Novel xylan degrading enzymes from polysaccharide utilizing loci of Prevotella copri DSM18205

Prevotella copri is a bacterium that can be found in the human gastrointestinal tract (GIT). The role of P. copri in the GIT is unclear, and elevated numbers of the microbe have been reported both in dietary fiber-induced improvement in glucose metabolism but also in conjunction with certain inflammatory conditions. These findings raised our interest in investigating the possibility of P. copri to grow on xylan, and identify the enzyme systems playing a role in digestion of xylan-based dietary fibers. Two xylan degrading polysaccharide utilizing loci (PUL10 and 15) were found in the genome, with three and eight glycoside hydrolase (GH) -encoding genes, respectively. Three of them were successfully produced in Escherichia coli: One extracellular enzyme from GH43 (subfamily 12, in PUL10, 60 kDa) and two enzymes from PUL15, one extracellular GH10 (41 kDa), and one intracellular GH43 (subfamily 137 kDa). Based on our results, we propose that in PUL15, GH10 (1) is an extracellular endo-1,4-β-xylanase, that hydrolazes mainly glucuronosylated xylan polymers to xylooligosaccharides (XOS); while, GH43_1 in the same PUL, is an intracellular β-xylosidase, catalyzing complete hydrolysis of the XOS to xylose. In PUL10, the characterized GH43_12 is an arabinofuranosidase, with a role in degradation of arabinoxylan, catalyzing removal of arabinose-residues on xylan.Fil: Linares Pastén, Javier A. Lund University. Biotechnology Department; SueciaFil: Hero, Johan Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Pisa, José Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Teixeira, Cristina. Lund University. Biotechnology Department; SueciaFil: Nyman, Margareta. Department Food Technology, Engineering And Nutrition; SueciaFil: Adlercreutz, Patrick. Lund University. Biotechnology Department; SueciaFil: Martinez, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Nordberg Karlsson, Eva. Universidad Nacional de Tucumán. Facultad de Ciencias Exactas y Tecnología; Argentin

Comparison of the enzymatic depolymerization of polyethylene terephthalate and AkestraTM using Humicola insolens cutinase

The enzymatic depolymerization of synthetic polyesters has become of great interest in recycling plastics. Most of the research in this area focuses on the depolymerization of polyethylene terephthalate (PET) due to its widespread use in various applications. However, the enzymatic activity on other commercial polyesters is less frequently investigated. Therefore, AkestraTM attracted our attention, which is a copolymer derived from PET with a partially biobased spirocyclic acetal structure. In this study, the activity of Humicola insolens cutinase (HiCut) on PET and AkestraTM films and powder was investigated. HiCut showed higher depolymerization activity on amorphous PET films than on Akestra™ films. However, an outstanding performance was achieved on AkestraTM powder, reaching 38% depolymerization in 235h, while only 12% for PET powder. These results are consistent with the dependence of the enzymes on the crystallinity of the polymer since Akestra™ is amorphous while the PET powder has 14% crystallinity. On the other hand, HiCut docking studies and molecular dynamic simulations (MD) suggested that the PET-derived mono (hydroxyethyl)terephthalate dimer (MHET)2 is a hydrolyzable ligand, producing terephthalic acid (TPA), while the Akestra™-derived TPA-spiroglycol ester is not, which is consistent with the depolymerization products determined experimentally. MD studies also suggest ligand-induced local conformational changes in the active site

Modeled 3D-Structures of Proteobacterial Transglycosylases from Glycoside Hydrolase Family 17 Give Insight in Ligand Interactions Explaining Differences in Transglycosylation Products

The structures of glycoside hydrolase family 17 (GH17) catalytic modules from modular proteins in the ndvB loci in Pseudomonas aeruginosa (Glt1), P. putida (Glt3) and Bradyrhizobium diazoefficiens (previously B. japonicum) (Glt20) were modeled to shed light on reported differences between these homologous transglycosylases concerning substrate size, preferred cleavage site (from reducing end (Glt20: DP2 product) or non-reducing end (Glt1, Glt3: DP4 products)), branching (Glt20) and linkage formed (1,3-linkage in Glt1, Glt3 and 1,6-linkage in Glt20). Hybrid models were built and stability of the resulting TIM-barrel structures was supported by molecular dynamics simulations. Catalytic amino acids were identified by superimposition of GH17 structures, and function was verified by mutagenesis using Glt20 as template (i.e., E120 and E209). Ligand docking revealed six putative subsites (−4, −3, −2, −1, +1 and +2), and the conserved interacting residues suggest substrate binding in the same orientation in all three transglycosylases, despite release of the donor oligosaccharide product from either the reducing (Glt20) or non-reducing end (Glt1, Gl3). Subsites +1 and +2 are most conserved and the difference in release is likely due to changes in loop structures, leading to loss of hydrogen bonds in Glt20. Substrate docking in Glt20 indicate that presence of covalently bound donor in glycone subsites −4 to −1 creates space to accommodate acceptor oligosaccharide in alternative subsites in the catalytic cleft, promoting a branching point and formation of a 1,6-linkage. The minimum donor size of DP5, can be explained assuming preferred binding of DP4 substrates in subsite −4 to −1, preventing catalysis

Study on oligomerization of glutamate decarboxylase from Lactobacillus brevis using asymmetrical flow field-flow fractionation (AF4) with light scattering techniques

In this work, asymmetrical flow field-flow fractionation (AF4) coupled with UV/Vis, multi-angle light scattering (MALS), and differential refractive index (dRI) detectors (AF4-UV-MALS-dRI) was employed for analysis of glutamate decarboxylase (LbGadB) from Lactobacillus brevis (L. brevis). AF4 provided molecular weight (MW) (or size)-based separation of dimer, hexamer, and aggregates of LbGadB. The effect of pH on oligomerization of LbGadB was investigated, and then AF4 results were compared to those from molecular modeling. The MWs measured by AF4-UV-MALS-dRI for dimeric and hexameric forms of LbGadB were 110 and 350 kDa, respectively, which are in good agreements with those theoretically calculated (110 and 330 kDa). The molecular sizes determined by AF4-UV-MALS-dRI were also in good agreement with those obtained from molecular modeling (6 and 10 nm, respectively, for dimeric and hexameric from AF4-UV-MALS-dRI and 6.4 × 7.6 and 7.6 × 13.1 nm from molecular modeling). The effects of temperature, salt type, and salt concentration on oligomerization of LbGadB were also investigated using dynamic light scattering (DLS). It was found that the hexameric form of LbGadB was most stable at pH 6 and in presence of NaCl or KCl. The results indicate that AF4, in combination of various online detectors mentioned above, provides an effective tool for monitoring of oligomerization of LbGadB under different conditions, such as temperature, pH, type of salts, and salt concentrations

Endo-xylanases as tools for production of substituted xylooligosaccharides with prebiotic properties

Xylan has a main chain consisting of β-1,4-linked xylose residues with diverse substituents. Endoxylanases cleave the xylan chain at cleavage sites determined by the substitution pattern and thus give different oligosaccharide product patterns. Most known endoxylanases belong to glycoside hydrolase (GH) families 10 and 11. These enzymes work well on unsubstituted xylan but accept substituents in certain subsites. The GH11 enzymes are more restricted by substituents, but on the other hand, they are normally more active than the GH10 enzymes on insoluble substrates, because of their smaller size. GH5 endoxylanases accept arabinose substituents in several subsites and require it in the − 1 subsite. This specificity makes the GH5 endoxylanases very useful for degradation of highly arabinose-substituted xylans and for the selective production of arabinoxylooligosaccharides, without formation of unsubstituted xylooligosaccharides. The GH30 endoxylanases have a related type of specificity in that they require a uronic acid substituent in the − 2 subsite, which makes them very useful for the production of uronic acid substituted oligosaccharides. The ability of dietary xylooligosaccharides to function as prebiotics in humans is governed by their substitution patterns. Endoxylanases are thus excellent tools to tailor prebiotic oligosaccharides to stimulate various types of intestinal bacteria and to cause fermentation in different parts of the gastrointestinal tract. Continuously increasing knowledge on the function of the gut microbiota and discoveries of novel endoxylanases increase the possibilities to achieve health-promoting effects

Interaction between Myricetin Aggregates and Lipase under Simplified Intestinal Conditions

Myricetin, a flavonoid found in the plant kingdom, has previously been identified as a food molecule with beneficial effects against obesity. This property has been related with its potential to inhibit lipase, the enzyme responsible for fat digestion. In this study, we investigate the interaction between myricetin and lipase under simplified intestinal conditions from a colloidal point of view. The results show that myricetin form aggregates in aqueous medium and under simplified intestinal condition, where it was found that lipase is in its monomeric form. Although lipase inhibition by myricetin at a molecular level has been reported previously, the results of this study suggest that myricetin aggregates inhibit lipase by a sequestering mechanism as well. The size of these aggregates was determined to be in the range of a few nm to >200 nm

Investigation of Structural Features of Two Related Lipases and the Impact on Fatty Acid Specificity in Vegetable Fats

One of the indispensable applications of lipases in modification of oils and fats is the possibility to tailor the fatty acid content of triacylglycerols (TAGs), to meet specific requirements from various applications in food, nutrition, and cosmetic industries. Oleic acid (C18:1) and stearic acid (C18:0) are two common long fatty acids in the side chain of triglycerides in plant fats and oils that have similar chemical composition and structures, except for an unsaturated bond between C9 and C10 in oleic acid. Two lipases from Rhizomucor miehei (RML) and Rhizopus oryzae (ROL), show activity in reactions involving oleate and stearate, and share high sequence and structural identity. In this research, the preference for one of these two similar fatty acid side chains was investigated for the two lipases and was related to the respective enzyme structure. From transesterification reactions with 1:1 (molar ratio) mixed ethyl stearate (ES) and ethyl oleate (EO), both RML and ROL showed a higher activity towards EO than ES, but RML showed around 10% higher preference for ES compared with ROL. In silico results showed that stearate has a less stable interaction with the substrate binding crevice in both RML and ROL and higher tendency to freely move out of the substrate binding region, compared with oleate whose structure is more rigid due to the existence of the double bond. However, Trp88 from RML which is an Ala at the identical position in ROL shows a significant stabilization effect in the substrate interaction in RML, especially with stearate as a ligand

Synthesis of novel oligomeric anionic alkyl glycosides using laccase/TEMPO oxidation and cyclodextrin glucanotransferase (CGTase)‐catalyzed transglycosylation

Modification of alkyl glycosides, to alter their properties and widen the scope of potential applications, is of considerable interest. Here, we report the synthesis of new anionic alkyl glycosides with long carbohydrate chains, using two different approaches: laccase/TEMPO oxidation of a long‐carbohydrate‐chain alkyl glycoside, and cyclodextrin glucanotransferase (CGTase)‐catalysed elongation of anionic alkyl glycosides. The laccase/TEMPO oxidation of dodecyl β‐D‐maltooctaoside proceeded efficiently with the formation of aldehyde and acid products. However, depolymerization occurred to a large extent, limiting the product yield and purity. On the other hand, CGTase‐catalysed coupling/disproportionation reactions with α‐cyclodextrin and dodecyl β‐D‐maltoside diuronic acid (DDM‐2COOH) or octyl β‐D‐glucuronic acid (OG‐COOH) as substrates gave high conversions, especially when the CGTase Toruzyme was used. It was found that pH had a strong influence on both the enzyme activity and the acceptor specificity. With non‐ionic substrates (dodecyl β‐D‐maltoside and octyl β‐D‐glucoside), Toruzyme exhibited high catalytic activity at pH 5‐6, but for the acidic substrates (DDM‐2COOH and OG‐COOH) the activity was highest at pH 4. This is most likely due to the enzyme favoring the protonated forms of DDM‐2COOH and OG‐COOH, which exist at lower pH (pKa about 3)

Synthesis of novel oligomeric anionic alkyl glycosides using laccase/TEMPO oxidation and cyclodextrin glucanotransferase (CGTase)‐catalysed transglycosylation

Modification of alkyl glycosides, to alter their properties and widen the scope of potential applications, is of considerable interest. Here, we report the synthesis of new anionic alkyl glycosides with long carbohydrate chains, using two different approaches: laccase/TEMPO oxidation of a long‐carbohydrate‐chain alkyl glycoside, and cyclodextrin glucanotransferase (CGTase)‐catalysed elongation of anionic alkyl glycosides. The laccase/TEMPO oxidation of dodecyl β‐D‐maltooctaoside proceeded efficiently with the formation of aldehyde and acid products. However, depolymerization occurred to a large extent, limiting the product yield and purity. On the other hand, CGTase‐catalysed coupling/disproportionation reactions with α‐cyclodextrin and dodecyl β‐D‐maltoside diuronic acid (DDM‐2COOH) or octyl β‐D‐glucuronic acid (OG‐COOH) as substrates gave high conversions, especially when the CGTase Toruzyme was used. It was found that pH had a strong influence on both the enzyme activity and the acceptor specificity. With non‐ionic substrates (dodecyl β‐D‐maltoside and octyl β‐D‐glucoside), Toruzyme exhibited high catalytic activity at pH 5‐6, but for the acidic substrates (DDM‐2COOH and OG‐COOH) the activity was highest at pH 4. This is most likely due to the enzyme favoring the protonated forms of DDM‐2COOH and OG‐COOH, which exist at lower pH (pKa about 3)

Novel Function of CtXyn5A from Acetivibrio thermocellus: Dual Arabinoxylanase and Feruloyl Esterase Activity Occur in the Same Active Site

Uncharacterized side activities of enzymes can have significant negative effects on reaction products and yields. Hence, their identification and characterization is crucial for the development of successful reaction systems. Here, we report the presence of feruloyl esterase activity in CtXyn5A from Acetivibrio thermocellus besides its well-known arabinoxylanase activity for the first time. Both reaction types appear to be catalysed in the same active site in two subsequential steps. The ferulic acid substituent is cleaved off first, followed by the hydrolysis of the xylan backbone. The esterase activity on complex carbohydrates was found to be higher than the one of a designated ferulic acid esterase (E-FAERU). Therefore, we conclude that the enzyme exhibits a dual function rather than an esterase side activity