Study And Application Of Immobilized Nuclease P1 Enzymatic Membrane Reactor

As an intercross research of many subjects,enzymatic membrane reactor(EMR) combines the high catalytic activity of bio-enzyme and perm selectivity of separation membrane.EMR achieves the simultaneous process of reaction and separation,displaying super production efficiency.Hollow fiber ultrafiltration membranes are fabricated using novel high performance heterocyclic poly(phthazinone ether sulfone ketone)(PPESK)as membrane materials.Based on this work,IEMR is designed and studied systemically and applied in producing 5'-nucleotide.The relative experiments and results are summarized as follow.Some enzymatic properties of immobilized nuclease P1 are studied in the experiment. The experimental results show that,the optimum pH of immobilized nuclease P1 is 5.4 which improved 0.2 units compared with free nuclease P1 and immobilized nuclease P1 has better pH stability.The optimum temperature of immobilized nuclease P1 is 70℃which improved 10℃compared with free nuclease P1 and immobilized nuclease P1 is greatly improved in aspect of thermal stability.Immobilized nuclease P1 has more than 30%activity after 4 hours in 700℃.A certain dose of Zn²⁺has activation or strengthening effect on activity of nuclease P1.But the enzyme would be mainly no activity if the dosage of Zn²⁺is too great.The immobilized condition is selected in the experiment.The effect of enzyme concentration,gelatin concentration,glutaraldehyde concentration,immobilizing time and immobilizing temperature on the activity of immobilized enzyme are studied.The best immobilizing conditions are that enzyme concentration 200mg·L^-1,pH 5.2,immobilizing temperature 35℃,immobilizing time 30min,respectively.Immobilized enzymatic membrane reactor(IEMR)is designed.The effect of reaction temperature,time,pressure,pH,concentration of Zn²⁺,substrate concentration and load of enzyme on the activity of enzyme on the working performance of IEMR are studied.The results show that after running 5 hours,the conversion of RNA achieves 85%when the temperature,pressure,pH,concentration of Zn²⁺,substrate concentration and load of enzyme are 65℃,0.1MPa,5.4,0.0006mol·L^-1,2%,200mg·L^-1,respectively. The membrane fouling conditions are studied and the reason is analyzed.The fouled membranes are cleaned by 60℃hot alkali whose mass fraction is 2%under the pressure of 0.1MPa by 30 minutes.The reusability of IEMR is also studied.The results show that,the conversion reached the maximum when IEMR is used repeatedly by the second time.The conversion reduces only 7%when IEMR is used repeatedly 4 times which can meet the requirements for industrial production.