Paenibacillus sp. strain JDR-2 and XynA1: a novel system for methylglucuronoxylan utilization

Environmental and economic factors predicate the need for efficient processing of renewable sources of fuels and chemicals. To fulfill this need, microbial biocatalysts must be developed to efficiently process the hemicellulose fraction of lignocellulosic biomass for fermentation of pentoses. The predominance of methylglucuronoxylan (MeGAXn), a beta-1,4 xylan in which 10% to 20% of the xylose residues are substituted with alpha-1,2-4-O-methylglucuronate residues, in hemicellulose fractions of hardwood and crop residues has made this a target for processing and fermentation. A Paenibacillus sp. (strain JDR-2) has been isolated and characterized for its ability to efficiently utilize MeGAXn. A modular xylanase (XynA1) of glycosyl hydrolase family 10 (GH 10) was identified through DNA sequence analysis that consists of a triplicate family 22 carbohydrate binding module followed by a GH 10 catalytic domain followed by a single family 9 carbohydrate binding module and concluding with C-terminal triplicate surface layer homology (SLH) domains. Immunodetection of the catalytic domain of XynA1 (XynA1 CD) indicates that the enzyme is associated with the cell wall fraction, supporting an anchoring role for the SLH modules. With MeGAXn as substrate, XynA1 CD generated xylobiose and aldotetrauronate (MeGAX3) as predominant products. The inability to detect depolymerization products in medium during exponential growth of Paenibacillus sp. strain JDR-2 on MeGAXn, as well as decreased growth rate and yield with XynA1 CD-generated xylooligosaccharides and aldouronates as substrates, indicates that XynA1 catalyzes a depolymerization process coupled to product assimilation. This depolymerization/assimilation system may be utilized for development of biocatalysts to efficiently convert MeGAXn to alternative fuels and biobased products.     

Structure, function, and regulation of the aldouronate utilization gene cluster from Paenibacillus sp. strain JDR-2

Direct bacterial conversion of the hemicellulose fraction of hardwoods and crop residues to biobased products depends upon extracellular depolymerization of methylglucuronoxylan (MeGAX_n), followed by assimilation and intracellular conversion of aldouronates and xylooligosaccharides to fermentable xylose. Paenibacillus sp. strain JDR-2, an aggressively xylanolytic bacterium, secretes a multimodular cell-associated GH10 endoxylanase (XynA1) that catalyzes depolymerization of MeGAX_n and rapidly assimilates the principal products, β-1,4-xylobiose, β-1,4-xylotriose, and MeGAX₃, the aldotetrauronate 4-O-methylglucuronosyl-α-1,2-xylotriose. Genomic libraries derived from this bacterium have now allowed cloning and sequencing of a unique aldouronate utilization gene cluster comprised of genes encoding signal transduction regulatory proteins, ABC transporter proteins, and the enzymes AguA (GH67 α-glucuronidase), XynA2 (GH10 endoxylanase), and XynB (GH43 β-xylosidase/α-arabinofuranosidase). Expression of these genes, as well as xynA1 encoding the secreted GH10 endoxylanase, is induced by growth on MeGAX_n and repressed by glucose. Sequences in the yesN, lplA, and xynA2 genes within the cluster and in the distal xynA1 gene show significant similarity to catabolite responsive element (cre) defined in Bacillus subtilis for recognition of the catabolite control protein (CcpA) and consequential repression of catabolic regulons. The aldouronate utilization gene cluster in Paenibacillus sp. strain JDR-2 operates as a regulon, coregulated with the expression of xynA1, conferring the ability for efficient assimilation and catabolism of the aldouronate product generated by a multimodular cell surface-anchored GH10 endoxylanase. This cluster offers a desirable metabolic potential for bacterial conversion of hemicellulose fractions of hardwood and crop residues to biobased products.     

Genetic Engineering of Enterobacter asburiae Strain JDR-1 for Efficient Production of Ethanol from Hemicellulose Hydrolysates

Dilute acid pretreatment is an established method for hydrolyzing the methylglucuronoxylans of hemicellulose to release fermentable xylose. In addition to xylose, this process releases the aldouronate methylglucuronoxylose, which cannot be metabolized by current ethanologenic biocatalysts. Enterobacter asburiae JDR-1, isolated from colonized wood, was found to efficiently ferment both methylglucuronoxylose and xylose in acid hydrolysates of sweet gum xylan, producing predominantly ethanol and acetate. Transformation of E. asburiae JDR-1 with pLOI555 or pLOI297, each containing the PET operon containing pyruvate decarboxylase (pdc) and alcohol dehydrogenase B (adhB) genes derived from Zymomonas mobilis, replaced mixed-acid fermentation with homoethanol fermentation. Deletion of the pyruvate formate lyase (pflB) gene further increased the ethanol yield, resulting in a stable E. asburiae E1(pLOI555) strain that efficiently utilized both xylose and methylglucuronoxylose in dilute acid hydrolysates of sweet gum xylan. Ethanol was produced from xylan hydrolysate by E. asburiae E1(pLOI555) with a yield that was 99% of the theoretical maximum yield and at a rate of 0.11 g ethanol/g (dry weight) cells/h, which was 1.57 times the yield and 1.48 times the rate obtained with the ethanologenic strain Escherichia coli KO11. This engineered derivative of E. asburiae JDR-1 that is able to ferment the predominant hexoses and pentoses derived from both hemicellulose and cellulose fractions is a promising subject for development as an ethanologenic biocatalyst for production of fuels and chemicals from agricultural residues and energy crops.Lignocellulosic resources, including forest and agricultural residues and evolving energy crops, offer benign alternatives to petroleum-based resources for production of fuels and chemicals. As renewable resources, these lignocellulosic materials are expected to decrease dependence on exhaustible supplies of petroleum and mitigate the net release of carbon dioxide into the atmosphere. The development of economically acceptable bioconversion processes requires pretreatments that release the maximal quantities of hexoses (predominantly glucose released from cellulose) and pentoses (arabinose and xylose) from hemicelluloses and also requires microbial biocatalysts that efficiently convert these compounds to a single targeted product.As one of three main components of lignocellulosics, hemicellulose contains polysaccharides comprised of pentoses, hexoses and sugar acids that account for 20 to 35% of the total biomass from different sources (21). Methylglucuronoxylans (MeGAX_n), consisting of long chains of as many as 70 β-xylopyranose residues linked by β-1,4-glycosidic bonds (25), are the predominant components in the hemicellulose fractions of agricultural residues and energy crops, including corn stover, sugarcane bagasse, poplar, and switchgrass (7, 18, 23, 24). In hardwood and softwood xylans, a 4-O-methylglucuronic acid is attached at the 2′ position of every sixth to eighth xylose residue (12, 15). Dilute acid hydrolysis is commonly used to make the monosaccharides comprising hemicellulose accessible for fermentation (7, 22). However, the α-1,2 glucuronosyl linkage in xylan is resistant to dilute acid hydrolysis, which results in the release of methylglucuronoxylose (MeGAX) along with xylose and other monosaccharides. MeGAX is not fermented by bacterial biocatalysts currently used to convert hemicellulose to ethanol, such as Escherichia coli KO11 (2, 6). In sweet gum xylan, as much as 27% of the carbohydrate may be in this unfermentable fraction after dilute acid pretreatment (2, 20). Complete utilization of all hemicellulosic sugars can improve the efficiency of conversion of lignocellulosic materials to fuel ethanol and other value-added products.Our previous research on the processing of hemicelluloses for fermentation led to isolation of Enterobacter asburiae strain JDR-1. This isolate performed mixed-acid fermentation of the principal hexoses and pentoses that can be derived from cellulose and hemicellulose fractions of lignocellulosic biomass and exhibited a novel metabolic potential based on its ability to ferment MeGAX and xylose to ethanol and acetate as major fermentation products from sweet gum MeGAX_n hydrolysates generated by dilute acid pretreatment (2). This strain has been genetically modified to produce d-(−)-lactate as the predominant product from acid hydrolysates of MeGAX_n (3).In this study, the PET operon containing the pdc, adhA, and adhB genes from Zymomonas mobilis (10, 11) was incorporated into a pflB E. asburiae JDR-1 isolate by plasmid transformation to construct homoethanologenic strains. The resulting recombinant strains were compared with E. asburiae wild-type strain JDR-1 and the ethanologenic strain E. coli KO11 to evaluate their efficiencies of production of ethanol from dilute acid hydrolysates of sweet gum MeGAX_n.     

Genetic engineering of <Emphasis Type="Italic">Enterobacter asburiae</Emphasis> strain JDR-1 for efficient <Emphasis Type="SmallCaps">d</Emphasis>(−) lactic acid production from hemicellulose hydrolysate

In the dilute acid pretreatment of lignocellulose, xylose substituted with α-1,2-methylglucuronate is released as methylglucuronoxylose (MeGAX), which cannot be fermented by biocatalysts currently used to produce biofuels and chemicals. Enterobacter asburiae JDR-1, isolated from colonized wood, efficiently fermented both MeGAX and xylose in acid hydrolysates of sweetgum xylan. Deletion of pflB and als genes in this bacterium modified the native mixed acid fermentation pathways to one for homolactate production. The resulting strain, Enterobacter asburiae L1, completely utilized both xylose and MeGAX in a dilute acid hydrolysate of sweetgum xylan and produced lactate approximating 100% of the theoretical maximum yield. Enterobacter asburiae JDR-1 offers a platform to develop efficient biocatalysts for production of fuels and chemicals from hemicellulose hydrolysates of hardwood and agricultural residues.     

Complete Fermentation of Xylose and Methylglucuronoxylose Derived from Methylglucuronoxylan by Enterobacter asburiae Strain JDR-1

Acid pretreatment is commonly used to release pentoses from the hemicellulose fraction of cellulosic biomass for bioconversion. The predominant pentose in the hemicellulose fraction of hardwoods and crop residues is xylose in the polysaccharide methylglucuronoxylan, in which as many as one in six of the β-1,4-linked xylopyranose residues is substituted with α-1,2-linked 4-O-methylglucuronopyranose. Resistance of the α-1,2-methylglucuronosyl linkages to acid hydrolysis results in release of the aldobiuronate 4-O-methylglucuronoxylose, which is not fermented by bacterial biocatalysts currently used for bioconversion of hemicellulose. Enterobacter asburiae strain JDR-1, isolated from colonized hardwood (sweetgum), efficiently ferments both methylglucuronoxylose and xylose, producing predominantly ethanol and acetate. ¹³C-nuclear magnetic resonance studies defined the Embden-Meyerhof pathway for metabolism of glucose and the pentose phosphate pathway for xylose metabolism. Rates of substrate utilization, product formation, and molar growth yields indicated methylglucuronoxylose is transported into the cell and hydrolyzed to release methanol, xylose, and hexauronate. Enterobacter asburiae strain JDR-1 is the first microorganism described that ferments methylglucuronoxylose generated along with xylose during the acid-mediated saccharification of hemicellulose. Genetic definition of the methylglucuronoxylose utilization pathway may allow metabolic engineering of established gram-negative bacterial biocatalysts for complete bioconversion of acid hydrolysates of methylglucuronoxylan. Alternatively, Enterobacter asburiae strain JDR-1 may be engineered for the efficient conversion of acid hydrolysates of hemicellulose to biofuels and chemical feedstocks.     

GH115 α-glucuronidase and GH11 xylanase from Paenibacillus sp. JDR-2: potential roles in processing glucuronoxylans

Paenibacillus sp. JDR-2 (Pjdr2) has been studied as a model for development of bacterial biocatalysts for efficient processing of xylans, methylglucuronoxylan, and methylglucuronoarabinoxylan, the predominant hemicellulosic polysaccharides found in dicots and monocots, respectively. Pjdr2 produces a cell-associated GH10 endoxylanase (Xyn10A₁) that catalyzes depolymerization of xylans to xylobiose, xylotriose, and methylglucuronoxylotriose with methylglucuronate-linked α-1,2 to the nonreducing terminal xylose. A GH10/GH67 xylan utilization regulon includes genes encoding an extracellular cell-associated Xyn10A₁ endoxylanase and an intracellular GH67 α-glucuronidase active on methylglucuronoxylotriose generated by Xyn10A₁ but without activity on methylglucuronoxylotetraose generated by a GH11 endoxylanase. The sequenced genome of Pjdr2 contains three paralogous genes potentially encoding GH115 α-glucuronidases found in certain bacteria and fungi. One of these, Pjdr2_5977, shows enhanced expression during growth on xylans along with Pjdr2_4664 encoding a GH11 endoxylanase. Here, we show that Pjdr2_5977 encodes a GH115 α-glucuronidase, Agu115A, with maximal activity on the aldouronate methylglucuronoxylotetraose selectively generated by a GH11 endoxylanase Xyn11 encoded by Pjdr2_4664. Growth of Pjdr2 on this methylglucuronoxylotetraose supports a process for Xyn11-mediated extracellular depolymerization of methylglucuronoxylan and Agu115A-mediated intracellular deglycosylation as an alternative to the GH10/GH67 system previously defined in this bacterium. A recombinantly expressed enzyme encoded by the Pjdr2 agu115A gene catalyzes removal of 4-O-methylglucuronate residues α-1,2 linked to internal xylose residues in oligoxylosides generated by GH11 and GH30 xylanases and releases methylglucuronate from polymeric methylglucuronoxylan. The GH115 α-glucuronidase from Pjdr2 extends the discovery of this activity to members of the phylum Firmicutes and contributes to a novel system for bioprocessing hemicelluloses.

     

Characterization of XynC from Bacillus subtilis subsp. subtilis strain 168 and analysis of its role in depolymerization of glucuronoxylan

Secretion of xylanase activities by Bacillus subtilis 168 supports the development of this well-defined genetic system for conversion of methylglucuronoxylan (MeGAXn [where n represents the number of xylose residues]) in the hemicellulose component of lignocellulosics to biobased products. In addition to the characterized glycosyl hydrolase family 11 (GH 11) endoxylanase designated XynA, B. subtilis 168 secretes a second endoxylanase as the translated product of the ynfF gene. This sequence shows remarkable homology to the GH 5 endoxylanase secreted by strains of Erwinia chrysanthemi. To determine its properties and potential role in the depolymerization of MeGAXn, the ynfF gene was cloned and overexpressed to provide an endoxylanase, designated XynC, which was characterized with respect to substrate preference, kinetic properties, and product formation. With different sources of MeGAXn as the substrate, the specific activity increased with increasing methylglucuronosyl substitutions on the beta-1,4-xylan chain. With MeGAXn from sweetgum as a preferred substrate, XynC exhibited a Vmax of 59.9 units/mg XynC, a Km of 1.63 mg MeGAXn/ml, and a k(cat) of 2,635/minute at pH 6.0 and 37 degrees C. Matrix-assisted laser desorption ionization-time of flight mass spectrometry and 1H nuclear magnetic resonance data revealed that each hydrolysis product has a single glucuronosyl substitution penultimate to the reducing terminal xylose. This detailed analysis of XynC from B. subtilis 168 defines the unique depolymerization process catalyzed by the GH 5 endoxylanases. Based upon product analysis, B. subtilis 168 secretes both XynA and XynC. Expression of xynA was subject to MeGAXn induction; xynC expression was constitutive with growth on different substrates. Translation and secretion of both GH 11 and GH 5 endoxylanases by the fully sequenced and genetically malleable B. subtilis 168 recommends this bacterium for the introduction of genes required for the complete utilization of products of the enzyme-catalyzed depolymerization of MeGAXn. B. subtilis may serve as a model platform for development of gram-positive biocatalysts for conversion of lignocellulosic materials to renewable fuels and chemicals.     

Engineering the Xylan Utilization System in Bacillus subtilis for Production of Acidic Xylooligosaccharides

Xylans are the predominant polysaccharides in hemicelluloses and an important potential source of biofuels and chemicals. The ability of Bacillus subtilis subsp. subtilis strain 168 to utilize xylans has been ascribed to secreted glycoside hydrolase family 11 (GH11) and GH30 endoxylanases, encoded by the xynA and xynC genes, respectively. Both of these enzymes have been defined with respect to structure and function. In this study, the effects of deletion of the xynA and xynC genes, individually and in combination, were evaluated for xylan utilization and formation of acidic xylooligosaccharides. Parent strain 168 depolymerizes methylglucuronoxylans (MeGX_n), releasing the xylobiose and xylotriose utilized for growth and accumulating the aldouronate methylglucuronoxylotriose (MeGX₃) with some methylglucuronoxylotetraose (MeGX₄). The combined GH11 and GH30 activities process the products generated by their respective actions on MeGX_n to release a maximal amount of neutral xylooligosaccharides for assimilation and growth, at the same time forming MeGX₃ in which the internal xylose is substituted with methylglucuronate (MeG). Deletion of xynA results in the accumulation of β-1,4-xylooligosaccharides with degrees of polymerization ranging from 4 to 18 and an average degree of substitution of 1 in 7.2, each with a single MeG linked α-1,2 to the xylose penultimate to the xylose at the reducing terminus. Deletion of the xynC gene results in the accumulation of aldouronates comprised of 4 or more xylose residues in which the MeG may be linked α-1,2 to the xylose penultimate to the nonreducing xylose. These B. subtilis lines may be used for the production of acidic xylooligosaccharides with applications in human and veterinary medicine.     

Xylan Utilization Regulon in Xanthomonas citri pv. citri Strain 306: Gene Expression and Utilization of Oligoxylosides

Xanthomonas citri pv. citri strain 306 (Xcc306), a causative agent of citrus canker, produces endoxylanases that catalyze the depolymerization of cell wall-associated xylans. In the sequenced genomes of all plant-pathogenic xanthomonads, genes encoding xylanolytic enzymes are clustered in three adjacent operons. In Xcc306, these consecutive operons contain genes encoding the glycoside hydrolase family 10 (GH10) endoxylanases Xyn10A and Xyn10C, the agu67 gene, encoding a GH67 α-glucuronidase (Agu67), the xyn43E gene, encoding a putative GH43 α-l-arabinofuranosidase, and the xyn43F gene, encoding a putative β-xylosidase. Recombinant Xyn10A and Xyn10C convert polymeric 4-O-methylglucuronoxylan (MeGX_n) to oligoxylosides methylglucuronoxylotriose (MeGX₃), xylotriose (X₃), and xylobiose (X₂). Xcc306 completely utilizes MeGX_n predigested with Xyn10A or Xyn10C but shows little utilization of MeGX_n. Xcc306 with a deletion in the gene encoding α-glucuronidase (Xcc306 Δagu67) will not utilize MeGX₃ for growth, demonstrating the role of Agu67 in the complete utilization of GH10-digested MeGX_n. Preferential growth on oligoxylosides compared to growth on polymeric MeGX_n indicates that GH10 xylanases, either secreted by Xcc306 in planta or produced by the plant host, generate oligoxylosides that are processed by Xyn10 xylanases and Agu67 residing in the periplasm. Coordinate induction by oligoxylosides of xyn10, agu67, cirA, the tonB receptor, and other genes within these three operons indicates that they constitute a regulon that is responsive to the oligoxylosides generated by the action of Xcc306 GH10 xylanases on MeGX_n. The combined expression of genes in this regulon may allow scavenging of oligoxylosides derived from cell wall deconstruction, thereby contributing to the tissue colonization and/or survival of Xcc306 and, ultimately, to plant disease.     

Arabinoxylan Oligosaccharide Hydrolysis by Family 43 and 51 Glycosidases from Lactobacillus brevis DSM 20054

Due to their potential prebiotic properties, arabinoxylan-derived oligosaccharides [(A)XOS] are of great interest as functional food and feed ingredients. While the (A)XOS metabolism of Bifidobacteriaceae has been extensively studied, information regarding lactic acid bacteria (LAB) is still limited in this context. The aim of the present study was to fill this important gap by characterizing candidate (A)XOS hydrolyzing glycoside hydrolases (GHs) identified in the genome of Lactobacillus brevis DSM 20054. Two putative GH family 43 xylosidases (XynB1 and XynB2) and a GH family 43 arabinofuranosidase (Abf3) were heterologously expressed and characterized. While the function of XynB1 remains unclear, XynB2 could efficiently hydrolyze xylooligosaccharides. Abf3 displayed high specific activity for arabinobiose but could not release arabinose from an (A)XOS preparation. However, two previously reported GH 51 arabinofuranosidases from Lb. brevis were able to specifically remove α-1,3-linked arabinofuranosyl residues from arabino-xylooligosaccharides (AXHm3 specificity). These results imply that Lb. brevis is at least genetically equipped with functional enzymes in order to hydrolyze the depolymerization products of (arabino)xylans and arabinans. The distribution of related genes in Lactobacillales genomes indicates that GH 43 and, especially, GH 51 glycosidase genes are rare among LAB and mainly occur in obligately heterofermentative Lactobacillus spp., Pediococcus spp., members of the Leuconostoc/Weissella branch, and Enterococcus spp. Apart from the prebiotic viewpoint, this information also adds new perspectives on the carbohydrate (i.e., pentose-oligomer) metabolism of LAB species involved in the fermentation of hemicellulose-containing substrates.     

Molecular and biochemical characterization of a new alkaline active multidomain xylanase from alkaline wastewater sludge

A xylanase gene, xyn-b39, coding for a multidomain glycoside hydrolase (GH) family 10 protein was cloned from the genomic DNA of the alkaline wastewater sludge of a paper mill. Its deduced amino acid sequence of 1,481 residues included two carbohydrate-binding modules (CBM) of family CBM_4_9, one catalytic domain of GH 10, one family 9 CBM and three S-layer homology (SLH) domains. xyn-b39 was expressed heterologously in Escherichia coli, and the recombinant enzyme was purified and characterized. Xyn-b39 exhibited maximum activity at pH 7.0 and 60 °C, and remained highly active under alkaline conditions (more than 80 % activity at pH 9.0 and 40 % activity at pH 10.0). The enzyme was thermostable at 55 °C, retaining more than 90 % of the initial activity after 2 h pre-incubation. Xyn-b39 had wide substrate specificity and hydrolyzed soluble substrates (birchwood xylan, beechwood xylan, oat spelt xylan, wheat arabinoxylan) and insoluble substrates (oat spelt xylan and wheat arabinoxylan). Hydrolysis product analysis indicated that Xyn-b39 was an endo-type xylanase. The K _m and V _max values of Xyn-b39 for birchwood xylan were 1.01 mg/mL and 73.53 U/min/mg, respectively. At the charge of 10 U/g reed pulp for 1 h, Xyn-b39 significantly reduced the Kappa number (P < 0.05) with low consumption of chlorine dioxide alone.     

First Structural Insights into α-l-Arabinofuranosidases from the Two GH62 Glycoside Hydrolase Subfamilies

α-l-Arabinofuranosidases are glycoside hydrolases that specifically hydrolyze non-reducing residues from arabinose-containing polysaccharides. In the case of arabinoxylans, which are the main components of hemicellulose, they are part of microbial xylanolytic systems and are necessary for complete breakdown of arabinoxylans. Glycoside hydrolase family 62 (GH62) is currently a small family of α-l-arabinofuranosidases that contains only bacterial and fungal members. Little is known about the GH62 mechanism of action, because only a few members have been biochemically characterized and no three-dimensional structure is available. Here, we present the first crystal structures of two fungal GH62 α-l-arabinofuranosidases from the basidiomycete Ustilago maydis (UmAbf62A) and ascomycete Podospora anserina (PaAbf62A). Both enzymes are able to efficiently remove the α-l-arabinosyl substituents from arabinoxylan. The overall three-dimensional structure of UmAbf62A and PaAbf62A reveals a five-bladed β-propeller fold that confirms their predicted classification into clan GH-F together with GH43 α-l-arabinofuranosidases. Crystallographic structures of the complexes with arabinose and cellotriose reveal the important role of subsites +1 and +2 for sugar binding. Intriguingly, we observed that PaAbf62A was inhibited by cello-oligosaccharides and displayed binding affinity to cellulose although no activity was observed on a range of cellulosic substrates. Bioinformatic analyses showed that UmAbf62A and PaAbf62A belong to two distinct subfamilies within the GH62 family. The results presented here provide a framework to better investigate the structure-function relationships within the GH62 family.     

An arabinogalacto(4-O-methylglucurono)xylan from the leaves of Hordeum vulgare

An arabinogalacto(4-O-methylglucurono)xylan with a $\text{DP}$_n of ca. 96 has been isolated from the leaves of barley. Based on structural studies it is proposed that the hemicellulose consists of a main chain of β (1→4)-linked d-xylopyranosyl residues to which are attached an average of 8·1 l-arabinofuranosyl residues, 3·8 galactopyranosyl-(1→4)-d-xylopyranosyl-(1→2)-l-arabinofuranosyl residues and 4·4 4-O-methyl-d-glucopyranuronosyl residues.     

The GH130 Family of Mannoside Phosphorylases Contains Glycoside Hydrolases That Target β-1,2-Mannosidic Linkages in Candida Mannan

The depolymerization of complex glycans is an important biological process that is of considerable interest to environmentally relevant industries. β-Mannose is a major component of plant structural polysaccharides and eukaryotic N-glycans. These linkages are primarily cleaved by glycoside hydrolases, although recently, a family of glycoside phosphorylases, GH130, have also been shown to target β-1,2- and β-1,4-mannosidic linkages. In these phosphorylases, bond cleavage was mediated by a single displacement reaction in which phosphate functions as the catalytic nucleophile. A cohort of GH130 enzymes, however, lack the conserved basic residues that bind the phosphate nucleophile, and it was proposed that these enzymes function as glycoside hydrolases. Here we show that two Bacteroides enzymes, BT3780 and BACOVA_03624, which lack the phosphate binding residues, are indeed β-mannosidases that hydrolyze β-1,2-mannosidic linkages through an inverting mechanism. Because the genes encoding these enzymes are located in genetic loci that orchestrate the depolymerization of yeast α-mannans, it is likely that the two enzymes target the β-1,2-mannose residues that cap the glycan produced by Candida albicans. The crystal structure of BT3780 in complex with mannose bound in the −1 and +1 subsites showed that a pair of glutamates, Glu²²⁷ and Glu²⁶⁸, hydrogen bond to O₁ of α-mannose, and either of these residues may function as the catalytic base. The candidate catalytic acid and the other residues that interact with the active site mannose are conserved in both GH130 mannoside phosphorylases and β-1,2-mannosidases. Functional phylogeny identified a conserved lysine, Lys¹⁹⁹ in BT3780, as a key specificity determinant for β-1,2-mannosidic linkages.     

Cloning and characterization of a xylanase,KRICT PX1 from the strain Paenibacillus sp. HPL-001

The KRICT PX1 gene (GB: FJ380951) consisting of 996 bp encoding a protein of 332 amino acids (38.1 kDa) from the recently isolated Paenibacillus sp. strain HPL-001 (KCTC11365BP) has been cloned and expressed in Escherichia coli. The xylanase KRICT PX1 showed high activity on birchwood xylan, and was active over a pH range of 5.0 to 11.0, with two optima at pH 5.5 and 9.5 at 50 °C with K_m value of 5.35 and 3.23, respectively. The xylanase activity was not affected by most salts, such as NaCl, LiCl, KCl, NH₄Cl, CaCl₂, MgCl₂, MnCl₂, and CsCl₂ at 1 mM, but affected by CuSO₄, ZnSO₄, and FeCl₃. One mM of EDTA, 2-mercaptoethanol, and PMSF did not affect the xylanase activity. TLC analysis of the catalyzed products after reaction with birchwood xylan revealed that xylobiose was the major product with smaller amounts of xylotriose and xylose. A similarity analysis of the amino acids in KRICT PX1 resulted 72% identity with xylanase from Geobacillus stearothermophilus (GB: ZP_03040360), 70% identity with intracellular xylanase from an uncultured bacterium (GB: AAP51133), 68% identity with endo-1-4-xylanse from Paenibacillus sp. (GB: ZP_02847150). In addition, the amino acid alignment of KRICT PX1 with glycosyl hydralase (GH) family 10 xylanases revealed a high degree of homology in highly conserved regions including the catalytic sites, and this was confirmed through PROSITE scan. These results imply that KRICT PX1 is a new xylanase gene, and this alkaline xylanase belongs to GH family 10.