Discovery and characterization of enzymes acting on chitin

In the shift from a fossil-based to a bio-based economy, exploration of renewable recourses is needed. Chitin is considered the second most abundant polysaccharide on Earth, after cellulose, and its water-soluble derivatives chitosan and chitooligosaccharides (CHOS) have several applications, for example in medicine, agriculture, and the food industry. Today, the extraction of chitin from chitin-rich biomasses and the subsequent production of chitosan and CHOS involve harsh chemicals. It is of interest to replace the current chemical processing technology with enzyme-driven processes, since this would be more environmentally friendly. In addition, enzymes can be used to produce well-defined chitosans and CHOS, which is of interest, since the bioactivity of these compounds depends on properties such as the fraction of acetylation (FA), the degree of polymerization (DP) and the pattern of acetylation (PA). Investigation of proteins utilized by microorganisms during growth on chitin might provide insight into natural chitin conversion and may yield enzymes that can aid in industrial valorization of chitin-rich biomasses. Paper I describes the characterization of a carbohydrate esterase family 4 (CE4) deacetylase, which was selected because of its potential application in the production of CHOS with defined FA and PA. To utilize these enzymes in an optimal way, good understanding of their substrate interactions and specificities is needed. Paper I includes the first enzyme-substrate complex of a CE4 deacetylase with an open active site, providing valuable insight into how the enzyme interacts with its substrate. The enzyme is able to deacetylate a variety of substrates at varying positions. This broad specificity and the presence of seemingly few subsites occupied by the substrate indicate that it may be difficult to use or develop this type of CE4 enzymes for enzymatic tailoring of the PA of CHOS. The genome of Cellvibrio japonicus encodes a large array of carbohydrate-active enzymes, including several putative chitinases and other enzymes possibly involved in chitin degradation. Whether these enzymes are actually involved in chitin utilization by this Gram-negative bacterium had not been investigated at the start of the work described in this thesis. Paper II describes a study of proteins that C. japonicus secretes during growth on chitin, using a novel, plate-based proteomics approach which yielded secretome samples with a relatively low fraction of cytoplasmic proteins. This study revealed that the four glycosyl hydrolase family 18 (GH18) chitinases encoded in the C. japonicus genome are produced in high amounts, indicating that these enzymes are all involved in natural chitin turnover. Chitin degradation studies showed that C. japonicus has considerable chitinolytic power. The proteomics study revealed several proteins without an obvious role in chitin degradation that also are produced in high amounts during growth on chitin, thus providing a list of proteins that could be targeted in future searches for proteins that degrade chitin-rich biomass. Paper III describes an in-depth investigation of the GH18 chitinases encoded by C. japonicus. Knockout studies showed that one of the chitinases, CjChi18D, is crucial for the bacterium’s ability to utilize chitin as a carbon source. Biochemical characterization showed that CjChi18D is the most efficient chitin degrader, which could explain its crucial role. Comparative studies of the four enzymes indicated different and putatively complementary functions, as exemplified by CjChi18C having the by far highest activity against chitohexaose. Indeed, when combining enzymes, synergistic effects on chitin degradation efficiency were observed. Transcriptomic analysis showed that the four GH18 chitinases and a chitin-active LPMO, CjLPMO10A, are strongly up-regulated when C. japonicus grows on chitin, along with several other putatively chitin-active enzymes as well as a few proteins of unknown function, which are up-regulated to a lesser extent. Serratia marcescens produces one the best studied chitinolytic machineries, involving three chitinases, a lytic polysaccharide monooxygenase, and a chitobiase. However, the genome sequence of one of the most frequently studied S. marcescens strains was not available at the start of this thesis work, and possible involvement of other proteins in chitin utilization had not been investigated. Paper IV describes the genome sequence of S. marcescens BJL200 and a proteomics investigation of proteins secreted during growth on chitin. The genome sequence showed that S. marcescens encodes a fourth chitinase, SmChiD, but the proteomics data indicated that this chitinase is not important in chitin utilization. Indeed, biochemical characterization of SmChiD supported the notion that this enzyme is not important for chitin conversion and, thus, likely has another, yet unknown, biological role. Taken together, the results presented in this thesis provide novel insight into chitin-active enzymes encoded by bacteria. Paper I provides insights into the substrate binding of CE4 deacetylases with an open active site. Papers II-IV reveal chitin-active enzymes, in particular hydrolases, that play key roles in natural chitin conversion. Additionally, Papers II-IV yield a list with proteins without an obvious role in chitin degradation, which may be targeted in future studies of the degradation of chitin-rich biomasses. Further studies on tailoring CE4 deacetylases for modification of chitosan and CHOS and on more efficient chitin conversion using enzymes derived from S. marcescens and C. japonicus are currently in progress