Immobilization of Maleate Cis-Trans Isomerase and Synthesis of Fumarate

Abstract:

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.