Identification and characterisation of an enzyme from Monoglobus pectinilyticus associated with degradation of pectin from kiwiberry : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biological Sciences at Massey University, Palmerston North, New Zealand

Abstract

Pectin is a complex polysaccharide and a very important source of dietary soluble fibre, present in a variety of fruit and vegetables. Pectin possesses a wide range of contributions to a healthy diet and a healthy human gut, such as lowering cholesterol, protecting against intestinal inflammation and maintaining digestive health. Yet pectin is unable to be degraded by human gastrointestinal enzymes, arriving in the large intestine mostly intact. Limited research has showed the domination of Gram-negative bacteria which possess a range of secreted extracellular cell-bound and cell-free pectin degrading enzymes. These enzymes attack the pectin backbone and the accessory side chains resulting in the isolation of mono/oligosaccharides for subsequent uptake. The recent novel discovery of Monoglobus pectinilyticus, a Gram-positive bacterium, expanded the narrow knowledge regarding specific pectinolytic degraders. Genomic studies showed this bacterium contains an arsenal of enzymes specifically for pectin degradation. However, several putative pectin-degrading enzymes produced by M. pectinilyticus were unable to be identified against up-to-date databases, thus suggesting that there may be potentially novel classes of CAZyme(s) to be discovered. The proposed study will attempt to identify such enzyme(s) using New Zealand cultivar kiwiberries as the source of pectin. The initial aim of this study was to investigate the enzyme(s) involved in β-1,4-galactan by growing M. pectinilyticus with a mixture of pectin substrates (0.5% citrus pectin, 0.5% kiwiberry pectin and 0.5% potato galactan), in hope to identify such enzyme(s). Unfortunately, no β-galactosidase/β-galactanase was found. However a potentially novel binding enzyme, protein 0050 was isolated, cloned and expressed, which may be involved in a pectin degrading version of a cellulosome complex. Further research into isolating a variety of secreted proteins activated in presence of galactan during M. pectinilyticus cultivation is needed to understand the complexity of pectin degradation.