Genetically Encoded Multienzyme Particles for the Biosynthesis of Putrescine from l-Arginine

Abstract

Immobilized enzyme biocatalysts have wide industrial applications, but the multiple steps involved in their preparation significantly increase the time, effort, and cost to produce them. Our group has previously developed a platform for the direct immobilization of enzymes based on the crystal-forming Cry3Aa protein. The application of this platform has been demonstrated for the direct immobilization of individual enzymes, with a molecular size limitation of approximately 50 kDa. However, the biosynthesis of many compounds involve cascade reactions utilizing multiple enzymes of various sizes including those larger than this limit. Herein, we report the use of a larger Cry homologue, Cry1Ab, for the in vivo coimmobilization of two enzymes involved in the biosynthesis of putrescine�human arginase I (hArg, 35 kDa) and ornithine decarboxylase (ODC, 79 kDa). We show that Cry1Ab-mediated immobilization enhanced the thermostability of these enzymes, allowing for the efficient and recyclable conversion of arginine to putrescine, yielding 30 mM putrescine per cycle at nearly 100% conversion in 30 min for the first seven cycles. Furthermore, the putrescine produced could be directly converted to the thermostable polyamide nylon-4,6.