Trans-α-xylosidase and trans-β-galactosidase activities, widespread in plants, modify and stabilize xyloglucan structures

Cell‐wall components are hydrolysed by numerous plant glycosidase and glycanase activities. We investigated whether plant enzymes also modify xyloglucan structures by transglycosidase activities. Diverse angiosperm extracts exhibited transglycosidase activities that progressively transferred single sugar residues between xyloglucan heptasaccharide (XXXG or its reduced form, XXXGol) molecules, at 16 μm and above, creating octa‐ to decasaccharides plus smaller products. We measured remarkably high transglycosylation:hydrolysis ratios under optimized conditions. To identify the transferred monosaccharide(s), we devised a dual‐labelling strategy in which a neutral radiolabelled oligosaccharide (donor substrate) reacted with an amino‐labelled non‐radioactive oligosaccharide (acceptor substrate), generating radioactive cationic products. For example, 37 μm [Xyl‐³H]XXXG plus 1 mm XXLG‐NH₂ generated ³H‐labelled cations, demonstrating xylosyl transfer, which exceeded xylosyl hydrolysis 1.6‐ to 7.3‐fold, implying the presence of enzymes that favour transglycosylation. The transferred xylose residues remained α‐linked but were relatively resistant to hydrolysis by plant enzymes. Driselase digestion of the products released a trisaccharide (α‐[³H]xylosyl‐isoprimeverose), indicating that a new xyloglucan repeat unit had been formed. In similar assays, [Gal‐³H]XXLG and [Gal‐³H]XLLG (but not [Fuc‐³H]XXFG) yielded radioactive cations. Thus plants exhibit trans‐α‐xylosidase and trans‐β‐galactosidase (but not trans‐α‐fucosidase) activities that graft sugar residues from one xyloglucan oligosaccharide to another. Reconstructing xyloglucan oligosaccharides in this way may alter oligosaccharin activities or increase their longevity in vivo. Trans‐α‐xylosidase activity also transferred xylose residues from xyloglucan oligosaccharides to long‐chain hemicelluloses (xyloglucan, water‐soluble cellulose acetate, mixed‐linkage β‐glucan, glucomannan and arabinoxylan). With xyloglucan as acceptor substrate, such an activity potentially affects the polysaccharide’s suitability as a substrate for xyloglucan endotransglucosylase action and thereby modulates cell expansion. We conclude that certain proteins annotated as glycosidases can function as transglycosidases.     

Development of a noninvasive reporter system for gene expression in Porphyromonas gingivalis

Several reports have supported the association of Porphyromonas gingivalis with periodontal disease. Genetic studies are vital for understanding the relative importance of virulence factors in this organism. Thus, gene reporters may prove useful for the study of gene expression in this organism. We have investigated the use of the green fluorescent protein (GFP), bacterial luciferase, and bifunctional xylosidase/arabinosidase enzyme (XA) as reporters of gene expression in P. gingivalis. Fusion cassettes containing the promoterless tetracycline resistant gene [tetA(A)Q2] and the promoterless gfp, luxAB, or xa gene were placed under the control of the rgpA promoter in P. gingivalis W83 using recombinational allelic exchange. The rgpA gene encodes for an arginine-specific protease in P. gingivalis. No GFP activity was detected in P. gingivalis isogenic mutants carrying the rgpA::gfp-tetA(Q)2 fusion construct. Luciferase activity in P. gingivalis mutants carrying the rgpA::luxAB-tetA(Q)2 fusion was only detected in the presence of exogenous FMNH(2). xa gene expression in P. gingivalis with the rgpA::xa-tetA(Q)2 fusion construct was detected in crude extracts using rho-nitrophenol derivatives as substrate and on agar plates with methylumbelliferyl derivatives under long-wave ultraviolet light. This indicates that both luxAB and xa genes can be used as reporters of gene expression in P. gingivalis. However, only the xa gene can be used as a noninvasive reporter gene.     

Significant enhancement in the binding of p-nitrophenyl-β- -xylobioside by the E128H mutant F/10 xylanase from Streptomyces olivaceoviridis E-86

Mutagenesis studies were carried out to examine the effects of replacement of either the nucleophile Glu-236 or the acid/base Glu-128 residue of the F/10 xylanase by a His residue. To our surprise, the affinity for the p-nitrophenyl-β- -xylobioside substrate was increased by 10³-fold in the case of the mutant E128H enzyme compared with that of the wild-type F/10 xylanase. The catalytic activity of the mutant enzymes was low, despite the fact that the distance between the nucleophilic atom (an oxygen in the native xylanase and a nitrogen in the mutant) and the α-carbon was barely changed. Thus, the alteration of the acid/base functionality (Glu-128 to His mutation) provided a significantly favorable interaction within the E128H enzyme/substrate complex in the ground state, accompanying a reduction in the stabilization effect in the transition state.     

牦牛瘤胃元基因组文库中木聚糖酶基因的分析

【目的】了解牦牛瘤胃微生物木聚糖酶多样性及其降解特征,为木聚糖降解提供新的基因资源。【方法】根据对已构建的瘤胃微生物元基因组细菌人工染色体(BAC)克隆文库高通量测序结果的注释,筛选其中编码木聚糖酶的基因并进行多样性分析;对其中一个木聚糖酶基因及其连锁的木糖苷酶基因进行克隆表达和酶学性质表征,分析其协同作用。【结果】共筛选到14个木聚糖酶基因,均编码GH10家族木聚糖酶,其氨基酸序列之间的相似性为20.5%-91.3%;其中7个木聚糖酶基因所在的不同的DNA片段(contig)上存在木糖苷酶基因,编码的木糖苷酶属于GH43或GH3糖苷水解酶家族。将其中一对连锁的木聚糖酶(Xyn32)和木糖苷酶基因(Xyl33)分别克隆、表达和纯化。纯化后的木聚糖酶比活为1.98 IU/mg,但不具有阿魏酸酯酶活性;木糖苷酶比活为0.07 U/mg,且具有α-阿拉伯呋喃糖苷酶活性。体外实验证明,木糖苷酶Xyl33对与之连锁的木聚糖酶Xyn32的木聚糖降解具有协同作用。     

Phylogenetic variation in glycosidases and glycanases acting on plant cell wall polysaccharides, and the detection of transglycosidase and trans-β-xylanase activities

Wall polysaccharide chemistry varies phylogenetically, suggesting a need for variation in wall enzymes. Although plants possess the genes for numerous putative enzymes acting on wall carbohydrates, the activities of the encoded proteins often remain conjectural. To explore phylogenetic differences in demonstrable enzyme activities, we extracted proteins from 57 rapidly growing plant organs with three extractants, and assayed their ability to act on six oligosaccharides ‘modelling’ selected cell‐wall polysaccharides. Based on reaction products, we successfully distinguished exo‐ and endo‐hydrolases and found high taxonomic variation in all hydrolases screened: β‐d ‐xylosidase, endo‐(1→4)‐β‐d ‐xylanase, β‐d ‐mannosidase, endo‐(1→4)‐β‐d ‐mannanase, α‐d ‐xylosidase, β‐d ‐galactosidase, α‐l ‐arabinosidase and α‐l ‐fucosidase. The results, as GHATAbase, a searchable compendium in Excel format, also provide a compilation for selecting rich sources of enzymes acting on wall carbohydrates. Four of the hydrolases were accompanied, sometimes exceeded, by transglycosylase activities, generating products larger than the substrate. For example, during β‐xylosidase assays on (1→4)‐β‐d ‐xylohexaose (Xyl₆), Marchantia, Selaginella and Equisetum extracts gave negligible free xylose but approximately equimolar Xyl₅ and Xyl₇, indicating trans‐β‐xylosidase activity, also found in onion, cereals, legumes and rape. The yield of Xyl₉ often exceeded that of Xyl_7–8, indicating that β‐xylanase was accompanied by an endotransglycosylase activity, here called trans‐β‐xylanase, catalysing the reaction 2Xyl₆→ Xyl₃ + Xyl₉. Similar evidence also revealed trans‐α‐xylosidase, trans‐α‐arabinosidase and trans‐α‐arabinanase activities acting on xyloglucan oligosaccharides and (1→5)‐α‐l ‐arabino‐oligosaccharides. In conclusion, diverse plants differ dramatically in extractable enzymes acting on wall carbohydrate, reflecting differences in wall polysaccharide composition. Besides glycosidase and glycanase activities, five new transglycosylase activities were detected. We propose that such activities function in the assembly and re‐structuring of the wall matrix.     

Enzymatic hydrolysis of water-soluble wheat arabinoxylan. 1. Synergy between alpha-L-arabinofuranosidases,endo-1,4-beta-xylanases,and beta-xylosidase activities

Hydrolysis of arabinoxylan is an important prerequisite for improved utilization of wheat hemicellulose in the ethanol fermentation industry. This study investigates the individual and combined efficiencies of three commercial, cellulytic and hemicellulytic enzyme preparations, Celluclast 1.5 L, Ultraflo L, and Viscozyme L, in catalyzing the liberation of arabinose and xylose from water-soluble wheat arabinoxylan. Ultraflo L was the best enzyme preparation for releasing arabinose, liberating 53 wt% of the theoretical maximum after 48 h of reaction (10 wt% enzyme/substrate ratio, 40 degrees C, pH 6). Celluclast 1.5 L was superior to the other enzyme preparations in releasing xylose, liberating 26 wt% of the theoretical maximum after 48 h of reaction (10 wt% enzyme/substrate ratio, 50 degrees C, pH 5). The 50:50 mixtures of the enzyme preparations showed no synergistic cooperation in arabinose release, but a synergistic interaction in xylose release was found between Ultraflo L and Celluclast 1.5 L. On the basis of high-performance anion exchange chromatography (HPAEC) analysis of the hydrolysates after enzymatic reaction, we propose that the observed synergism between Celluclast 1.5 L and Ultraflo L is the result of positive interaction between alpha-L-arabinofuranosidase and endo-1,4-beta-xylanase activities present in Ultraflo L that released arabinose, xylobiose and xylotriose, and beta-xylosidase activities in Celluclast 1.5 L, capable of catalyzing the hydrolysis of xylobiose and xylotriose to xylose.     

A beta-glucosidase/xylosidase from the phytopathogenic oomycete, Phytophthora infestans

An 85-kDa beta-glucosidase/xylosidase (BGX1) was purified from the axenically grown phytopathogenic oomycete, Phytophthora infestans. The bgx1 gene encodes a predicted 61-kDa protein product which, upon removal of a 21 amino acid leader peptide, accumulates in the apoplastic space. Extensive N-mannosylation accounts for part of the observed molecular mass difference. BGX1 belongs to family 30 of the glycoside hydrolases and is the first such oomycete enzyme deposited in public databases. The bgx1 gene was found in various Phytophthora species, but is apparently absent in species of the related genus, Pythium. Despite significant sequence similarity to human and murine lysosomal glucosylceramidases, BGX1 demonstrated neither glucocerebroside nor galactocerebroside-hydrolyzing activity. The native enzyme exhibited glucohydrolytic activity towards 4-methylumbelliferyl (4-MU) beta-D-glucopyranoside and, to lesser extent, towards 4-MU-D-xylopyranoside, but not towards 4-MU-beta-D-glucopyranoside. BGX1 did not hydrolyze carboxymethyl cellulose, cellotetraose, chitosan or xylan, suggesting high substrate specificity and/or specific cofactor requirements for enzymatic activity.     

Thermostable β-xylosidase from Thermomonospora curvata

Thermomonospora curvata produced a thermostable β-xylosidase during growth on birch xylan. The enzyme, extracted by sonication of early stationary phase mycelia, was purified by isoelectric focusing and size exclusion HPLC. The isoelectric point was pH 4.8. The molecular weight was estimated to be 102 000 by size exclusion HPLC and 112 000 by SDS-PAGE. Maximal activity occurred at pH 6–7 and 60–68°C. K _m values for xylobiose and p-nitrophenyl-β -D-xylopyranoside were 4.0 M and 0.6 M respectively. The enzyme was sensitive to low levels of Hg²⁺ (50% inhibition at 0.2 μM), but was stimulated by Co²⁺ and Pb²⁺. Addition of the xylosidase to a xylanase reaction mixture increased the liberation of xylose equivalents from xylan and decreased the proportion of xylobiose in the hydrolysate. Received 14 April 1997/ Accepted in revised form 21 October 1997     

嗜碱芽孢杆菌C-125木糖苷酶基因的表达与酶特征鉴定

嗜碱芽孢杆菌(Bacillus halodurans)C-125菌株的基因组中,一个编码木糖苷酶的基因(BH1068)被克隆并在大肠杆菌中获得高效表达。通过全面分析纯化蛋白,确证了它的木糖苷酶功能。该酶在pH4～9的范围内保持稳定,最适pH值为中性,有较宽的最适温度(35°C～45°C),且能在45°C范围内保持稳定。这些特性使得该酶可在较为宽广的条件下对木聚糖进行酶促降解。该酶对人工合成底物对硝基苯-β-木糖苷(p-nitrophenyl-β-xylose,pNPX)的比活力为174mU/mg蛋白质,且木糖对其反馈抑制较弱(抑制常数Ki为300mmol/L)。结果显示该酶是活性较高且较耐木糖抑制的细菌源木糖苷酶。该酶与商品化的木聚糖酶一起水解山毛举木聚糖(Beechwood xylan)时显示了增效作用,且水解率可获40%。该酶最适pH为中性,对木糖耐受等特性与大多数来源于真菌、最适pH为酸性、对木糖敏感的木糖苷酶将有较好的互补。结果表明该酶在木聚糖或含木聚糖多糖的单糖化过程可能发挥重要作用。