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

DOI：10.3969/j.issn.1673-1689.2018.08.001

中文关键词: 马来酸顺反异构酶 富马酸 R5肽段 固定化酶

英文关键词: maleate cis-trans isomerate,fumarate,R5 peptide,immobilized enzyme

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中文摘要:

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。

英文摘要:

In order to prepare a stable and efficient immobilized enzyme to produce fumarate from maleate，maleate cis-trans isomerase（MaiA） fromSerratia marcescenswas 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.

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