固定化马来酸顺反异构酶合成富马酸

Immobilization of Maleate Cis-Trans Isomerase and Synthesis of Fumarate

为了制备稳定高效的固定化酶转化底物马来酸来生产富马酸,本文将粘质沙雷氏菌(Serratia marcescens)来源的马来酸顺反异构酶与R5肽段融合表达,融合酶比酶活可达42 U/mg。将细胞破碎上清液用40%硫酸铵沉淀,再用终体积分数为0.1%的戊二醛在室温下交联1 h形成交联酶聚集体,最后用1 mol/L正硅酸甲酯包埋,固定化酶的酶活回收率达到60%。在55℃下,固定化酶的半衰期可达4 h(游离酶仅为0.5 h),进行8次重复催化反应后,可保留78%的初始酶活。将固定化马来酸顺反异构酶装入填充床反应器,连续转化10个批次后富马酸的转化率可保持在95%以上。该研究为马来酸顺反异构酶生产富马酸的工业化应用提供了借鉴。

In order to prepare a stable and efficient immobilized enzyme to produce fumarate from maleate,maleate cis-trans isomerase(MaiA)from Serratia marcescens was used as a model enzyme in which a silica-precipitating peptide R5 were fused to its N-terminal.The specific activity of the purified fusion enzyme(R5-MaiA)could reach 42 U/mg.The optimum conditions for preparing the immobilized enzyme could be summarized as:cell crude extract was precipitated with 40%-saturation ammonia sulfate followed by cross-linking with 0.1%glutaraldehyde for 1 h at room temperature;then the cross-linking enzyme aggregates(CLEAs)was encapsulated through biosilicification by rapidly mixing with 1 mol/L hydrolyzed tetramethoxysilane(TMOS).The immobilized enzyme Si-CLEAs retained 60%specific activity of the free enzyme.Si-CLEAs had higher thermostability(t1/2=4 h)than the free enzyme(t1/2=1.5 h)at 55℃.Si-CLEAs retained about 78%of the initial activities after eight catalytic batches.When loaded in a packed bed reactor,the packed-bed reactor showed high stability and conversion ratio of 95%after reused for 10 times.The study will be useful for industrial applications of fumarate synthesis using immobilized maleate cis-trans isomerase.