Production of xylanase by the ruminal anaerobic fungus Neocallimastix frontalis

Xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) production was investigated in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released principally into the culture fluid and had pH and temperature optima of 5.5 and 55 degrees C, respectively. In the presence of low concentrations of substrate, the enzyme was stabilized at 50 degrees C. Xylobiose was the principal product of xylanase action, with lesser amounts of longer-chained xylooligosaccharides. No xylose was detected, indicating that xylobiase activity was absent. Activities of xylanase up to 27 U ml-1 (1 U represents 1 micromol of xylose equivalents released min-1) were obtained for cultures grown on xylan (from oat spelt) at 2.5 mg ml-1 in shaken cultures. No growth occurred in unshaken cultures. Xylanase production declined with elevated concentrations of xylan (less than 2.5 mg ml-1), and this was accompanied by an accumulation of xylose and, to a lesser extent, arabinose. Addition of either pentose to cultures grown on low levels of xylan in which neither sugar accumulated suppressed xylanase production, and in growth studies with the paired substrates xylan-xylose, active production of the enzyme occurred during growth on xylan only after xylose had been preferentially utilized. When cellobiose, glucose, and xylose were tested as growth substrates for the production of xylanase (each initially at 2.5 mg ml-1), they were found to be less effective than xylan, and use of xylan from different origins (birch wood or larch wood) as the growth substrate or in the assay system resulted in only marginal differences in enzyme activity. However, elevated production of xylanase occurred during growth on crude hemicellulose (barley straw leaf). The results are discussed in relation to the role of the anaerobic fungi in the ruminal ecosystem, and the possible application of the enzyme in bioconversion processes is also considered.     

Production of xylanase by the ruminal anaerobic fungus Neocallimastix frontalis.

Xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) production was investigated in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released principally into the culture fluid and had pH and temperature optima of 5.5 and 55 degrees C, respectively. In the presence of low concentrations of substrate, the enzyme was stabilized at 50 degrees C. Xylobiose was the principal product of xylanase action, with lesser amounts of longer-chained xylooligosaccharides. No xylose was detected, indicating that xylobiase activity was absent. Activities of xylanase up to 27 U ml-1 (1 U represents 1 micromol of xylose equivalents released min-1) were obtained for cultures grown on xylan (from oat spelt) at 2.5 mg ml-1 in shaken cultures. No growth occurred in unshaken cultures. Xylanase production declined with elevated concentrations of xylan (less than 2.5 mg ml-1), and this was accompanied by an accumulation of xylose and, to a lesser extent, arabinose. Addition of either pentose to cultures grown on low levels of xylan in which neither sugar accumulated suppressed xylanase production, and in growth studies with the paired substrates xylan-xylose, active production of the enzyme occurred during growth on xylan only after xylose had been preferentially utilized. When cellobiose, glucose, and xylose were tested as growth substrates for the production of xylanase (each initially at 2.5 mg ml-1), they were found to be less effective than xylan, and use of xylan from different origins (birch wood or larch wood) as the growth substrate or in the assay system resulted in only marginal differences in enzyme activity. However, elevated production of xylanase occurred during growth on crude hemicellulose (barley straw leaf). The results are discussed in relation to the role of the anaerobic fungi in the ruminal ecosystem, and the possible application of the enzyme in bioconversion processes is also considered.     

Characterization of a bacterial xylanase resistant to repression by glucose and xylose

Summary Bacillus subtilis CD4, when grown in nutrient broth or minimal medium in presence of xylan, produced extracellular xylanase that hydrolyzed xylan optimally at pH 5. The enzyme was induced by xylan, xylose and glucose. Addition of xylose or glucose in xylan containing medium did not affect enzyme production. The structural gene encoding xylanase was cloned and expressed in E. coli. The recombinant enzyme exhibited similar properties like that of native enzyme including resistance to repression by xylose and glucose.     

The regulation of xylanolytic enzyme formation by Butyrivibrio fibrisolvens NCFB 2249

Butyrivibrio fibrisolvens NCFB 2249 formed xylan-degrading enzymes on a wide range of carbohydrate growth substrates. The specific activities of α-L-arabinofuranosidase and β-D-xylosidase were increased (up 20-fold) after growth on xylan or xylose-containing saccharides. Xylose was not an effective substrate for xylanase production although its formation was induced on xylobiose and higher DP xylose-containing saccharides. Acetyl esterase activity was also highest after growth on xylan. The synthesis of xylanase and β-xylosidase was repressed by glucose and hemicellulosic pentoses and although α-L-arabinofuranosidase formation was also subject to catabolite regulation, xylose did not repress its synthesis.     

Inducible character of β‐xylanase in a hyperproducing mutant of Thermomyces lanuginosus

Ten strains of Thermomyces lanuginosus from various culture collections were evaluated for extracellular endo‐β‐1,4‐xylanase production. The best xylanase producer (5771±173 nkat/mL) T. lanuginosus SK, was subjected to UV and N‐methyl‐N‐nitro‐N‐nitrosoguanidine mutagenesis. A mutant strain T. lanuginosus MC134, that showed on oatspelts xylan a 1.5 fold higher xylanase production than the parent strain SK, was subjected to a study of the regulation of xylanase synthesis during growth on various carbohydrates and during induction in glucose‐grown cells. In the growth experiments the highest production of xylanase was observed in the presence of xylans, however, an appreciable amount of the enzyme, about 10%, was also produced during growth on xylose. Xylobiose was found to be the most efficient xylanase inducer in the glucose‐grown cells. Its induction efficiency was followed by xylose, beechwood and birchwood xylan. Xylanase induction by polysaccharides started several hours later but proceeded for a longer time than that induced by the low molecular mass inducers, indicating that the polysaccharides serve as more sustainable source of inducers and that they have to be first hydrolyzed by the low level of constitutively synthesized xylanase. The repression of the induction of xylanase by glucose confirmed that the xylanase synthesis in the mutant strain is similar to the parent strain and exhibits an induction‐repression regulation mechanism.     

Relationships between activities of xylanases and xylan structures

Structures of five water-soluble xylans have been determined. Four purified xylanase enzymes have been studied for the hydrolysis of the xylans. Different xylanases have different activities against various xylan structures. The key factors that influence the rate of xylan hydrolysis are chain length and degree of substitution. Two family 11 xylanases, Orpinomyces pc2 xylanase and Trichoderma longibrachiatum xylanase, can rapidly hydrolyze xylans that have a chain length greater than 8 xylose residues, and their hydrolytic rates are not sensitive to substituents on the xylan backbone. A family 11 xylanase from Aureobasidium pullulans is most effective on xylans that have a long chain (greater than 19 xylose residues), and also is effective against substituent groups. Although Thermatoga maritima xylanase is also more active on a long xylan chain (greater than 19 xylose residues), its hydrolytic rate is greatly reduced by substituents on xylan backbones.     

Xylanase ofChaetomium cellulolyticum: Its nature of production and hydrolytic potential

Summary Maximum xylanase production byChaetomium cellulolyticum was obtained in the culture supernatant after 30 h of growth at 37°C in basal medium containing 1% xylan at pH maintained between 6.5 and 7.5. Addition of 0.05% Tween 80 to the medium increased the enzyme production considerably. Xylanase production was found to be growth associated. The optimal conditions for enzymatic hydrolysis of xylan were found to be pH 6.0 and 50°C. During enzymatic hydrolysis, xylose, xylobiose and other xylooligosaccharides were liberated from xylan. The pH values for xylanase production and for xylan hydrolysis were closely related to the utilization of hemicelluloses of aspen wood for fungal protein production by this organism as reported in our earlier work.     

Saccharification of xylan by an amyloglucosidase of Termitomyces clypeatus and synergism in the presence of xylanase

Summary An amyloglucosidase from a mycelial culture of the mushroom Termitomyces clypeatus hydrolysed larch wood xylan independently and synergistically with an endo-(14) xylanase of the same fungus. The glucoamylase saccharified xylan predigested with xylanase at a faster rate compared to that of xylanase acting on amylase-digested xylan. However, overall saccharification of xylan in both cases was the same. Only glucose was liberated from xylan by amylase digestion whereas xylose, xylobiose and other oligosaccharides were liberated during xylanase digestion. The synergistic response of enzyme combinations was reflected in the liberation of glucose from xylan, rather than xylose. Glucoamylase and xylanase activities on soluble and insoluble fractions of larch wood xylan with different xylose and glucose contents suggested that synergism in xylanolysis by the presence of glucoamylase was dependent on the activity of the participating xylanase on the xylan preparation. It is suggested that possibly -glucosidic linkages are present in xylan and that amyloglucosidase might be involved in xylanolysis. Correspondence to: S. Sengupta     

Induction of cellulase and hemicellulase activities of Thermoascus aurantiacus by xylan hydrolyzed products

Thermoascus aurantiacus is able to secrete most of the hemicellulolytic and cellulolytic enzymes. To establish the xylanase inducers of T. aurantiacus, the mycelia were first grown on glucose up until the end of the exponential growth phase, followed by washing and re-suspension in a basal medium without a carbon source. Pre-weighed amounts of xylose (final concentration of 3.5 mg/ml), xylobiose (7 mg/ml) and hydrolyzed xylan from sugarcane bagasse (HXSB) which contained xylose, xylobiose and xylotriose (6.8 mg/ml) were evaluated as inducers of xylanase. It was observed that xylose did not suppress enzyme induction of T. aurantiacus when used in low concentrations, regardless of whether it was inoculated with xylobiose. Xylobiose promoted fast enzyme production stopping after 10 h, even at a low consumption rate of the carbon source; therefore xylobiose appears to be the natural inducer of xylanase. In HXSB only a negligible xylanase activity was determined. Xylose present in HXSB was consumed within the first 10 h while xylobiose was partially hydrolyzed at a slow rate. The profile of α-arabinofuranosidase induction was very similar in media induced with xylobiose or HXSB, but induction with xylose showed some positive effects as well. The production profile for the xylanase was accompanied by low levels of cellulolytic activity. In comparison, growth in HXSB resulted in different profiles of both xylanase and cellulase production, excluding the possibility of xylanase acting as endoglucanases.     

Purification and characterization of two xylanases from Trichoderma longibrachiatum.

Two endoxylanases were purified from the culture medium of Trichoderma longibrachiatum. Both enzymes were highly basic, and lacked activity on carboxymethyl-cellulose. An enzyme of 21.5 kDa (xylanase A) had a specific activity of 510 U/mg protein, a Km of 0.15 mg soluble xylan/ml, possessed transglycosidase activity and generated xylobiose and xylotriose as the major endproducts from xylan or xylose oligomers. A larger enzyme of 33 kDa (xylanase B) had a specific activity of 131 U/mg protein, a Km of 0.19 mg soluble xylan/ml, lacked detectable transglycosidase activity and generated xylobiose and xylose as major endproducts from xylan and xylose oligomers. Xylotriose was the smallest oligomer attacked by both enzymes. In addition, xylotriose inhibited hydrolysis of xylopentanose by both enzymes, while xylobiose appeared to inhibit xylanase B, but not xylanase A.     

细菌木聚糖酶高产菌的选育及产酶条件

木聚糖是一种在植物体内大量存在的半纤维素,是在自然界中含量仅次于纤维素的一种可再生植物纤维。木聚糖酶(ｘｙｌａｎａｓｅ,ＥＣ3.2.1.8)是一类能够特异降解木聚糖的酶类。近年来,人们将其广泛用于造纸工业的纸浆生物处理,与其他消化酶类一起用作饲料添加剂,以及应用于食品加工工业和纺织工业等。木聚糖酶可以由许多种微生物产生[1],我国多集中于霉菌木聚糖酶的研究。本文报告了一株细菌木聚糖酶产生菌的筛选及产酶条件的研究结果。1　材料和方法11　菌株本实验室分离、保存的木聚糖酶产生菌ＷＸＵＬＩ11及其突变株ＷＬＵＮ024。12　培养…     

Conversion of xylan to ethanol by ethanologenic strains of Escherichia coli and Klebsiella oxytoca.

A two-stage process was evaluated for the fermentation of polymeric feedstocks to ethanol by a single, genetically engineered microorganism. The truncated xylanase gene (xynZ) from the thermophilic bacterium Clostridium thermocellum was fused with the N terminus of lacZ to eliminate secretory signals. This hybrid gene was expressed at high levels in ethanologenic strains of Escherichia coli KO11 and Klebsiella oxytoca M5A1(pLOI555). Large amounts of xylanase (25 to 93 mU/mg of cell protein) accumulated as intracellular products during ethanol production. Cells containing xylanase were harvested at the end of fermentation and added to a xylan solution at 60 degrees C, thereby releasing xylanase for saccharification. After cooling, the hydrolysate was fermented to ethanol with the same organism (30 degrees C), thereby replenishing the supply of xylanase for a subsequent saccharification. Recombinant E. coli metabolized only xylose, while recombinant K. oxytoca M5A1 metabolized xylose, xylobiose, and xylotriose but not xylotetrose. Derivatives of this latter organism produced large amounts of intracellular xylosidase, and the organism is presumed to transport both xylobiose and xylotriose for intracellular hydrolysis. By using recombinant M5A1, approximately 34% of the maximal theoretical yield of ethanol was obtained from xylan by this two-stage process. The yield appeared to be limited by the digestibility of commercial xylan rather than by a lack of sufficient xylanase or by ethanol toxicity. In general form, this two-stage process, which uses a single, genetically engineered microorganism, should be applicable for the production of useful chemicals from a wide range of biomass polymers.     

Conversion of xylan to ethanol by ethanologenic strains of Escherichia coli and Klebsiella oxytoca.

A two-stage process was evaluated for the fermentation of polymeric feedstocks to ethanol by a single, genetically engineered microorganism. The truncated xylanase gene (xynZ) from the thermophilic bacterium Clostridium thermocellum was fused with the N terminus of lacZ to eliminate secretory signals. This hybrid gene was expressed at high levels in ethanologenic strains of Escherichia coli KO11 and Klebsiella oxytoca M5A1(pLOI555). Large amounts of xylanase (25 to 93 mU/mg of cell protein) accumulated as intracellular products during ethanol production. Cells containing xylanase were harvested at the end of fermentation and added to a xylan solution at 60 degrees C, thereby releasing xylanase for saccharification. After cooling, the hydrolysate was fermented to ethanol with the same organism (30 degrees C), thereby replenishing the supply of xylanase for a subsequent saccharification. Recombinant E. coli metabolized only xylose, while recombinant K. oxytoca M5A1 metabolized xylose, xylobiose, and xylotriose but not xylotetrose. Derivatives of this latter organism produced large amounts of intracellular xylosidase, and the organism is presumed to transport both xylobiose and xylotriose for intracellular hydrolysis. By using recombinant M5A1, approximately 34% of the maximal theoretical yield of ethanol was obtained from xylan by this two-stage process. The yield appeared to be limited by the digestibility of commercial xylan rather than by a lack of sufficient xylanase or by ethanol toxicity. In general form, this two-stage process, which uses a single, genetically engineered microorganism, should be applicable for the production of useful chemicals from a wide range of biomass polymers.     

Xylanase production by a new alkali-tolerant isolate of Bacillus

The xylanolytic system of an alkali-tolerant Bacillus sp. consists of several xylanases ranging from 22 to 120 kDa and pI values from 7.0 to 9.0. Crude xylanase retained 72% of initial activity after 5 h at pH 9.0 and 45°C. Xylanase production was induced by xylose and xylan and was maximum at 42°C and pH 7.8. Crude xylanase released xylotriose and xylotetraose as main products of xylan hydrolysis. Xylose was not detected. © Rapid Science Ltd. 1998     

Genetic analysis of a locus on the Bacteroides ovatus chromosome which contains xylan utilization genes.

Bacteroides ovatus, a gram-negative obligate anaerobe found in the human colon, can utilize xylan as a sole source of carbohydrate. Previously, a 3.8-kbp segment of B. ovatus chromosomal DNA, which contained genes encoding a xylanase (xylI) and a bifunctional xylosidase-arabinosidase (xsa), was cloned, and expression of the two genes was studied in Escherichia coli (T. Whitehead and R. Hespell, J. Bacteriol. 172:2408-2412, 1990). In the present study, we have used segments of the cloned region to construct insertional disruptions in the B. ovatus chromosomal locus containing these two genes. Analysis of these insertional mutants demonstrated that (i) xylI and xsa are probably part of the same operon, with xylI upstream of xsa, (ii) the true B. ovatus promoter was not cloned on the 3.5-kbp DNA fragment which expressed xylanase and xylosidase in E. coli, (iii) there is at least one gene upstream of xylI which could encode an arabinosidase, and (iv) xylosidase rather than xylanase may be a rate-limiting step in xylan utilization. Insertional mutations in the xylI-xsa locus reduced the rate of growth on xylan, but the concentration of residual sugars at the end of growth was the same as that with the wild type. Thus, a slower rate of growth on xylan was not accompanied by less extensive digestion of xylan. Mutants in which xylI had been disrupted still expressed some xylanase activity. This second activity was associated with membranes and produced xylose from xylan, whereas the xylI gene product partitioned primarily with the soluble fraction and produced xylobiose from xylan.     

Xylanases of thermophilic bacteria from Icelandic hot springs

Thermophilic, aerobic bacteria isolated from Icelandic hot springs were screened for xylanase activity. Of 97 strains tested, 14 were found to be xylanase positive. Xylanase activities up to 12 nkat/ml were produced by these strains in shake flasks on xylan medium. The xylanases of the two strains producing the highest activities (ITI 36 and ITI 283) were similar with respect to temperature and pH optima (80°C and pH 8.0). Xylanase production of strain ITI 36 was found to be induced by xylan and xylose. Xylanase activity of 24 nkat/ml was obtained with this strain in a laboratory-scale-fermentor cultivation on xylose medium. -Xylosidase activity was also detected in the culture filtrate. The thermal half-life of ITI 36 xylanase was 24 h at 70°C. The highest production of sugars from hydrolysis of beech xylan was obtained at 70°C, although xylan depolymerization was detected even up to 90°C. Correspondence to: M. Rättö     

Genetic analysis of a locus on the Bacteroides ovatus chromosome which contains xylan utilization genes.

Bacteroides ovatus, a gram-negative obligate anaerobe found in the human colon, can utilize xylan as a sole source of carbohydrate. Previously, a 3.8-kbp segment of B. ovatus chromosomal DNA, which contained genes encoding a xylanase (xylI) and a bifunctional xylosidase-arabinosidase (xsa), was cloned, and expression of the two genes was studied in Escherichia coli (T. Whitehead and R. Hespell, J. Bacteriol. 172:2408-2412, 1990). In the present study, we have used segments of the cloned region to construct insertional disruptions in the B. ovatus chromosomal locus containing these two genes. Analysis of these insertional mutants demonstrated that (i) xylI and xsa are probably part of the same operon, with xylI upstream of xsa, (ii) the true B. ovatus promoter was not cloned on the 3.5-kbp DNA fragment which expressed xylanase and xylosidase in E. coli, (iii) there is at least one gene upstream of xylI which could encode an arabinosidase, and (iv) xylosidase rather than xylanase may be a rate-limiting step in xylan utilization. Insertional mutations in the xylI-xsa locus reduced the rate of growth on xylan, but the concentration of residual sugars at the end of growth was the same as that with the wild type. Thus, a slower rate of growth on xylan was not accompanied by less extensive digestion of xylan. Mutants in which xylI had been disrupted still expressed some xylanase activity. This second activity was associated with membranes and produced xylose from xylan, whereas the xylI gene product partitioned primarily with the soluble fraction and produced xylobiose from xylan.     

The effect of carbohydrates on the expression of the Prevotella ruminicola 1,4-β-D-endoglucanase ☆

The β-1,4-endoglucanase of the ruminai bacterium, Prevotella ruminicola B₁4, hydrolysed carboxymethylcellulose and barley glucan but not xylan or mannan. Endoglucanase activity was present in 88- and 82-kDa proteins, and there was at least a 20-fold variation in endoglucanase activity when P. ruminicola B₁4 was grown on different sugars. The highest activities were observed with mannose, cellobiose or xylose and little activity was observed with sucrose, arabinose or rhamnose. P. ruminicola B₁4 also had significant xylanase and mannanase activities, but these activities were present in proteins that had lower molecular masses than the endoglucanase and these proteins did not cross-react with antibody made against the endoglucanase. Mannanase activity has a similar pattern of expression to the endoglucanase, while the xylanase was not induced or repressed by the same sugars or combinations of sugars. The xylanase activity was greatest when xylan was the energy source for growth, but xylose was a very poor inducer of xylanase activity.     

Production of alkaline xylanase by a newly isolated alkaliphilic Bacillus sp. strain 41M-1

Alkaliphilic Bacillus sp. strain 41M-1, isolated from soil, produced xylan-degrading enzymes extracellularly. Optimum pH for the crude xylanase preparation was about pH 9, confirming the production of novel alkaline xylanase(s) by the isolate. Xylanases were induced by xylan, but were not produced in the presence of xylose, arabinose or glucose. Xylanase productivity was influenced by culture pH, and production at pH 10.5 was higher than that at pH 8.0. Zymogram analysis of the culture supernatant showed the alkaline xylanase with a molecular mass of 36 kDa.