Characterization of a novel GH30 non-specific endoxylanase AcXyn30B from Acetivibrio clariflavus.

The xylanolytic enzymes Clocl_1795 and Clocl_2746 from glycoside hydrolase (GH) family 30 are highly abundant in the hemicellulolytic system of Acetivibrio clariflavus (Hungateiclostridium, Clostridium clariflavum). Clocl_1795 has been shown to be a xylobiohydrolase AcXbh30A releasing xylobiose from the non-reducing end of xylan and xylooligosaccharides. In this work, biochemical characterization of Clocl_2746 is presented. The protein, designated AcXyn30B, shows low sequence similarity to other GH30 members and phylogenetic analysis revealed that AcXyn30B and related proteins form a separate clade that is proposed to be a new subfamily GH30_12. AcXyn30B exhibits similar specific activity on glucuronoxylan, arabinoxylan, and aryl glycosides of linear xylooligosaccharides suggesting that it is a non-specific xylanase. From polymeric substrates, it releases the fragments of degrees of polymerization (DP) 2-6. Hydrolysis of different xylooligosaccharides indicates that AcXyn30B requires at least four occupied catalytic subsites for effective cleavage. The ability of the enzyme to hydrolyze a wide range of substrates is interesting for biotechnological applications. In addition to subfamilies GH30_7, GH30_8, and GH30_10, the newly proposed subfamily GH30_12 further widens the spectrum of GH30 subfamilies containing xylanolytic enzymes. KEY POINTS: Bacterial GH30 endoxylanase from A. clariflavus (AcXyn30B) has been characterized AcXyn30B is non-specific xylanase hydrolyzing various xylans and xylooligosaccharides Phylogenetic analysis placed AcXyn30B in a new GH30_12 subfamily.

Cellulose- and xylan-degrading yeasts: Enzymes, applications and biotechnological potential.

Microbes and their carbohydrate-active enzymes are central for depolymerization of complex lignocellulosic polysaccharides in the global carbon cycle. Their unique abilities to degrade and ferment carbohydrates are also utilized in many industrial processes such as baking, brewing and production of biofuels and drugs. Effective degradation and utilization of cellulose and hemicelluloses is important for the shift towards green bioeconomy, and requires microbes equipped with proper sets of carbohydrate-active enzymes (CAZymes). Knowledge of cellulolytic and xylanolytic CAZymes has mainly been generated from bacteria and filamentous fungi, while yeasts have been largely overlooked and may represent an untapped resource in natural CAZymes with industrial relevance. Cellulose and xylan-degrading yeasts with the ability to ferment saccharides are also promising candidates for consolidated bioprocesses (CBPs), as they can degrade lignocellulose and utilize its constituents to produce desired products at the same time. Cellulolytic yeasts able to utilize insoluble crystalline cellulose are rare while xylanolytic yeasts are rather widespread in nature. The lack of particular enzymes in yeasts can be remediated by introducing the missing enzymes into strains having outstanding product-forming attributes. In this review, we provide a comprehensive overview of the cellulose- and xylan-degrading ascomycetous and basidiomycetous yeasts known to date. We describe how these yeasts can be identified through bioprospecting and bioinformatic approaches and summarize available growth and enzymatic assays for strain characterization. Known and predicted CAZymes are extensively analyzed, both in individual species and in a phylogenetic perspective. We also describe the strategies used for construction of recombinant cellulolytic and xylanolytic strains as well as current applications for polysaccharide-degrading yeasts. Finally, we discuss the great potential of these yeasts as industrial cell factories, identify open research questions and provide suggestions for future investigations.

Yeast GH30 Xylanase from Sugiyamaella lignohabitans Is a Glucuronoxylanase with Auxiliary Xylobiohydrolase Activity.

Xylanases are the enzymes that catalyze the breakdown of the main hemicellulose present in plant cell walls. They have attracted attention due to their biotechnological potential for the preparation of industrially interesting products from lignocellulose. While many xylanases have been characterized from bacteria and filamentous fungi, information on yeast xylanases is scarce and no yeast xylanase belonging to glycoside hydrolase (GH) family 30 has been described so far. Here, we cloned, expressed and characterized GH30 xylanase SlXyn30A from the yeast Sugiyamaella lignohabitans. The enzyme is active on glucuronoxylan (8.4 U/mg) and rhodymenan (linear ß-1,4-1,3-xylan) (3.1 U/mg) while its activity on arabinoxylan is very low (0.03 U/mg). From glucuronoxylan SlXyn30A releases a series of acidic xylooligosaccharides of general formula MeGlcA2Xyln. These products, which are typical for GH30-specific glucuronoxylanases, are subsequently shortened at the non-reducing end, from which xylobiose moieties are liberated. Xylobiohydrolase activity was also observed during the hydrolysis of various xylooligosaccharides. SlXyn30A thus expands the group of glucuronoxylanases/xylobiohydrolases which has been hitherto represented only by several fungal GH30-7 members.

Catalytic Diversity of GH30 Xylanases.

Catalytic properties of GH30 xylanases belonging to subfamilies 7 and 8 were compared on glucuronoxylan, modified glucuronoxylans, arabinoxylan, rhodymenan, and xylotetraose. Most of the tested bacterial GH30-8 enzymes are specific glucuronoxylanases (EC 3.2.1.136) requiring for action the presence of free carboxyl group of MeGlcA side residues. These enzymes were not active on arabinoxylan, rhodymenan and xylotetraose, and conversion of MeGlcA to its methyl ester or its reduction to MeGlc led to a remarkable drop in their specific activity. However, some GH30-8 members are nonspecific xylanases effectively hydrolyzing all tested substrates. In terms of catalytic activities, the GH30-7 subfamily is much more diverse. In addition to specific glucuronoxylanases, the GH30-7 subfamily contains nonspecific endoxylanases and predominantly exo-acting enzymes. The activity of GH30-7 specific glucuronoxylanases also depend on the presence of the MeGlcA carboxyl, but not so strictly as in bacterial enzymes. The modification of the carboxyl group of glucuronoxylan had only weak effect on the action of predominantly exo-acting enzymes, as well as nonspecific xylanases. Rhodymenan and xylotetraose were the best substrates for exo-acting enzymes, while arabinoxylan represented hardly degradable substrate for almost all tested GH30-7 enzymes. The results expand current knowledge on the catalytic properties of this relatively novel group of xylanases.

Non-Specific GH30_7 Endo-ß-1,4-xylanase from Talaromyces leycettanus.

This study describes the catalytic properties of a GH30_7 xylanase produced by the fungus Talaromyces leycettanus. The enzyme is an ando-ß-1,4-xylanase, showing similar specific activity towards glucuronoxylan, arabinoxylan, and rhodymenan (linear ß-1,3-ß-1,4-xylan). The heteroxylans are hydrolyzed to a mixture of linear as well as branched ß-1,4-xylooligosaccharides that are shorter than the products generated by GH10 and GH11 xylanases. In the rhodymenan hydrolyzate, the linear ß-1,4-xylooligosaccharides are accompanied with a series of mixed linkage homologues. Initial hydrolysis of glucuronoxylan resembles the action of other GH30_7 and GH30_8 glucuronoxylanases, resulting in a series of aldouronic acids of a general formula MeGlcA2Xyln. Due to the significant non-specific endoxylanase activity of the enzyme, these acidic products are further attacked in the unbranched regions, finally yielding MeGlcA2Xyl2-3. The accommodation of a substituted xylosyl residue in the -2 subsite also applies in arabinoxylan depolymerization. Moreover, the xylose residue may be arabinosylated at both positions 2 and 3, without negatively affecting the main chain cleavage. The catalytic properties of the enzyme, particularly the great tolerance of the side-chain substituents, make the enzyme attractive for biotechnological applications. The enzyme is also another example of extraordinarily great catalytic diversity among eukaryotic GH30_7 xylanases.

Xylanases of glycoside hydrolase family 30 - An overview.

Xylan is the most abundant hemicellulose in nature and as such it is a huge source of renewable carbon. Its bioconversion requires a battery of xylanolytic enzymes. Of them the most important are the endo-ß-1,4-xylanases which depolymerize the polysaccharide into smaller fragments. Most of the xylanases are members of glycoside hydrolase (GH) families 10 and 11, although they are classified in some other GH families. The relatively new xylanases of GH30 are of special interest. Initially, they appeared to be specific glucuronoxylanases, however, other specificities were found later among prokaryotic and in particular eukaryotic enzymes. This review gives an overview of the substrate and product specificities observed for the GH30 xylanases characterized to date. An emphasis is given to the structure-activity relationship in order to explain how minor differences in catalytic centre and its vicinity can alter catalytic properties from the endoxylanase into the reducing end xylose releasing exoxylanase or into the non-reducing end xylobiohydrolase. Biotechnological potential of the GH30 xylanases is also considered.

A novel bacterial GH30 xylobiohydrolase from Hungateiclostridium clariflavum.

Typical bacterial GH30 xylanases are glucuronoxylanases requiring 4-O-methylglucuronic acid (MeGlcA) substitution of a xylan main chain for their action. They do not exhibit a significant activity on neutral xylooligosaccharides, arabinoxylan (AraX), or rhodymenan (Rho). In this work, the biochemical characterization of the bacterial Clocl_1795 xylanase from Hungateiclostridium (Clostridium) clariflavum DSM 19732 (HcXyn30A) is presented. Amino acid sequence analysis of HcXyn30A revealed that the enzyme does not contain amino acids known to be responsible for MeGlcA coordination in the -2b subsite of glucuronoxylanases. This suggested that the catalytic properties of HcXyn30A may differ from those of glucuronoxylanases. HcXyn30A shows similar specific activity on glucuronoxylan (GX) and Rho, while the specific activity on AraX is about 1000 times lower. HcXyn30A releases Xyl2 as the main product from the non-reducing end of different polymeric and oligomeric substrates. Catalytic properties of HcXyn30A resemble the properties of the fungal GH30 xylobiohydrolase from Acremonium alcalophilum, AaXyn30A. HcXyn30A is the first representative of a prokaryotic xylobiohydrolase. Its unique specificity broadens the catalytic diversity of bacterial GH30 xylanases. KEY POINTS: • Bacterial GH30 xylobiohydrolase from H. clariflavum (HcXyn30A) has been characterized. • HcXyn30A releases xylobiose from the non-reducing end of different substrates. • HcXyn30A is the first representative of bacterial xylobiohydrolase.

A novel GH30 xylobiohydrolase from Acremonium alcalophilum releasing xylobiose from the non-reducing end.

Xylanases of the GH30 family are grouped to subfamilies GH30-7 and GH30-8. The GH30-8 members are of bacterial origin and well characterized, while the GH30-7 members are from fungal sources and their properties are quite diverse. Here, a heterologous expression and characterization of the GH30-7 xylanase AaXyn30A from a cellulolytic fungus Acremonium alcalophilum is reported. From various polymeric and oligomeric substrates AaXyn30A generates xylobiose as the main product. It was proven that xylobiose is released from the non-reducing end of all tested substrates, thus the enzyme behaves as a typical non-reducing-end acting xylobiohydrolase. AaXyn30A is active on different types of xylan, exhibiting the highest activity on rhodymenan (linear ß-1,3-ß-1,4-xylan) from which also an isomeric xylotriose Xyl-ß-1,3-Xyl-ß-1,4-Xyl is formed. Production of xylobiose from glucuronoxylan is at later stage accompanied by a release of aldouronic acids differing from those liberated by the bacterial GH30-8 glucuronoxylanases.

Glucuronoxylan recognition by GH 30 xylanases: A study with enzyme and substrate variants.

XynA from Erwinia chrysanthemi (EcXyn30A), belonging to glycoside hydrolase family 30 subfamily 8, is specialized for hydrolysis of 4-O-methylglucuronoxylan (GX). Carboxyl group of 4-O-methylglucuronic acid serves as a substrate recognition element interacting ionically with positively charged Arg293 of the enzyme. We determined kinetic parameters of EcXyn30A on GX, its methyl ester (GXE) and 4-O-methylglucoxylan (GXR) and compared them with behavior of the enzyme variant in which Arg293 was replaced by Ala. The modifications of the substrate carboxyl groups resulted in several thousand-fold decrease in catalytic efficiency of EcXyn30A. In contrast, the R293A replacement reduced catalytic efficiency on GX only 18-times. The main difference was in catalytic rate (kcat) which was much lower for EcXyn30A acting on the modified substrates than for the variant which exhibited similar kcat values on all three polymers. The R293A variant cleaved GX, GXE and GXR on the second glycosidic bond from branch towards the reducing end, similarly to EcXyn30A. The R293A replacement caused 15-times decrease in specific activity on MeGlcA3Xyl4, but it did not influence low activity on linear xylooligosaccharides. Docking experiments showed that MeGlcA3Xyl4 and its esterified and reduced forms were bound to both enzymes in analogous way but with different binding energies.