MANUFACTURE OF COMMERCIAL ENZYMES

VI. MANUFACTURE OF COMMERCIAL ENZYMES

For the most part, this discussion focuses on microbial enzymes rather than on enzymes from plant and animal sources. Plant and animal strains, breeds, and cultivars are not selected specifically for enzyme production. Rather, enzymes are byproducts recovered from plants and animals selected, bred, or propagated for other reasons. Additionally, strain and breed selection in plants and animals is a difficult, time-consuming, tedious process. Microorganisms, on the other hand, do not pose these limitations, and strain selection and the propagation of selected strains are rapidly achieved because of their short generation times (Aunstrup, 1980).

Microorganisms can be isolated from such natural sources as soil, compost, and rotting wood or can be obtained from culture collections. Enrichment cultures (e.g., using the substrate of interest as a sole carbon source or nitrogen source) select organisms from a Microorganisms can be isolated from such natural sources as soil, compost, and rotting wood or can be obtained from culture collections. Enrichment cultures (e.g., using the substrate of interest as a sole carbon source or nitrogen source) select organisms from a

The ultimate objectives in strain selection are to isolate microbial strains that grow on inexpensive media and give a high, constant yield of enzyme in a short time. The ideal strain should produce a minimal amount of secondary metabolites and secondary enzyme activities in the growth media. Recovery of the produced enzyme should be simple and inexpensive and should lead to a stable product that can be handled safely. The process for producing the enzyme must be safe to the plant operators, and the effluents should not be hazardous.

The selected strain is propagated and maintained in such a manner that the mutated properties are retained. For commercial manufacture of enzymes, the strains are grown either as submerged cultures or as semisolid fermentation products. Numerous factors that affect the performance of cultures during fermentation have been discussed by Frost (1986). Growth of the culture is synonymous with product secretion in fermentations. The product has to be isolated and partially purified. In such processes, the first operation is to separate insolubles from solubles. Insolubles in submerged fermentation may be cell mass, but in semisolid fermentation this can include unutilized growth media constituents in addition to the cell mass. For enzymes secreted into the growth medium the insoluble material is a byproduct, but in instances where the enzyme may be located within the cells, the insoluble material is of interest and the supernatant is considered to be a by- product (Frost, 1986).

Endocellular enzymes are isolated by rupturing the cells using such methods as ball milling, freeze-thawing, pH, temperature, or osmotic shocking, high-pressure homogeniza- tion, or cell lysis with lysozyme. The resulting materials have to be separated from the cell debris, leaving cell-free extracts. Exocellular enzymes have already been made cell- free by separation from the growth medium.

Cell-free extracts can be purified by a series of consecutive fractionation and concen- tration steps including ion exchange chromatography, reverse osmosis, or ultrafiltration; or the protein can be precipitated by treatment with organic solvents, polyethylene glycol, dextran, polyacrylic acid, or ammonium sulfate. Precipitation of the protein is one method by which enzyme can be separated from carbohydrates in solution.

Enzymes in crude form can then be dried to obtain powders, or they can be manufac- tured as a concentrated liquid. Enzymes for use in food processing have to meet several other safety guidelines. For example, microbiological specifications such as freedom from Salmonella and Escherichia coli can be met by filtering enzyme solutions through 0.45 µm or smaller filters and by preventing contamination in the remaining operations as well. For drying enzymes, not only should the product be free from bacteria, but it should also

be nonhygroscopic and dust-free. For either type of preparation, the material has to be be nonhygroscopic and dust-free. For either type of preparation, the material has to be