Screening of cellulase-free fungal xylanases and evaluation of their performance on sulfite dissolving pulp

Cellulase-free xylanase preparations of different fungal origin lowered the xylan content of acid sulfite dissolving pulp from 5% (w/v) to less than 4%. This was not, though, related to the amount of reducing sugars released. The pulp bleachability improved in samples where xylan had been mostly degraded, however, the increment in brightness declined (from 2.0–2.9 to 1.0–1.2 brightness points) when the subsequent number of chemical bleaching steps increased from 1 to 5.     

Production and properties of xylanases from Aspergillus terricola Marchal and Aspergillus ochraceus and their use in cellulose pulp bleaching

Aspergillus terricola and Aspergillus ochraceus, isolated from Brazilian soil, were cultivated in Vogel and Adams media supplemented with 20 different carbon sources, at 30 °C, under static conditions, for 120 and 144 h, respectively. High levels of cellulase-free xylanase were produced in birchwood or oat spelt xylan-media. Wheat bran was the most favorable agricultural residue for xylanase production. Maximum activity was obtained at 60 °C and pH 6.5 for A. terricola, and 65 °C and pH 5.0 for A. ochraceus. A. terricola xylanase was stable for 1 h at 60 °C and retained 50% activity after 80 min, while A. ochraceus xylanase presented a t ₅₀ of 10 min. The xylanases were stable in an alkali pH range. Biobleaching of 10 U/g dry cellulose pulp resulted in 14.3% delignification (A. terricola) and 36.4% (A. ochraceus). The brightness was 2.4–3.4% ISO higher than the control. Analysis in SEM showed defibrillation of the microfibrils. Arabinase traces and β-xylosidase were detected which might act synergistically with xylanase.     

The role of two Trichoderma reesei xylanases in the bleaching of pine kraft pulp

Summary The two major xylanases of Trichoderma reesei with different pI values and pH optima were compared for increasing the bleachability of pine kraft pulp. The efficiencies of the two enzymes acting on pulp substrate were very similar in hydrolysis yield, extraction kappa number or final brightness value. Only slight synergism between the two enzymes was observed in both hydrolysis and bleaching tests. The pH optimum of the pI 5.5 xylanase was similar in pulp treatment and in the hydrolysis of isolated substrates, and the bleaching result also correlated well with the hydrolysis of pulp xylan. By contrast, the pI 9.0 xylanase acted differently on pulp than on isolated xylans at different pH values and the pH optimum on pulp was increased. The bleachability of pulp by the pI 9.0 xylanase was improved more than expected at pH 7.0, although the hydrolysis of pulp xylan was substantially decreased. A similar phenomenon was also observed when the hydrolysis was performed in water instead of buffer. It thus appears that the degree of hydrolysis needed to obtain improved bleachability with pI 9.0 xylanase can be minimized by proper adjustment of the hydrolysis conditions. Correspondence to: J. Buchert     

Natural and recombinant fungal laccases for paper pulp bleaching

Three laccases, a natural form and two recombinant forms obtained from two different expression hosts, were characterized and compared for paper pulp bleaching. Laccase from Pycnoporus cinnabarinus, a well known lignolytic fungus, was selected as a reference for this study. The corresponding recombinant laccases were produced in Aspergillus oryzae and A. niger hosts using the lacI gene from P. cinnabarinus to develop a production process without using the expensive laccase inducers required by the native source. In flasks, production of recombinant enzymes by Aspergilli strains gave yields close to 80 mg l^–1. Each protein was purified to homogeneity and characterized, demonstrating that the three hosts produced proteins with similar physico-chemical properties, including electron paramagnetic resonance spectra and N-terminal sequences. However, the recombinant laccases have higher Michaelian (K _m) constants, suggesting a decrease in substrate/enzyme affinity in comparison with the natural enzyme. Moreover, the natural laccase exhibited a higher redox potential (around 810 mV), compared with A. niger (760 mV) and A. oryzae (735 mV). Treatment of wheat straw Kraft pulp using laccases expressed in P. cinnabarinus or A. niger with 1-hydroxybenzotriazole as redox mediator achieved a delignification close to 75%, whereas the recombinant laccase from A. oryzae was not able to delignify pulp. These results were confirmed by thioacidolysis. Kinetic and redox potential data and pulp bleaching results were consistent, suggesting that the three enzymes are different and each fungal strain introduces differences during protein processing (folding and/or glycosylation).     

Production of cellulase-free thermostable xylanases by an isolated strain of Aspergillus niger PPI, utilizing various lignocellulosic wastes

A strain of Aspergillus niger PPI having prolific xylanolytic potential was isolated and the optimum conditions for maximum xylanase production was studied, resulting in the following: 4% substrate concentration, 10% v/v inoculum size, 72 h of incubation and pH 3.5–4.5 at 28 °C. The production profile of xylanase was examined with various lignocellulosics and maximum yield was achieved with oat. The hemicellulose content of wastes was also determined and oatmeal was found to have maximum hemicellulose content followed by wheat straw, sugarcane bagasse, rice husk and gram residue respectively. The enzyme showed maximum activity at pH 4 and temperature 60 °C. However, maximum stability was achieved at pH 3.5 and temperature 55 °C. Cellulase activity was found altogether absent in the enzyme broth.     

Production,isolation and partial purification of xylanases from anAspergillus sp.

AnAspergillus sp., isolated from a rubbish dump, produced 10.6 IU ml^-1 xylanase activity. Two xylanases were recognized and each was purified to homogeneity by two-stage chromatography on DEAE-and CM-Sepharose. Xylanase I had a pI of 7.2 and anM _r of 26 kDa whereas xylanase II had a pI of 4.7 and anM _r of 21 kDa. At 50°C, xylanase I was stable for 2.5 h but xylanase II was only stable for 1 h.P. Khanna is with the National Environmental Engineering Research Institute, Nehru Marg, Nagpur 440 020, India. S. Sivakami Sundari and N. Jothi Kumar are with the National Environmental Engineering Research Institute, Madras Zonal Laboratory, CSIR Madras Complex, Taramani 600 113, India.     

The performance of fungal xylan-degrading enzyme preparations in elemental chlorine-free bleaching for Eucalyptus pulp

Cellulase-free xylan-degrading enzyme preparations from Acrophialophora nainiana, Humicola grisea var. thermoidea and two Trichoderma harzianum strains were used as bleaching agents for Eucalyptus kraft pulp, prior to a chlorine dioxide and alkaline bleaching sequence. In comparison to the control sequence (performed without xylanase pretreatment), the sequence incorporating enzyme treatment was more effective. Removal of residual lignin was indicated by a reduction in kappa number. Overall, enzyme preparations from T. harzianum were marginally more effective in reducing pulp viscosity and chlorine chemical consumption and improving the brightness of the kraft pulp. However, the highest reduction in pulp viscosity was mediated by the xylanase preparation from A. nainiana. Xylanase pretreatment compares very favorably with that of chemical pulping. Journal of Industrial Microbiology & Biotechnology (2002) 28, 204–206 DOI: 10.1038/sj/jim/7000227 Received 27 April 2001/ Accepted in revised form 03 November 2001     

Production and characterization of xylanases of a Bacillus strain isolated from soil

Xylanase production was performed by growing a Bacillus isolate on agricultural by-products, wheat straw, wheat bran, corn cobs and cotton bagasse. A maximum xylanase activity of 180 U/ml was obtained together with a cellulase activity of 0.03 U/ml on 4 (w/v) corn cobs. Electrophoretic analysis showed the presence of three endo--1, 4-xylanases having molecular weights of about 22, 23 and 40 kDa. Xylanolytic activity was stable up to 50 °C in the pH range of 4.5–10 and the highest activity was observed at 70 °C and pH 6.5.     

Production of melanin bleaching enzyme of fungal origin and its application in cosmetics

We screened wild fungal isolates for melanolytic activity and found thatSporotrichum pruinosum was the most promising of the very limited number of fungi that decolourised synthetic melanin. We used a submerged aerobic process to produce a skin depigmentation enzyme by this strain, and found that in the medium the presence of Mn²⁺ ions was necessary, the limitation of carbon source was beneficial, and Zn²⁺ ions were inhibitory. Cultivation in a stirred bioreactor required immobilization of mycelium and use of low stirring velocity. A partially purified enzyme was prepared and tested for depigmentation of human skin corneocytes and whole epidermis of phototypes III and V. This is the first study demonstrating the effective enzymatic degradation of the skin melanin rather than inhibition of its synthesis. This opens the possibility of using melanolytic enzymes in cosmetic skin lightening.     

Bleaching of eucalyptus Kraft pulp with commercial xylanases

Three commercially available xylanases were tested in the bleaching of Kraft pulps from Eucaliptus globulus and Eucaliptus nitens. These xylanase preparations, in combination with the sequence D 0 E OP D 1 1 , led to savings of chlorine dioxide that were in the range of 12 to 40% without affecting the target brightness of 90% ISO. Enzyme-treated pulps required slightly higher beating to reach the same freeness than the controls, although the strength properties of both pulps were similar.     

Production of thermostable pectinase and xylanase for their potential application in bleaching of kraft pulp

A very high level of alkalophilic and thermostable pectinase and xylanase has been produced from newly isolated strains of Bacillus subtilis and Bacillus pumilus respectively. Enzyme production for pectinase was carried out under SSF using combinations of cheap agricultural residues while xylanase was produced under submerged fermentation using wheat bran as substrate to minimize the cost of production of these enzymes Among the various substrates tested, the highest yield of pectinase production was observed by using combination of WB + CW (6592 U/g of dry substrate) supplemented with 4% yeast extract when incubated at 37 °C for 72 h using deionized water of pH 7.0 as moistening agent. The biobleaching effect of these cellulase free enzymes on kraft pulp was determined. Both xylanase and pectinase showed stability over a broad range of pH from 6 to 10 and temperature from 55 to 70 °C. The bleaching efficiency of the pectinase and xylanase on kraft pulp was maximum after 150 min at 60 °C using enzyme dosage of 5 IU/ml of each enzyme at 10% pulp consistency with about 16% reduction in kappa number and 84% reduction in permanganate number. Enzyme treated pulp when subjected to CDED₁D₂ steps, 25% reduction in chlorine consumption and upto 19% reduction in consumption of chlorine dioxide was observed for obtaining the same %ISO brightness. Also an increase of 22 and 84% in whiteness and fluorescence respectively and a decrease of approximately 19% in the yellowness of the biotreated pulp were observed by pretreatment of the pulp with our enzymatic mixture.     

Production, characterization, and use of serpin antibodies

Serine protease inhibitors (serpins) constitute a still expanding superfamily of structural similar proteins, which are localized extracellularly and intracellularly. Serpins play a central role in the regulation of a wide variety of (patho) physiological processes including coagulation, fibrinolysis, inflammation, development, tumor invasion, and apoptosis. Serpins have a unique mechanism of inhibition that involves a profound change in conformational state upon interaction with their protease. This conformational change enables the production of monoclonal antibodies specific for native, complexed, and inactivated serpins. Antibodies, and assays based on these antibodies, have been helpful in elucidating the (patho) physiological function of serpins in the last decade. Serpin-specific antibodies can be used for: (1) structure-function studies such as detection of conformational changes; (2) identification of target-proteases; (3) the detection and quantification of serpin and serpin-protease complexes in bodily fluids by immunoassays such as ELISA, RIA or FACS; (4) detection of serpins in tissues by immunohistochemistry; and (5) possible therapeutical interventions. This review summarizes the techniques we have used to obtain and screen antibodies against extra- and intracellular serpins, as well as the use of these antibodies for some of the above-mentioned purposes.     

Molecular cloning of fungal xylanases: an overview

Xylanases have received great attention in the development of environment-friendly technologies in the paper and pulp industry. Their use could greatly improve the overall lignocellulosic materials for the generation of liquid fuels and chemicals. Fungi are widely used as xylanase producers and are generally considered as more potent producers of xylanases than bacteria and yeasts. Large-scale production of xylanases is facilitated with the advent of genetic engineering. Recent breakthroughs in genomics have helped to overcome the problems such as limited enzyme availability, substrate scope, and operational stability. Genes encoding xylanases have been cloned in homologous and heterologous hosts with the objectives of overproducing the enzyme and altering its properties to suit commercial applications. Owing to the industrial importance of xylanases, a significant number of studies are reported on cloning and expression of the enzymes during the last few years. We, therefore, have reviewed recent knowledge regarding cloning of fungal xylanase genes into various hosts for heterologous production. This will bring an insight into the current status of cloning and expression of the fungal xylanases for industrial applications.     

Production and properties of xylanases from actinomycetes

The extracellular xylanase activity of 15 actinomycete strains, representing a range of taxa, was examined. Enzyme production was correlated with growth in all cases and product analysis demonstrated that degradation was the result of endoxylanase activity. This was subject to end-product inhibition, probably by xylobiose, but was not inhibited by cellobiose or monomeric sugars. Gel electrophoresis showed that up to six separate endoxylanases were produced but only one was identified in strains that exhibited poor activity. Activity against xylan was found to be specific in that no cross-reactions with endoglucanase activity were detected. The xylan-degrading systems of actinomycetes are clearly complex and non-uniform, although there is some evidence of conservation within Streptomyces.     

Totally chlorine-free bleaching of flax pulp

Invertase and urease are enzyme entities highly associated with the cells of the astaxanthin-producer yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma) during any stage of its cell growth cycle. In this study cellobiose was a more efficient carbon source than sucrose or its hexose counterparts for invertase expression. Extensive ultrasonication or abrasion with glass pearls were required in order to promote enzyme release. In contrast to the yeast whose growth declines above 27 degrees C, the released enzymes displayed a higher optimum temperature range when assayed in vitro. Isoforms from both enzymes could be resolved either by FPLC on DEAE-Sepharose or by an affinity approach on immobilized Concanavalin. The zymogram for invertase showed a pI somewhat less acidic than that of the similar enzyme from S. cerevisiae.     

Bifunctional xylanases and their potential use in biotechnology

Plant cell walls are comprised of cellulose, hemicellulose and other polymers that are intertwined. This complex structure acts as a barrier to degradation by single enzyme. Thus, a cocktail consisting of bi and multifunctional xylanases and xylan debranching enzymes is most desired combination for the efficient utilization of these complex materials. Xylanases have prospective applications in the food, animal feed, and paper and pulp industries. Furthermore, in order to enhance feed nutrient digestibility and to improve wheat flour quality xylanase along with other glycohydrolases are often used. For these applications, a bifunctional enzyme is undoubtedly much more valuable as compared to monofunctional enzyme. The natural diversity of enzymes provides some candidates with evolved bifunctional activity. Nevertheless most resulted from the in vitro fusion of individual enzymes. Here we present bifunctional xylanases, their evolution, occurrence, molecular biology and potential uses in biotechnology.     

Thermostable microbial xylanases for pulp and paper industries: trends,applications and further perspectives

World Journal of Microbiology and Biotechnology - As obligate aerobic soil organisms, the ability of Azotobacter species to fix nitrogen is unusual given that the nitrogenase complex requires a...     

Production and properties of xylanases from thermophilic actinomycetes

30 strains of xylanolytic thermophilic actinomycetes were isolated from composted grass and cattle manure and identified as members of the generaThermomonospora, Saccharomonospora, Microbispora, Streptomyces andActinomadura. Screening of these strains for extracellular xylanase indicated that strains ofSaccharomonospora andMicrobispora generally were poor xylanase producers (0.5–1.5 U/ml) whereas relatively high activities were observed in cultures ofStreptomyces andActionomadura (4–12 U/ml).A preliminary characterization of the enzymes of strains of the latter genera suggested that xylanases of all the strains ofActinomadura exhibited higher thermostabilities than those ofStreptomyces. To evaluate the potential of thermophilicActinomadura for industrial applications, xylanases of three strains were studied in more detail. The highest activity levels for xylanases were observed in cultures grown on xylan and wheat bran. The optimal pH and temperature for xylanase activities ranged from 6.0 to 7.0 and 70 to 80°C. The enzymes exhibited considerable thermostability at their optimum temperature. The half-lives at 75°C were in the range from 6.5 to 17h. Hydrolysis of xylan by extracellular xylanases yielded xylobiose, xylose and arabinose as principal products. Estimated by the amount of reducing sugars liberated the degree of hydrolysis was 55 to 65%. Complete utilization of xylan is presumably achieved by -xylosidase activities which could be shown to be largely cell-associated in the 3Actinomadura strains.     

Production of cellulases and xylanases by low-temperature basidiomycetes

Three of four isolates, representing phylogenetically distinct groupings of low-temperature basidiomycetes (LTB), were capable of utilizing wheat straw, and to a lesser extent conifer wood at 15 degrees C. A cottony snow mould LTB (LRS 013) and a fruit rot LTB (LRS 241) grown on straw significantly degraded filter paper, carboxymethylcellulose (CMC), p-nitrophenyl beta-glucopyranoside (i.e., beta-glucosidases), and xylan. Enzymes produced by Coprinus psychromorbidus (LRS 067) were limited to xylanases from straw and wood and beta-glucosidases from wood. A sclerotia-forming LTB (LRS 131) exhibited poor growth on both substrates, and did not produce detectable quantities of extracellular enzymes. None of the LTB isolates tested degraded avicel. The temperature optima of CMCases and xylanases in the filtrates from the straw medium ranged from 25 degrees C to 55 degrees C, and with the exception of LRS 067, significant activity was observed at 5 degrees C. Two cellulases (25 and 31 kDa) and two xylanases (24 and 34 kDa) were observed on zymograms for LRS 013 and 241. Reduction of enzymes with 2-mercaptoethanol adversely affected their activity on zymograms, and an additional cellulase band was observed for non-reduced samples. This study indicates that LTB produce an array of cellulolytic and xylanolytic enzymes, and that some of these enzymes possess low-temperature optima which may facilitate degradation of plant fibre under low-temperature conditions.     

Stability of xylanases in the presence of methanol and its evaluation on the bleaching capacity

A commercial (Cartazyme) and non-commercial (Asperzyme) xylanases were studied. Cartazyme stability in a 0–70% (v/v) methanol at 50°C and 65°C was carried out. No deactivation was found for Cartazyme in the presence of 15% methanol at 50°C. Half-life activity decay (t1/2) of Cartazyme at 50°C in 30%, 50% and 70% methanol solutions were 4.0 h, 2.3 h and 1.2 h, respectively. At 65°C, which is the ozone-alkali-peroxide (ZEP) bleaching temperature, only significant results on Kappa number reduction and selectivity were only observed in 15% methanol (t1/2 30 min) at the Z stage. For the Asperzyme, a t1/2 of 36.5 min at 50°C was found. In the Z stage with Asperzyme in the presence of 25% of methanol, a 20% Kappa number reduction and an improvement of the ZEP sequence of the brightness of 3.1 points were obtained. These results were correlated with the xylanase stability.