TBL38 atypical homogalacturonan-acetylesterase activity and cell wall microdomain localization in Arabidopsis seed mucilage secretory cells.

Plant cell walls constitute complex polysaccharidic/proteinaceous networks whose biosynthesis and dynamics implicate several cell compartments. The synthesis and remodeling of homogalacturonan pectins involve Golgi-localized methylation/acetylation and subsequent cell wall-localized demethylation/deacetylation. So far, TRICHOME BIREFRINGENCE-LIKE (TBL) family members have been described as Golgi-localized acetyltransferases targeting diverse hemicelluloses or pectins. Using seed mucilage secretory cells (MSCs) from Arabidopsis thaliana, we demonstrate the atypical localization of TBL38 restricted to a cell wall microdomain. A tbl38 mutant displays an intriguing homogalacturonan immunological phenotype in this cell wall microdomain and in an MSC surface-enriched abrasion powder. Mass spectrometry oligosaccharide profiling of this fraction reveals an increased homogalacturonan acetylation phenotype. Finally, TBL38 displays pectin acetylesterase activity in vitro. These results indicate that TBL38 is an atypical cell wall-localized TBL that displays a homogalacturonan acetylesterase activity rather than a Golgi-localized acetyltransferase activity as observed in previously studied TBLs. TBL38 function during seed development is discussed.

Characterization of a novel strain of Aspergillus aculeatinus: From rhamnogalacturonan type I pectin degradation to improvement of fruit juice filtration.

Aspergillus spp. are well-known producers of pectinases commonly used in the industry. Aspergillus aculeatinus is a recently identified species but poorly characterized. This study aimed at giving a comprehensive characterization of the enzymatic potential of the O822 strain to produce Rhamnogalacturonan type I (RGI)-degrading enzymes. Proteomic analysis identified cell wall degrading enzymes (cellulases, hemicellulases, and pectinases) that accounted for 92 % of total secreted proteins. Twelve out of fifty proteins were identified as RGI-degrading enzymes. NMR and enzymatic assays revealed high levels of arabinofuranosidase, arabinanase, galactanase, rhamnogalacturonan hydrolases and rhamnogalacturonan acetylesterase activities in aqueous extracts. Viscosity assays carried out with RGI-rich camelina mucilage confirmed the efficiency of enzymes secreted by O822 to hydrolyze RGI, by decreasing viscosity by 70 %. Apple juice trials carried out at laboratory and pilot scale showed an increase in filtration flow rate and yield, paving the way for an industrial use of enzymes derived from A. aculeatinus.

Three novel rhamnogalacturonan I- pectins degrading enzymes from Aspergillus aculeatinus: Biochemical characterization and application potential.

Rhamnogalaturonans I (RGI) pectins, which are a major component of the plant primary cell wall, can be recalcitrant to digestion by commercial enzymatic cocktails, in particular during fruit juice clarification process. To overcome these problems and get better insights into RGI degradation, three RGI degrading enzymes (RHG: Endo-rhamnogalacturonase; ABF: α-Arabinofuranosidases; GAN: Endo-ß-1,4-galactanase) from Aspergillus aculeatinus were expressed in Pichia pastoris, purified and fully biochemically characterized. All three enzymes showed acidic pH optimum, and temperature optima between 40-50 °C. The Km values were 0.5 mg.ml-1, 1.64 mg.ml-1 and 3.72 mg.ml-1 for RHG, ABF, GAN, respectively. NMR analysis confirmed an endo-acting mode of action for RHG and GAN, and exo-acting mode for ABF. The application potential of these enzymes was assessed by measuring changes in viscosity of RGI-rich camelina mucilage, showing that RHG-GAN enzymes induced a decrease in viscosity by altering the structures of the RGI backbone and sidechains.