Exploration of halogenases and chitinases for the production of halogenated compounds and the remediation of chitin-containing waste

Abstract

This work aims to tackle two different subject matters with the utilisation of enzymes. The first part aims to illustrate the potential of flavin-dependent halogenases when applied to the synthesis of halogenated organic molecules, which have become important tools in the pharmaceutical and agricultural industries. Here we use a combination of in silico methods to identify five potential FDHs and subsequently designed and generated constructs for testing. Large-scale production and purification was carried out for three candidates which were subsequently tested in vitro using a substrate library. Initial results suggest that there is a degree of halogenation observed even in reactions which did not contain halogenase. These findings have now also been observed by others. Subsequent work with one of the enzymes showed that it was capable of halogenating several substrates with good conversion under modified conditions. Part two aims to tackle the ongoing problem of seafood waste, which is often discarded into the ocean, onto beaches or burned. Chitinases are enzymes which are capable of breaking down the chitin matrix and could be applied to remediating such waste products to produce value-added products. Here we identify a chitinase from the mosquito parasite, Crithidia fasciculata. We then sought to test the ability of this chitinase (CfCHT1) to break down chitin and its analogues. We found CfCHT1 to be active on short oligomers, but unable to break down colloidal chitin, precluding its use for degrading larger polymeric units of chitin, as found in seafood waste. Nevertheless, we determined that CfCHT1 is primarily an endochitinase, but also possesses a small amount of exochitinase activity. We also tested its optimum pH, temperature and stability to pH and temperature. Finally, we attempted to obtain crystallographic data to aid our understanding of CfCHT1. Moreover, we believe that CfCHT1 may not be a true chitinase.