Identification of a novel xanthan-binding module of a multi-modular Cohnella sp. xanthanase.

A new strain of xanthan-degrading bacteria identified as Cohnella sp. has been isolated from a xanthan thickener for food production. The strain was able to utilize xanthan as the only carbon source and to reduce the viscosity of xanthan-containing medium during cultivation. Comparative analysis of the secretomes of Cohnella sp. after growth on different media led to the identification of a xanthanase designated as CspXan9, which was isolated after recombinant production in Escherichia coli. CspXan9 could efficiently degrade the ß-1,4-glucan backbone of xanthan after previous removal of pyruvylated mannose residues from the ends of the native xanthan side chains by xanthan lyase treatment (XLT-xanthan). Compared with xanthanase from Paenibacillus nanensis, xanthanase CspXan9 had a different module composition at the N- and C-terminal ends. The main putative oligosaccharides released from XLT-xanthan by CspXan9 cleavage were tetrasaccharides and octasaccharides. To explore the functions of the N- and C-terminal regions of the enzyme, truncated variants lacking some of the non-catalytic modules (CspXan9-C, CspXan9-N, CspXan9-C-N) were produced. Enzyme assays with the purified deletion derivatives, which all contained the catalytic glycoside hydrolase family 9 (GH9) module, demonstrated substantially reduced specific activity on XLT-xanthan of CspXan9-C-N compared with full-length CspXan9. The C-terminal module of CspXan9 was found to represent a novel carbohydrate-binding module of family CBM66 with binding affinity for XLT-xanthan, as was shown by native affinity polyacrylamide gel electrophoresis in the presence of various polysaccharides. The only previously known binding function of a CBM66 member is exo-type binding to the non-reducing fructose ends of the ß-fructan polysaccharides inulin and levan.

Proteomic analysis of Viscozyme L and its major enzyme components for pectic substrate degradation.

Enzymatic degradation of plant biomass requires the coordinated action of various enzymes. In this study, the production of reducing sugars from pectic substrates and sugar beet pulp (SBP) was investigated and compared using commercial enzyme preparations, including M2, pectinase (E1), Viscozyme L (V-L) and L-40. V-L, a cellulolytic enzyme mix produced by Aspergillus sp. was further evaluated as the most robust enzyme cocktail with the strongest SBP degradation ability in terms of the release of monosaccharides, methanol, and acetate from SBP. Mass-spectrometry-based proteomics analysis of V-L revealed 156 individual proteins. Of these, 101 proteins were annotated as containing a carbohydrate-active enzyme module. Notably, of the 50 most abundant proteins, ca. 44 % were predicted to be involved in pectin degradation. To reveal the role of individual putative key enzymes in pectic substrate decomposition, two abundant galacturonases (PglA and PglB), were heterologously expressed in Pichia pastoris and further characterized. PglA and PglB demonstrated maximum activity at 57 °C and 68 °C, respectively, and exhibited endo-type cleavage patterns towards polygalacturonic acid. Further studies along this line may lead to a better understanding of efficient SBP degradation and may help to design improved artificial enzyme mixtures with lower complexity for future application in biotechnology.

Arabinan saccharification by biogas reactor metagenome-derived arabinosyl hydrolases.

BACKGROUND: Plant cell walls represent the most plentiful renewable organic resource on earth, but due to their heterogeneity, complex structure and partial recalcitrance, their use as biotechnological feedstock is still limited. RESULTS: In order to identify efficient enzymes for polysaccharide breakdown, we have carried out functional screening of metagenomic fosmid libraries from biogas fermenter microbial communities grown on sugar beet pulp, an arabinan-rich agricultural residue, or other sources containing microbes that efficiently depolymerize polysaccharides, using CPH (chromogenic polysaccharide hydrogel) or ICB (insoluble chromogenic biomass) labeled polysaccharide substrates. Seventy-one depolymerase-encoding genes were identified from 55 active fosmid clones by using Illumina and Sanger sequencing and dbCAN CAZyme (carbohydrate-active enzyme) annotation. An around 56 kb assembled DNA fragment putatively originating from Xylanivirga thermophila strain or a close relative was analyzed in detail. It contained 48 ORFs (open reading frames), of which 31 were assigned to sugar metabolism. Interestingly, a large number of genes for enzymes putatively involved in degradation and utilization of arabinose-containing carbohydrates were found. Seven putative arabinosyl hydrolases from this DNA fragment belonging to glycoside hydrolase (GH) families GH51 and GH43 were biochemically characterized, revealing two with endo-arabinanase activity and four with exo-α-L-arabinofuranosidase activity but with complementary cleavage properties. These enzymes were found to act synergistically and can completely hydrolyze SBA (sugar beet arabinan) and DA (debranched arabinan). CONCLUSIONS: We screened 32,776 fosmid clones from several metagenomic libraries with chromogenic lignocellulosic substrates for functional enzymes to advance the understanding about the saccharification of recalcitrant lignocellulose. Seven putative X. thermophila arabinosyl hydrolases were characterized for pectic substrate degradation. The arabinosyl hydrolases displayed maximum activity and significant long-term stability around 50 °C. The enzyme cocktails composed in this study fully degraded the arabinan substrates and thus could serve for arabinose production in food and biofuel industries.

Biochemical and Structural Characterization of Thermostable GH159 Glycoside Hydrolases Exhibiting α-L-Arabinofuranosidase Activity.

Functional, biochemical, and preliminary structural properties are reported for three glycoside hydrolases of the recently described glycoside hydrolase (GH) family 159. The genes were cloned from the genomic sequences of different Caldicellulosiruptor strains. This study extends the spectrum of functions of GH159 enzymes. The only activity previously reported for GH159 was hydrolytic activity on ß-galactofuranosides. Activity screening using a set of para-nitrophenyl (pNP) glycosides suggested additional arabinosidase activity on substrates with arabinosyl residues, which has not been previously reported for members of GH159. Even though the thermophilic enzymes investigated-Cs_Gaf159A, Ch_Gaf159A, and Ck_Gaf159A-cleaved pNP-α-l-arabinofuranoside, they were only weakly active on arabinogalactan, and they did not cleave arabinose from arabinan, arabinoxylan, or gum arabic. However, the enzymes were able to hydrolyze the α-1,3-linkage in different arabinoxylan-derived oligosaccharides (AXOS) with arabinosylated xylose at the non-reducing end (A3X, A2,3XX), suggesting their role in the intracellular hydrolysis of oligosaccharides. Crystallization and structural analysis of the apo form of one of the Caldicellulosiruptor enzymes, Ch_Gaf159A, enabled the elucidation of the first 3D structure of a GH159 member. This work revealed a five-bladed ß-propeller structure for GH159 enzymes. The 3D structure and its substrate-binding pocket also provides an explanation at the molecular level for the observed exo-activity of the enzyme. Furthermore, the structural data enabled the prediction of the catalytic amino acids. This was supported by the complete inactivation by mutation of residues D19, D142, and E190 of Ch_Gaf159A.

Characterization of Two α-l-Arabinofuranosidases from Acetivibrio mesophilus and Their Synergistic Effect in Degradation of Arabinose-Containing Substrates.

Arabinofuranosidases are important accessory enzymes involved in the degradation of arabinose-containing poly- and oligosaccharides. Two arabinofuranosidases from the recently described novel anaerobic cellulolytic bacterium Acetivibrio mesophilus, designated AmAraf51 and AmAraf43, were heterologously expressed in Escherichia coli and biochemically characterized. AmAraf51 not only removed arabinose moieties at O-3, O-2 and terminal O-5 positions of arabinose-containing oligosaccharides, but also exhibited exo-ß-xylosidase side activity. In comparison, AmAraf43 preferably cleaved 1,3-linkages from arabinosyl disubstitutions. AmAraf51 and AmAraf43 demonstrated maximum activity at 70 °C and 57 °C, respectively. Judging from the genetic context and substrate specificity, AmAraf51 may decompose internalized arabino/xylo-oligosaccharides. The embedding of the AmAraf43 gene between genes for several putative xylanolytic enzymes, along with its enzymatic properties suggests that AmAraf43 cleaves arabinose decorations from heteroxylans extracellularly. The enzymes revealed completely converse activity profiles towards arabinan/arabinoxylan: AmAraf51 displayed strong activity on arabinan, while AmAraf43 prefers arabinoxylan. AmAraf51 dramatically stimulated the saccharification level of wheat arabinoxylan (WAX-RS) and sugar beet arabinan when administered along with xylanase M_Xyn10 or arabinanase PpAbn43, respectively. For WAX-RS degradation, the yield of arabinose and xylose was boosted 13.77-fold and 4.96-fold, respectively. The bifunctional activity, thermostability and high catalytic efficiency make AmAraf51 an interesting candidate for industrial applications.

Strategic aromatic residues in the catalytic cleft of the xyloglucanase MtXgh74 modifying thermostability, mode of enzyme action, and viscosity reduction ability.

The thermostable endo-processive xyloglucanase MtXgh74 from Myceliophthora thermophila was used to study the influence of aromatic amino acids in the catalytic cleft on the mode of action and the ability of enzyme to reduce xyloglucan viscosity. The enzyme derivative Mut I with mutations W64A/W67A in the "negative" subsites of the catalytic cleft resulted in a 5.5-fold increase of the Km value. Mut I produced oligosaccharides of various lengths in addition to xyloglucan building blocks. The W320A/W321A substitutions in the "positive" subsites of the mutated enzyme Mut II catalytic cleft increased the Km value 54-fold and resulted in an endo-dissociative mode of action. The ability of Mut II to reduce the viscosity of xyloglucan at 50 °C was much better than that of other MtXgh74 variants. Besides, Mut II efficiently reduced viscosity of a natural substrate, the pulp of xyloglucan-containing tamarind seed flour. The Km, Vmax, and kcat values and viscosity reduction ability of the enzyme derivative Mut III (W320A/W321A/G446Y) returned to levels close to that of MtXgh74. The pattern of xyloglucan hydrolysis by Mut III was typical for endo-processive xyloglucanases. The thermostability of Mut I and Mut II at 60 °C decreased significantly compared to the wild type, whereas the thermostability of Mut III at 60 °C restored almost to the MtXgh74-wt value. All mutants lost the ability to cleave the backbone of xyloglucan building blocks which was a characteristic of MtXgh74. Instead they acquired a low branch removing activity. Molecular dynamics simulations revealed the role of mutated amino acids in the complex action mechanism of GH74 enzymes. KEY POINTS: • Endo-processive mode of action of the xyloglucanase MtXgh74 was altered by rational design. • The endo-dissociative mutant Mut II (W320A/W321A) efficiently reduced XyG viscosity. • The substitutions W320A/W321A/G446Y in Mut III recovered the endo-processive mode. • Mut II can be used to reduce the viscosity of biomass slurries containing tamarind seed flour.

Studies on the gushing potential of Penicillium expansum.

Gushing describes the spontaneous excessive over-foaming of carbonated beverages leading to considerable economic losses and reputational damages to the beverage industry. Surface-active proteins produced by filamentous fungi are involved in the induction of gushing. In the current study, the role of Penicillium expansum in sparkling wine gushing was investigated. Almost 40 P. expansum strains were analyzed regarding their ability to secrete surface-active proteins and to induce gushing in carbonated water as a model system and in sparkling wine. The majority of the strains produced surface-active compounds and induced gushing. The severity of gushing depended on the volume of culture supernatant added to carbonated liquids. Moreover, sparkling wine showed more severe gushing than carbonated water. A protein with a molecular mass of 20 kDa was prominent in gushing-inducing P. expansum culture supernatants. It was identified as PEX2_044840 from P. expansum. This protein was heterologously expressed in Pichia pastoris (Komagataella phaffi). The purified recombinant protein induced gushing in sparkling wine after addition of at least 30 µg/mL of protein sample.

Biochemical characterisation of four rhamnosidases from thermophilic bacteria of the genera Thermotoga, Caldicellulosiruptor and Thermoclostridium.

Carbohydrate active enzymes are classified in databases based on sequence and structural similarity. However, their function can vary considerably within a similarity-based enzyme family, which makes biochemical characterisation indispensable to unravel their physiological role and to arrive at a meaningful annotation of the corresponding genes. In this study, we biochemically characterised the four related enzymes Tm_Ram106B, Tn_Ram106B, Cb_Ram106B and Ts_Ram106B from the thermophilic bacteria Thermotoga maritima MSB8, Thermotoga neapolitana Z2706-MC24, Caldicellulosiruptor bescii DSM 6725 and Thermoclostridium stercorarium DSM 8532, respectively, as α-L-rhamnosidases. Cobalt, nickel, manganese and magnesium ions stimulated while EDTA and EGTA inhibited all four enzymes. The kinetic parameters such as Km, Vmax and kcat were about average compared to other rhamnosidases. The enzymes were inhibited by rhamnose, with half-maximal inhibitory concentrations (IC50) between 5 mM and 8 mM. The α-L-rhamnosidases removed the terminal rhamnose moiety from the rutinoside in naringin, a natural flavonone glycoside. The Thermotoga sp. enzymes displayed the highest optimum temperatures and thermostabilities of all rhamnosidases reported to date. The four thermophilic and divalent ion-dependent rhamnosidases are the first biochemically characterised orthologous enzymes recently assigned to glycoside hydrolase family 106.

The hemicellulose-degrading enzyme system of the thermophilic bacterium Clostridium stercorarium: comparative characterisation and addition of new hemicellulolytic glycoside hydrolases.

BACKGROUND: The bioconversion of lignocellulosic biomass in various industrial processes, such as the production of biofuels, requires the degradation of hemicellulose. Clostridium stercorarium is a thermophilic bacterium, well known for its outstanding hemicellulose-degrading capability. Its genome comprises about 50 genes for partially still uncharacterised thermostable hemicellulolytic enzymes. These are promising candidates for industrial applications. RESULTS: To reveal the hemicellulose-degrading potential of 50 glycoside hydrolases, they were recombinantly produced and characterised. 46 of them were identified in the secretome of C. stercorarium cultivated on cellobiose. Xylanases Xyn11A, Xyn10B, Xyn10C, and cellulase Cel9Z were among the most abundant proteins. The secretome of C. stercorarium was active on xylan, ß-glucan, xyloglucan, galactan, and glucomannan. In addition, the recombinant enzymes hydrolysed arabinan, mannan, and galactomannan. 20 enzymes are newly described, degrading xylan, galactan, arabinan, mannan, and aryl-glycosides of ß-d-xylose, ß-d-glucose, ß-d-galactose, α-l-arabinofuranose, α-l-rhamnose, ß-d-glucuronic acid, and N-acetyl-ß-d-glucosamine. The activities of three enzymes with non-classified glycoside hydrolase (GH) family modules were determined. Xylanase Xyn105F and ß-d-xylosidase Bxl31D showed activities not described so far for their GH families. 11 of the 13 polysaccharide-degrading enzymes were most active at pH 5.0 to pH 6.5 and at temperatures of 57-76 °C. Investigation of the substrate and product specificity of arabinoxylan-degrading enzymes revealed that only the GH10 xylanases were able to degrade arabinoxylooligosaccharides. While Xyn10C was inhibited by α-(1,2)-arabinosylations, Xyn10D showed a degradation pattern different to Xyn10B and Xyn10C. Xyn11A released longer degradation products than Xyn10B. Both tested arabinose-releasing enzymes, Arf51B and Axh43A, were able to hydrolyse single- as well as double-arabinosylated xylooligosaccharides. CONCLUSIONS: The obtained results lead to a better understanding of the hemicellulose-degrading capacity of C. stercorarium and its involved enzyme systems. Despite similar average activities measured by depolymerisation tests, a closer look revealed distinctive differences in the activities and specificities within an enzyme class. This may lead to synergistic effects and influence the enzyme choice for biotechnological applications. The newly characterised glycoside hydrolases can now serve as components of an enzyme platform for industrial applications in order to reconstitute synthetic enzyme systems for complete and optimised degradation of defined polysaccharides and hemicellulose.