Study On The Immobilization And Application Of Enoate Reductases

Enoate reductases(ERs)is a type of enzymes with great potential application value,from the‘old yellow enzyme'family,widely distributed in microorganisms and plants.It can selectively reduce C-C double bond for?,?-unsaturated compounds,such as aldehydes,ketones and nitroalkenes.Those products of catalytic reaction are the key intermediates in pesticide,food additive and medicine.The adcantages of ER in catalytic reaction include less environmental pollution and mild reaction conditions and so on.Despite all this,there are still some limitations,such as consuming expensive coenzyme and losing activity easily.In response to the above problems,three different immobilization methods were adopted to regenerate the coenzyme in the catalytic reaction,and the stability of ER was improved significantly.Those methods were cross-linked enzyme aggregates(CLEAs),biomimetic immobilization(BI)and cellulose covalent immobilization(CCI),respectively.In the preparation of ER-GDH-CLEAs(co-immobilized enzymes),ammonium sulfate solution(final concentration 4M)and oxidized dextran(15%,v/v)was precipitant and crosslinking agent,respectively.Moreover,the amount of ER and glucose dehydrogenase(GDH)were 11 U and 50 U.Under the above optimal preparation conditions,immobilization efficiency of ER-GDH-CLEAs was 94.2%,activity recovery of ER and GDH in immobilized enzyme were44.1%and 18.8%respectively.Different structural characteristics of ER-GDH-CLEAs were shown in scanning electron microscopy(SEM).Compared to aldehyde glutaraldehyde crosslinking agent,oxidized dextran made structure more porous and adding BSA also enhanced this trend.Combined with the experimental data,it is found that the porous structure might be more conducive to the contact between substrate and enzyme.Furthermore,the stability of enzyme was significantly improved.After 8 h of heat treatment at 50°C,relative activity of ER in ER-GDH-CLEAs maintained 65.2%,while that of free ER was only 9.2%.In addition,the reusability of ER-GDH-CLEAs was remarkable.After fourteen times of recycling,ER-GDH-CLEAs could maintain 114%of its initial activity,more than 100%.Maybe protein conformation was changed in reuse.With regard to preparation of ER-GDH-SPs.Enzymes were trapped within a silica support by hydrolyzing a silicic acid precursor(tetramethyl orthosilicate,TMOS,final concentration 4M).The optimal buffer solution was sodium phosphate buffer(50 mM,pH 7.0),immobilized time was2 h,the amount of ER and GDH were 11 U and 50 U,respectively.Under the above conditions,immobilization efficiency of ER-GDH-SPs was 94.7%,activity recovery of ER and GDH respectively were 44.5%and 22.3%.The SEM showed a granular aggregate in the field of view,combined with experimental data of the immobilization efficiency of ER-GDH-SPs(higher than94%)and the immobilization principle,indicating that the enzyme protein was entrapped in nano silicon dioxide.Due to the nano-silica coating,ER-GDH-SPs obtained outstanding stability.After heat treatment at 50°C for 8 h,the relative activity of ER in ER-GDH-SPs retained 55.4%,while that of free ER was almost completely lost.Furthermore,ER-GDH-SPs could retain nearly 100%of initial activity in the first six cycles.In the preparation of ER-or GDH-CCI,the optimum conditions were pretreatment of lignocellulose with DESs,and surface modification was made by using sodium periodate,ethylenediamine(final concentration 300 mM)and glutaraldehyde(final concentration 100mM).When ER amount was 30 mg,immobilization efficiency and activity recovery reached95.5%and 47.5%,respectively.SEM showed that lignocellulose became rough and porous because most lignin was removed by DESs.After further modification,the protein was combined with carrier and obtained excellent thermal stability.Free and ER-CCI were incubated at a temperature of 50°C for 8 h,ER-CCI still remained 82.3%of its initial activity,whereas free ER lost 90.8%.In addition,immobilized enzyme could retain 80%of initial activity in the first eight cycles.In order to confirm the effect of coenzyme regeneration in catalytic reaction with immobilized enzymes.The reduction reaction of 4-(4-Methoxyphenyl)-3-buten-2-one was used as the model reaction,and the continuous conversion of substrate was carried out.Five times of substrate(100?M per time)and NAD~+(only once,100?M)were added during the experiment.Based on principle of redox reaction of ER,it was obtained that the consumption ratio of NAD~+to substrate was 2:1.When the concentration of the substrate was 100?M,the product concentration of the reaction in the ideal state was 50?M.In the actual experiment,the concentration of product in ER-GDH-CLEAs,ER-GDH-SPs,ER-and GDH-CCI catalytic systems were 215?M,155?M and 105?M which were about four,three and two times of the ideal concentration respectively.Above results indicated that the three immobilization methods achieved coenzyme regeneration effectively.In conclusion,the immobilization efficiency of ER prepared by three immobilization methods were higher than 94%.Although the activity recovery was low(45%),the data was at the same level with the relevant reports of oxidation-reduction immobilized enzymes.At present,the immobilization of ER has not been reported.