Microbial enzymes are predominantly produced by submerged fermentations, although some solid-substrate fermentations are used, particularly for the production of extracellular fungal enzymes (Solidsubstrate fermentations).
In fact, the first commercial microbial enzyme preparation was produced via solidsubstrate fermentation. This enzyme, ‘Takadiastase’, a fungal amylase, was produced by culturing Aspergillus oryzae on moist rice or wheat bran. The process was initially developed by Dr Jokichi Takamine and patented in the USA in 1884. However, large-scale production of microbial enzymes was not generally feasible until after the middle of the 20th century. It became possible only after the vast improvements to submerged fermentation technology that followed the development of penicillin fermentations in the 1940s.
Most industrial enzymes are products of batch processes and few are currently produced via continuous fermentation. The fermenters for bulk enzyme production are up to 100m3 capacity, but fine enzymes may be produced on smaller scales of a few hundred litres or less. Most fermenters are stirred tank reactors that are operated under aseptic conditions and use low-cost undefined complex media.
As in the development of any fermentation process, enzyme production processes traditionally begin with the search for a suitable producer organism. Use of GRAS-listed (generally regarded as safe) organisms is an important consideration for enzymes that are to be used in food or medical applications. A programme of microorganism screening and selection is necessary, to determine enzyme properties, such as optimum pH and heat resistance, and examination of the ability to secrete the target enzyme.
Enzymes from thermophiles generally provide several advantages. They are thermostable, able to operate at higher temperatures than enzymes of mesophiles, thereby increasing diffusion rates and solubility, and decreasing both viscosity and the risk of microbial contamination. The fermentation system and conditions for maximum production of the enzyme per unit of biomass, using inexpensive carbon and nitrogen feedstocks, must then be determined.
Apart from enzyme productivity, a further consideration is enzyme stability, which can influence the timing, and operations used in, downstream processing. The level of purification applied varies considerably depending on whether the enzyme is intracellular or extracellular, and on its end use. Downstream processing involves separation, purification, stabilization and preservation for specific methods of protein purification).
Some important industrial microbial enzymes that are used in an immobilized form
| Enzyme | EC number | Source | Product/role |
| Aminoacylase | 3.5.1.14 | Aspergillus oryzae | L-amino acids |
| Amyloglucosidase (glucoamylase) | 3.2.1.3 | Aspergillus niger, Rhizopus niveus | Glucose production from starch |
| Glucose isomerase | 5.3.1.5 | Actinomyces missouriensis, Bacillus coagulans | High fructose corn syrup |
| Hydantoinase | 4.5.2.2 | Flavobacterium ammoniagenes | D- and L-amino acids |
| Invertase | 3.2.1.26 | Saccharomyces cerevisiae, Aspergillus niger | Invert sugar (glucose + fructose) |
| Lactase (β-galactosidase) | 3.2.1.23 | Aspergillus oryzae, Kluyveromyces fragilis | Lactose-free milk and whey |
| Lipase | 3.1.1.3 | Rhizopus arrhizus | Cocoa butter substitutes |
| Naringinase*(hesperidinase) | 3.2.1.40, 3.2.1.21 | Penicillium decumbens | Debittering of citrus fruit juice |
| Nitrile hydratase | 4.2.1.84 | Rhodococcus rhodochrous | Acrylamide |
| Penicillin acylase (penicillin amidase) | 3.5.2.6 | Escherichia coli, Bacillus subtilis | Penicillin side chain cleavage |
| Melibiase (raffinase) (α-galactosidase) | 3.2.1.32 | Aspergillus niger, Saccharomyces cerevisiae | Removal of raffinose from sugar beet extracts |
| Thermolysin (a zinc protease) | 3.4.24.27 | Bacillus thermoproteolyticus | Aspartame (L-aspartyl-L-phenylalanine methyl ester) a low-calorie sweetener |
*Consists of two enzymes, an α-rhamnosidase and a β-glucosidase.
Strain improvement may be attempted to further enhance enzyme productivity. In the past, this has often involved cycles of random mutagenesis and screening. However, other strategies are now available for both organism and protein engineering. Targets for improvement often include increased secretion efficiency for extracellular enzymes and overcoming of the organism’s own regulatory mechanisms.
The latter may involve attempts to relieve catabolite repression, a common regulatory mechanism for many hydrolytic enzymes. Other improvements may be achieved by enhancing mRNA half-life and increasing gene dosage through chromosomal amplification or by plasmid amplification, if the enzyme is plasmid encoded. Targets for enzyme (protein) engineering are enhancement of enzyme activity, improved stability, altered pH optima or temperature tolerance, modified specificity and general improvements to their industrial performance. However, such changes involve the manipulation of specific amino acid constituents and are dependent on prior knowledge of the amino acid sequence of the protein.
Today, if a useful enzyme is identified in a microorganism, plant or animal, which is itself difficult to culture, or if little is known of its physiology and biochemistry, other strategies for commercial production can be adopted. Rather than proceeding with extensive
research and development programmes to facilitate the production of the enzyme by the natural producer organism, the structural gene for the enzyme can be transferred into a selected, readily cultivated, ‘host’ microorganism, along with appropriate mechanisms
for control. Genetic engineering now makes possible the expression of the gene coding for almost any enzyme, no matter what its origin. This enables enzyme manufacturers to develop rapidly a process for producing large quantities of the enzyme.
Expression in GRAS-listed organisms provides obvious advantages. Prime candidates for this role are species within three genera of microorganisms, namely Bacillus, Aspergillus and Saccharomyces. Their biology is well understood and they have proved safe to handle, quick to grow and can produce high yields of enzymes, many of which can be excreted into the fermentation medium. A further advantage is that the medium and conditions under which they grow and perform well are already known, thus minimizing further costly experimentation to optimize fermentation conditions.
Reference Sources:
- https://www.microtek.ac.in/Adminpanel/MicroData/B.Sc/BOOK-BIOLOGICAL PRODUCTION OF
ENZYMES.pdf - https://www.sciencedirect.com/science/article/pii/B9780444636638000033
- https://link.springer.com/chapter/10.1007/978-3-031-42999-6_6
- https://www.researchgate.net/publication/344361569_Debittering_of_citrus_juice_by_different_processing_methods_A_novel_approach_for_food_industry_and_agro-industrial_sector
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