi,et al.The fresh-keeping effect of lysozyme on refrigerant pork was studied.The result showed that the marine lysozyme had a good effect on maintaining the sensory indexes of meat.And the lysozyme had a significant effect on controlling the reproduction of bacteria,and decelerating the rising of the value of TVB-N.An ideal effect was obtainded in the test as a result of the use of 0.5%marine lysozyme and 2%glycine.The results obtained suggested that these unique properties of the Marine Microorganism Lysozyme were in accordance with the environmental conditions that the marine strain S-12-86 lived.Therefor,exploring marine microorganism enzyme had a bright future.