Effect of xylanases on peroxide bleachability of eucalypt (E. globulus) kraft pulp

Industrial eucalypt (E. globulus L.) kraft pulp was treated with two commercial xylanase preparations Ecopulp^® TX-200A and Pulpzyme^® HC (endo-1,4-β-xylanase activity; EC 3.2.1.8) and bleached by totally chlorine-free (TCF) three-stage hydrogen peroxide bleaching sequence, without oxygen pre-delignification. The effect of enzymatic stage on pulp properties and bleachability has been studied and compared with reference (control) pulps, processed without enzyme addition. The similar mode of enzymatic action was noted for both xylanase preparations. Final brightness of 86% ISO was achieved after complete bleaching. Direct bleaching effect caused pulp brightening (by 1.2–1.5% ISO) and delignification (by 7–10%) immediately after the enzymatic stage. The maximal bleach boosting was shown after the first peroxide stage and then diminished, despite the progressive increase in delignification over the control. The loss in efficiency of xylanase treatment by the end of peroxide bleaching was associated with specific behavior of xylan-derived chromophores, i.e., hexenuronic acids.     

Cellulose degrading enzymes and their potential industrial applications

Bioconversion of cellulose to soluble sugars and glucose is catalyzed by a group of enzymes called cellulases. Microorganisms including fungi, bacteria and actinomycetes produce mainly three types of cellulase components—endo-1,4-β-D-glucanase, exo-1,4-β-D-glucanase and β-glucosidase—either separately or in the form of a complex. Over the last several decades, cellulases have become better understood at a fundamental level; nevertheless, much remains to be learnt. The tremendous commercial potential of cellulases in a variety of applications remains the driving force for research in this area. This review summarizes the present state of knowledge on microbial cellulases and their applications.     

Characterization of Cellulase Secretion and Cre1-Mediated Carbon Source Repression in the Potential Lignocellulose-Degrading Strain Trichoderma asperellum T-1

Trichoderma asperellum, a traditional bio-control species, was demonstrated to be an excellent candidate for lignocellulose degradation in this work. Comparing to the representatively industrial strain of Trichoderma reeseiQM6a, T. asperellum T-1 showed more robust growth, stronger spore production, faster secretion of lignocellulose-decomposing enzymes and better pH tolerance. The reducing sugar released by strain T-1 on the second day of fermentation was 87% higher than that of strain QM6a, although the maximum reducing sugar yield and the cellulase production persistence of the strain T-1 were lower. Our experiment found that the cellulase secretion was strongly inhibited by glucose, suggesting the existence of carbon source repression pathway in T. asperellum T-1. The inhibiting effect was enhanced with an increase in glucose concentration and was closely related to mycelium growth. SDS-PAGE and secondary mass-spectrum identification confirmed that the expression of endo-1,4-β-xylanase I in T. asperellum T-1 was down-regulated when glucose was added. The factor Cre1, which plays an important role in the down-regulation of the endo-1,4-β-xylanase I gene, was investigated by bioinformatics methods. The protein structure of Cre1, analyzed using multiple protein sequence alignment, indicates the existence of the Zn-fingers domain. Then, the binding sites of Cre1 on the endo-1,4-β-xylanase I gene promoter were further elucidated. This study is the first report about Cre1-mediated carbon repression in the bio-control strain T. asperellum T-1. All of the above results provided good references for better understanding T. asperellum T-1 and improving its application for lignocellulose degradation.     

Production and Properties of Xylan-Degrading Enzymes from Cellulomonas uda

Xylan degradation and production of β-xylanase and β-xylosidase activities were studied in cultures of Cellulomonas uda grown on purified xylan from birchwood. β-Xylanase activity was found to be associated with the cells, although in various degrees. The formation of β-xylanase activity was induced by xylotriose and repressed by xylose. β-Xylosidase activity was cell bound. Both constitutive and inducible β-xylosidase activities were suggested. β-Xylanase and β-xylosidase activities were inhibited competitively by xylose. β-Xylanase activity had a pronounced optimum pH of 5.8, whereas the optimum pH of β-xylosidase activity ranged from 5.4 to 6.1. The major products of xylan degradation by a crude preparation of β-xylanase activity, in decreasing order of amount, were xylobiose, xylotriose, xylose, and small amounts of xylotetraose. This pattern suggests that β-xylanase activity secreted by C. uda is of the endosplitting type. Supernatants of cultures grown on cellulose showed not only β-glucanase but also β-xylanase activity. The latter could be attributed to an endo-1,4-β-glucanase activity which had a low β-xylanase activity.     

Purification and characterization of an extracellular endo-1,4-β-xylanase from Fusarium oxysporum f. sp. melonis

Abstract Fusarium oxysporum f. sp. melonis produces extracellular endo-1,4-β-xylanase and β-xylosidase when grown in shaken culture at 26°C in a mineral salts medium containing oat spelt xylan and glucose as carbon sources. Endo-1,4-β-xylanase was purified 251 times from 5-day-old culture filtrates, by Sephacryl S-200, ion exchange and gel filtration HPLC. The purified sample yielded a single band in SDS polyacrylamide gels with a molecular mass of 80 kDa on electrophoretic mobility and 83 kDa by gel filtration behavior. High activity of the endo-1,4-β-xylanase against xylan was observed between 5 and 8 pH, and between 40 and 60°C, the optimum pH and temperature being 5.0 and 50°C, respectively. Kinetic properties of the enzyme are similar to those of other fungal xylanases, showing high affinity towards oat spelt xylan with a K _m of 1 mM expressed as xylose equivalent.     

Novel media for detection of microbial producers of cellulase and xylanase

Abstract Agar nutrient media containing 0.2% soluble hydroxyethylcellulose covalently dyed with Ostazin Brilliant Red H-3B or soluble beechwood 4-O-methyl- d -glucurono- d -xylan dyed with Remazol Brilliant Blue R were used for sensitive detection of microorganisms producing and secreting into the surrounding medium endo-1,4-β-glucanase and/or endo-1,4-β-xylanase. Pale clearing zones formed around the colonies grown on such media indicated the production of corresponding polysaccharide-hydrolases.     

短小芽孢杆菌木聚糖酶基因在毕赤酶母中的分泌表达及酶学性质研究

将短小芽孢杆菌HB030的内切-1,4-木聚糖酶基因克隆到毕赤酵母表达载体pPIC9K,得到重组质粒pH-BM220,将pHBM220经酶切后分别转化三株毕赤酵母KM71、GS115、SMD1168,该木聚糖酶基因在三株毕赤酵母中均实现了分泌表达,将重组毕赤酵母KM71（pHBM220）,GS115（pHBM220),GS115(pHBM220),SMD1168（pHBM220）分别诱导产酶,对重组酶进行相关的酶学性质分析表明,三的最适反应pH值约为5.5,最适反应温度约为60℃,在其最适反应条件下测得三粗酶液酶活分别为10.80IU/mL,11.63IU/mL,9.68IU/mL,重组毕赤酵母KM71（pHBM220）所产酶的热稳定性较好,而在pH稳定性方面三没有太大的差异。     

宇佐美曲霉木聚糖酶基因xynⅡ在不同毕赤酵母中的分泌表达

将宇佐美曲霉E001的内切-1,4-木聚糖酶基因克隆到毕赤酵母表达载体pPIC9K中,得到重组质粒pPXY-NII,将其经SalⅠ线性化后分别转化2株毕赤酵母GS115和KM71,xynⅡ基因通过同源重组被整合到毕赤酵母染色体上,并处于酵母α因子的下游,经筛选获得阳性重组菌PXGL98(Mut+)和PXKL29(Muts)。该木聚糖酶基因在2株毕赤酵母中均实现了分泌表达。同时对工程菌的发酵条件进行了优化,在甲醇诱导下,PXGL98与PXKL29培养物上清液中的酶活力分别可达1156.92 U/mL和1646.03 U/mL。     

Production of xylooligosaccharides from enzymatic hydrolysis of xylan by the white-rot fungi Pleurotus

The white-rot fungi basidiomycetes Pleurotus sp. BCCB068 and Pleurotus tailandia were used to degrade oat-spelt xylan under submerged fermentation over a period of 40 days. Activities of endo-1,4-β-xylanase and β-xylosidase and xylan degradation products were determined. Xylan degradation by Pleurotus sp. BCCB068 and P. tailandia reached 75.1% and 73.4%, respectively. The formation of xylooligosaccharides and the simple sugars xylose, arabinose, cellobiose, mannose, and maltose were observed for both strains. The xylan degradation exhibited by these Pleurotus strains indicates they have potential for use in biotechnological processes related to degradation of hemicellulose sources.     

Degradation of different [(glucurono)arabino]xylans by a combination of purified xylan-degrading enzymes

The degradation of [({4-O-methyl-}glucurono)arabino]xylans from rice bran, oat spelts, wheat flour, larchwood, and birchwood with two types of endo-(1,4)--xylanase (I and III), (1,4)--xylosidase, (1,4)--d-arabinoxylan arabinofuranohydrolase (AXH), and an acetyl xylan esterase (AE), single and in combinations was investigated. The endo-(1,4)--xylanases showed the highest initial release of reducing end-groups on oat spelt xylan, followed successively by larchwood xylan, wheat flour xylan, birchwood xylan and rice bran xylan. The extent of degradation governed by degree and pattern of substitution was highest for oat spelts, followed by wheat flour and larchwood xylan. The extent of hydrolysis for the commercially available birchwood xylan was low, due to the partly insoluble fraction. Rice bran arabinoxylan could only partly be degraded by the combined action of endo-(1,4)--xylanase and AXH. The combination of endo-(1,4)--xylanase I or III, with (1,4)--xylosidase and AXH, or AE, resulted in the highest degree of hydrolysis after 24 h of incubation. Correspondence to: A. G. J. Voragen     

Purification, biochemical characterization, and structure of recombinant endo-1,4-β-xylanase XylE

The gene xylE encoding endo-1,4-β-xylanase from the 10th family of glycosyl hydrolases produced by the mycelial fungus Penicillium canescens has been expressed under the control of the strong promoter of the bgaS gene encoding β-galactosidase from P. canescens. As a result, a strain-producer of endoxylanase XylE was developed. The recombinant enzyme was isolated and purified to homogeneity with specific activity of 50 U/mg. The physicochemical and biochemical properties of the endoxylanase were studied. The maximal enzymatic activity was observed at pH 6.0 and 70°C. Endoxylanase XylE was shown to be a highly thermostable enzyme with half-inactivation period τ_1/2 of 7 h at 60°C. The kinetic parameters were 0.52 mg/ml (K _m) and 75 μmol/min per mg (V _max) using birch xylan as the substrate. Crystals of endoxylonase XylE were obtained, and the 3D structure was solved at 1.47 ? resolution. The 3D structure of an endo-1,4-β-xylanase from the 10th family containing carbohydrate and unique cyclic structure located at the C-terminus of the polypeptide chain was obtained for the first time.     

Probing the Role of Sigma π Interaction and Energetics in the Catalytic Efficiency of Endo-1,4-β-Xylanase

Chaetomium globosum endo-1,4-β-xylanase (XylCg) is distinguished from other xylanases by its high turnover rate (1,860 s⁻¹), the highest ever reported for fungal xylanases. One conserved amino acid, W48, in the substrate binding pocket of wild-type XylCg was identified as an important residue affecting XylCg''s catalytic efficiency.     

Induction of the Synthesis of Endo-1,4-β-xylanase and β-Galactosidase in Original and Recombinant Strains of the Fungus Penicillium canescens

The induction of synthesis of the secreted enzymes endo-1,4--xylanase (EC 3.2.1.8) and -galactosidase (EC 3.2.1.23) in original and recombinant Penicillium canescens strains has been studied. In all producer strains, the synthesis of these enzymes was induced by arabinose and its metabolite arabitol. The two enzymes differed in the concentration of arabinose required for induction: the synthesis of -galactosidase was most pronounced at 1 mM, whereas maximum synthesis of endo-1,4--xylanase was observed at 5–10 mM. An increase in the number of endo-1,4--xylanase copies in the high-copy-number strain of the fungus suppressed the synthesis of -galactosidase; the synthesis of endo-1,4--xylanase in the high-copy-number recombinant producing -galactosidase was affected to a lesser extent. The amount of enzymes synthesized did not depend on the saccharide used as the sole source of carbon for growing the mycelium prior to its transfer to the inducer-containing medium.     

Sequence analysis,cloning and over-expression of an endoxylanase from the alkaliphilic <Emphasis Type="BoldItalic">Bacillus halodurans</Emphasis>

The BhMIR32 xyn11A gene, encoding an extracellular endoxylanase of potential interest in bio-bleaching applications, was amplified from Bacillus halodurans MIR32 genomic DNA. The protein encoded is an endo-1,4-β-xylanase belonging to family 11 of glycosyl hydrolases. Its nucleotide sequence was analysed and the mature peptide was subcloned into pET22b(+) expression vector. The enzyme was over-expressed in a high density Escherichia coli culture as a soluble and active protein, and purified in a single step by immobilised metal ion affinity chromatography with a specific activity of 3073 IU mg⁻¹.     

Study on the use of soya bean polysaccharide degrading enzymes in broiler nutrition

Several non-starch polysaccharide (NSP)-degrading enzymes were studied for their suitability as feed enzymes in combination with soya bean meal (SBM) in broiler nutrition. In vitro experiments with SBM and several enzymes isolated from an Aspergillus aculeatus multi-enzyme preparation or respective enzyme specificities showed that only enzymes of the 1,4-β-arabinogalactanase type led to effective solubilization of dry matter from SBM, while other enzymes needed very high concentrations to invoke this effect. 1,4-β-galactomannanase activity also showed a high rate of sugar release from SBM, but the release of sugars from treated SBM (simulated stomach digestion with pepsin) was reduced, while an 1,4-β-arabinogalactanase from Humicola insolens still led to a high release of sugars on the same substrate. Synergistic effects on the release of sugars were observed when both galactanase and galactomannanase enzymes were used. Synergistic effects of galactanase and mannanase were also noted for two different endopolygalacturonases, partly purified from the multi-enzyme preparation.

In feeding trials with broilers, increasing concentrations of the multi-enzyme preparation with numerous NSP-degrading enzymes showed beneficial effects on feed intake, but high in feed concentrations were needed. Further feeding trials with the galactanase preparation from H. insolens alone and in combination with a mannanase preparation (Gammanase) were conducted. However, due to weak in vivo stability of the galactanase, only a small effect on feed conversion was noticed. Additional supplementation of the mannanase reduced live weight gain and feed intake.

It is concluded that enzymes of the 1,4-β-arabinogalactanase type are the most likely candidates for a SBM-specific feed enzyme in broiler nutrition, possibly in combination with endopolygalacturonases. However, while a partial degradation of soya NSP may enhance digestibility, a high degree of hydrolysis may produce oligo- and monomers with anti-nutritive effects such as osmotic pressure or microbial fermentation in the small intestine.     

Changes in levels of mRNAs of transforming growth factor (TGF)-β1, -β2, -β3, TGF-β type II receptor and sulfated glycoprotein-2 during apoptosis of mouse uterine epithelium

To examine the roles played by transforming growth factors (TGF)-β1, -β2, -β3, and TGF-β type II receptors in the induction of apoptosis in the mouse uterine epithelium after estrogen deprivation, we investigated the expression of their mRNAs and the mRNA of sulfated glycoprotein-2 (SGP-2). Pellets containing 100 μg estradiol-17β (E₂) were implanted into ovariectomized mice and removed four days later. Apoptotic indices (percentage of apoptotic cells) of both luminal and glandular epithelia increased after E₂ pellets were removed, but administration of progesterone (P), 5-dihydrotestosterone (DHT), or continued implantation of E₂ pellets suppressed this increase. Levels of mRNAs of TGF-β1, -β2, and -β3, and SGP-2 did not increase after estrogen deprivation. However, estrogen deprivation caused a gradual increase in the level of TGF-β type II receptor mRNA, and its level increased about six-fold six days later. Moreover, E₂, P, and DHT markedly decreased the level of TGF-β type II receptor mRNA. In situ hybridization demonstrated that mRNAs of TGF-β1, -β2, -β3 and TGF-β type II receptor were localized to the epithelium. Exogenous administration of TGF-β1 into the uterine stroma induced apoptosis in the epithelium, a finding that suggests that signals produced by TGF-βs can induce apoptosis. Therefore, the present results suggest that increased sensitivity of uterine epithelial cells to TGF-βs, as demonstrated by an increase in TGF-β type II receptor mRNA, is involved in the induction of apoptosis after estrogen deprivation, although signals produced by TGF-βs do not appear sufficient to induce apoptosis.     

Thermostable xylanolytic enzymes from Rhodothermus marinus grown on xylan

The thermophilic eubacterium Rhodothermus marinus was cultivated in a fermentor and studied with respect to activities of induced xylanolytic enzymes. Growth in the fermentor on xylan occurred with a maximum specific growth rate of 0.43 h^–1 for a batch culture. The final cell concentration was 4 g cell dry weight (CDW)/l for cells grown on xylan compared to 2 g CDW/l for cells grown without xylan in the cultivation medium. At least two xylanolytic enzymes, endo-1,4--xylanase and xylan 1,4--xylosidase, were secreted into the culture medium when cells were cultivated on xylan. Of the three cellulolytic enzymes tested for activity, -glucosidase activity was in the range of the xylanolytic enzyme activities whereas cellulose-1,4--cellobiosidase and cellulase activities were hardly detectable. The expression of endo-1,4--xylanase activities during cultivation indicates the existance of more than one xylanase in R. marinus. This is also observed in fractions from gel filtration. The xylanolytic enzymes are heat-stable. At 90°C and at pH 7.0 the half-life of the endo-1,4--xylanase was about 14 h and that of xylan 1,4--xylosidase was 45 min. Correspondence to: L. Dahlberg     

Catalytic properties of the endoxylanase I from Thermoascus aurantiacus

Endo-β-1,4-xylanase I previously purified from Thermoascus aurantiacus solid state culture was further characterized. The enzyme had a molecular weight of 33 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and 31 kDa by gel filtration. Thin layer chromatography (TLC) analysis showed that endoxylanase liberates aldotetrauronic acid MeGlcA-1,2-Xylβ-1,4-Xylβ-1,4-Xyl as the shortest acidic fragment from glucuronoxylan and an isomeric xylotriose (Xyl₃) of the structure Xylβ1-3Xylβ1-4Xyl from rhodymenan. The enzyme performed ideally on O-acetyl-4-O-methylglucuronoxylan, liberating large amounts of short acetylated and non-acetylated fragments. Also, the enzyme was capable to hydrolyse arabinoxylan to arabinose (Arab), xylose (Xyl) and xylobiose (Xyl₂). The enzyme degraded pNPX (4-nitrophenyl β- -xylopyranoside) by a complex reaction pathway that involved both hydrolysis and glycosyl transfer reactions. The enzyme tolerates the replacement of β-xylopyranosyl units in several artificial substrates by β-glucopyranosyl, - -arabinopyranosyl and - -arabinofuranosyl units and was active on pNPC (4-nitrophenyl β- -cellobioside), pNP-Arap (4-nitrophenyl - -arabinopyranoside) and pNPAraf (4-nitrophenyl - -arabinofuranoside). The enzyme also hydrolysed the 4-methylumbelliferyl glycosides of β- -xylobiose and β- -xylotriose at the agluconic linkage. The results suggested that the xylanase I from T. aurantiacus has catalytic properties similar to those belonging to family 10.     

Xylanase of the micromycete <Emphasis Type="Italic">Rhizopus</Emphasis> var. <Emphasis Type="Italic">microsporus</Emphasis> 595: Preparation,structural and functional characteristics,and application

Procedures for the production of endo-1,4-β-xylanase have been developed. An active producer—Rhizopus var. microsporus BKMF-595—has been chosen, and the conditions of surface and submerged cultivation, as well as the composition of the culture medium for this strain, have been optimized to ensure maximum yield of the target enzyme. Activity of xylomicrosporin Px equaled 123 U/g, while the activity of xylomicrosporin Gx equaled 25 U/cm³. Homogeneous enzyme preparations, purified 59.44-fold and 72.6-fold, have been obtained. The dependence of endo-1,4-β-xylanase catalytic activity on temperature and pH of the reaction medium has been studied. The enzyme has been shown to be most stable in the pH range 5.0–6.0 and to be thermostable. Amino acid composition and subunit structure of the enzyme were determined; the molecular masses of the subunits equaled 50 and 56 kDa. Carboxyl groups of glutamic and aspartic acid residues of the active center were experimentally shown to play an important role in catalysis. The potential of this enzyme for beer production has been demonstrated.     

Functional display of family 11 endoxylanases on the surface of phage M13

Two family 11 endoxylanases (EC 3.2.1.8) were functionally displayed on the surface of bacteriophage M13. The genes encoding endo-1,4-xylanase I from Aspergillus niger (ExlA) and endo-1,4-xylanase A from Bacillus subtilis (XynA) were fused to the gene encoding the minor coat protein g3p in phagemid vector pHOS31. Phage rescue resulted in functional monovalent display of the enzymes as was demonstrated by three independent tests. Firstly, purified recombinant phage particles showed a clear hydrolytic activity in an activity assay based on insoluble, chromagenic arabinoxylan substrate. Secondly, specific binding of endoxylanase displaying phages to immobilized endoxylanase inhibitors was demonstrated by interaction ELISA. Finally, two rounds of selection and amplification in a biopanning procedure against immobilized endoxylanase inhibitor were performed. Phages displaying endoxylanases were strongly enriched from background phages displaying unrelated proteins. These results open perspectives to use phage display for analysing protein-protein interactions at the interface between endoxylanases and their inhibitors. In addition, this technology should enable engineering of endoxylanases into novel variants with altered binding properties towards endoxylanase inhibitors.