Cellulases and xylanase of an anaerobic rumen fungus grown on wheat straw, wheat straw holocellulose, cellulose, and xylan

The activities of cellulolytic and xylanolytic enzymes produced by an anaerobic fungus (R1) which resembled Neocallimastix sp. were investigated. Carboxymethylcellulase (CMCase), cellobiase, and filter paper (FPase) activities had pH optima of 6.0, 5.5, and 6.0, respectively. CMCase and cellobiase activities both had a temperature optimum of 50 degrees C, whereas FPase had an optimum of 45 degrees C. The pH and temperature optima for xylanase activity were pH 6.0 and 50 degrees C, respectively. Growth of the fungus on wheat straw, wheat straw holocellulose, or cellulose resulted in substantial colonization, with at least 43 to 58% losses in substrate dry matter and accumulation of comparable amounts of formate. This end product was correlated to apparent loss of substrate dry weight and could be used as an indicator of fungal growth. Milling of wheat straw did not enhance the rate or extent of substrate degradation. Growth of the R1 isolate on the above substrates or xylan also resulted in accumulation of high levels of xylanase activity and lower cellulase activities. Of the cellulases, CMCase was the most active and was associated with either low or trace amounts of cellobiase and FPase activities. During growth on xylan, reducing sugars, including arabinose and xylose, rapidly accumulated in the medium. Xylose and other reducing sugars, but not arabinose, were subsequently used for growth. Reducing sugars also accumulated, but not as rapidly, when the fungus was grown on wheat straw, wheat straw holocellulose, or cellulose. Xylanase activities detected during growth of R1 on media containing glucose, xylose, or cellobiose suggested that enzyme production was constitutive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycosidases of the rumen anaerobic fungus Neocallimastix frontalis grown on cellulosic substrates.

The rumen anaerobic fungus Neocallimastix frontalis was grown on cellulosic substrates, and the cellular distribution and types of glycosidases produced by the organism were studied. Fungal cultures were fractionated into extracellular, insoluble (membrane), and intracellular fractions and assayed for glycosidase activity by using Avicel, carboxymethylcellulose, xylan, starch, polygalacturonic acid, and the p-nitrophenyl derivatives of galactose, glucose, and xylose as substrates. Enzymic activity was highest in the extracellular fraction; however, the membrane fraction also displayed appreciable activity. The intracellular fraction was inactive towards all substrates. Polygalacturonic acid was the only substrate not hydrolyzed by the active fractions, indicating that pectinase was absent. The results show that N. frontalis, a common rumen anaerobic fungus, produces enzymes for degrading cellulose and hemicellulose, key components of plant fiber.     

Glycosidases of the rumen anaerobic fungus Neocallimastix frontalis grown on cellulosic substrates

The rumen anaerobic fungus Neocallimastix frontalis was grown on cellulosic substrates, and the cellular distribution and types of glycosidases produced by the organism were studied. Fungal cultures were fractionated into extracellular, insoluble (membrane), and intracellular fractions and assayed for glycosidase activity by using Avicel, carboxymethylcellulose, xylan, starch, polygalacturonic acid, and the p-nitrophenyl derivatives of galactose, glucose, and xylose as substrates. Enzymic activity was highest in the extracellular fraction; however, the membrane fraction also displayed appreciable activity. The intracellular fraction was inactive towards all substrates. Polygalacturonic acid was the only substrate not hydrolyzed by the active fractions, indicating that pectinase was absent. The results show that N. frontalis, a common rumen anaerobic fungus, produces enzymes for degrading cellulose and hemicellulose, key components of plant fiber.     

Degradation and utilization of cellulose and straw by three different anaerobic fungi from the ovine rumen.

Three different ruminal fungi, a Neocallimastix sp. (strain LM-1), a Piromonas sp. (strain SM-1), and a Sphaeromonas sp. (strain NM-1), were grown anaerobically in liquid media which contained a suspension of either 1% (wt/vol) purified cellulose or finely milled wheat straw as the source of fermentable carbon. Fungal biomass was estimated by using cell wall chitin or cellular protein in cellulose cultures and chitin in straw cultures. Both strains LM-1 and SM-1 degraded cellulose with a concomitant increase in fungal biomass. Maximum growth of both fungi occurred after incubation for 4 days, and the final yield of protein was the same for both fungi. Cellulose degradation continued after growth ceased. Strain NM-1 failed to grow in the cellulose medium. All three anaerobic fungi grew in the straw-containing medium, and loss of dry weight from the cultures indicated degradation of straw to various degrees (LM-1 greater than SM-1 greater than NM-1). The total fiber component and the cellulose component of the straw were degraded in similar proportions, but the lignin component remained undegraded by any of the fungi. Maximum growth yield on straw occurred after 4 days for strain LM-1 and after 5 days for strains SM-1 and NM-1. The calculated yield of cellular protein for strain LM-1 was twice that of both strains SM-1 and NM-1. The cellular protein yield of strain SM-1 was the same in both cellulose and straw cultures. In contrast to cellulose, straw degradation ceased after the end of the growth phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of pretreatment severity on xylan solubility and enzymatic breakdown of the remaining cellulose from wheat straw

The effect of process conditions used for wheat straw pretreatments on the liquor- and residue-composition was studied. Hereto, the pretreatment conditions were expressed in a 'combined severity R(0)(')-factor'. The higher the combined severity factor (R(0)(')) the more xylan was released from the wheat straw, but the more xylan decomposed and furfural formation occurred. The percentage of residual xylan present after pretreatment appeared to be a good indicator concerning cellulose degradability or bio-ethanol production. Namely, cellulose degradation by using commercial enzymes was higher at higher severities corresponding to a lower amount of residual xylan. The xylan release and degradation was studied in more detail by using HPSEC and MALDI-TOF mass spectrometry. The more severe the treatment the more (acetylated) xylose oligomers with a DP lower than nine were analysed. The presence of (acetylated) xylans with a DP of 9-25 increased slightly from low to medium severity. The quantification of the DP-distribution of the (acetylated) xylans released proved to be a good tool to predict cellulose degradability.     

Production of a thermostable alkali-tolerant xylanase from Bacillus circulans AB 16 grown on wheat straw

Bacillus circulans AB 16 was able to produce 50 IU/ml of xylanase, with negligible cellulase activity when grown on untreated wheat straw. The pH optimum of the crude enzyme was 6–7 with a temperature optimum of 80 C. The enzyme showed high pH and thermal stability retaining 100% activity at 60 C, pH 8 and 9 after 2.5 h of incubation. The residual activity at 70 C after 2.5 h was 62% and 45% at pH 8 and 9, respectively. At 75 C only 22.2% activity remained at pH 8 after 1 h incubation. Since Kraft pulp is alkaline this enzyme could be used for prebleaching of pulp at temperatures up to 70 C without pH adjustment.     

Degradation and utilization of cellulose and straw by three different anaerobic fungi from the ovine rumen

Three different ruminal fungi, a Neocallimastix sp. (strain LM-1), a Piromonas sp. (strain SM-1), and a Sphaeromonas sp. (strain NM-1), were grown anaerobically in liquid media which contained a suspension of either 1% (wt/vol) purified cellulose or finely milled wheat straw as the source of fermentable carbon. Fungal biomass was estimated by using cell wall chitin or cellular protein in cellulose cultures and chitin in straw cultures. Both strains LM-1 and SM-1 degraded cellulose with a concomitant increase in fungal biomass. Maximum growth of both fungi occurred after incubation for 4 days, and the final yield of protein was the same for both fungi. Cellulose degradation continued after growth ceased. Strain NM-1 failed to grow in the cellulose medium. All three anaerobic fungi grew in the straw-containing medium, and loss of dry weight from the cultures indicated degradation of straw to various degrees (LM-1 greater than SM-1 greater than NM-1). The total fiber component and the cellulose component of the straw were degraded in similar proportions, but the lignin component remained undegraded by any of the fungi. Maximum growth yield on straw occurred after 4 days for strain LM-1 and after 5 days for strains SM-1 and NM-1. The calculated yield of cellular protein for strain LM-1 was twice that of both strains SM-1 and NM-1. The cellular protein yield of strain SM-1 was the same in both cellulose and straw cultures. In contrast to cellulose, straw degradation ceased after the end of the growth phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Simple method for cryopreservation of an anaerobic rumen fungus using ethylene glycol and rumen fluid

Abstract The cryopreservation of an anaerobic rumen fungus, Piromyces communis OTS1, was examined at −84 °C using dimethyl sulfoxide, propylene glycol or ethylene glycol as cryoprotectants. Ethylene glycol was the most effective agent, combining high survival and low toxicity, followed by dimethyl sulfoxide and propylene glycol. Cell-free rumen fluid in the cryopreservation medium decreased the toxicity of the cryoprotectant agents and also had a protective action per se. A survival of 80% after 1 year storage was obtained when samples with an initial zoospore density of 5 × 10⁴ zoospores/ml were equilibrated for 15 min in medium containing 0.64 M ethylene glycol and 5% cell-free rumen fluid, then frozen with dry ice and stored at −84 °C.     

Characterization of an acetyl xylan esterase from the anaerobic fungus Orpinomyces sp. strain PC-2.

A 1,067-bp cDNA, designated axeA, coding for an acetyl xylan esterase (AxeA) was cloned from the anaerobic rumen fungus Orpinomyces sp. strain PC-2. The gene had an open reading frame of 939 bp encoding a polypeptide of 313 amino acid residues with a calculated mass of 34,845 Da. An active esterase using the original start codon of the cDNA was synthesized in Escherichia coli. Two active forms of the esterase were purified from recombinant E. coli cultures. The size difference of 8 amino acids was a result of cleavages at two different sites within the signal peptide. The enzyme released acetate from several acetylated substrates, including acetylated xylan. The activity toward acetylated xylan was tripled in the presence of recombinant xylanase A from the same fungus. Using p-nitrophenyl acetate as a substrate, the enzyme had a K(m) of 0.9 mM and a V(max) of 785 micromol min(-1) mg(-1). It had temperature and pH optima of 30 degrees C and 9.0, respectively. AxeA had 56% amino acid identity with BnaA, an acetyl xylan esterase of Neocallimastix patriciarum, but the Orpinomyces AxeA was devoid of a noncatalytic repeated peptide domain (NCRPD) found at the carboxy terminus of the Neocallimastix BnaA. The NCRPD found in many glycosyl hydrolases and esterases of anaerobic fungi has been postulated to function as a docking domain for cellulase-hemicellulase complexes, similar to the dockerin of the cellulosome of Clostridium thermocellum. The difference in domain structures indicated that the two highly similar esterases of Orpinomyces and Neocallimastix may be differently located, the former being a free enzyme and the latter being a component of a cellulase-hemicellulase complex. Sequence data indicate that AxeA and BnaA might represent a new family of hydrolases.     

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.     

The effect of lasalocid on glucose uptake, hydrogen production and the solubilization of straw by the anaerobic rumen fungus Neocallimastix frontalis

Three isolates of Neocallimastix frontalis grown in pure culture with glucose as substrate differed in their response to the presence of lasalocid in the growth medium. For two strains (RE1 and RK21) hydrogen production and glucose uptake were reduced to 50% or less of the control values in the presence of lasalocid at 0–25 μg/ ml. A third strain (PNK2) retained significant fermentative ability in the presence of at least four times this amount of lasalocid. Lasalocid was rather less inhibitory to the solubilization of straw by these fungi, PNK2 again providing the most resistant strain. It is concluded that the use of lasalocid in ruminant feeds is likely to have a strain selective effect on the rumen population of Neocallimastix.     

Growth and fermentation of an anaerobic rumen fungus on various carbon sources and effect of temperature on development.

An anaerobic fungus (strain R1) resembling Neocallimastix spp. was isolated from sheep rumen. When grown on defined medium, the isolate utilized a wide range of polysaccharides and disaccharides, but of the eight monosaccharides tested only fructose, glucose, and xylose supported growth. The organism had doubling times of 5.56 h on glucose and 6.67 h on xylose, and in each case fermentation resulted in production of formate, acetate, lactate, and ethanol. During active growth, formate was a reliable indicator of fungal biomass. Growth on a medium containing glucose and xylose resulted in a doubling time of 8.70 h, but diauxic growth did not occur since both sugars were utilized simultaneously. The optimum temperature for zoospore and immature plant development was 39 degrees C, and no development occurred below 33 degrees C or above 41 degrees C.     

Effects of xylan and starch on secretome of the basidiomycete Phanerochaete chrysosporium grown on cellulose

Lignocellulosic biomass contains cellulose and xylan as major structural components, and starch as a storage polysaccharide. In the present study, we have used comparative secretomic analysis to examine the effects of xylan and starch on the expression level of proteins secreted by the basidiomycete Phanerochaete chrysosporium grown on cellulose,. Forty-seven spots of extracellular proteins expressed by P. chrysosporium separated by two-dimensional electrophoresis were identified by liquid chromatography-tandem mass spectrometry analysis. Addition of starch to the cellulolytic culture did not affect fungal growth significantly, but did decrease the production of total extracellular enzymes, including cellulases and xylanases. In contrast, addition of xylan increased mycelial volume and the production of extracellular proteins. Xylan increased synthesis of several glycoside hydrolase (GH) family 10 putative endoxylanases and a putative glucuronoyl esterase belonging to carbohydrate esterase family 15, for which plant cell wall xylan may be a substrate. Moreover, cellobiose dehydrogenase and GH family 61 proteins, which are known to promote cellulose degradation, were also increased in the presence of xylan. These enzymes may contribute to degradation by the fungus of not only cellulose but also complex carbohydrate components of the plant cell wall.     

Utilization of cellulose,starch, xylan,and other hemicelluloses for growth by the rumen phycomyceteNeocallimastix frontalis

The rumen phycomyceteNeocallimastix frontalis was found to utilize for growth a wide range of plant polysaccharides, including cellulose, starch, and xylan. The ability to utilize polysaccharides was absent after prolonged culture in vitro on glucose, but present after subculture on grass particles. Grass-grown organisms were capable of growing at the expense of cellobiose, maltose, and xylose, capacities absent before exposure to grass particles.N. frontalis cultures digested at least 41% of the dry weight of water-insoluble grass tissue, and up to 75% of the dry weight of filter paper.     

Strategies of xylanase supplementation for an efficient saccharification and cofermentation process from pretreated wheat straw

Ethanol production from lignocellulosic raw materials includes a pretreatment step before enzymatic hydrolysis (EH). Pretreated substrates contain complex hemicelluloses in the solid fraction that can protect the cellulose from enzymatic attack. In addition, soluble xylooligomers are contained in the pretreated materials and may have an inhibitory effect on cellulase activity. In this context, several approaches for xylanase supplementation have been studied to increase EH yields. In this study, the whole slurry obtained after steam explosion pretreatment of wheat straw has been used as substrate. EH experiments were performed using commercial cellulase preparations supplemented with an endoxylanase (XlnC) from Aspergillus nidulans. Among different strategies of XlnC supplementation, the 24‐h xylanase treatment before cellulase addition yielded an increase of 40.1 and 10.1% in glucose and xylose production, respectively. Different XlnC addition strategies were integrated in a simultaneous saccharification and cofermentation process (SSCF) using the xylose fermenting strain Saccharomyces cerevisiae F12. Ethanol production in SSCF was 28.4% higher when comparing to a simultaneous saccharification and fermentation process. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Transformation and expression of an anaerobic fungal xylanase in several strains of the rumen bacterium Butyrivibrio fibrisolvens

AIMS: To obtain reliable transformation of a range of Butyrivibrio fibrisolvens strains and to express a Neocallimastix patriciarum xylanase gene in the recipients. METHODS AND RESULTS: Eight strains (H17c, E14, LP1309, LP1028, AR11a, OB156, LP210B and LP461A) of Bu. fibrisolvens were transformed by the Gram-positive vector pUB110. A xylanase expression/secretion cassette containing Bu. fibrisolvens promoter and signal peptide elements fused to catalytic domain II of the N. patriciarum xylanase A cDNA (xynANp) was inserted into pUB110 to create the plasmid pUBxynA. pUBxynA was used to transform seven of the Bu. fibrisolvens strains transformed by pUB110. In strain H17c pUBxynA, which produced native xylanase, 2.46 U mg-1 total xylanase activity was produced with 45% extracellular xylanase. In strain H17c pUMSX, 0.74 U mg-1 total xylanase activity was produced with 98% extracellular xylanase. H17c pUBxynA exhibited increased (28.7%) degradation of neutral detergent fibre compared with unmodified H17c; however, progressive loss of pUBxynA was observed in long-term cultivation. CONCLUSIONS: A stable transformation system was developed that was applicable for a range of Bu. fibrisolvens strains and high levels of expression of a recombinant xylanase were obtained in H17c which lead to increased fibre digestion. SIGNIFICANCE AND IMPACT OF THE STUDY: This stable transformation system with the accompanying recombinant plasmids will be a useful tool for further investigation aimed at improving ruminal fibre digestion.     

Studies on the capacity of the cellulase of the anaerobic rumen fungus Piromonas communis P to degrade hydrogen bond-ordered cellulose

The anaerobic rumen fungus Piromonas communis, when cultured on cotton fibre as the carbon source, produces an extracellular cellulase that is capable of solubilizing crystalline hydrogen-bond-ordered cellulose, in the form of the cotton fibre, at a rate that is greater than that of any other cellulases reported in the literature hitherto. The cell-free culture fluid is also very rich in xylan-degrading enzymes. The activity towards crystalline cellulose resides in a high-molecular-mass (approximately 700–1000 kDa) component (so-called crystalline-cellulose-solubilizing component, CCSC) that comprises endo (1 4)--D-gluconase (carboxymethylcellulase), -D-glucosidase and another enzyme that appears to be important for the breakdown of hydrogen-bond-ordered cellulose. The CCSC is associated with only a small amount of the endo-(1 4)--D-glucanase (1.9%), -D-glucosidase (0.7%) and protein (0.5%) found in the crude cell-free cellulase preparation. The CCSC, unlike the bulk of the endo-(1 4)--D-glucanase and -D-glucosidase, is very strongly absorbed on the microcrystalline cellulose, Avicel.     

Effects of rhamnolipid on the cellulase and xylanase in hydrolysis of wheat straw

The effects of biosurfactant rhamnolipid (RL) and chemical surfactant Triton X-100 on the production of cellulases and xylanase from Penicillium expansum (P. expansum) in untreated, acid- and alkali-pretreated wheat straw submerged fermentations were studied, and the influences on the activity and stability of Cellulase R-10 were also investigated. The results showed that RL and Triton X-100 enhanced the activities of cellulases and xylanase to different extents and the stimulatory effects of RL were superior to those of Triton X-100. During the peak enzyme production phase, RL (60 RE mg/l) increased cellulases activities by 25.5-102.9%, in which the raise of the same enzyme in acid-pretreated straw broths was the most. It was found that the reducing sugars by hydrolyzing wheat straw with Cellulase R-100 were not visibly increased after adding RL. However, it distinctly protected Cellulase R-10 from degradation or inactivation, keeping the reducing sugars yield at about 17%.     

Secretome analysis of the fungus Trichoderma harzianum grown on cellulose

Trichoderma harzianum is a mycoparasitic filamentous fungus that produces and secretes a wide range of extracellular hydrolytic enzymes used in cell wall degradation. Due to its potential in biomass conversion, T. harzianum draws great attention from biofuel and biocontrol industries and research. Here, we report an extensive secretome analysis of T. harzianum. The fungus was grown on cellulose medium, and its secretome was analyzed by a combination of enzymology, 2DE, MALDI-MS and -MS/MS (Autoflex II), and LC-MS/MS (LTQ-Orbitrap XL). A total of 56 proteins were identified using high-resolution MS. Interestingly, although cellulases were found, the major hydrolytic enzymes secreted in the cellulose medium were chitinases and endochitinases, which may reflect the biocontrol feature of T. harzianum. The glycoside hydrolase family, including chitinases (EC 3.2.1.14), endo-N-acetylglucosaminidases (EC 3.2.1.96), hexosaminidases (EC 3.2.1.52), galactosidases (EC 3.2.1.23), xylanases (EC 3.2.1.8), exo-1,3-glucanases (EC 3.2.1.58), endoglucanases (EC 3.2.1.4), xylosidases (EC 3.2.1.37), α-L-arabinofuranosidase (EC 3.2.1.55), N-acetylhexosaminidases (EC 3.2.1.52), and other enzymes represented 51.36% of the total secretome. Few representatives were classified in the protease family (8.90%). Others (17.60%) are mostly intracellular proteins. A considerable part of the secretome was composed of hypothetical proteins (22.14%), probably because of the absence of an annotated T. harzianum genome. The T. harzianum secretome composition highlights the importance of this fungus as a rich source of hydrolytic enzymes for bioconversion and biocontrol applications.     

Effect of heterotrophic ruminal bacteria on xylan metabolism by the anaerobic fungus Piromyces communis

Six rumen bacteria were cocultured with the rumen fungus Piromyces communis and the effects on xylanolysis determined. The rate and extent of xylan utilization was enhanced in cocultures with Prevotella ruminicola or Succinivibrio dextrinosolvens. The positive effects of Suc. dextrinosolvens and Prev. ruminicola on xylanolysis by P. communis correlated with effective cross-feeding by the bacteria on arabinose and xylose released from xylan. Xylanolysis was not enhanced in cocultures of P. communis with Streptococcus bovis, Veillonella parvula or Ruminococcus flavefaciens. A comparison of fermentation product profiles and of extracellular enzyme activities showed that whereas saccharolytic species and Butyrivibrio fibrisolvens were dominant in cocultures, P. communis dominated in the culture with R. flavefaciens. Extracellular xylanase and β-xylosidase activities were not increased by cocultivation of P. communis with any of the heterotrophic bacteria.