Insights into the enzymatic degradation of mannan (in situ) and pectin utilization by Bacteroides thetaiotaomicron

Abstract

The cell walls of plants are extremely complex and dynamic, and serve as the most abundant organic carbon source on the planet. These cell walls represent a potential alternative to fossil substrate for several industries such as the biofuel and chemical sectors. Plant cell walls also represent a significant nutrient for the microorganisms that inhabit the human distal gut. Understanding how the plant cell wall is degraded by microbial enzymes is critical to the industrial use of these composite structures, but also in the design of human promoting novel pre- and probiotics. In this thesis work is described that has analysed the importance of non-catalytic carbohydrate binding modules (CBMs) in the enzymatic degradation of -mannanas in plant cell walls, and the mechanism by which complex pectins are utilized by gut bacteria. Plant cell wall degrading enzymes often contain one or more CBMs. Work presented in this thesis evaluated the contribution of the specificities displayed by the catalytic modules and CBMs of endo-1,4-mannanases (mannanases) and esterases, to the enzymatic degradation of intact plant cell walls of both Nicotiana tabacum (tobacco) and Physcomitrella patens (moss). The data showed that CBMs greatly potentiated the activity of mannanases and esterases against intact plant cell walls. The cellulose and mannan binding CBMs have the greatest impact on the removal of mannan from tobacco and Physcomitrella cell walls, respectively. Finally, rather than improving the efficiency of mannan degradation by promoting enzyme substrate proximity, CBM32, which binds to the non-reducing end of mannan chains, enhanced the hydrolytic activity of a mannanase by preventing transglycosylation reactions. This work provides insights into the biological significance in vivo for the complex array of hydrolytic enzymes expressed by plant cell wall degrading organisms. Chapter 4 and 5 focused on the mechanism by which a prominent human gut bacterium, Bacteroides thetaiotaomicron, degraded the complex pectins rhamnogalacturonan I (RGI) and rhamnogalacturonan II (RGII). RGII is particularly complex consisting of 13 different monosaccharides and over 20 linkages and is believed to be highly resistant to microbial digestion. In this study proteins encoded by the RGI and RGII utilization loci of B. thetaiotaomicron were expressed in Escherichia coli, and their activity against RGI and RGII were assessed. With respect to RGII BT1010 was shown to be an -L-galactosidase that removes the terminal sugar on chain A. BT3662is an -L-arabinofuranosidase that hydrolysed the -1,3-linkage between arabinofuranosyl units and the polygalacturonic acid backbone. Both enzymes displayed novel activities for their respective CAZy families, GH95 (BT1010) and GH43 (BT3662). With respect to RGI a model for the complete depolymerization of the polysaccharide was generated. Briefly, the enzyme consortium that degraded the GalA-Rha backbone included three rhamnogalacturonan lyases and four glycoside hydrolases; two GH105 4,5unsaturated rhamnogalacturonase, a rhamnosidase and an RGI-specific galacturonosidase. The kinetic parameters of the lyases and GH105 enzymes revealed distinct but complementary specificities. The short galactan side chains remaining after endo-galactanase attack were removed by three -galactosidases. The model is completed by the identification of an esterase that was shown to play an important role in the capacity of the lyases and glycoside hydrolases to access the RGI backbone. Whole cell assays showed that significant RGI degradation occurred on the bacterial surface and unsaturated tetrasaccharides induced the expression of the RGI degrading enzymes. The data presented in this thesis underpins the use of CBMs in the industrial utilization of integral cell wall polysaccharides, while the data presented for pectin degradation by a prominent gut bacterium identifies opportunities for developing novel functional foods.