The industrial preparation of paper is a multistep process highly energy demanding and producing large volumes of pollutants (Pokhrel and Viraraghavan, 2004). One of the most energy consuming and costly challenges within the procedure remains the elimination of lignin present in the wood (Sierra-Alvarez R, Lettinga G, 1991). Lignin forms a gangue with cellulose and hemicelluloses, it is responsible of the natural color of pulp and it is a principal component of wastewater increasing significantly COD levels (Luisa et al., 1996). Delignification of wood pulp is traditionally done by non-nature friendly oxidants containing chlorine or oxygen (Weinstock et al., 1996).
In addition, bleaching releases high levels of lignin in wastewater which treatment demands rising costs for elimination of more and more difficult pollutants to isolate (Zhang et al., 2002). Therefore elimination of lignin through cost-effective and nature friendly methods to replace traditional expensive and polluting procedures have being showing increasing interest (Sena-Martins et al., 2008). Usage of enzyme in pulp and paper industry is investigated and the multiple potential applications of laccase in this field of industry define this particular enzyme as a promising alternative to existing costly and polluting processes (Barreca et al., 2003; Shi, 2006). Laccases are enzymes belonging to the multicopper oxidases class. These enzymes are involved in several oxidative functions and particularly in delignification. Laccases, together with mediators, are able to delignify wood pulp by an oxidation chain reaction leading to lignin oxidation respecting the integrity of cellulose (Gamelas et al., 2005).
Laccase are also used in biopulping which is applicable to both mechanical and chemical pulps. Advantages of using laccase mediator system (LMS) in biopulping results in reduction of refining energy, increase of mill throughput in mechanical pulping, enhanced paper strength properties, reduction of pitch problems, improved yield, and reduced environmental impact. Finally, recent studies showed that laccase used to treat white water from TMP reduced pitch by 50% and reduced lignans, lignin and other hydrophilic extractives by 90% (Windsten and Kandelbauer, 2008).
Further application of laccase in pulp and paper industry showed applicability in deinking paper when recycled, in manufacturing of lignocellulose based composite materials such as fiberboards and in grafting various phenolics acid derivatives onto kraft pulp fibers (Lund and Ragauskas, 2001; Chandra and Ragauskas, 2002). Historically, fungal laccases were largely studied because of their high redox potential and enzymatic activity (Cho et al., 2004). However, industrial usages of fungal laccases are limited since the yield of fungal protein production and the fermentation time of fungi are not economically viable. In the other hand, bacterial laccases are easy to produce in large quantity but are less active than fungal ones (Kunamneni A et al., 2008).
Current developments in biotechnology and microbiology allow yield improvements of bacterially produced enzymes together with considerable production price reduction allowing economically viable applications in industry (Gavrilescu and Chisti, 2005). Other challenges faced when enzymes are wished to be implemented within an industrial process are the conditions at which the process is conducted. Differently to chemicals, enzymes perform optimally in conditions rather different than the industrial ones. The pH, reaction temperature, solvents and reaction time rarely match with natural conditions at which microorganisms are growing. At MetGen Oy, we use a technology platform to modify enzymes properties by mutations of their amino acid sequence in order to increase their activity and fit the conditions required by industry (pH, temperature, solvents tolerance...).
An intelligent design of mutations to be introduced within the sequence and a subsequent screen of the various mutants in industrial conditions permit the selection of the best clone to be produced in large scale and implemented in the industrial process.
References:
Barreca AM, Fabbrini M, Galli C, Gentili P, Ljunggren S. Laccase/mediated oxidation of a lignin model for improved delignification procedures. J Mol Catal B-Enzym (2003); 26: 105-10
Chandra, RP, Ragauskas, AJ. Evaluating Laccase-Facilitated Coupling of Phenolic Acids to High-Yield Kraft Pulps. Enzyme Microbiol. Techn. (2002); 30(7) 855-861
Cho NS, Shin W, Jeong SW, Leonowicz A. Degradation of Lignosulfonate by Fungal Laccase with Low Molecular Mediators. Bull. Korean Chem. Soc. (2004) 25, 10, 1551-1554
Gamelas JAF, Tavares APM, Evtuguin DV, Xavier AMB. Oxygen bleaching of kraft pulp with polyoxometalates and laccase applying a novel multi-stage process. J Mol Catal B-Enzym (2005); 33: 57-64
Gavrilescu M, Chisti Y. Biotechnology-a sustainable alternative for chemical industry. Biotechnol Adv. (2005) Nov;23(7-8):471-99
Kunamneni A , Camarero S, García-Burgos C, Plou FJ, Ballesteros A, Alcalde M. Engineering and Applications of fungal laccases for organic synthesis. Microbial Cell Factories 2008, 7:32
Luisa M, Goncalves FC, Steiner W. Purification and characterization of laccase from a newly isolated wood-decaying fungus. Enzymes for pulp and paper processing. Eds.:Jeffries TW, Viikari IL. American Chemical Society. Washington, USA, (1996); 258-66
Lund, M, Ragauskas, AJ. Enzymatic Modification of Kraft Lignin Through Oxidative Coupling with Water-Soluble Phenols. Appl. Microbiol. Biotechnol. (2001); 55, 699
Pokhrel D, Viraraghavan T. Treatment of pulp and paper mill wastewater - a review. Science of The Total Environment (2004) Volume 333, Issues 1-3, 15 October 2004, Pages 37-58
Sena-Martins G, Almeida-Vara E, Duarte JC. Eco-friendly new products from enzymatically modified industrial lignins Industrial Crops and Products (2008) 27, 2, 189-195
Shi J: CN1844572 A (2006)
Sierra-Alvarez R, Lettinga G. The Methanogenic Toxicity of Wastewater Lignins and Lignin Related Compounds. J. Chem. Tech. Biotechnol. (1991), 50, 443-455
Weinstock IA, Atalla RH, Reiner RS, Moen MA, Hammel KE. A new environmentally benign technology and approach to bleaching kraft pulp. Polyoxometalates for selective delignification and waste mineralization. New J. Chem. (1996) 20,269-275
Widsten P, Kandelbauer A. Laccase applications in the forest products industry: A review. Enzyme and Microbial Technology (2008); 42, 4 : 293-307
Zhang X, Stebbing DW, Soong G, Saddler JN, Beatson RP. A combined fungal and enzyme treatment system to remove TMP/newsprint mill white water substances. Tappi J (2002);1:26-32
