The Modification Of Titanium Dioxide And Its Application In Immobilization Of Penicillin G Acylase

Penicillin series of antibiotics are a sort of important antibiotics,they have a very broad application prospects in the field of antibacterial anti-inflammatory.however,because of abuse and overproduction,large amounts of penicillin had been emitted into the environment,which had lead to the drug-fast of germs and inefficacy of original penicillin.In order to decrease the trend,the common modification method was hydrolyzed the original penicillin to produce 6-aminopenicillanic acid?6-APA?first,and then chang the side chain to prepare semi-synthetic penicillin.Thus,6-APA had become the most important precursor for the synthesis of penicillin antibiotics.There were two main ways to obtain 6-APA from penicillin G,i.e.chemical hydrolysis and enzyme catalytic hydrolysis.Chemical hydrolysis method was a long way,relative low efficiency,serious environmental pollution and large amount of side reactions process,and had been casted away.Compared with chemical hydrolysis,enzyme catalytic hydrolysis method was a high selectivity and specificity,mild reaction condition,non-polluting,and high yield process,which had attracted many interesting,and had replaced the chemical hydrolysis method and widely used in industry.However,the use of free PGA for the conversion of penicillin G also presented some disadvantages,such as poor stability,weak environmental tolerance,poor reusability,and difficulty in purification of the product.In order to improve the efficiency of enzyme-catalyzed hydrolysis,the enzyme was always immobilized.However,PGA immobilization will lead to the loss of PGA activity.In addition,after the discarded of immobilized PGA,it would be difficult to spread due to poor exercise ability,and the decomposition products will cause serious problems such as regional environmental,social and visual angle pollution.Therefore,the systematic study of immobilization was still necessary.Titanium dioxide?TiO₂?particle possessed super-hydrophilic surface,which would be beneficial for maintaining the active center of PGA according to our research result.Besides,TiO₂ also had a high refraction and masking effect to short wavelength ultraviolet,which meant immobilized PGA would be protected and avoid the damage of sun.What's more,TiO₂ in the sun could exhibit good catalytic activity,which would degrade the wasted immobilized PGA and realizae harmless treatment.Just for above reasons,TiO₂ was sonscripted in this paper as the carrier to immobilize PGA.During this process,conditions of the modification of TiO₂ and the immobilization of PGA were explored,and some meaningful results were obtained and summarized below.?1?TiO₂ particle was modified with glutaraldehyde,and modification conditions,i.e.particle size?d?,active temperature?T_a?,modification temperature?T_m?,modification time?t_m?,p H,the ratio of aldehyde to ethanol?R_a?andthe ratio of ethanol to water?R_e?,were studied and optimized.Using grafting rate?G?as the optimizing index,ultraviolet-visible spectrophotometry as characterization method,and the obtained optimal modification conditions were d=3.4?m,T_a=120^?,T_m=45^?,t_m=30h,p H=6.0,R_a=1:10 and R_e=1.Under the optimal conditions,G of glutaraldehyde arrived at 50%.Thereafter,a series of modified TiO₂ obtained from different modification conditions were adopted to immobilized PGA.During this process,the ultraviolet-visible spectrophotometry of 6-aminopenicillanic acid was conscripted as characterization method.The enzyme loading capacity?ELC?,enzyme activity?EA?and enzyme activity retaining ratio?EAR?were studied.The results show that the ELC arrived 8780 U,EA arrived 14872 U/g,EA arrived 88%.?2?Effects of immobilization conditions on EA,ELC,and EAR were investigated and optimzed.The optimal immobilization conditions were obtained?immobilization time t_i=24 h,immobilization temperature T_i=35°C,immobilized p H=8,immobilized enzyme concentration C_i=2.5%?.At the same time,the stability of the immobilized enzyme?temperature stability,p H stability,and time stability?was explored.The results showed that the stability of the immobilized enzyme was greatly improved compared to the free enzyme.The reusability of the immobilized enzyme has also been improved.?3?TiO₂ was modified by different functional groups to preliminary study the influence of microenviroment on EA,ELC,and EAR of the immobilized PGA throuth thesurfacestructuraldesign,andresultsshowthat3-glycidoxypropyltrimethoxysilane modified TiO₂ and immobilized PGA showed the besteffectinprimarymodification.Glutaraldehydeand3-glycidoxypropyltrimethoxysilane modified TiO₂ in turn and then immobilized PGA showed the best effect in secondary modification.The reusability of immobilized PGA was explored under different microenvironment.The results showed that the effect of reusability was the best under the aldehyde microenvironment,the effect of reusability was the worst under the amino microenvironment.This paper will provide technical support for the design of the PGA immobilized carrier,the purpose was prepared the carrier with high enzyme activity,high loading capacity and high enzyme activity retention ratio.