Modified Agarose Immobilized Enzymes And Its Applications

Acrylamide and plasticizers,which have potential carcinogenicity and reproductive toxicity to humans,are frequently generated in the foods.At present,the more effective and feasible strategies are pretreatment of food with L-asparaginase to inhibit the formation of acrylamide,and the use of lipase to synthesize a migration resistant plasticizer..Immobilization of enzyme not only enhances its catalytic ability,but also enables the enzyme to be recycled.In food industry,safety,efficiency and costs should be considered comprehensively.Agarose gel is widely used in immobilized enzymes because of its safety,non-toxicity,well biocompatibility,strong modifiable properties,and stable chemical properties.In this paper,aiming at the two hazards of acrylamide and plasticizer,the modified agarose gel and coupling method with high safety and well immobilization effect were screened,and L-asparaginase and T1 lipase were immobilized in this way,respectively.Furtherly,the application performance of the modified agarose immobilized enzyme was explored in the actual application system.The results are as follows:(1)The agarose gel was modified with epoxy,amino and carboxyl groups,respectively.Combining the optimization of immobilization methods,a NHS modified agarose gel was successfully prepared,which is safe,rapid and efficient.The protein loading rate reached 60.45%after 2 hours'conjection,and the highest protein loading was 0.91 mg·g^-1 gel.In this method,L-ASNase and T1 lipase were immobilized and optimized and obtained excellent immobilizations.The recovery rate of L-ASNase activity was 60%?70%,and the highest specific activity could reach 33.43 U·g^-1 gel.The protein loading rate of T1 lipase was60%?80%,and the highest specific activity was 143.23 U·g^-1 gel.(2)The catalytic performances of NHS-agarose immobilization with L-ASNase was studied.After immobilization,L-ASNase has a wider temperature and p H adaptability,its stability was significantly improved,and the value of Km decreased,indicating the increased affinity to the substrate.The thermal stability of immobilized L-ASNase at 47°C was 7.4 times higher than that of the free.After ten recycles,the residual activity still remained 73.51%.Compared with the 5 days'shelf time of L-ASNase,the immobilized L-ASNase could be stored for 28 days and retain 71.11%initial activity,which reflected the excellent performance of the modified agarose immobilized enzyme.A packed bed enzyme reactor that simulated continuous production was constructed with immobilized L-ASNase,and the optimal parameters of the reactor were determined through single factor experiments:at 35°C,the substrate solution flows through the reactor which is occupied with 8 g of 12 U·g^-1 immobilized L-ASNase at the rate of 1 m L·min^-1 under 35°C for continuous treatment,resulting in an 81%and 89%reduction in asparagine and acrylamide,respectively.And the packed bed enzyme reactor dynamics was preliminary explored,providing guidance for industial applications.(3)The catalytic performance of T1 lipase immobilized on NHS-agarose was studied.Although the optimal temperature dropped slightly after immobilization,the operability and stability increased significantly.After incubating at 50°C for 12 h,the immobilized T1 lipase could remain 72.25%activity.After six recycles,it still retained 77.58%of its initial activity.The immobilized T1 lipase had a shelf life of 15 days in tertbutanol,retaining 67.54%of its initial activitty,which showed excellent stability and application potential.Then we explored the application performance of immobilized T1 lipase to synthesize polyester plasticizer.And the polyester synthesis process was determined:at 50°C,the ratio of acetone to toluene was1:1,the addition of immobilized T1 lipase was 0.8%wt,the molar ratio ofsubstrate(TMP:DMBA)is 1:9 and 1%wt addition of molecular sieve.A polyester plasticizer with an average molecular weight of 1416 and a polydispersity of 1.10 was obtained.Blend it with PVC,its plasticizing efficiency on the glass transition temperature and tensile elongation of the PVC piece is equivalent to 72.84%and 87.10%of DOP,respectively.In addition,the polyester plasticized PVC piece has better thermal stability.and its mobility in the simulated alcohol and ester extraction environment is 47.3%and 14.71%of DOP,respectively,showing excellent resistance to migration and safety.