重组极耐热木聚糖酶和重组极耐热漆酶协同漂白麦草浆

利用来自海栖热袍茵的重组极耐热木聚糖酶XynB和来自嗜热栖热菌Thermus thermophilus HB27的重组极耐热漆酶Tth-laccase对麦草浆进行协同漂白。结果表明,当未漂浆经XL漂序处理(X:重组木聚糖酶用量20 U/g绝干浆,pH 5.8,温度90℃,浆浓8%,处理时间2 h;L:重组漆酶用量3 U/g绝干浆,pH 4.5,温度90℃,浆浓8%,处理时间1.5 h),可获得最佳漂白效果。与对照浆比较,XL处理使浆料白度提升11.5%ISO,卡伯值降低6.9。双酶协同处理在改善浆料可漂性的同时,对纸浆纤维强度无负面影响。在后续过氧化氢漂白段中,当漂终白度相近时,XL预处理浆可节省约50%H_2O_2消耗量。     

Production of a recombinant xylanase in plants and its potential for pulp biobleaching applications

The purpose of this study was to produce recombinant xylanase in transgenic plants and to test its potential application for pulp bleaching. The xynII xylanase gene from Trichoderma reesei was inserted into the Arabidopsis genome. Many transgenic plants produced biologically active XYNII and accumulated in leaves at level of 1.4-3.2% of total soluble proteins. The bleaching ability of XYNII on Kraft pulp was demonstrated by a reduction in the kappa number and the residual lignin contents. The bleaching efficiency of transgenic plant produced XYNII was similar to commercial xylanase on unbleached Kraft pulp. The effect of xylanase treatment on Kraft pulp was also investigated by SEM. Clear physical change on the pulp fiber surface was observed and was related to the amount xylan removed and microfibrils were visible on the fiber surface. This report demonstrates the potential application of plant produced recombinant xylanase for pulp and paper bleaching.     

Characterization of a cellulase-free, neutral xylanase from Thermomyces lanuginosus CBS 288.54 and its biobleaching effect on wheat straw pulp

A xylanase purified from the thermophilic fungus Thermomyces lanuginosus CBS 288.54 was characterized and its potential application in wheat straw pulp biobleaching was evaluated. Xylanase was purified 33.6-fold to homogeneity with a recovery yield of 21.5%. It appeared as a single protein band on SDS-PAGE gel with a molecular mass of approx. 26.2 kDa. The purified xylanase had a neutral optimum pH ranging from pH 7.0 to pH 7.5, and it was also stable over pH 6.5-10.0. The optimal temperature of the xylanase was 70-75 degrees C and it was stable up to 65 degrees C. The purified xylanase was found to be not glycosylated. The xylanase was highly specific towards xylan, but did not exhibit other enzyme activity. Apparent Km values of the xylanase for birchwood, beechwood, soluble oat-spelt and insoluble oat-spelt xylans were 4.0, 4.7, 2.0 and 23.4 mg ml-1, respectively. The potential application of the xylanase was further evaluated in biobleaching of wheat straw pulp. The brightness of bleached pulps from the xylanase pretreated wheat straw pulp was 1.8-7.79% ISO higher than that of the control, and showed slightly lower tensile index and breaking length than the control. Although chlorine consumption was reduced by 28.3% during bleaching, the xylanase pretreated pulp (15 U g-1 pulp) still maintained its brightness at the control level. Besides, pretreatment of pulp with the xylanase was also effective at an alkaline pH as high as pH 10.0.     

Production of a <Emphasis Type="Italic">Trichoderma reesei</Emphasis> QM9414 xylanase in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and its application in biobleaching of wheat straw pulp

The purpose of this study was to produce a Trichoderma reesei xylanase (XYN2) in Pichia pastoris and to test its potential application for pulp bleaching. The recombinant xylanase was purified by a two-step process of ultrafiltration and gel filtration chromatography. The molecular mass of the recombinant enzyme was 21 and 25 kDa by SDS–PAGE analysis, due to different glycosylation of the native protein. The optimum pH and temperature of the recombinant XYN2 was 5.0 and 50 °C. Enzyme activity was stable at 50 °C and at pH 5.0–7.0. The bleaching ability of the recombinant xylanase was also studied at 50 °C and pH 6.0, using wheat straw pulp. Biobleaching of the xylanase produced chlorine dioxide savings of up to 60%, while retaining brightness at the control level and led to a lower kappa number and small enhancements in tensile, burst and tear strength of pulp fibers.     

Differential and synergistic effects of xylanase and laccase mediator system (LMS) in bleaching of soda and waste pulps

AIMS: Investigation of waste pulps and soda pulp bleaching with xylanase (X) and laccase mediator system (LMS) alone and in conjunction (one after the other) (XLMS). METHODS AND RESULTS: Soda and different grades of waste pulp fibres [used for making three-layered duplex sheets - top layer (TL), protective layer (PL) and bottom layer (BL)] when pretreated with either xylanase (40.0 IU g(-1)) or LMS (up to 200.0 U g(-1)) alone and in combination (one after the other) (XLMS) exhibited an increase in release of reducing sugars [up to 881.0% soda pulp; up to 736.6% (TL), up to 215.7% (PL) and up to 198.0% (BL) waste pulp], reduction in kappa number [up to 17.6% soda pulp; up to 14.0% (TL), up to 25.3% (PL) and up to 10.9% (BL), waste pulp], improvement in brightness [up to 20.4% soda pulp; up to 23.6% (TL), up to 8.6% (PL) and up to 5.0% (BL), waste pulp] when compared with the respective controls. The usage of XLMS along with 15% reduced level of hypochlorite at CEHHXLMS/EHHXLMS bleaching stage reduced kappa number [5.5% soda pulp; 11.4% (TL), 7.9% (PL), waste pulp] and improved brightness [1.0% soda pulp; 0.9% (TL), 1.4% (PL) waste pulp] when compared with the controls. Scanning electron microscopic studies revealed development of cracks, flakes, pores and peeling off the fibres in the enzyme-treated pulp samples. These modifications of the fibre surface during enzymatic bleaching in turn indicated the removal of lignin and derived compounds from the fibre cell wall. CONCLUSIONS: The work describes synergistic action of xylanase with LMS for bleaching of waste and nonwood pulps for eco-friendly production of paper and thus reveals a new unexploited arena for enzyme-based pulp bleaching. SIGNIFICANCE AND IMPACT OF THE STUDY: The drastic improvement in pulp properties obtained after xylanase and LMS treatment would improve the competitiveness of enzyme-based, environmentally benign processes over chemicals both economically and environmentally.     

Comparison of different fungal enzymes for bleaching high-quality paper pulps

Wild and recombinant hydrolases and oxidoreductases with a potential interest for environmentally sound bleaching of high-quality paper pulp (from flax) were incorporated into a totally chlorine free (TCF) sequence that also included a peroxide stage. The ability of feruloyl esterase (from Aspergillus niger) and Mn2+-oxidizing peroxidases (from Phanerochaete chrysosporium and Pleurotus eryngii) to decrease the final lignin content of flax pulp was shown. Laccase from Pycnoporus cinnabarinus (without mediator) also caused a slight improvement of pulp brightness that was increased in the presence of aryl-alcohol oxidase. However, the best results were obtained when the laccase treatment was performed in the presence of a mediator, 1-hydroxybenzotriazol (HBT), enabling strong delignification of pulps. The enzymatic removal of lignin resulted in high-final brightness values that are difficult to attain by chemical bleaching of this type of pulp. A partial inactivation of laccase by HBT was observed but this negative effect was strongly reduced in the presence of pulp. The good results obtained with the same laccase expressed in A. niger at bioreactor scale, revealed the feasibility of using recombinant laccase for bleaching high-quality non-wood pulps in the presence of a mediator.     

Comparing the efficiency of the laccase–NHA and laccase–HBT systems in eucalyptus pulp bleaching

Laccase–mediator systems have the disadvantage that the mediator is expensive and potentially toxic. In this work, we used N-hydroxyacetanilide (NHA) in combination with laccase for the first time to bleach eucalypt pulp and found it to be a very promising, advantageous alternative to 1-hydroxybenzotriazole (HBT) as mediator. Thus, NHA is efficiently oxidized by laccase to a radical that absorbs light at 350 nm. Also, NHA is a better substrate for laccase than is HBT. An innovative result is that the enzyme is inactivated to a similar extent by both mediators under the typical treatment conditions of the bleaching step (L). This adverse effect, however, is strongly reduced in the presence of pulp. Moreover, the laccase–NHA system is as efficient as the laccase–HBT system in reducing the kappa number of eucalyptus pulp. Using a xylanase pretreatment or unbleached pulp boosts kappa number reduction and bleaching with the laccase–mediator system. Based on the results of cyclic voltammetry tests, NHA has a slightly lower redox potential than HBT, which further supports use of the former; also, unlike HBT, NHA is oxidized in a reversible, pH-dependent manner. Interestingly, the laccase–NHA system provides more efficient bleaching of eucalyptus pulp at pH 5 than it does at pH 4.     

Biobleaching of wheat straw-rich soda pulp with alkalophilic laccase from gamma-proteobacterium JB: optimization of process parameters using response surface methodology

An alkalophilic laccase from gamma-proteobacterium JB was applied to wheat straw-rich soda pulp to check its bleaching potential by using response surface methodology based on central composite design. The design was employed by selecting laccase units, ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) concentration and pH as model factors. The results of second order factorial design experiments showed that all three independent variables had significant effect on brightness and kappa number of laccase-treated pulp. Optimum conditions for biobleaching of pulp with laccase preparation (specific activity, 65 nkat mg(-1) protein) were 20 nkat g(-1) of pulp, 2mM ABTS and pH 8.0 which enhanced brightness by 5.89% and reduced kappa number by 21.1% within 4h of incubation at 55 degrees C, without further alkaline extraction of pulp. Tear index (8%) and burst index (18%) also improved for laccase-treated pulp as compared to control raw pulp. Treatment of chemically (CEH1H2) bleached pulp with laccase showed significant effect on release of chromophores, hydrophobic and reducing compounds. Laccase-prebleaching of raw pulp reduced the use of hypochlorite by 10% to achieve brightness of resultant hand sheets similar to the fully chemically bleached pulp.     

Thermogravimetry study of xylanase- and laccase/mediator-treated eucalyptus pulp fibres

Thermogravimetric analyses (TGA) was applied to study the effects of enzymatic bleaching of eucalyptus pulp with xylanase and a laccase-mediator system. The thermal degradation profile of the pulps was sensitive to the enzymatic treatments. Xylanase treatment produced an ordered and clean microfibril, whereas laccase oxidized surface cellulose chains and increased the amorphous (paracrystalline) cellulose content. In this case, pulp viscosity decreased from 972 to 859 mL/g and apparent pulp crystallinity calculated from TGA data decreased almost 50%. Alkaline extraction was necessary to recover pulp crystallinity and to remove oxidized lignin in the laccase-treated samples. TGA data allowed differentiating and quantifying crystalline and amorphous cellulose. This thermogravimetric approach is a simple method in order to monitor superficial changes in cellulosic microfibrils.     

Investigation of hydroxamic acids as laccase-mediators for pulp bleaching

A number of hydroxamic acids have been synthesized and investigated as laccase-mediators for pulp bleaching. As compared with N-hydroxyacetanilide (NHA), one of the most effective laccase-mediators reported so far, N-(4-cyanophenyl)acetohydroxamic acid (NCPA), resulted in the highest brightness and lowest kappa number of hardwood kraft pulp of all the laccase-mediators studied. The bleaching efficacy of a laccase/7-cyano-4-hydroxy-2H-1,4-benzoxazin-3-one system was also comparable with that of a laccase/NHA system. A laccase/NCPA system was further studied for the bleaching of unbleached softwood kraft pulp. The effects of pulp consistency, laccase dosage, NCPA dosage, incubation time, and oxygen pressure on the bleaching efficacy of a laccase/NCPA system were studied.     

Biobleaching of wheat straw pulp with recombinant laccase from the hyperthermophilic <Emphasis Type="Italic">Thermus thermophilus</Emphasis>

The recombinant laccase from Thermus thermophilus was applied to the biobleaching of wheat straw pulp. The best bleaching effect was when the pulp was treated with 3 U laccase g⁻¹ dry pulp at 90°C, pH 4.5, 8% consistency for 1.5 h. Under these conditions, the pulp brightness was increased by 3.3% ISO, and the pulp kappa number was decreased by 5.6 U. Enzymatic treatment improved the bleachability of wheat straw pulp but caused no damage to the pulp fibers. The use of enzyme-treated pulp saved 25% H₂O₂ consumption in subsequent peroxide bleaching without decreasing the final brightness. Pulp biobleaching in the presence of 5 mM ABTS further increased the pulp brightness by 1.5% ISO. This is the first report on the application of laccase from T. thermophilus in the pulp and paper sector.     

The role of xylanases and laccases on hexenuronic acid and lignin removal

The variation of the contents in hexenuronic acids (HexA) and lignin in Eucalyptus kraft pulp during sequences with the laccase–mediator treatment with or without xylanase pretreatment was studied. The laccase–HBT system (HBT: 1-hydroxybenzotriazole) initially oxidized lignin alone but altered cellulose in the pulp as well after some time. Once all accessible lignin was removed, the system acted on HexA. As a result, the laccase–mediator treatment reduced the HexA content of the pulp, especially if a xylanase pretreatment was applied before. A previously unseen effect was observed here: HexA removal was found to depend on the laccase and HBT doses, but not on the reaction time. In addition, the xylanase pretreatment was found to strongly boost the effects of the laccase–HBT system by facilitating their access to HexA without affecting the lignin content.     

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).     

Modification of high-lignin kraft pulps with laccase. Part 2. Xylanase-enhanced strength benefits

The effects of xylanase pretreatment of high lignin content softwood (SW) kraft pulp on subsequent pulp treatment with laccase in combination with gallic acid were investigated. Although xylanase pretreatment was ineffective in enhancing the laccase-facilitated biografting of gallic acid to kraft fibers, it was beneficial for subsequent treatment with laccase exclusively. Treating pulp fibers with xylanase followed by laccase provided a collective 25% and 46% increase in dry and wet tensile strength properties, respectively.     

Enhanced production of cellulase-free thermostable xylanase by Bacillus pumilus ASH and its potential application in paper industry

A very high level of cellulase-free, thermostable xylanase has been produced from newly isolated strain of Bacillus pumilus under submerged fermentation in a basal medium supplemented with wheat bran (2%, w/v) pH 8.0 and at 37 °C. After optimization of various production parameters, an increase of nearly 13-fold in xylanase production (5407 IU/ml) was achieved. The produced xylanase is stable in neutral to alkaline pH region at 70 °C. The suitability of this xylanase for use in the bioleaching of eucalyptus Kraft pulp was investigated. A xylanase dose of 5 IU/g of oven dried pulp of 10% consistency exhibited the optimum bleach boosting of the pulp at pH 7.0 and 60 °C after 180 min of treatment. An increase of 5% in brightness along with an increase of 21% and 28% in whiteness and fluorescence respectively, whereas 18% decrease in the yellowness of the biotreated pulp was observed. Enzyme treated pulp when subjected to chemical bleaching, resulted in 20% reduction in chlorine consumption and up to 10% reduction in consumption of chlorine dioxide. Also a reduction of about 16% in kappa number and 83% in permanganate number, along with a reduction in COD value and significant improvement in various pulp properties, viz. viscosity, tensile strength, breaking length, burst factor, burstness, tear factor and tearness were observed in comparison to the conventional chemical bleaching.     

Production of cellulase‐free xylanase by the recombinant Bacillus subtilis and its applicability in paper pulp bleaching

A metagenomic xylanase gene (Mxyl) was successfully cloned into shuttle vector pWH1520 and expressed in Bacillus subtilis extracellularly. On induction with xylose, recombinant xylanase secretion commenced after 6 h. Identifying critical variables for recombinant xylanase production by one‐variable‐at‐time approach followed by optimization of the selected variables (xylose, inoculum density, incubation density) by response surface methodology (RSM) led to three‐fold enhancement in extracellular xylanase production (119 U mL^?1). When the pulp was treated with recombinant xylanase at 80°C and pH 9.0, kappa number of the pulp was reduced with concomitant increase in brightness and 24% reduction in chlorine consumption. This is the first report on the expression of metagenomic xylanase gene in Bacillus subtilis extracellularly and its utility in developing an environment‐friendly pulp bleaching process. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1441–1447, 2013     

Enhanced production of a thermostable xylanase from Streptomyces sp. QG-11-3 and its application in biobleaching of eucalyptus kraft pulp

A thermostable and cellulase-free xylanase has been produced from Streptomyces sp. QG-11-3 in solid substrate fermentation using wheat bran and eucalyptus kraft pulp as the prime solid substrates. The maximum xylanase yield obtained using these two substrates were 2360 U/g and 1200 U/g dry solid substrate at substrate:moisture ratios of 1:3 and 1:2.5, respectively. In immobilized cell system using polyurethane foam (PUF) and three nonwoven fabrics, namely, polyester, silk, and cotton, the xylanase yields were enhanced by 2.5-fold (203 U/ml), 1.91-fold (155 U/ml), 1.54-fold (125 U/ml), and 1.47-fold (119 U/ml), respectively, compared to the xylanase yield in liquid-batch fermentation (81 U/ml). In the biobleaching experiments, the xylanase dose of 3.5 U/g moisture free pulp exhibited the optimum bleach boosting of eucalyptus kraft pulp at pH 8.5 and 50 degrees C after 2 h of treatment. When xylanase treated pulp was subsequently treated with 4.5% chlorine, it resulted in reduction of kappa number by 25%, enhanced the brightness (%ISO) by 20% and improved the pulp properties such as tensile strength and burst factor by up to 63% and 8%, respectively.     

Xylanase and Mannanase enzymes from Streptomyces galbus NR and their use in biobleaching of softwood kraft pulp

Enzymatic pretreatment of softwood kraft pulp was investigated using xylanase and mannanase, singly or in combination, either sequentially or simultaneously. Enzymes were obtained from Streptomyces galbus NR that had been cultivated in a medium, containing either xylan of sugar cane bagasse or galactomannan of palm-seeds, when they were used as sole carbon sources from local wastes in fermentation media. No cellulase activity was detected. Incubation period, temperature, initial pH values and nature of nutritive constituents were investigated. Optimum production of both enzymes was achieved after 5 days incubation on a rotary shaker (200 rpm) at 35 degrees C and initial pH 7.0. Partial purification of xylanase and mannanase in the cultures supernatant were achieved by salting out at 40-60 and 60-80% ammonium sulphate saturation with a purification of 9.63- and 8.71-fold and 68.80 and 62.79% recovery, respectively. The xylanase and mannanase from S. galbus NR have optimal activity at 50 and 40 degrees C, respectively. Both enzymes were stable at a temperature up to 50 degrees C. Xylanase and mannanase showed highest activity at pH 6.5 and were stable from 5.0 to 8.0 and from 5.5 to 7.5, respectively. The partial purified enzymes preparations of xylanase and mannanase enzymes showed high bleaching activity, which is an important consideration for industry. Xylanase was found to be more effective for paper-bleaching than mannanase. When xylanase and mannanase were dosed together (simultaneously), both enzymes were able to enhance the liberation of reducing sugars and improve pulp bleachability, possibly as a result of nearly additive interactions. The simultaneous addition of both enzymes was more effective in pulp treatment than their sequential addition.     

Laccase for biobleaching of eucalypt kraft pulp by means of a modified industrial bleaching sequence

Biobleaching of kraft pulp is a possible application of laccase, but it has not been described in detail for complete industrial bleaching sequences yet. Therefore, in this work, the biobleaching of Eucalyptus globulus kraft pulp was performed using a modified industrial totally chlorine‐free sequence. The modification consisted in the substitution of an enzymatic delignification stage, based on the application of laccase from Trametes villosa, for the first alkaline extraction one. The enzymatic stage was performed with several synthetic and natural mediators, namely 1‐hydroxybenzotriazole (HBT), violuric acid (VA), methyl syringate, and syringaldehyde. Several pulp properties were analyzed after each stage of the bleaching process—kappa number, ISO brightness, viscosity, and optical properties of CIEL*a*b* system. The new biobleaching sequence improved the pulp properties, in comparison to the conventional bleaching sequence, if HBT or VA was used as mediators. VA was selected as the best mediator of those tested and the effect of its concentration in the enzymatic stage was subsequently studied. Reducing the initial concentration by 30%, the same pulp quality was obtained, but if the reduction attained 60%, an important decrease in pulp integrity was detected. The modified bleaching sequence could improve the bleached pulp properties (kappa number 10%, ISO brightness 1%, and viscosity 5%) in comparison to the mill sequence. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Xylanases of marine fungi of potential use for biobleaching of paper pulp

Microbial xylanases that are thermostable, active at alkaline pH and cellulase-free are generally preferred for biobleaching of paper pulp. We screened obligate and facultative marine fungi for xylanase activity with these desirable traits. Several fungal isolates obtained from marine habitats showed alkaline xylanase activity. The crude enzyme from NIOCC isolate 3 (Aspergillus niger), with high xylanase activity, cellulase-free and unique properties containing 580 U l^–1 xylanase, could bring about bleaching of sugarcane bagasse pulp by a 60 min treatment at 55°C, resulting in a decrease of ten kappa numbers and a 30% reduction in consumption of chlorine during bleaching. The culture filtrate showed peaks of xylanase activity at pH 3.5 and pH 8.5. When assayed at pH 3.5, optimum activity was detected at 50°C, with a second peak of activity at 90°C. When assayed at pH 8.5, optimum activity was seen at 80°C. The crude enzyme was thermostable at 55°C for at least 4 h and retained about 60% activity. Gel filtration of the 50–80% ammonium sulphate-precipitated fraction of the crude culture filtrate separated into two peaks of xylanase with specific activities of 393 and 2,457 U (mg protein)^–1. The two peaks showing xylanase activity had molecular masses of 13 and 18 kDa. Zymogram analysis of xylanase of crude culture filtrate as well as the 50–80% ammonium sulphate-precipitated fraction showed two distinct xylanase activity bands on native PAGE. The crude culture filtrate also showed moderate activities of -xylosidase and -l-arabinofuranosidase, which could act synergistically with xylanase in attacking xylan. This is the first report showing the potential application of crude culture filtrate of a marine fungal isolate possessing thermostable, cellulase-free alkaline xylanase activity in biobleaching of paper pulp.