Chapter 7
Approaches to Cellulase Purification Christian Paech
1
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Cellulases comprise a mixture of enzymes whose complexity is determined by gene structure and by post-translational events. They bring about the enzymatic degradation of cellulose the rate of which depends on the synergistic interaction of individual components involved. To study this phenomenon isolated, purified enzymes must be available. The merits of techniques employed in cellulase purification are discussed and a table is provided which lists the methods used in the preparation of a particular cellulase. The need for purifying enzymes has been eloquently advocated by Arthur Kornberg (7) based on Ephraim Racker's dogma "don't waste clean thinking on dirty enzymes." The extent to which an enzyme can be purified is set by the sensitivity of detection methods although with rapid advances in analytical techniques this limit may be difficult to reach. The burden of enzyme preparation can be eased i f only those impurities are eliminated that interfere with the analyte. W a l k i n g the fine line between removing and tolerating impurities is a practical approach to answering these questions: what degree of purity, stability, and authenticity is necessary, and what quantity of a protein is needed to unambiguously determine its performance and its molecular characteristics. These considerations should precede the bench work as they shape the separation strategy and determine the outcome of the attempted purification. Often analogy to an existing method is the best approach to purifying enzymes. In many cases, however, there is no precedence because the enzyme has never been described before or it is from a significantly different source material (e.g., a protease from an extracellular microbial broth versus a liquid laundry detergent). Then, general advice on how to practice the art and science of protein purification may be found i n one of several excellent reviews on this subject (2-5). 1
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In Enzymatic Conversion of Biomass for Fuels Production; Himmel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.
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7. P A E C H
Approaches to Cellulase
Purification
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Regardless of the approach, the first step i n protein purification has the greatest impact on its outcome. Here, efforts should be made to remove non-proteinaceous contaminants - even at the expense of yield - as they might seriously interfere with subsequent steps. Equally important is to remember that the percentage of the desired protein lost in every step increases as purification proceeds, i.e. the total number of purification steps must be kept at a minimum. A n overall yield of less than 1% after 5 to 6 steps is a sobering experience. In any source material quite a few enzymes are unique i n their physical-chemical properties and provide for easy purification. For example, alkaline proteases from the extracellular space of microbes often have an isoelectric point >10 and are recovered in pure form simply by cation exchange chromatography (9). Ribulose 1,5bisphosphate carboxylase/oxygenase from higher plants is a soluble protein with a mass of 520,000 D a which lends itself to rapid purification by sucrose gradient centrifugation (10,11). There are no such advantages when the desired protein resides matrix-bound, is part of an isozyme mixture, or is subject to multiple posttranslational modifications by proteases and de-glycosylating enzymes. A case in point are the enzymes involved i n cellulose depolymerization. First, microorganisms accommodated the topographical diversity of the substrate, cellulose, by producing cellulases, that are structurally different but catalyze the same chemical reaction, the hydrolysis of a 6-1,4-glucosylic bond. Thus, one should a priori not expect a great diversity of protein structure among cellulases. Indeed, cellobiohydrolase II and endoglucanase I from Trichoderma reesei could only be separated by immunoadsorption (72). Second, some microorganisms coped with environmental adversity by forming tightly aggregated cellulolytic complexes which resist fragmentation into active components (13-16). Whether it is structural multiplicity or protein aggregation, nature clearly did not have the needs of a biochemist in mind when evolving these enzymes. Third, the ubiquitous protein recycling tools, proteases (and de-glycosylating enzymes in a supportive role), add another dimension to enzyme multiplicity. Successive removal of glyco residues and nicking of the protein structure rapidly degenerates the enzyme spectrum, a process particularly prevalent among cellulases. Clearly, fractionation of cellulases to determine genetic or process-related multiplicity presents a rather demanding separation problem. The original observation by Reese and coworkers (17,18) on the accelerated cellulose degradation by T. viride enzymes has laid the foundation for the hypothesis that cellulases are acting synergistically on cellulose (19-37). This mutually supportive function of cellulases is difficult to measure without access to the components i n pure form. However, the information is needed to improve on the commercial application of cellulases. T h e B a s i c C e l l u l o l y t i c System The term 'cellulases' is a rather global expression for a consortium of enzymes which collectively brings about the degradation of cellulose to glucose. Cellulases have been identified in many organisms: fungi, bacteria, protozoa, nematodes, plants and many other organisms (38). W h i l e this list is constandy being amended or corrected (e.g. (25)) and though new ideas may come from understanding how various organisms have solved for themselves the problem of utilizing cellulose, it is clear that, for
In Enzymatic Conversion of Biomass for Fuels Production; Himmel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.
132
ENZYMATIC CONVERSION OF BIOMASS FOR FUELS PRODUCTION
practical reasons, initially only microorganisms are being viewed as promising candidates for the production and the commercial application of their cellulases. This may change, however, as more genes for cellulases become available and the technology i n metabolic engineering advances. M o s t of the early knowledge on cellulases is derived from studies of fungal systems, of which Trichoderma reesei (formerly T. viride) has become the predominant industrial producer. The specific activity of fungal cellulases is generally low (
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