Studies On Two Kinds Of Novel Lipase Immobilization Technologies And Their Application For Chiral Resolution

Lipase is a kind of green,safe and efficient biocatalyst with many excellent catalytic properties such as esterification,esterification and transesterification,which make it to have a wide applications in food,medical,bioenergy and other industrial fields.However,the fermented free enzyme usually exhibit poor environmental stability and no reusability,resulting the high production cost.Thus,to meet the standards of industrial application,further modification is necessary for the free enzyme.Immobilization is one of the effective methods to improve catalytic efficiency,stability and reusability of enzyme.And the efficiency is usually influenced by many factors such as immobilized carrier,crosslinking medium,immobilization conditions and reaction solvent.So far,a large number of lipase immobilization technologies has been reported in literature,but most of the strategies are traditional and similar.The most used immobilized carriers are concentrated on the sol-gels,molecular sieves,macroporous resins and other traditional materials.It can be seen that immobilization technology has met a development bottleneck,so the research and exploration on new immobilization strategies are the most crucial and urgent issues in nowadays immobilization field.With the development of chemistry and material science,along with the increase of academic crosslinking between biotechnology and other disciplines,the immobilization strategies based on new immobilization materials and preparation methods have been gradually increased,and these provide new driving force for the development of enzymatic immobilization.Therefore,in this study,two new lipase immobilization strategies were explored,and the prepared immobilized BCL(Burkholderia cepacia lipase,BCL)were further applied to catalytic resolution of(R,S)-1-phenylethyl alcohol.The lipase catalytic efficiency and operational stability have been greatly improved.The main research work and the results are summarized as follows:1.To explore the application of a one step inorganic-protein immobilization method in lipase immobilization,a nano-flower like immobilized BCL was prepared and the immobilization conditions were optimized.When the enzyme loading was 24 mg/mL and the buffer concentration was 20 mM,the morphology of the immobilized BCL observed by SEM was the most clear and stable,and the highest immobilization and catalytic efficiency were also obtained.In the application of chiral resolution of 1-phenylethyl alcohol,the recovery enzyme activity remained at 70%after storage for 10 days,and about 80%after being reused for 5 times.2.To discuss the effect of carrier pH sensitivity on lipase immobilization,a pH-sensitive immobilized BCL was prepared and the immobilization conditions were optimized.When the enzyme loading was 2:1(BCL:carrier)and the buffer pH was 6.5,the prepared immobilized BCL had a higher immobilization rate and exhibited the highest pH sensitivity and the catalytic efficiency.In the application of chiral resolution of1-phenylethyl alcohol,the recovery enzyme activity remained at 80%after storage for 10days,and was about 90%after being reused for 5 times.It can be seen that the immobilized BCL performed good operational stability and reusability,which is conducive to the practical applications.3.A comparison between the two kinds of immobilized BCL and other immobilized enzymes reported in related papers was conducted.The effects of immobilized BCL on the chiral resolution of(R,S)1-phenylethyl alcohol was evaluated through three key factors,reaction equilibrate time,conversion and ee_s value.The results showed that the prepared immobilized BCL could greatly shorten the required time to reach reaction equilibrium and exhibit good operational stability.Thus,the immobilized BCL has a great potential in the application of biocatalysis of chiral drugs.