Prepration Of Magnetic Nanoparticals Cross-linked Enzeyme Aggregates And Its Application In Microtubular Reactor

Recently,cross-linked enzyme aggregates（CLEAs）have been received lots of attention,however,the application of conventional CLEAs is greatly restricted owing to its small particle size,high viscosity and hard reuse.In this work,the magnetic cross-linked enzyme aggregates（M-CLEAs）were prepared by adding nano-Fe₃O₄ in lipase CLEAs in order to improve the structure and properties of the M-CLEAs and reduce the difficulity of CLEAs reuse.M-CLEAs were used to synthesize butyl oleate and degrade diesel oil in water in microtubule reactor in which substrates and products were immediately separated through rotating magnetic field produced by magnets.Moreover,the preparation conditions of M-CLEAs and its structure and enzymatic properties were also studied.The main results are as follows:（1）M-CLEAs with the highest activity（2897.69 U·g-1）was prepared under the conditions:free lipase 70 mg mL-1,nano-Fe₃O₄ 9 mg·mL-1,crosslinking agent 2.075%（v/v）,precipitant volume 12 times of free lipase solution volume,cross-linking time 120 min at 4 ℃ and precipitation time 30 min.（2）Analysis of morphological structure showed that,compared with CLEAs without nano-Fe₃O₄,M-CLEAs had bigger particle size,more cavities,higher intensity of UV absorption and slight red shift.In the second-structure of M-CLEAs,a-Helix,β-Sheet and β-Turn increased 3.5%,1.1%and 2.6%respectively,but the random coil decreased 8.7%.This indicated that M-CLEAs rigidity increased but its flexibility decreased in some extent.Moreover,the fluorescence intensity of two chromophore group in the M-CLEAs decreased,especially the peak 1（Tryptophan residue）.（3）Compared with free lipase enzyme,M-CLEAs had lower sensitivity to external environment factors,such as high or low temperature,acid,alkali and organic denaturant.M-CLEAs did not show loss of enzyme activity in the buffer（pH 6-9）after 2 h,and the enzyme activity retained 95%in 4℃ after 30 days.In addition,M-CLEAs showed great operation stability.M-CLEAs activity retained 88%and above 50%when it was continuously used in reaction for 3 and 6 batches,respectively.In microtubule reactor,structure of M-CLEAs was not changed after 4 cycles（18 h/cycle）,while the second structure and fluorescence intensity of M-CLEAs was slightly changed.Study on reaction dynamics showed that M-CLEAs had low affinity for triolein than free lipase but its Vmax was higher than free lipase.（4）In the synthesis of butyl oleate,the highest yield was 84%under the following reaction conditions:M-CLEAs dosage 9 mg·mL-1,reaction time 15 h and reaction temperature 45 ℃.Yield of butyl oleate was higher by 16%（reaction time 18 h）when the reaction was catalyzed by three layers of M-CLEAs than by two layers of M-CLEAs.Yield of butyl oleate did not decrease after 4 cycles（18 h/cycle）.Experiment on diesel oil degradation showed that the degradation rate was 91%after 12 h when temperature was 25 ℃ and diesel oil concentration was 2000 mg·mL-1.Similarly,the degradation rate was 93%after 8 h when diesel oil concentration was 500 mg·mL-1 and temperature was 25 ℃.In a word,addition of nano-Fe₃O₄ not only improves performance and structure of CLEAs but also facilitate its resue.This study provides the reference for the application of CLEAs in industry.