Whole-cell Immobilization Of Lipase Displayed On The Surface Of Staphylococcus Carnosus

Staphylococcus carnosus is a recognized safe food-grade Gram-positive bacterium.The extracellular protease hydrolysis activity of S.carnosus is extremely low,so it can be used as a good host bacteria for genetic engineering.Lipase is an interfacial enzyme that hydrolyzes triacylglycerol.Widely used in biodiesel,food,nutrition and health products,degummed oil,detergent,environmental management and biosensors.Free lipase is expensive and has poor stability and the substrate of lipase is usually insoluble in water.This problem limits its practicality.Compared with the immobilization of enzymes,the immobilization of cells has the advantages of fast reaction,strong resistance to pollution,stronger resistance to poisoning,and higher reuse rate.As an environmentally-friendly renewable"green fuel",biodiesel is an alternative to high-quality petrochemical diesel.Among them,bio-enzymatic preparation of biodiesel has attracted the attention of many countries at home and abroad due to its mild reaction conditions,less alcohol consumption,and no environmental pollution.In this study,the expression vector p BT2-SPP-LIP-CBD-XM was constructed first,and it was successfully electroporated into Staphylococcus carnosum for expression.A transformant with high enzyme activity was screened and named M5.The success of surface display of fusion protein was proved by SDS-PAGE observation of purified bacteria and lysozyme treatment of strain S.carnosus/p BT2-SPP-LIP-CBD-XM.Secondly,three immobilization methods were used to immobilize the cells with fusion proteins displayed on the surface,and a method for fixing cells with microcrystalline cellulose was screened.The enzymatic properties of the immobilized whole-cell enzymes were investigated.After whole cell enzyme immobilization,the optimum temperature of lipase can reach 45℃,which is more resistant to high temperature than free cell lipase,and can still retain about 80%of the enzyme activity after incubation at 40℃ for 3 h.The temperature stability is good;the optimum p H is 9.0,the stability is good between p H8.0-9.0,and about 70%of the enzyme activity can still be retained after treatment for 3 h;n-hexane and ethyl acetate are effective for immobilized whole-cell enzymes The activity of Mg²⁺,K⁺,Co²⁺,Li⁺can activate the whole cell enzyme activity after immobilization,and the relative enzyme activity is 125%,113%,117%and 110%,while Na⁺,Ca²⁺,Cu²⁺,Mn²⁺,Fe²⁺and Zn²⁺have different degrees of inhibition on enzyme activity.Finally,the optimal conditions for preparing biodiesel from immobilized cells were explored.The reaction was performed for 5 h,the alcohol-oil ratio was 3:1,the amount of immobilized cells was 0.8 g,and when the reaction temperature was 50°C,the glycerol yield used as the biodiesel conversion rate reached the highest value of 77%.