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.

Xanthan: enzymatic degradation and novel perspectives of applications.

The extracellular heteropolysaccharide xanthan, synthesized by bacteria of the genus Xanthomonas, is widely used as a thickening and stabilizing agent across the food, cosmetic, and pharmaceutical sectors. Expanding the scope of its application, current efforts target the use of xanthan to develop innovative functional materials and products, such as edible films, eco-friendly oil surfactants, and biocompatible composites for tissue engineering. Xanthan-derived oligosaccharides are useful as nutritional supplements and plant defense elicitors. Development and processing of such new functional materials and products often necessitate tuning of xanthan properties through targeted structural modification. This task can be effectively carried out with the help of xanthan-specific enzymes. However, the complex molecular structure and intricate conformational behavior of xanthan create problems with its enzymatic hydrolysis or modification. This review summarizes and analyzes data concerning xanthan-degrading enzymes originating from microorganisms and microbial consortia, with a particular focus on the dependence of enzymatic activity on the structure and conformation of xanthan. Through a comparative study of xanthan-degrading pathways found within various bacterial classes, different microbial enzyme systems for xanthan utilization have been identified. The characterization of these new enzymes opens new perspectives for modifying xanthan structure and developing innovative xanthan-based applications. KEY POINTS: • The structure and conformation of xanthan affect enzymatic degradation. • Microorganisms use diverse multienzyme systems for xanthan degradation. • Xanthan-specific enzymes can be used to develop xanthan variants for novel applications.

Unusual substrate specificity in GH family 12: structure-function analysis of glucanases Bgh12A and Xgh12B from Aspergillus cervinus, and Egh12 from Thielavia terrestris.

In this study, we compared the properties and structures of three fungal GH12 enzymes: the strict endoglucanase Bgh12A and the xyloglucanase Xgh12B from Aspergillus cervinus, and the endoglucanase Egh12 from Thielavia terrestris combining activity on linear ß-glucan and branched xyloglucan. Egh12 from T. terrestris was produced in Pichia pastoris, purified, and characterized as a thermostable enzyme with maximal activity at 70 ºC and a half-life time of 138 min at 65 °C. We for the first time demonstrated that the GH12 endoglucanases Egh12 and Bgh12A, but not the strict xyloglucanase Xgh12B, hydrolyzed (1,3)-ß-linkages in (1,3;1,4)-ß-D-glucooligosaccharides and had transglycosylase activity on (1,3)-ß-D-glucooligosaccharides. Phylogenetic analysis indicated that Egh12 from T. terrestris and Bgh12A from A. cervinus are more related than Bgh12A and Xgh12B isolated from one strain. The X-ray structure of Bgh12A was determined with 2.17 Å resolution and compared with 3D-homology models of Egh12 and Xgh12B. The enzymes have a ß-jelly roll structure with a catalytic cleft running across the protein. Comparative analysis and a docking study demonstrated the importance of endoglucanase-specific loop 1 partly covering the catalytic cleft for correct placement of the linear substrates. Variability in substrate specificity between the GH12 endoglucanases is determined by non-conservative residues in structural loops framing the catalytic cleft. A residue responsible for the thermostability of Egh12 was predicted. The key structural elements and residues described in this study may serve as potential targets for modification aimed at the improvement of enzymatic properties. KEY POINTS: • Thermostable endoglucanase Egh12 from T. terrestris was produced in P. pastoris, purified, and characterized • The X-ray structure of GH12 endoglucanase Bgh12A from A. cervinus was resolved • GH12 endoglucanases, but not GH12 xyloglucanases, hydrolyze (1,3)-ß-linkages in (1,3;1,4)-ß-D-glucooligosaccharides.

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.

Novel endo-(1,4)-ß-glucanase Bgh12A and xyloglucanase Xgh12B from Aspergillus cervinus belong to GH12 subgroup I and II, respectively.

In spite of intensive exploitation of aspergilli for the industrial production of carbohydrases, little is known about hydrolytic enzymes of fungi from the section Cervini. Novel glycoside hydrolases Bgh12A and Xgh12B from Aspergillus cervinus represent examples of divergent activities within one enzyme family and belong to the GH12 phylogenetic subgroup I (endo-(1,4)-ß-glucanases) and II (endo-xyloglucanases), respectively. The bgh12A and xgh12B genes were identified in the unsequenced genome of A. cervinus using primers designed for conservative regions of the corresponding subgroups and a genome walking approach. The recombinant enzymes were heterologously produced in Pichia pastoris, purified, and characterized. Bgh12A was an endo-(1,4)-ß-glucanase (EC 3.2.1.4) hydrolyzing the unbranched soluble ß-(1,4)-glucans and mixed linkage ß-(1,3;1,4)-D-glucans. Bgh12A exhibited maximum activity on barley ß-glucan (BBG), which amounted to 614 ± 30 U/mg of protein. The final products of BBG and lichenan hydrolysis were glucose, cellobiose, cellotriose, 4-O-ß-laminaribiosyl-glucose, and a range of higher mixed-linkage gluco-oligosaccharides. In contrast, the activity of endo-xyloglucanase Xgh12B (EC 3.2.1.151) was restricted to xyloglucan, with 542 ± 39 U/mg protein. The enzyme cleaved the (1,4)-ß-glycosidic bonds of the xyloglucan backbone at the unsubstituted glucose residues finally generating cellotetraose-based hepta-, octa, and nona-oligosaccharides. Bgh12A and Xgh12B had maximal activity at 55 °C, pH 5.0. At these conditions, the half-time of Xgh12B inactivation was 158 min, whereas the half-life of Bgh12A was 5 min. Recombinant P. pastoris strains produced up to 106 U/L of the target enzymes with at least 75% of recombinant protein in the total extracellular proteins. The Bgh12A and Xgh12B sequences show 43% identity. Strict differences in substrate specificity of Bgh12A and Xgh12B were in congruence with the presence of subgroup-specific structural loops and substrate-binding aromatic residues in the catalytic cleft of the enzymes. Individual composition of aromatic residues in the catalytic cleft defined variability in substrate selectivity within GH12 subgroups I and II.

Thermostable multifunctional GH74 xyloglucanase from Myceliophthora thermophila: high-level expression in Pichia pastoris and characterization of the recombinant protein.

A xyloglucanase of the GH74 family was identified in the thermophilic fungus strain Myceliophthora thermophila VKPM F-244, and its gene sequence was optimized for cloning and expression in Pichia pastoris. The recombinant xyloglucanase MtXgh74 exhibited the highest activity toward tamarind seed xyloglucan with a K M value of 0.51 ± 0.06 mg/mL. The activities on barley ß-glucan and carboxymethylcellulose were about 4 and 2%, respectively, compared to xyloglucan. Maximum xyloglucanase activity was observed at 70-75 °C and pH 6.5. After pre-incubation at 50 °C, pH 6.0 for 3 h, the enzyme retained 100% of its activity. The half-life of MtXgh74 at 60 °C, pH 6.0 was 40 min. In P. pastoris, MtXgh74 was produced in glycosylated form. The enzyme production in a 1 L bioreactor resulted in a yield of 118 U/mL or 5.3 g/L after 51 h fermentation. Kinetic studies of the hydrolysis product formation suggest that MtXgh74 has an endo-processive mode of action. The final products were the standard xyloglucan building blocks XXXG, XXLG, XLXG, and XLLG. Additionally, MtXgh74 hydrolyzed various linkages within the xyloglucan building blocks XXXG, XXLG, and XLXG (except XLLG) producing diverse low molecular weight oligosaccharides which may be identified by MALDI-TOF as XG, XX, XXG/GXX/XGX, XXX, LG, LX/XL, XLX/XXL, LLG, GXXXG, GXLLG, XLLGX. The unique combination of different activities within one enzyme along with its high thermostability and specificity toward xyloglucan makes MtXgh74 a promising candidate enzyme for industrial applications.