Engineering of xylanases for the development of biotechnologically important characteristics

Xylanases are the main biocatalysts used for the reduction of the xylan backbone from hemicellulose, randomly splitting off β-1,4-glycosidic linkages between xylopyranosyl residues. Xylanase market has been annually estimated at 500 million US Dollars and they are potentially used in broad industrial process ranges such as paper pulp biobleaching, xylo-oligosaccharide production, and biofuel manufacture from lignocellulose. The highly stable xylanases are preferred in the downstream procedure of industrial processes because they can tolerate severe conditions. Almost all native xylanases can not endure adverse conditions thus they are industrially not proper to be utilized. Protein engineering is a powerful technology for developing xylanases, which can effectively work in adverse conditions and can meet requirements for industrial processes. This study considered state-of-the-art strategies of protein engineering for creating the xylanase gene diversity, high-throughput screening systems toward upgraded traits of the xylanases, and the prediction and comprehensive analysis of the target mutations in xylanases by in silico methods. Also, key molecular factors have been elucidated for industrial characteristics (alkaliphilic enhancement, thermal stability, and catalytic performance) of GH11 family xylanases. The present review explores industrial characteristics improved by directed evolution, rational design, and semi-rational design as protein engineering approaches for pulp bleaching process, xylooligosaccharides production, and biorefinery & bioenergy production.     

Kraft pulp biobleaching and mediated oxidation of a nonphenolic substrate by laccase from Streptomyces cyaneus CECT 3335

A new laccase (EC 1.10.3.2) produced by Streptomyces cyaneus CECT 3335 in liquid media containing soya flour (20 g per liter) was purified to homogeneity. The physicochemical, catalytic, and spectral characteristics of this enzyme, as well as its suitability for biobleaching of eucalyptus kraft pulps, were assessed. The purified laccase had a molecular mass of 75 kDa and an isoelectric point of 5.6, and its optimal pH and temperature were 4.5 and 70 degrees C, respectively. The activity was strongly enhanced in the presence of Cu(2+), Mn(2+), and Mg(2+) and was completely inhibited by EDTA and sodium azide. The purified laccase exhibited high levels of activity against 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and 2,6-dimethoxyphenol and no activity against tyrosine. The UV-visible spectrum of the purified laccase was the typical spectrum of the blue laccases, with an absorption peak at 600 nm and a shoulder around 330 to 340 nm. The ability of the purified laccase to oxidize a nonphenolic compound, such as veratryl alcohol, in the presence of ABTS opens up new possibilities for the use of bacterial laccases in the pulp and paper industry. We demonstrated that application of the laccase from S. cyaneus in the presence of ABTS to biobleaching of eucalyptus kraft pulps resulted in a significant decrease in the kappa number (2.3 U) and an important increase in the brightness (2.2%, as determined by the International Standard Organization test) of pulps, showing the suitability of laccases produced by streptomycetes for industrial purposes.     

Laccase production at reactor scale by filamentous fungi

Laccases have received much attention from researchers during the past decades due to their broad substrate specificity and to the fact that they use molecular oxygen as the final electron acceptor instead of hydrogen peroxide as used by peroxidases. This makes laccases highly interesting for a wide variety of processes, such as textile dye decolouration, pulp bleaching, effluent detoxification, biosensors and bioremediation.

The successful application of laccases to the above-mentioned processes requires the production of large quantities of enzyme at low cost. Filamentous fungi are able to produce laccases in high amounts, however, an efficient production system at bioreactor scale is still lacking. This is mainly due to the fact that laccase production by wild-type strains of filamentous fungi is linked to secondary metabolism, which implies that the following drawbacks must be overcome: uncontrolled fungal growth, the formation of polysaccharides around mycelia and the secretion of certain compounds (i.e. proteases) that inactivate laccases. This review summarizes the current status of laccase production by wild-type strains of filamentous fungi at the bioreactor scale.     

制浆造纸生物技术研究进展

制浆造纸工业是国民经济的主要支柱之一,但也是资源消耗和环境污染的大户。近年来,国外用于制浆造纸工业的生物技术研究异常活跃,除废水生物处理外,木聚糖酶助漂、脂肪酶控制树脂、木片真菌预处理和酶法废纸脱墨等工艺已经在生产中得到实际应用,生物制浆、漆酶漂白工艺也已进入中试阶段。结合以草浆为主的特点,我国的制浆造纸生物技术研究也已日趋活跃起来。     

Biological bleaching of chemical pulps

Use of biotechnology in pulp bleaching has attracted considerable attention and achieved interesting results in recent years. Enzymes of the hemicellulolytic type, particularly xylan-attacking enzymes, xylanases are now used commercially in the mills for pulp treatment and subsequent incorporation into bleach sequences. The aims of the enzymatic treatment depend on the actual mill conditions and may be related to environmental demands, reduction of chemical costs or maintenance or even improvement of product quality. The use of oxidative enzymes from white-rot fungi, that can directly attack lignin, is a second-generation approach, which could produce larger chemical savings than xylanase but has not yet been developed to the full scale. It is being studied in several laboratories in Canada, Japan, the U.S.A. and Europe. Certain white-rot fungi can delignify kraft pulps increasing their brightness and their responsiveness to brightening with chemicals. The fungal treatments are too slow but the enzyme manganese peroxidase and laccase can also delignify pulps and enzymatic processes are likely to be easier to optimize and apply than the fungal treatments. Development work on laccase and manganese peroxidase continues. This article presents an overview of developments in the application of hemicellulase enzymes, lignin-oxidizing enzymes and white-rot fungi in bleaching of chemical pulps. The basic enzymology involved and the present knowledge of the mechanisms of the action of enzymes as well as the practical results and advantages obtained on the laboratory and industrial scale are discussed.     

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     

嗜热和嗜碱木聚糖酶研究进展

木聚糖酶是降解半纤维素主要成分木聚糖的关键酶,广泛应用在食品、饲料、制浆造纸、生物脱胶等行业。特别是在造纸工业中,木聚糖酶显示出巨大的应用潜力,已成为国内外研究的热点。纸浆漂白工艺中需要酶在高温碱性条件下发挥作用。目前,主要通过筛选野生型木聚糖酶资源和对现有中性中温木聚糖酶分子改造的方法获得嗜热碱木聚糖酶。文中就嗜热嗜碱木聚糖酶的筛选、嗜热嗜碱机制研究及分子改造进展进行了综述,并对其前景进行了展望。     

Kraft Pulp Biobleaching and Mediated Oxidation of a Nonphenolic Substrate by Laccase from Streptomyces cyaneus CECT 3335

A new laccase (EC 1.10.3.2) produced by Streptomyces cyaneus CECT 3335 in liquid media containing soya flour (20 g per liter) was purified to homogeneity. The physicochemical, catalytic, and spectral characteristics of this enzyme, as well as its suitability for biobleaching of eucalyptus kraft pulps, were assessed. The purified laccase had a molecular mass of 75 kDa and an isoelectric point of 5.6, and its optimal pH and temperature were 4.5 and 70°C, respectively. The activity was strongly enhanced in the presence of Cu²⁺, Mn²⁺, and Mg²⁺ and was completely inhibited by EDTA and sodium azide. The purified laccase exhibited high levels of activity against 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and 2,6-dimethoxyphenol and no activity against tyrosine. The UV-visible spectrum of the purified laccase was the typical spectrum of the blue laccases, with an absorption peak at 600 nm and a shoulder around 330 to 340 nm. The ability of the purified laccase to oxidize a nonphenolic compound, such as veratryl alcohol, in the presence of ABTS opens up new possibilities for the use of bacterial laccases in the pulp and paper industry. We demonstrated that application of the laccase from S. cyaneus in the presence of ABTS to biobleaching of eucalyptus kraft pulps resulted in a significant decrease in the kappa number (2.3 U) and an important increase in the brightness (2.2%, as determined by the International Standard Organization test) of pulps, showing the suitability of laccases produced by streptomycetes for industrial purposes.     

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).     

Heterologous laccase production and its role in industrial applications

Laccases are blue multicopper oxidases, catalyzing the oxidation of an array of aromatic substrates concomitantly with the reduction of molecular oxygen to water. These enzymes are implicated in a variety of biological activities. Most of the laccases studied thus far are of fungal origin. The large range of substrates oxidized by laccases has raised interest in using them within different industrial fields, such as pulp delignification, textile dye bleaching, and bioremediation. Laccases secreted from native sources are usually not suitable for large-scale purposes, mainly due to low production yields and high cost of preparation/purification procedures. Heterologous expression may provide higher enzyme yields and may permit to produce laccases with desired properties (such as different substrate specificities, or improved stabilities) for industrial applications. This review surveys researches on heterologous laccase expression focusing on the pivotal role played by recombinant systems towards the development of robust tools for greening modern industry.     

Molecular cloning and characterization of a novel metagenome-derived multicopper oxidase with alkaline laccase activity and highly soluble expression

Lac591, a gene encoding a novel multicopper oxidase with laccase activity, was identified through activity-based functional screening of a metagenomic library from mangrove soil. Sequence analysis revealed that lac591 encodes a protein of 500 amino acids with a predicted molecular mass of 57.4 kDa. Lac591 was overexpressed heterologously as soluble active enzyme in Escherichia coli and purified, giving rise to 380 mg of purified enzyme from 1 l induced culture, which is the highest expression report for bacterial laccase genes so far. Furthermore, the recombinant enzyme demonstrated activity toward classical laccase substrates syringaldazine (SGZ), guaiacol, and 2, 6-dimethoxyphenol (2, 6-DMP). The purified Lac591 exhibited maximal activity at 55°C and pH 7.5 with guaiacol as substrate and was found to be stable in the pH range of 7.0–10.0. The substrate specificity on different substrates was studied with the purified enzyme, and the optimal substrates were in the order of 2, 6-DMP > catechol > α-naphthol > guaiacol > SGZ > 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). The alkaline activity and highly soluble expression of Lac591 make it a good candidate of laccases in industrial applications for which classical laccases are unsuitable, such as biobleaching of paper pulp and dyestuffs processing.     

Screening for novel laccase-producing microbes

AIMS: To discover novel laccases potential for industrial applications. METHODS AND RESULTS: Fungi were cultivated on solid media containing indicator compounds that enabled the detection of laccases as specific colour reactions. The indicators used were Remazol Brilliant Blue R (RBBR), Poly R-478, guaiacol and tannic acid. The screening work resulted in isolation of 26 positive fungal strains. Liquid cultivations of positive strains confirmed that four efficient laccase producers were found in the screening. Biochemical characteristics of the four novel laccases were typical for fungal laccases in terms of molecular weight, pH optima and pI. The laccases showed good thermal stability at 60 degrees C. CONCLUSIONS: Plate-test screening based on polymeric dye compounds, guaiacol and tannic acid is an efficient way to discover novel laccase producers. The results indicated that screening for laccase activity can be performed with guaiacol and RBBR or Poly R-478. SIGNIFICANCE AND IMPACT OF THE STUDY: Laccases have many potential industrial applications including textile dye decolourization, delignification of pulp and effluent detoxification. It is essential to find novel, efficient enzymes to further develop these applications. This study showed that relatively simple plate test screening method can be used for discovery of novel laccases.     

Reutilization of effluents from laccase-mediator treatments of kraft pulp for biobleaching

Several effluents from laccase-mediator treatments of kraft pulp were recovered and subsequently reused with fresh pulp in order to simulate recirculation of effluents during biobleaching. The effluents were used as a new bleaching stage without any modification except enzyme addition. Pulp treated with effluents were afterwards chemically bleached by using the simple sequence LQPo, where L represents the treatment with effluent and laccase addition, Q is a chelating stage and Po is an alkaline peroxide stage. This system showed a promising potential on delignification, with kappa number ranging from 5.5 to 6.6 after LQPo sequence, depending on the type of effluent employed in L stage. Improvements on pulp brightness were also reported compared with control experiment.     

Insights into laccase producing organisms,fermentation states,purification strategies,and biotechnological applications

Laccases are phenol oxidases belonging to the superfamily of multicopper oxidases and are found in bacteria, fungi, lichens, higher plants, and insects. Over the past few decades, laccases and laccase mediator systems (LMS) have found uses in a wide range of technological applications such as textile dye decolorization, industrial wastewater detoxification, pulp bleaching, chemical synthesis, and development of miniaturized biosensors. This has encouraged numerous studies to find and purify laccases with exploitable characteristics. The main aim of the present review is to summarize the rich literature data gained in recent years from the studies on laccases, focusing on the organisms that produce them, the methods used for screening, laccase activity assays, purification strategies, and the application of laccases as eco‐friendly biocatalysts. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1443–1463, 2015     

Reactivities of Various Mediators and Laccases with Kraft Pulp and Lignin Model Compounds

Laccase-catalyzed oxygen delignification of kraft pulp offers some potential as a replacement for conventional chemical bleaching and has the advantage of requiring much lower pressure and temperature. However, chemical mediators are required for effective delignification by laccase, and their price is currently too high at the dosages required. To date, most studies have employed laccase from Trametes versicolor. We have found significant differences in reactivity between laccases from different fungi when they are tested for pulp delignification in the presence of the mediators 2,2(prm1)-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) and 1-hydroxybenzotriazole (HBT). A more detailed study of T. versicolor laccase with ABTS and HBT showed that HBT gave the most extensive delignification over 2 h but deactivated the enzyme, and therefore a higher enzyme dosage was required. Other mediators, including 1-nitroso-2-naphthol-3,6-disulfonic acid, 4-hydroxy-3-nitroso-1-naphthalenesulfonic acid, promazine, chlorpromazine, and Remazol brilliant blue, were also tested for their ability to delignify kraft pulp. Studies with dimeric model compounds indicated that the mechanisms of oxidation by ABTS and HBT are different. In addition, oxygen uptake by laccase is much slower with HBT than with ABTS. It is proposed that the dication of ABTS and the 1-oxide radical of HBT, with redox potentials in the 0.8- to 0.9-V range, are required for pulp delignification.     

Recent developments and applications of immobilized laccase

Laccase is a promising biocatalyst with many possible applications, including bioremediation, chemical synthesis, biobleaching of paper pulp, biosensing, textile finishing and wine stabilization. The immobilization of enzymes offers several improvements for enzyme applications because the storage and operational stabilities are frequently enhanced. Moreover, the reusability of immobilized enzymes represents a great advantage compared with free enzymes. In this work, we discuss the different methodologies of enzyme immobilization that have been reported for laccases, such as adsorption, entrapment, encapsulation, covalent binding and self-immobilization. The applications of laccase immobilized by the aforementioned methodologies are presented, paying special attention to recent approaches regarding environmental applications and electrobiochemistry.     

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.     

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.     

Modification of paper properties by the pretreatment of wastepaper pulp with Graphiumputredinis, Trichodermaharzianum and fusant xylanases

Graphiumputredinis, Trichodermaharzianum and fusant were used in the present study to produce extracellular xylanases, an important industrial enzyme used in pulp and paper industry produced in a minimal medium supplemented with oat spelt xylan (1%, w/v) pH 7.0 at 27+/-2 degrees C. The enzyme was purified to homogeneity by DEAE-Cellulose and Superdex 75 FPLC column, respectively. The enzyme was found to be a monomer as determined by SDS gel electrophoresis. The optimum pH and temperature for purified G. putredinis, T. harzianum and fusant xylanases were 5.0-6.0 and 50-70 degrees C, respectively. Pretreatment of paper pulp with G. putredinis, T. harzianum and fusant xylanases decreased pulp kappa number. Xylanases particularly that of fusant at 5 IU/g pulp concentration and 1.5% pulp consistency at 60 degrees C for 18 h followed by EDED process yielded good quality paper from waste paper pulp. A significant increase in pulp brightness and improvement in various pulp properties, viz. burst capacity, thickness and bulkness of the treated pulp were observed in comparison to the conventional chemical bleaching. Easy purification and high stability of these enzymes makes it amicable for industrial applications.