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3.4. Large-Scale Production of Enzymes

Among various enzymes produced at large scale are proteases (subtilisin, rennet), hydrolases (pectinase, lipase, lactase), isomerases (glucose isomerase), and oxidases (glucose oxidase). These enzymes are produced using overproducing strains of certain organisms. Separation and purification of an enzyme from an organism require disruption of cells, removal of cell debris and nucleic acids, precipitation of proteins, ultrafiltration of the desired enzyme, chromatographic separations (optional), crystallization, and drying. The process scheme varies depending on whether the enzyme is intracellular or extracellular. In some cases, it may be more advantageous to use inactive (dead or resting) cells with the desired enzyme activity in immobilized form. This approach eliminates costly enzyme separation and purification steps and is therefore economically more feasible. Details of protein separations are covered in Chapter 11, “Recovery and Purification of Products.”

The first step in the large-scale production of enzymes is to cultivate the organisms producing the desired enzyme. Enzyme production can be regulated and fermentation conditions can be optimized for overproduction of the enzyme. Proteases are produced by using overproducing strains of Bacillus, Aspergillus, Rhizopus, and Mucor; pectinases are produced by Aspergillus niger; lactases are produced by yeast and Aspergillus; lipases are produced by certain strains of yeasts and fungi; glucose isomerase is produced by Flavobacterium arborescens or Bacillus coagulans. After the cultivation step, cells are separated from the media usually by filtration or sometimes by centrifugation. Depending on the intracellular or extracellular nature of the enzyme, either the cells or the fermentation broth is further processed to separate and purify the enzyme. The recovery of intracellular enzymes is more complicated and involves the disruption of cells and removal of cell debris and nucleic acids. Figure 3.24 depicts a schematic of an enzyme plant producing intracellular enzymes.

Figure 3.24.

Figure 3.24. A flowsheet for the production of an extracellular enzyme.

In some cases, an enzyme may be both intracellular and extracellular, which requires processing of both broth and cells. Intracellular enzymes may be released by increasing the permeability of the cell membrane. Certain salts, such as CaCl2, and other chemicals, such as dimethylsulfoxide (DMSO), and pH shift may be used for this purpose. If enzyme release is not complete, then cell disruption may be essential.

The processes used to produce these industrial enzymes have much in common with the processes used to make proteins from recombinant DNA, discussed later.

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