Cellulase recovery

Recovery of cellulases from wheat-straw hydrolysis fermentation broth [83]... 

However, we have observed that values obtained with crude extracts were only qualitative. Often, they did not accurately estimate the quantities of the individual enzymes present. Inhibitors were typically present that caused the underestimation of certain enzymes ie.g., ligninases Table II) and that could potentially mask less dominant enzymes. Also, certain polysaccharidases e.g., hemicellulases) were often overestimated due to the action of non-specific or synergistic enzymes e.g., other hemicellulases or cellulases) (9,14), This artifact resulted in low apparent recovery of a given activity and only moderate increases in specific activity upon purification of the major corresponding enzyme present, in spite of the fact that SDS polyacrylamide gels indicated good recovery and substantial removal of contaminants (14),... 

Once the crude cellulase solution was obtained, it was concentrated and at the same time separated from most of the salts (left from the fermentation) in the enzyme solution. The steps which accomplished this were (1) addition of ammonium sulfate (75% saturation) to precipitate protein (2) recovery of the protein as a pellet by centrifugation at 12,000 rpm for 15 min (3) redissolution of the protein in 0.1M sodium phos-phate-0.2mM EDTA buffer (pH 6.8) (4) desalting on a 1.5 X 45 cm Sephadex G-25 column and (5) lyophilization to obtain concentrated enzyme followed by a final ammonium sulfate precipitation to obtain precipitated enzyme. 

The development of the sequential elution methods makes it possible not only to cleanly fractionate the three cellulase components, but to do the fractionation with very little loss of enzyme. The total recovery of major enzyme components, summarized in Table III, is considerably higher than those reported previously by other researchers (8,9). Table III also gives the molecular weights of the three enzyme components. 

A number of relatively new methods are being investigated to improve the recovery of small molecules. These methods include elec-trokinetic separators with bipolar membranes, simulated moving-bed chromatography and supercritical fluid extraction. The latter is practiced for food components. It has also been described for proteins but has not yet found wide acceptance in this field. A fastgrowing field is the production of bioethanol via fermentation processes either from milled com or from recycled biomass. The fermentation and saccharification processes can occur simultaneously in the fermenting tank by means of saccharification enzymes (amylases, cellulases). 

Solutions containing active enzymatic proteins (protease, lipase, trypsin, pepsin, prophase, or cellulase) or their mixtures, adjusted to the nature of the matrix of the solid material of biological origin [79, 83, 84]. The aim of the procedure is to break up proteins, polysaccharides, or fat chains and release the constituent amino acids, sugars, or short aliphatic chains. Enzymatic decomposition of the matrix can be considerably enhanced by application of ultrasound the process can, for example, increase the efficiency of disintegration of cell walls in yeast and thus improve the recovery of selenium by as much as 20 % [85]. 

Cellulase enzymes break down cellulose and beta-glucan associated with the cell walls, thereby decreasing the viscosity of the mash and increasing the ease of the juice recovery. The conversion of cellulose and beta-glucans into soluble... 

Fruit juice clarification by pectinases and cellulases is another interesting application. In the conventional process after the enzymatic reaction in the pulp treatment step takes place, filtration over diatomaceous earth follows. This filtration-type process produces a lot of solid waste, and results in costly enzyme loss. MBR are appropriate for such application either for enzyme recovery and recycle or in the form of a more compact CMR type system, with the biocatalyst immobilized on the membrane itself [4.58]. 

For the concentration of enzyme solutions it is important to use methods giving the highest possible yield of enzymic activity—i.e., loss of activity should be brought to a minimum. None of the above methods will in all cases fulfill these requirements since denaturation and consequently low yield of enzymic activity are reported for all these methods. To avoid denaturation owing to precipitation as well as owing to freezedrying and evaporation, Petterson et al. (39) introduced the dextran gel concentration method of Flodin et al. (12) into cellulase research. This method was found to give essentially quantitative recovery of cellulase activity from culture solutions of Polyporus versicolor. 

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