MOLECULAR BIOTECHNOLOGY Volume XX, 2002 PLEASE PROVIDE SHORT TITLE 1 Uncorrected Proof Copy Uncorrected Proof Copy Uncorrected Proof Copy REVIEW 1 Molecular Biotechnology 2002 Humana Press Inc. All rights of any nature whatsoever reserved. 1073–6085/2002XX:XXXX–XXX/$xx.xx *Author to whom all correspondence and reprint requests should be addressed: Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001, Lisboa, Portugal. E-mail: paula.pereira@mail.ineti.pt Abstract A New Look at Xylanases An Overview on Purification Strategies Paula Sá-Pereira,* Helena Paveia, Maria Costa-Ferreira, and Maria Raquel Aires-Barros Interest on xylanases from different sources has increased markedly in the past decade, in part because of the application of these enzymes in the pulp and paper industry. Purity and purification costs are becoming important issues in modern biotechnology as the industry matures and competitive products reach the mar- ketplace. Thus, new paths for successful and efficient xylanase recovery have to be followed. This article reviews the isolation and purification methods used for the recovery of microbial xylanases. Origins and applications of xylanases are described, highlighting the special features of this class of en- zymes, such as the carbohydrate-binding domains (CBDs) and their importance in the development of affin- ity methodologies to increase and facilitate xylanase purification. Implications of recombinant DNA technology for the isolation and purification of xylanases are evaluated. Several purification procedures are analyzed, taking into consideration the sequence of the methods used in each and the number of times each method is used. New directions to improve xylanase separation and purification from fermentation media are described. Index Entries: Xylanase; purification strategies; CBD; biospecific affinity. 1. Introduction Wood and pulp fibers constitute renewable raw materials that can be processed with biological catalytic systems. Xylan, the major component of hemicellulose, and cellulose, account for more than 50% of all plant biomass. Both polymers to- gether constitute the most abundant organic car- bon source on Earth (1–11). The energy content of both xylan and cellulose, based on the total plant biomass on Earth, is equivalent to almost 640-billion tons of oil (11). The development of biotechnological methods based on xylan- and cellulose-degrading enzymes could provide a renewable resource of recycled biomolecules for application in several industrial sectors, such as the chemical and pharmaceutical industries. Interest in xylanases from different sources has increased markedly in the past decade, partly be- cause of the application of these enzymes in the pulp and paper industries, where they have been used for the selective removal of hemicelluloses from kraft pulp prior to pulp bleaching. Before their application, however, the separation and pu- rification of xylanases from complex fermentation media has to be achieved. Purification of xylanases to homogeneity is necessary for detailed biochemical and molecular studies, and for the successful determination of their primary amino-acid sequences and their three-dimensional structures. Several purification techniques for protein purification have undergone intense commercial promotion and use that has led to their wide dis- semination. Protein purification varies from a simple one-step purification procedure to large- scale purification processes. The key to obtaining successful and efficient protein-purification strat- egies is the selection of appropriate techniques AU: First page OK as edited? AU: Proofread chapter care- fully, many edits made to manuscript.