Fermentation Opitmization And Characterization Of Marine Microorganism Extremozymes

The sea is an unique ecological environment,including oligotrophic, high salt, highpressure, low light, low temperature （especially deep sea）, no light and local high temperature,etc. kinds of extreme environment. The prospect application of the resources of marinemicroorganisms that makes the world and organization do a lot of research on it.Themicroorganisms from the sea are used to the most extreme environmental conditions,whichcan produce all sorts of unique biological active substance. The diversity of marinemicroorganisms is rich, discovered groups mainly includeing: bacteria, fungi, bacteria, themicro algae and the virus. The species diversity decided their metabolism product diversity,therefore, the sea is extremely important sources of the bio-enzymes.The strain SY-7was isolated from Tianjin offshore salt pan. Through physiological andphylogentic analysis,16S rRNA gene sequences showed that it belonged to the genusThalassobacillus, having the highest similarity （98.88%） with Thalassobacillus devorans.Cells were Gram-positive. It grew best at30℃, pH7.5and with the presence of10%NaCl.Study on the extracellular protease of SY-7shows that the optimum condition for activity is45℃，pH7.5-9.0，respectively, it is thermal-stable at40℃．The activity is restrained bySDS,PMSF,AEBSF and EDTA, while affected little by beta-mercaptoethanol. Ca²⁺,Mg²⁺ enhance the lipase activity whereas the effect of Na⁺、K+on the enzyme is little. Theenzyme can be repressed by Co²⁺, Cu²⁺, Zn²⁺, Fe³⁺, Fe²⁺,Ni⁺whereas Hg²⁺ shows strongsuperession. The protease can be repressed by EDTA. Three statistically significantparameters （quantity of glucose, liquid volume, inoculum volume） were determined by PBdesign. With the hybrid design, we generated response surfaces and obtained the responsesurface function. The production of protease was increased significantly to1390U/ml from870U/ml by the optimal levels of the fermentation conditions. The test data enzyme activityreached98.5%of the calculated data, indicating that the optimization was effective.One clone （LIP001） producing cold-active lipase was selected from metagenomic libraryof deep sea sediment for the optimization study of fermentation conditions. The effects ofmain factors on the growth and lipase-producing of LIP001were determined by single-factortest. The results showed that the optimal condition were30℃, pH7.0, and inoculum size5%, liquid volume50ml in250ml flask, respectively. The fermentation conditions for thelipase-producing of LIP001were further optimized by orthogonal experiment on the basis ofsingle factor test. The major factors and their concentrations were as follows: olive oil （1%）,yeast extract （0.5%）, peptone （1%）,（NH₄）₂SO₄（0.5%）, phosphate （0.5%）, MgSO₄（0.2%）,and chloromycetin （12.5μg/ml）, respectively. Under the optimized condition, the lipaseactivity was1980U/ml which was increased by54.7%. For5L fermentor research, the lipaseactivity was2420U/ml. For50L fermentor research, the lipase activity was5620U/ml.For500L fermentor research, the lipase activity was5120U/ml. The optimum condition of theenzyme for activity is30℃，pH8.5-9.0，respectively, it is thermal-unstable at50℃or highertemperature, indicate it belongs to alkaline cold-active lipase.The activity is restrained by highconcentration of SDS,urea,Tween-80, guanidine hydrochloride and EDTA, while affectedlittle by beta-mercaptoethanol．Ca²⁺、Mg²⁺、Sr²⁺enhance the lipase activity whereas Hg+showsstrong supperession．...