Evaluation of a novel bifunctional xylanase–cellulase constructed by gene fusion

An artificial bifunctional enzyme, xylanase–cellulase, has been prepared by gene fusion. Three chimeric genes were constructed that encoded fusion proteins of different lengths. The fusion proteins exhibited both xylanase (XynX) and cellulase (Cel5Z::Ω) activity when cel5Z::Ω was fused downstream of xynX, but not when xynX was fused downstream of cel5Z::Ω. Activities of bifunctional enzymes decreased when a shorter xylanase peptide was fused. Three fusion enzymes were purified, and the molecular weights of the enzymes were estimated by CMC-SDS-PAGE and XYN-SDS-PAGE to be 149, 129, and 87 kDa, respectively. The fusion enzymes displayed optimum cellulase activity at pH 8.0 and 50 °C and optimum xylanase activity at pH 8.0 and 70 °C.     

Streptomyces thermocerradoensis I3 secretes a novel bifunctional xylanase/endoglucanase under solid-state fermentation

Lignocellulosic wastes can be potentially converted into several bioproducts such as glucose, xylo-oligosaccharides, and bioethanol. Certain processes, such as enzymatic hydrolysis, are generally needed to convert biomass into bioproducts. The present study investigated the production of xylanases and cellulases by Streptomyces thermocerradoensis I3 under solid-state fermentation (SSF), using wheat bran as a low-cost medium. The activities of xylanase and carboxymethyl cellulase (CMCase) were evaluated until 96 hr of incubation. The highest enzyme activity was observed after 72 hr of incubation. The crude enzyme extract was sequentially filtered, first using a 50 kDa filter, followed by a 30 kDa filter. Fraction 3 (F3) exhibited activities of both xylanase and CMCase. Xylanase and CMCase showed optimum activity at 70°C and pH 6.0 and 55°C and pH 6.0, respectively. The zymogram analysis showed a single activity band with a molecular mass of approximately 17 kDa. These findings provide strong evidence that the enzyme is a bifunctional xylanase/endoglucanase. This enzyme improved the saccharification of sugarcane bagasse by 1.76 times that of commercial cellulase. This enzyme has potential applications in various biotechnological procedures.     

A bifunctional cellulase–xylanase of a new Chryseobacterium strain isolated from the dung of a straw‐fed cattle

A new cellulolytic strain of Chryseobacterium genus was screened from the dung of a cattle fed with cereal straw. A putative cellulase gene (cbGH5) belonging to glycoside hydrolase family 5 subfamily 46 (GH5_46) was identified and cloned by degenerate PCR plus genome walking. The CbGH5 protein was overexpressed in Pichia pastoris, purified and characterized. It is the first bifunctional cellulase–xylanase reported in GH5_46 as well as in Chryseobacterium genus. The enzyme showed an endoglucanase activity on carboxymethylcellulose of 3237 μmol min^?1 mg^?1 at pH 9, 90 °C and a xylanase activity on birchwood xylan of 1793 μmol min^?1 mg^?1 at pH 8, 90 °C. The activity level and thermophilicity are in the front rank of all the known cellulases and xylanases. Core hydrophobicity had a positive effect on the thermophilicity of this enzyme. When similar quantity of enzymatic activity units was applied on the straws of wheat, rice, corn and oilseed rape, CbGH5 could obtain 3.5–5.0× glucose and 1.2–1.8× xylose than a mixed commercial cellulase plus xylanase of Novozymes. When applied on spent mushroom substrates made from the four straws, CbGH5 could obtain 9.2–15.7× glucose and 3.5–4.3× xylose than the mixed Novozymes cellulase+xylanase. The results suggest that CbGH5 could be a promising candidate for industrial lignocellulosic biomass conversion.     

Molecular cloning of a xylanase gene from Bacteroides succinogenes and its expression in Escherichia coli

A gene coding for xylanase synthesis in Bacteroides succinogenes was isolated by cloning, with Escherichia coli HB101 as the host. After partial digestion of B. succinogenes DNA with Sau3A, fragments were ligated into the BamHI site of pBR322 and transformed into E. coli HB101. Of 14,000 colonies screened, 4 produced clear halos on Remazol brilliant blue-xylan agar. Plasmids from two stable clones recovered exhibited identical restriction enzyme patterns, with the same 9.4-kilobase-pair (kbp) insert. The plasmid was designated pBX1. After subcloning of restriction enzyme fragments, a 3-kbp fragment was found to code for xylanase activity in either orientation when inserted into pUC18 and pUC19. The original clone possessed approximately 10-fold higher xylanase activity than did clones harboring the 3-kbp insert in pUC18, pUC19, or pBR322. The enzyme was partially secreted into the periplasmic space of E. coli. The periplasmic enzyme of the BX1 clone had 2% of the activity on carboxymethyl cellulose and less than 0.2% of the activity on p-nitrophenyl xyloside and a range of other substrates that it exhibited on xylan. The xylanase gene was not subject to catabolite repression by glucose or induction by either xylan or xylose. The xylanase activity migrated as a single broad band on nondenaturing polyacrylamide gels. The Km of the pBX1-encoded enzyme was 0.22% (wt/vol) of xylan, which was similar to that for the xylanase activity in an extracellular enzyme preparation from B. succinogenes. Based on these data it appears that the xylanase gene expressed in E. coli is fully functional and codes for an enzyme with properties similar to the B. succinogenes enzyme(s).     

Molecular cloning of a xylanase gene from Bacteroides succinogenes and its expression in Escherichia coli.

A gene coding for xylanase synthesis in Bacteroides succinogenes was isolated by cloning, with Escherichia coli HB101 as the host. After partial digestion of B. succinogenes DNA with Sau3A, fragments were ligated into the BamHI site of pBR322 and transformed into E. coli HB101. Of 14,000 colonies screened, 4 produced clear halos on Remazol brilliant blue-xylan agar. Plasmids from two stable clones recovered exhibited identical restriction enzyme patterns, with the same 9.4-kilobase-pair (kbp) insert. The plasmid was designated pBX1. After subcloning of restriction enzyme fragments, a 3-kbp fragment was found to code for xylanase activity in either orientation when inserted into pUC18 and pUC19. The original clone possessed approximately 10-fold higher xylanase activity than did clones harboring the 3-kbp insert in pUC18, pUC19, or pBR322. The enzyme was partially secreted into the periplasmic space of E. coli. The periplasmic enzyme of the BX1 clone had 2% of the activity on carboxymethyl cellulose and less than 0.2% of the activity on p-nitrophenyl xyloside and a range of other substrates that it exhibited on xylan. The xylanase gene was not subject to catabolite repression by glucose or induction by either xylan or xylose. The xylanase activity migrated as a single broad band on nondenaturing polyacrylamide gels. The Km of the pBX1-encoded enzyme was 0.22% (wt/vol) of xylan, which was similar to that for the xylanase activity in an extracellular enzyme preparation from B. succinogenes. Based on these data it appears that the xylanase gene expressed in E. coli is fully functional and codes for an enzyme with properties similar to the B. succinogenes enzyme(s).     

Biochemical Characterization and Structural Analysis of a Bifunctional Cellulase/Xylanase from Clostridium thermocellum

We expressed an active form of CtCel5E (a bifunctional cellulase/xylanase from Clostridium thermocellum), performed biochemical characterization, and determined its apo- and ligand-bound crystal structures. From the structures, Asn-93, His-168, His-169, Asn-208, Trp-347, and Asn-349 were shown to provide hydrogen-bonding/hydrophobic interactions with both ligands. Compared with the structures of TmCel5A, a bifunctional cellulase/mannanase homolog from Thermotoga maritima, a flexible loop region in CtCel5E is the key for discriminating substrates. Moreover, site-directed mutagenesis data confirmed that His-168 is essential for xylanase activity, and His-169 is more important for xylanase activity, whereas Asn-93, Asn-208, Tyr-270, Trp-347, and Asn-349 are critical for both activities. In contrast, F267A improves enzyme activities.     

Cloning,purification and characterization of an alkali-stable endoxylanase from thermophilic <Emphasis Type="Italic">Geobacillus</Emphasis> sp. 71

The gene encoding a xylanase from Geobacillus sp. 71 was isolated, cloned, and sequenced. Purification of the Geobacillus sp 7.1 xylanase, XyzGeo71, following overexpression in E. coli produced an enzyme of 47 kDa with an optimum temperature of 75°C. The optimum pH of the enzyme is 8.0, but it is active over a broad pH range. This protein showed the highest sequence identity (93%) with the xylanase from Geobacillus thermodenitrificans NG80-2. XyzGeo71 contains a catalytic domain that belongs to the glycoside hydrolase family 10 (GH10). XyzGeo71 exhibited good pH stability, remaining stable after treatment with buffers ranging from pH 7.0 to 11.0 for 6 h. Its activity was partially inhibited by Al³⁺ and Cu²⁺ but strongly inhibited by Hg²⁺. The enzyme follows Michaelis–Menten kinetics, with K_m and V_max values of 0.425 mg xylan/ml and 500 μmol/min.mg, respectively. The enzyme was free from cellulase activity and degraded xylan in an endo fashion. The action of the enzyme on oat spelt xylan produced xylobiose and xylotetrose.     

Differential expression of cellulases and xylanases by <Emphasis Type="Italic">Cellulomonas flavigena</Emphasis> grown on different carbon sources

The diversity of cellulases and xylanases secreted by Cellulomonas flavigena cultured on sugar cane bagasse, Solka-floc, xylan, or glucose was explored by two-dimensional gel electrophoresis. C. flavigena produced the largest variety of cellulases and xylanases on sugar cane bagasse. Multiple extracellular proteins were expressed with these growth substrates, and a limited set of them coincided in all substrates. Thirteen proteins with carboxymethyl cellulase or xylanase activity were liquid chromatography/mass spectrometry sequenced. Proteins SP4 and SP18 were identified as products of celA and celB genes, respectively, while SP20 and SP33 were isoforms of the bifunctional cellulase/xylanase Cxo recently sequenced and characterized in C. flavigena. The rest of the detected proteins were unknown enzymes with either carboxymethyl cellulase or xylanase activities. All proteins aligned with glycosyl hydrolases listed in National Center for Biotechnology Information database, mainly with cellulase and xylanase enzymes. One of these unknown enzymes, protein SP6, was cross-induced by sugar cane bagasse, Solka-floc, and xylan. The differences in the expression maps of the presently induced cultures revealed that C. flavigena produces and secretes multiple enzymes to use a wide range of lignocellulosic substrates as carbon sources. The expression of these proteins depends on the nature of the cellulosic substrate.     

Untersuchungen zur Bildung extrazellulärer Hemicellulasen durch Pseudocercosporella herpotrichoides in vitro

Investigations on the production of extracellular hemicellulases by Pseudocercosporella herpotrichoides in vitro For all 15 isolates of Pseudocercosporella herpotrichoides investigated, xylanase as well as arabanase activity could be demonstrated. After cultivation of 3 weeks, the activity of the enzymes reached a peak. The activity of xylanase was considerably increased by addition of xylan in comparison to Maltzin as the sole source of carbohydrate. Also the arabanase activity could be increased significantly by addition of araban or xylan as compared to the Maltzin variant. The optimum temperature with regard to activity and stability of xylanase ranged at 50°C. The pH-optimum for xylanase activity was found to be at pH 5.0, and the enzyme was stable in ° range between pH4.0 and 8.0 (9.0). In case of arabanase, the temperature optimum varied between 40 and 50°C; up to this temperature, the enzyme was also stable. At pH 5.0, the arabanase activity reached its optimum; stability was observed in - pH range between 4.0 and 9.0. In extracts prepared from autoclaved wheat coleoptiles which were inoculated with Pseudocercosporella herpotrichoides, the presence of the enzymes xylanase, arabanase, cellulase and polymethylgalacturonase could be demonstrated. The enzyme activities of the inoculated samples were considerably higher than those of non-inoculated controls. The differences, in most cases, were statistically significant. Der Deutschen Forchungsgemeinschaft danken wir für finanzielle Unterstützung.     

Simultaneous purification and immobilization of Aspergillus niger xylanase on the reversibly soluble polymer Eudragit(TM) L-100

The non-covalent immobilization of a commercial preparation of xylanase from A. niger was carried out on a reversibly soluble-insoluble enteric polymer Eudragit(TM) L-100. The immobilization of the xylanase activity by adsorption was simultaneously accompanied by removal of cellulase activity since the latter did not bind to the polymer. Thus, the soluble enzyme derivative may be useful for treatment of paper pulp bleaching in paper industry. The immobilized xylanase retained 60% of its activity toward xylan as the substrate. No change was observed in the pH optimum (5.5) of the enzyme upon immobilization. Only marginal increase in the K(m) of the free enzyme (3.6 mg ml(-1) to 5.0 mg ml(-1)) upon immobilization on the soluble polymer reflected that the enzyme-substrate binding continues to be efficient in spite of the macromolecular nature of the substrate. Fluorescence spectroscopy and UV difference spectroscopy were used to probe the change(s) in the enzyme structure upon immobilization. This change in structure was correlated with the "effectiveness factor" of the enzyme activity. CD spectra also showed that the enzyme undergoes drastic changes in the structure.     

Cloning and expression in Escherichia coli of a xylanase gene from Bacteroides ruminicola 23.

A gene coding for xylanase activity in the ruminal bacterial strain 23, the type strain of Bacteroides ruminicola, was cloned into Escherichia coli JM83 by using plasmid pUC18. AB. ruminicola 23 genomic library was prepared in E. coli by using BamHI-digested DNA, and transformants were screened for xylanase activity on the basis of clearing areas around colonies grown on Remazol brilliant blue R-xylan plates. Six clones were identified as being xylanase positive, and all six contained the same 5.7-kilobase genomic insert. The gene was reduced to a 2.7-kilobase DNA fragment. Xylanase activity produced by the E. coli clone was found to be greater than that produced by the original B. ruminicola strain. Southern hybridization analysis of genomic DNA from the related B. ruminicola strains, D31d and H15a, by using the strain 23 xylanase gene demonstrated one hybridizing band in each DNA.     

Cloning and expression in Escherichia coli of a xylanase gene from Bacteroides ruminicola 23

A gene coding for xylanase activity in the ruminal bacterial strain 23, the type strain of Bacteroides ruminicola, was cloned into Escherichia coli JM83 by using plasmid pUC18. AB. ruminicola 23 genomic library was prepared in E. coli by using BamHI-digested DNA, and transformants were screened for xylanase activity on the basis of clearing areas around colonies grown on Remazol brilliant blue R-xylan plates. Six clones were identified as being xylanase positive, and all six contained the same 5.7-kilobase genomic insert. The gene was reduced to a 2.7-kilobase DNA fragment. Xylanase activity produced by the E. coli clone was found to be greater than that produced by the original B. ruminicola strain. Southern hybridization analysis of genomic DNA from the related B. ruminicola strains, D31d and H15a, by using the strain 23 xylanase gene demonstrated one hybridizing band in each DNA.     

Studies on the alkalophilicStreptomyces with extracellular xylanolytic activity

Summary An alkalophilicStreptomyces which produced xylanase, isolated from soil, grew in a temperature range of 15–37°C. The pH optimum for growth was 10 and no growth occurred at pH 7. On a simple wheat bran medium the microorganism exhibited maximum enzyme secretion of 12 U/ml at pH 10. The enzyme had a broad pH optimum of 4.8–10 and the optimum temperature of 50°C. It was completely inactivated at 60°C in 2 h. The enzyme hydrolyzed xylan to a mixture of oligomeric products indicating that the main activity was of the endoxylanase type. The culture filtrate had no cellulase activity.     

Xylanase activity of an endo-cellulase of carboxymethyl-cellulase type from Irpex lacteus (Polyporus tulipiferae).

An endo-cellulase [EC 3.2.1.4.] of carboxymethyl-cellulase type (F-1) which was fractionated from culture filtrate of Irpex lacetus and purified to electrophoretic and ultracentrifugal homogeneity, was found to show xylanase [EC 3.2.1.8.] activity. The activity was not removed from any of the intermediate fractions during the purification of the initial F-I peak, and the radio of xylanase to cellulase activity remained almost unchanged through the purification processes. The xylanase activity of F-I showed not only the same optiomal pH, heat stability, and pH stability as its cellulase activity, but also the same mobility as the cellulase activity upon cellulose acetate film and starch zone electrophoreses. The overall rates of hydrolysis of mixtures of variouis concentrations of CM-cellulose and xylan by F-1 coincided well with those calculated from the Michaelis-Menten treatment of two substances competing for the same active site of the enzyme. These results indicate that the xylanase activity of F-1 is intrinsic to the cellulase itself.     

Cellulose and xylan degrading enzymes of Fusarium avenaceum

It was found that crude preparation obtained from the culture medium of Fusarium avenaceum degraded cellulose and xylan. After chromatography on CM-Sepharose CL-6B of this preparation six fraction were obtained. The eluted fractions II and V showed xylanase activity, fraction IV — cellulase activity and fraction III — xylanase and cellulase activity. The end products of xylan hydrolysis by all xylanase fractions (II, III, V) were xylobiose, xylose, xylotriose and xylotetrose. The end products of cellulose hydrolysis by fractions III and IV was cellobiose, glucose and cellotriose. The data from gel filtration on Sephacryl S-200 indicated a molecular weight of more than 250,000 for both cellulase IV and xylanase V. After gel filtration in the presence of urea disaggregation of those high molecular xylanase and cellulase particles was observed. Xylanase II in difference from the other fractions contained higher amount of sugar. Digestion of fraction II with cellulase-hemicellulase preparation from Phoma hibernica decreased the content of sugar from 17% to 8%, but did not change its enzymatic properties. Cellulase IV as well as xylanase V were inactivated by N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide and tetranitromethane, hence it is suggested that tryptophan and tyrosine are the essential for the activity of these enzymes.     

Studies of two low-molecular-weight endo-1(1→4)-β-d-xylanases constitutively synthesised by the cellulolytic fungus Trichoderma koningii

Two endo-(1→4)-β-d-xylanases (xylanases 1 and 2), which were constitutively synthesised by the fungus Trichoderma koningii, were purified to homogeneity on gel-filtration media and by isoelectric focusing. They had molecular weights of 29,000 (xylanase 1) and 18,000 (xylanase 2), and isoelectric pHs of 7.24 (xylanase 1) and 7.3 (xylanase 2); neither enzyme was associated with carbohydrate. Xylanase 1 had an optimum at the remarkably high temperature of 60–65°. Each enzyme liberated a different range of oligosaccharides from oat-straw arabinoxylan, but only xylanase 1 released l-arabinose and d-xylose. Both xylanases were free from cellulase activity.     

Assembling a novel bifunctional cellulase–xylanase from Thermotoga maritima by end-to-end fusion

An artificial, bifunctional, thermostable cellulase–xylanase enzyme from Thermotoga maritima by gene fusion. The fusion protein exhibited both cellulase and xylanase activity when xynA was fused downstream of cel5C but no activities were shown when xynA was fused upstream of cel5C. The enzyme was optimally active at pH 5.0 and 80°C over 30 min. E. coli expressed the fusion enzyme, with an apparent molecular mass of approximately 152 kDa by carboxymethyl cellulose- and xylan-SDS-PAGE.     

Production, Purification, and Characterization of beta-(1-4)-Endoxylanase of Streptomyces roseiscleroticus

Twelve species of Streptomyces that formerly belonged to the genus Chainia were screened for the production of xylanase and cellulase. One species, Streptomyces roseiscleroticus (Chainia rosea) NRRL B-11019, produced up to 16.2 IU of xylanase per ml in 48 h. A xylanase from S. roseiscleroticus was purified and characterized. The enzyme was a debranching beta-(1-4)-endoxylanase showing high activity on xylan but essentially no activity against acid-swollen (Walseth) cellulose. It had a very low apparent molecular weight of 5,500 by native gel filtration, but its denatured molecular weight was 22,600 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It had an isoelectric point of 9.5. The pH and temperature optima for hydrolysis of arabinoxylan were 6.5 to 7.0 and 60 degrees C, respectively, and more than 75% of the optimum enzyme activity was retained at pH 8.0. The xylanase had a K(m) of 7.9 mg/ml and an apparent V(max) of 305 mumol . min . mg of protein. The hydrolysis rate was linear for xylan concentrations of less than 4 mg/ml, but significant inhibition was observed at xylan concentrations of more than 10 mg/ml. The predominant products of arabinoxylan hydrolysis included arabinose, xylobiose, and xylotriose.     

Optimization for xylanase and cellulase production from Aspergillus niger ATTC 6275 in palm oil mill wastes and its application

Optimization of enzyme production from Aspergillus niger ATCC 6275 under both submerged and solid-substrate cultivation was investigated. Results from submerged cultivation using palm oil mill effluent revealed that pretreatment of ground palm cake did not improve enzyme production. Addition of 0.60g NH4NO3/l generated maximum activity of xylanase and cellulase (CMCase). The optimum aeration rate was 1.2 v/v min. Under solid-substrate cultivation, the results indicated that heating and alkali treatment of the ground palm cake gave no further improvement in enzyme production. The optimal N-source was 2% urea. Optimal initial moisture contents for xylanase and CMCase activities were 60% and 50% respectively, with temperature optima of 30°C and 35°C, respectively. The optimal inoculum size was 1× 108 spores/g palm cake with an initial pH of 4.5–5.0. The maximum activities of xylanase (282.9U/g) and CMCase (23.8U/g) were obtained under the optimum conditions. Solid-substrate cultivation was a better method for the production of enzyme, particularly xylanase, from A. niger ATCC 6275. The application of these enzymes to decanter effluent showed the separation of oil and grease and suspended solids from the effluent. This is comparable to the result achieved from using the commercial xylase preparation Meicelase and superior to the effect of Sumyzyme.     

The construction and characterization of two xylan-degrading chimeric enzymes

Degradation of xylan requires several enzymes. Two chimeric enzymes, xyln-ara and xyln-xylo, were constructed by linking the catalytic portion of a xylanase (xyln) to either an arabinofuranosidase (ara) or a xylosidase (xylo) with a flexible peptide linker. The recombinant parental enzymes and chimeras were produced in E. coli at high levels and purified for characterization of their enzymatic and kinetic properties as well as activities on natural substrates. The chimeras closely resemble the parental enzymes or their mixtures with regard to protein properties. They share similar temperature profiles and have similar catalytic efficiencies as the parental enzymes when assayed using substrates 4-nitrophenyl-alpha-L-arabinofuranoside or 2-nitrophenyl- beta-D-xylopyranoside. The chimeras also show unique enzymatic characteristics. In xylanase activity assays using Remazol Brilliant Blue-xylan, while the parental xylanase has a pH optimum of pH 8, the chimeras showed shifted pH optima as a consequence of significantly increased activity at pH 6 (the optimal pH for ara and xylo). Both chimeras exhibited additive effects of the parental enzymes when assayed at wide ranges of pH and temperatures. The xyln-xylo chimera had the same activities as the xyln/xylo mixture in hydrolyzing the natural substrates oat spelt xylan and wheat arabinoxylan. Compared to the xyln/ara mixture, the xyln-ara chimera released the same amounts of xylose from oat spelt xylan and approximately 30% more from wheat arabinoxylan at pH 6. Our results demonstrate the feasibility and advantages of generating bifunctional enzymes for the improvement of xylan bioconversion.