Hydrolysis of xyilans by a thermostable hybrid xylanase expressed in Escherichia coli

Escherichia coli-expressed a hybrid xylanase, Btx, encoded by a designed hybrid xylanase gene Btx was purified. The molecular mass of the enzyme was estimated to be 22 kDa. The K(m) and k(cat) values for Btx were 1.9 mg/ml and 140 s(-1), respectively. It hydrolyzed xylan principally to xylobiose and xylotriose, and was functionally similar to family 11 xylanases. As some differences were found in the hydrolytic products between birchwood xylan and wheat bran insoluble xylan, the xylan binding domains in xylanase Btx must have different effects on soluble and insoluble xylan.     

Fusion of family 2b carbohydrate-binding module increases the catalytic activity of a xylanase from Thermotoga maritima to soluble xylan

A family 2b carbohydrate-binding module from Streptomyces thermoviolaceus STX-II was fused at the carboxyl-terminus of XynB, a thermostable and single domain family 10 xylanase from Thermotoga maritima, to create a chimeric xylanase. The chimeric enzyme (XynB-CBM2b) was purified and characterized. It displayed a pH-activity profile similar to that of XynB and was stable up to 90 degrees C. XynB-CBM2b bound to insoluble birchwood and oatspelt xylan. Whereas its hydrolytic activities toward insoluble xylan and p-nitrophenyl-beta-xylopyranoside were similar to those of XynB, its activity toward soluble xylan was moderately higher than that of XynB.     

Hydrolysis of xylans by a thermostable hybrid xylanase expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Escherichia coli-expressed a hybrid xylanase, Btx, encoded by a designed hybrid xylanase gene btx was purified. The molecular mass of the enzyme was estimated to be 22 kDa. The K _m and k _cat values for Btx were 1.9 mg/ml and 140 s⁻¹, respectively. It hydrolyzed xylan principally to xylobiose and xylotriose, and was functionally similar to family 11 xylanases. As some differences were found in the hydrolytic products between birchwood xylan and wheat bran insoluble xylan, the xylan binding domains in xylanase Btx must have different effects on soluble and insoluble xylan.     

Purification and characterization of a new xylanase from Acrophialophora nainiana

A new xylanase activity (XynII) was isolated from liquid state cultures of Acrophialophora nainiana containing birchwood xylan as carbon source. XynII was purified to apparent homogeneity by gel filtration and ion exchange chromatographies. The enzyme was optimally active at 55 degrees C and pH 7.0. XynII had molecular mass of 22630+/-3.0 and 22165 Da, as determined by mass spectrometry and SDS-PAGE, respectively. The purified enzyme was able to act only on xylan as substrate. The apparent K(m) values on soluble and insoluble birchwood xylans were 40.9 and 16.1 mg ml(-1), respectively. The enzyme showed good thermal stability with half lives of 44 h at 55 degrees C and ca. 1 h at 60 degrees C The N-terminal sequence of XynII showed homology with a xylanase grouped in family G/11. The enzyme did not show amino acid composition similarity with xylanases from some fungi and Bacillus amyloliquefaciens.     

The recombinant xylanase B of Thermotoga maritima is highly xylan specific and produces exclusively xylobiose from xylans, a unique character for industrial applications

The xynB of a hyperthermophilic Eubacterium, Thermotoga maritima MSB8, coding xylanase B (XynB) was previously expressed in E. coli and the recombinant protein was characterized using the synthetic substrates [J. Biosci. Bioeng. 92 (2001) 423]. In this study, the same xylanase B was purified to homogeneity with a recovery yield of about 43% using heat treatment followed by the Ni-NTA affinity chromatography. The specificity of XynB towards different natural substrates was evaluated. XynB was highly specific towards xylans tested but exhibited low activities towards lichenan (19%), gellan gum (7.3%), laminarin (3.4%) and carboxymethylcellulose (CMC, 1.4%). The apparent K_m values of birchwood xylan and soluble oat-spelt xylan was 0.11 and 0.079 mg/ml, respectively. The XynB hydrolyzed xylooligosaccharides to yield predominantly xylobiose (X₂) and a small amount of xylose (X₁), suggesting that XynB was possibly an endo-acting xylanase. Analysis of the products from birchwood xylan degradation confirmed that the enzyme was an endo-xylanase with xylobiose and xylose as the main degradation products. HPLC results showed that hydrolyzed products of birchwood xylan by XynB yielded up to 66% of the total reaction product as xylobiose. These results clearly indicated that xylobiose could be mass-produced efficiently by the recombinant hyperthermostable XynB of T. maritima. Additionally, conversion of xylobiose (50 mM) to xylose was observed, while xylotriose (X₃) and xylotetraose (X₄) were detected in small amounts, indicating that the enzyme converted xylobiose to xylose based on the transglycosylation reaction. The increased binding ability of XynB to Avicel and/or insoluble xylan was also observed indicating the possibilities of roles of surface-aromatic amino acid residues for such action. However, further investigations are required to prove this speculation.     

Degradation and utilization by Butyrivibrio fibrisolvens H17c of xylans with different chemical and physical properties.

Hemicelluloses, mainly xylans, can be a major component of diets consumed by ruminants and undergo various degrees of microbial digestion in the rumen. The ability of Butyrivibrio fibrisolvens, a major xylanolytic ruminal species, to degrade and utilize nine chemically and physically different xylans for growth was examined. The arabinoxylans used included two isolated from corncobs (CCX-A and CCX-B), a native xylan excreted by corn cell tissue cultures (CX), an oxalic acid-treated, arabinose-depleted CX, and oat spelt xylan. Except for CCX-A, these xylans were extensively converted within 3 h of growth to acid-alcohol-soluble forms that remained at high levels for the duration of culture growth. These xylans contain mainly xylose and arabinose with small amounts of uronic acids. For a given xylan, all three components were used at about the same rate and extent. During the early stages of growth B. fibrisolvens also rapidly solubilized glucuronoxylans from birchwood, larchwood, 4-O-methylglucuronoxylan, and the xylose homopolymer xylan isolated from beechwood (BEWX). In contrast to the findings for the arabinoxylans, little acid-alcohol-soluble carbohydrate remained in these cultures after 9 h of growth, except for BEWX. Initially, with birchwood, larchwood, and 4-O-methylglucuronoxylan the uronic acid components were preferentially used over the xylose. Final xylan utilization measured at 72 h for all xylans varied from 57% for CCX-A to 92% for BEWX and was correlated with the initial 12-h utilization rate for a given xylan. Since CCX-A and BEWX are both highly water insoluble, this aspect did not appear to influence overall utilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

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     

The cellulolytic and hemi-cellulolytic system of Bacillus licheniformis SVD1 and the evidence for production of a large multi-enzyme complex

The cellulolytic and hemi-cellulolytic system of Bacillus licheniformis SVD1 was isolated and characterised in birchwood xylan cultures. The predominant activity in the crude culture was xylanase activity, but the crude culture also displayed Avicelase, carboxymethylcellulase (CMCase), mannanase, and pectinase activity. Most of the xylanase activity was found in the culture supernatant, but some activity was cell-associated. Using Sepharose 4B size exclusion chromatography, a 2000 kDa multi-enzyme complex (MEC) was purified. The MEC contained predominantly xylanase activity, as well as significant levels of mannanase and CMCase activity, but no Avicelase activity. SDS-PAGE revealed up to eight visible bands in the MEC while zymograms of the MEC displayed two xylanase active bands at 21 kDa and 45 kDa, and two CMCase active bands at 25 kDa and 30 kDa. More active bands were visible in the crude supernatant with an additional xylanase active band at 40 kDa and an additional CMCase active band at 55 kDa. Using thin layer chromatography (TLC), it was established that the crude fraction could release xylose from insoluble birchwood xylan, while the MEC was only able to produce xylobiose from this substrate. The MEC was further able to bind to insoluble xylan, but was unable to bind to crystalline cellulose. This MEC lacks many of the characteristic features of a cellulosome and is most likely a different type of complex. The presence of both high xylanase and mannanase activity makes this MEC unusual.     

Purification and Characterisation of Xylanolytic Enzymes of a Cellulase-free Thermophilic strain of Clostridium absonum CFR-702

Two endo-β-1-4-xylanases (EC 3.2.1.8), xylanase-I and xylanase-II, were purified fromClostridium absonum CFR-702 by ammonium sulphate precipitation and chromatographed on DEAE-Cellulose and phenyl-Sepharose. The enzymes in sodium dodecyl sulphate polyacrylamide gels resolved as proteins corresponding to molecular mass 150 and 95 kDa for xylanase-I and xylanase-II, respectively. The optimum pH and temperature ranges for the enzyme activities on birchwood xylan were between 6.5 and 7.5 and 75°C for xyl-I and 7.5 and 80°C for xyl-II. Xyl-I was stable up to 60°C whereas xyl-II was stable at 50°C. Both the enzymes liberated xylobiose, xylotriose and xylotetraose from birchwood xylan. Xyl-I and xyl-II with birchwood xylan had K_mvalues of 1.1 and 1.4%, and V_maxvalues of 454.54 and 363.63 μmol/min/mg protein respectively.     

Comparative characterization of deletion derivatives of the modular xylanase XynA of Thermotoga maritima

The modular Xylanase XynA from Thermotoga maritima consists of five domains (A1-A2-B-C1-C2). Two similar N-terminal domains (A1-A2-) are family 22 carbohydrate-binding modules (CBMs), followed by the catalytic domain (-B-) belonging to glycoside hydrolase family 10, and the C-terminal domains (-C1-C2), which are members of family 9 of CBMs. The gradual deletion of the non-catalytic domains resulted in deletion derivatives (XynAΔC; XynAΔA1C and XynAΔNC) with increased maximum activities (V _max) at 75°C, pH 6.2. Furthermore, these deletions led to a shift of the optimal NaCl concentration for xylan hydrolysis from 0.25 (XynA) to 0.5 M (XynAΔNC). In the presence of the family 22 CBMs, the catalytic domain retained more activity in the acidic range of the pH spectrum than without these domains. In addition to the deletion derivatives of XynA, the N-terminal domains A1 and A2 were produced recombinantly, purified, and investigated in binding studies. For soluble xylan preparations, linear β-1,4-glucans and mixed-linkage β-1,3-1,4-glucans, only the A2 domain mediated binding, not the A1 domain, in accordance with previous observations. The XynA deletion enzymes lacking the C domains displayed low affinity also to hydroxyethylcellulose and carboxymethylcellulose. With insoluble oat spelt xylan and birchwood xylan as the binding substrates, the highest affinity was observed with XynAΔC and the lowest affinity with XynAΔNC. Although the domain A1 did not bind to soluble xylan preparations, the insoluble oat spelt xylan-binding data suggest that this domain does play a role in substrate binding in that it improves the binding to insoluble xylans.     

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.     

Characterization and pH-dependent substrate specificity of alkalophilic xylanase from Bacillus alcalophilus

The gene of endo-beta-1-4 xylanase, xynT, was cloned from Bacillus alcalophilus AX2000 and expressed in Escherichia coli. This XynT, which belongs to glycoside hydrolase (GH) family 10, was found to have a molecular weight of approximately 37?kDa and exhibit optimal activity at pH 7-9 and 50?°C. It exhibits a high activity towards birchwood xylan and has the ability to bind avicel. Under optimal conditions, XynT hydrolyzes all xylooligomers into xylobiose as an end product with a preference for cleavage sites at the second or third glycosidic bond from the reducing end. XynT has a different substrate affinity on xylooligomers at pH 5.0, which contributes to its low activity toward xylotriose and its derived intermediate products. This low activity may be due to an unstable interaction with the amino acids that constitute subsites of the active site. Interestingly, the addition of Co(2+) and Mn(2+) led to a significant increase in activity by up to 40 and 50?%, respectively. XynT possesses a high binding affinity and hydrolytic activity toward the insoluble xylan, for which it exhibits high activity at pH 7-9, giving rise to its efficient biobleaching effect on Pinus densiflora kraft pulp.     

Characterization of a novel, ultra-large xylanolytic complex (xylanosome) from Streptomyces olivaceoviridis E-86

A novel, ultra-large xylanolytic complex (xylanosome) from Streptomyces olivaceoviridis E-86 was purified to homogeneity by ammonium sulfate precipitation and Sephacryl S-300 gel filtration chromatography. The purified xylanosome appeared as a single protein band on the non-denaturing (native) polyacrylamide gel electrophoresis (PAGE) gel with a molecular mass of approximately 1200 kDa. The optimal temperature and pH for xylanase activity was 60 °C and pH 6.0, respectively. The xylanase activity was stable within pH 4.1–10.3. It was stable up to 60 °C at pH 6.0. The xylanosome was highly specific towards oat-spelt xylan, and showed low activity towards corncob powder, but exhibited very low activity towards lichenan, CMC and p-nitrophenyl derivatives. Apparent K_m values of the xylansosome for birchwood, beechwood, soluble oat-spelt and insoluble oat-spelt xylans were 2.5, 3.6, 1.7 and 4.9 mg ml⁻¹, respectively. The main hydrolysis products of birchwood xylan were xylotriose, xylobiose and xylose. Analysis of the products from wheat arabinoxylan degradation by xylanosome confirmed that the enzyme had endoxylanase and debranching activities, with xylotriose, xylobiose, xylose and arabinose as the main degradation products. These unique properties of the purified xylanosome from Streptomyces olivaceoviridis E-86 make this enzymatic complex attractive for biotechnological applications.     

Expression of recombinant Aspergillus niger xylanase A in Pichia pastoris and its action on xylan

The mature peptide of Aspergillus niger xylanase A (AnxA) was successfully expressed in Pichia pastoris at high levels under the control of AOX1 promoter. The recombinant AnxA (reAnxA) was secreted into culture medium. After 96-h 0.25% methanol induction, the activity of reAnxA in the culture supernatant reached the peak, 175 U/mg, which was 1.9 times as high as that of the native AnxA (92 U/mg). Studies on enzymatic properties showed that the optimum temperature and optimum pH of reAnxA were 50 degrees C and 5.0, respectively. The reAnxA was very stable in a wide pH range of 3.0-8.0. After incubation at the pH 3.0-8.0, 25 degrees C for 1h, all the residual activities of reAnxA were over 80%. The K(m) and k(cat) values for reAnxA were 4.8 mg/ml and 123.2s(-1), respectively. HPLC analysis showed that xylotriose was the main hydrolysis product of birchwood xylan and bran insoluble xylan by reAnxA.     

Kinetic characterization of a glycoside hydrolase family 44 xyloglucanase/endoglucanase from Ruminococcus flavefaciens FD-1

Two forms of Ruminococcus flavefaciens FD-1 endoglucanase B, a member of glycoside hydrolase family 44, one with only a catalytic domain and the other with a catalytic domain and a carbohydrate binding domain (CBM), were produced. Both forms hydrolyzed cellotetraose, cellopentaose, cellohexaose, carboxymethylcellulose (CMC), birchwood and larchwood xylan, xyloglucan, lichenan, and Avicel but not cellobiose, cellotriose, mannan, or pullulan. Addition of the CBM increased catalytic efficiencies on both CMC and birchwood xylan but not on xyloglucan, and it decreased rates of cellopentaose and cellohexaose hydrolysis. Catalytic efficiencies were much higher on xyloglucan than on other polysaccharides. Hydrolysis rates increased with increasing cellooligosaccharide chain length. Cellotetraose hydrolysis yielded only cellotriose and glucose. Hydrolysis of cellopentaose gave large amounts of cellotetraose and glucose, somewhat more of the former than of the latter, and much smaller amounts of cellobiose and cellotriose. Cellohexaose hydrolysis yielded much more cellotetraose than cellobiose and small amounts of glucose and cellotriose, along with a low and transient amount of cellopentaose.     

Characterization of Xylan Utilization and Discovery of a New Endoxylanase in Thermoanaerobacterium saccharolyticum through Targeted Gene Deletions

The economical production of fuels and commodity chemicals from lignocellulose requires the utilization of both the cellulose and hemicellulose fractions. Xylanase enzymes allow greater utilization of hemicellulose while also increasing cellulose hydrolysis. Recent metabolic engineering efforts have resulted in a strain of Thermoanaerobacterium saccharolyticum that can convert C₅ and C₆ sugars, as well as insoluble xylan, into ethanol at high yield. To better understand the process of xylan solubilization in this organism, a series of targeted deletions were constructed in the homoethanologenic T. saccharolyticum strain M0355 to characterize xylan hydrolysis and xylose utilization in this organism. While the deletion of β-xylosidase xylD slowed the growth of T. saccharolyticum on birchwood xylan and led to an accumulation of short-chain xylo-oligomers, no other single deletion, including the deletion of the previously characterized endoxylanase XynA, had a phenotype distinct from that of the wild type. This result indicates a multiplicity of xylanase enzymes which facilitate xylan degradation in T. saccharolyticum. Growth on xylan was prevented only when a previously uncharacterized endoxylanase encoded by xynC was also deleted in conjunction with xynA. Sequence analysis of xynC indicates that this enzyme, a low-molecular-weight endoxylanase with homology to glycoside hydrolase family 11 enzymes, is secreted yet untethered to the cell wall. Together, these observations expand our understanding of the enzymatic basis of xylan hydrolysis by T. saccharolyticum.     

An acetylxylan esterase and a xylanase expressed from genes cloned from the ruminal fungus Neocallimastix patriciarum act synergistically to degrade acetylated xylans

A Neocallimastix patriciarum acetylxylan esterase (BnaA) was expressed from the cloned gene in Escherichia coli. Purified recombinant BnaA efficiently released acetate from soluble acetylated birchwood xylan (ABX), with a specific activity of 76 U mg⁻¹. In contrast, release of acetate was very inefficient from the insoluble substrates, spear grass and delignified spear grass. Addition of a recombinant xylanase, XynA, also expressed from a cloned N. patriciarum gene, had no effect on the release of acetate from ABX. However, the combination of recombinant BnaA and XynA released more acetate from spear grass and delignified spear grass than did BnaA alone. Significantly more reducing sugar was also released from all three substrates by the combination of recombinant XynA and BnaA than by XynA alone. Thus the extent of digestion of acetylated xylans by XynA appears to be limited by the acetylation. In this system BnaA does not appear to increase the rate of cleavage of insoluble substrates by XynA, but probably allows the release of shorter xylose oligomers from already solubilised acetylated xylan polymers. Received: 11 January 1999 / Accepted: 28 February 1999     

Expression of recombinant Bacillus licheniformis xylanase A in Pichia pastoris and xylooligosaccharides released from xylans by it

The mature peptide of Bacillus licheniformis xylanase A (BlxA) was successfully expressed in Pichia pastoris under the control of AOX1 promoter. After 96-h 0.25% methanol induction, the activity of recombinant B. licheniformis xylanase A (reBlxA) in culture supernatant was 122.9 U/mg. Enzymatic properties assays showed that the optimum temperature and pH for reBlxA were 60 degrees C and pH 6.0, respectively. When treated at 70 degrees C, pH 6.0 for 2 min, the residual activities of the reBlxA were 76%. Over 80% of reBlxA activity was retained after treatment of the enzyme by preincubation over a pH range of 5.0-9.0 for 1h at 25 degrees C. High performance liquid chromatography (HPLC) analysis revealed that xylotriose (X3) was the main hydrolysis product released from birchwood xylan and wheat bran insoluble xylan by reBlxA. The mode of action studies showed that reBlxA was an endo-acting xylanase and xylobiose (X2), xylotriose, xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6) could be hydrolyzed by it. This is the first report on the expression of reBlxA in yeast and on determining and quantifying the hydrolysis products released from xylans by reBlxA.