Optimization Of Fermentation And Enzymatic Characterization For Cold-active Lipase Producing Strains

In this study, lipase-producing strains were screened from oily soil samples ofHandan. After rescreening, the optimum temperature of enzyme and activity at0℃weremeasured by using5selected strains who had higher activity. A producingsstrain was named HEBUST LIP-L, and verified as cold-adapted lipase based on the results: optimum temperature was30℃and most active at0℃. It was further identified as Pseudomonas sp. according to its morphological and physiological-biochemical characteristics.By using Plackett-Burman and the statistical methods of combination of steepestascent and response surface experiments, three main factors: peptone、olive、Tween80were selected quickly and efficiently from the7suspect factors which could affect theproducing of cold-adapted lipase. The best combination of7factors was shown as:peptone26.4g/L, olive16.95mL/L, Tween8025.6mL/L, dextrin7.5g/L, KCl3mmol/LpH7.0, and seeded volume8%. The production of the lipase reached46.08U/mL,increased98%. The proposed model has a good prediction of the strain fermentation.The enzymes from Pseudomonas sp. HEBUST LIP-L were used in aqueoustwo-phase extraction, DEAE-cellulose ion exchange resin and Sephadex G-100columnfiltration experiments to separate and purify the lipase. The lipase was electrophoresispure and the molecular mass was35kD. The optimum pH and temperature of the purifiedenzyme were9.5and30℃respectively. Under these conditions, the lipase get maximumactivity and the activity was70%at0℃. The lipase belonged to the cold-adapted lipaseand have widen adaption of pH. It is stable at pH4.0-10.5and have good thermo stability.