초록
<P><B>ABSTRACT</B><P>The halogenase‐based catalysis is one of the most environmentally friendly methods for the synthesis of halogenated products, among which flavin‐dependent halogenases (FDHs) have attracted great interest as one of the most promising biocatalysts due to the remarkable site‐selectivity and wide substrate range. However, the complexity of constructing the NAD<SUP>+</SUP>‐NADH‐FAD‐FADH2 bicoenzyme cycle system has affected the engineering applications of FDHs. In this work, a coenzyme self‐sufficient tri‐enzyme fusion was constructed and successfully applied to the continuous halogenation of <I>L</I>‐tryptophan. <I>Sp</I>FDH was firstly identified derived from <I>Streptomyces pratensis</I>, a highly selective halogenase capable of generating 6‐chloro‐tryptophan from tryptophan. Then, using gene fusion technology, <I>Sp</I>FDH was fused with glucose dehydrogenase (GDH) and flavin reductase (FR) to form a tri‐enzyme fusion, which increased the yield by 1.46‐fold and making the coenzymes self‐sufficient. For more efficient halogenation of <I>L</I>‐tryptophan, a continuous halogenation bioprocess of <I>L</I>‐tryptophan was developed by immobilizing the tri‐enzyme fusion and attaching it to a continuous catalytic device, which resulted in a reaction yield of 97.6% after 12 h reaction. An FDH from <I>S. pratensis</I> was successfully applied in the halogenation and our study provides a concise strategy for the preparation of halogenated tryptophan mediated by multienzyme cascade catalysis.</P></P>