In this article we will discuss about the large scale production of enzymes by fermentation. Learn about:- 1. Sources of Enzymes 2. Selection of Microorganisms 3. Mechanisms of Enzyme Biosynthesis 4. Manipulation of Enzyme Biosynthesis 5. Kinetics of Enzyme Biosynthesis 6. Cultivation Techniques of Microorganisms.
- Sources of Enzymes
- Selection of Microorganisms
- Mechanisms of Enzyme Biosynthesis
- Manipulation of Enzyme Biosynthesis
- Kinetics of Enzyme Biosynthesis
- Cultivation Techniques of Microorganisms
1. Sources of Enzymes:
Industrial enzymes are produced from plants, animals, and microorganisms, but manufacture from the first two groups is limited for several reasons. Cultivation of plants is restricted to areas where climate is suitable. It is generally seasonal, impeding steady enzyme production.
As the concentration of enzymes in plant tissues is generally low, processing of large amounts of plant material is necessary. For example, approximately the annual yield of a tree is required for the production of 0.45 kg (lib) papaya latex. On the other hand, enzymes of animal origin are by-products of the meat industry and for this reason limited in supply. Moreover, they often compete with other end users for the supply of suitable glands.
In contrast, microbial enzymes can be produced in amounts meeting all demands of the market. Seasonal fluctuations of raw material do not count and there are possibilities for genetic and environmental manipulation of bacteria and fungi to give increased yields of desired enzymes in a way not possible with higher organisms. Moreover, the diversity of enzymes available from microorganisms is very great. Lastly, microbial enzymes present a wide spectrum of characteristics that makes them utilizable for quite specific applications.
The first step in the manufacture of an enzyme involves the selection of an organism suitable to produce the desired enzyme in amounts as large as possible.
The general aspects of this procedure can be outlined as follows:
(1) Extracellular enzymes are preferred, because difficult and costly methods of cell disruption are not necessary. As compared with intracellular enzymes, they are present in a relatively pure form in the culture liquor. Intracellular enzymes are industrially used to a lesser extent because of difficult procedures of cell disruption and separation of contaminating cell components.
(2) High yields of enzymes should be obtained with an economical time required for culture production.
(3) The strain must be stable with respect to productivity, requirement for culture conditions, and sporulation.
(4) The organism should be able to grow on cheap substrates.
(5) Synthetic activity should be as far as possible in the direction of the desired enzyme. Formation of interfering by-products should be low.
(6) Clarification of the culture liquor or extract should be possible without difficulties.
(7) The strain must not produce toxic substances and should be free of antibiotic activities. It should not belong to related strains that synthesize toxins.
Mostly, enzymes with particular properties, e.g., with respect to stability and activity, are desired. This requires special screening programs. As has been demonstrated, the technique of screening is also influenced by the method of cultivation of the organisms at the industrial production stage.
This is particularly valid for fungi. If the submerged culture technique is employed, an appropriate selection of strains should be done at an early stage of the program. It is worth mentioning that shaken cultures behave differently from deep cultures. For this reason shaken culture screens only permit a first rough selection. Further selection in deep culture is necessary.
Detection of mutants with increased productivity is difficult. Methods based on checking for halo formation is possible with extracellular enzymes but often worthless for industrial purposes. Therefore, attempts have been undertaken to find out correlations between production of a particular enzyme and one, or several, distinct physiological or morphological characteristics.
For example- Nasuno (1972) correlated formation of Aspergillus oryzae-tyye alkaline protease with smooth conidia and production of Aspergillus sojae-type alkaline protease with echinulate or tuberculate conidia. This correlation was valid for a large number of Aspergillus species tested. For detection of mutants with altered regulation mechanisms of enzyme biosynthesis see Demain (1971).
Metabolism is principally regulated by a change of the rate of enzyme reactions. Therefore, regulation of metabolism is mainly a problem of kinetics.
As outlined previously, the rate of most enzyme reactions can be described by the Michaelis-Menten equation:
Where [S] (substrate concentration), [Eo] (total enzyme concentration), k-2 (rate constant), and KM (Michaelis-Menten constant) are independent variables. The enzyme concentration is varied by two mechanisms, namely, by controlled protein synthesis and controlled protein degradation.
For control of enzyme synthesis the microbial cells bring into action the mechanisms of induction and repression. Since Jacob and Monod (1961) outlined their theory of enzyme regulation, these mechanisms have become increasingly understandable, and practical applications have led to sometimes drastic increases of enzyme production by environmental and genetic manipulations.
For a basic treatment of the regulation of enzyme biosynthesis see, e.g., Clarke (1971) and Demain (1972A).
There are only few enzymes synthesized in substantial concentration under all conditions of growth. These “constitutive” enzymes include, for example, the enzymes of the hexose-monophosphate pathway. Many of the enzymes used commercially fall into the inducible group. Their biosynthesis requires the presence of substrate in the medium.
For example- starch acts as an inducer for amylase. Dextrin, a degradation product of starch, was found to give 16% higher amylase production than did starch in Bacillus polymyxa, whereas maltose induced the synthesis of only 50% of the activity induced by starch. Often inducers are analogs or derivatives of the substrate, e.g., isopropyl-β-D-thiogalactoside for β-galac- tosidase.
In other cases compounds structurally similar to the substrate may serve as inducer, e.g., sophorose for cellulase. For polymer substrates which cannot enter the microbial cell, it has been shown that the dimer is the true inducer, such as cellobiose for cellulase. However, the dimers are active as inducers only when they are present in very low concentrations. At higher levels catabolite repression occurs.
Thus, it appears that the inductive effects of the polymers result from their slow hydrolysis to dimers which are consumed by the organism as rapidly as they are formed. The same result can be achieved when slowly metabolizable derivatives of the dimers are used, e.g., sucrose monopalmitate for invertase synthesis in yeasts and molds. Table 15.1 is a representation of inducers active toward commercial enzymes.
Repression Mechanisms—Feedback Repression:
Repression mechanisms are of the feedback or of the catabolite type. Feedback repression means that biosynthesis of an enzyme is inhibited when end products of a pathway are accumulated or added to the growth medium. For example, protease production in many bacilli is repressed in certain amino acid- containing media, and protease formation by Aspergillus niger is apparently sensitive to repression by sulfur-containing amino acids.
Catabolite repression is very important in commercial enzyme production since in many of the strains employed this type of regulation is effective. It occurs when cells are grown rapidly on readily metabolizable carbon sources. The classical example of catabolite repression is the repression of β-galactosidase in Escherichia coli by glucose (“glucose effect”).
The repressed enzymes can be of the constitutive or of the inducible type, but in most cases inducible enzymes are involved. By the intervention of catabolite repression it is ensured that when several substrates are present, only the enzymes acting on the best substrate will be formed and wasteful production of other enzymes avoided. Some examples of repressible enzymes are shown in Table 15.2.
The mechanism of carbon catabolite repression in bacteria is now largely understood. However, comparatively little is known about the mechanism(s) by which readily utilizable C sources repress the synthesis of many enzymes involved in the carbon metabolism of fungi; and still less is known about the way in which carbon catabolite repression is integrated with ammonium repression of enzymes involved in nitrogen metabolism.
Investigations by Perlman and Pastan (1969) indicate that inhibition of cyclic 3′,5′-adenosine monophosphate formation holds a key position in catabolite repression. In Escherichia coli, its intracellular concentration is depressed 1000-fold by growth on glucose, whereas the addition of this nucleotide reverses catabolite repression of many enzymes.
4. Manipulation of Enzyme Biosynthesis:
A number of methods are available to overcome any one of the control mechanisms which may exert an inhibiting effect on the production of large amounts of a given enzyme. These techniques can be divided into two main categories: manipulation of the genetic function of the organism, and manipulation of the environment of the organism.
The methods of genetic manipulation include the classical techniques of mutant formation and a class of novel techniques which is often termed “genetic engineering.” While mutant formation is quite usual in enzyme manufacture, genetic engineering techniques are restricted to research laboratories, unless easier handling permits their introduction into industrial practice.
There are two ways in which mutations can cause overproduction of enzymes. The first one is concerned with an alteration in the regulation mechanisms. Such mutational events effect removal of inducer requirement, resistance to end product repression, and resistance to catabolite repression. The second group of mutations leads to an increase in copies of the gene responsible for the production of the enzyme.
Genetic engineering means transfer of genes from one strain to another. Terms such as “plasmid transfer,” “phage escape synthesis,” etc., may be cited to characterize the methods employed.
Manipulations of the environment enable the biochemical engineer to overcome inhibition of enzyme biosynthesis as caused by regulatory mechanisms, by selection of suitable medium composition, or culture conditions.
In the following methods of genetic and environmental manipulations, which lead to substantial increases in enzyme production, are enumerated. For more details see Demain (1972B).
For inducible enzymes the application of two methods is possible- (1) mutation to constitutivity, or (2) incorporation of inducers into the medium. Often the most potent inducers are nonmetabolizable substrate analogs. For industrial practice it is important for a particularly expensive or not readily available inducer to be successfully replaced by compounds which can be converted by the organism to the required inducer.
End product repression of enzyme biosynthesis can be avoided by several means:
(1) Avoiding presence of end products as medium constituents. For instance, protease production by Aspergillus niger is derepressed under conditions of sulfate limitation.
(2) Limitation of end product accumulation is generally possible by adding an inhibitor of the pathway to the medium. External accumulation can further be limited by using mixed cultures with a second organism that metabolizes the repressive substance and by applying dialysis or ultrafiltration techniques in the fermentor system.
Internal buildup of end product co-repressors can be limited by starving an auxotroph mutant of the end product required for growth. There are several means: limited feeding of the end product, using slowly utilized derivatives of the required end product, growing partial auxotrophs (“leaky mutants”) in the absence of their end product requirement.
(3) Selection of regulatory mutants which are not repressed by end products (constitutive mutants). A commonly used method is selection for resistance to a toxic analog of the end product, but other methods are also available.
Catabolite repression is of great importance since many enzymes produced in industry are subject to this type of regulation.
Catabolite repression can be avoided by the following means:
(1) Avoidance of the use of repressing carbon sources in the medium. For example, replacement of fructose by glycerol increases a-amylase production of Bacillus stearothermophilus more than 25-fold.
(2) Derepression of the enzyme synthesis by growth limitation. Suitable means are slow feeding of the repressive substrate and use of slowly metabolizable analogs or derivatives of the substrate. For example- application of sucrose monopalmitate instead of sucrose was found to increase invertase production 80-fold. Cultivation at lower temperatures, addition of toxic substances to the medium, etc., are further methods of growth restriction.
(3) Mutation to resistance against catabolite repression may increase productivity considerably. For example- a yeast mutant has been obtained that produced 2% of its cellular protein in the form of invertase.
Rates of fermentation processes are desirable to know for both engineering and fundamental scientific reasons. Bioengineers are concerned with microbial dynamics from the point of view of process design and optimization, building on the experience in the classical chemical industry. Bio- scientists, on the other hand, are interested in the dynamic response of microorganisms as a tool for gaining insight into the mechanisms of microbial physiology.
However, biochemical processes are extremely complex and sensitive to a number of factors and rendering their mathematical modeling is most difficult. The available models for predicting the causal effects of changes of control variables are not simple and not accurate. This is the reason why commercial fermentation processes have not been significantly optimized in an engineering sense.
Excellent contributions to the development of kinetic models of enzyme formation stem from Terui and his associates (1967). The presented models refer to commercially produced hydrolases. They are based on the assumptions that the rate-limiting ability of the Enzyme-Forming System (EFS) corresponds to mRNA and that the specific rate of enzyme production (ϵ, units.mg-1.hr-1) is proportional to the quantity per cell of mRNA (r, quantity.mg-1), i.e., ϵ α aμ.
For growth-associated enzyme production (ϵ = aμ) the following hypothetical relationship was proposed:
Where μ is the specific growth rate (hr-1), k the monomolecular decay rate constant of the specific mRNA (hr-1), and a and b are the system constants. The second term on the right-hand side of the equation is based on the negative correlation of the change of e with that of μ. It expresses the rate of growth-associated repression exerted at the level of transcription. The third term represents the decay rate of mRNA or EFS.
The preceding model has been shown to be in accord with the actual fermentation processes for the production of α-amylase by Bacillus subtilis, and amyloglucosidase, acid protease, and polygalacturonase by Aspergillus niger. In other cases, such as production of amyloglucosidase, acid protease, polygalacturonase, and Cx-cellulase by Aspergillus niger and Cx-cellulase by Penicillium variabile, where enzyme formation in the stationary growth phase plays a major role in enzyme accumulation, another model was proposed. It concerns remaining mRNA formed in the preceding growing phase and turnover of RNA to mRNA in the non-growing phase.
This model has the form:
Where ϵm is the maximum rate of enzyme production at time tm, when growth has just ceased; is the monomolecular decay constant for cell RNA; and K1 is a system constant. To comment upon the terms on the right-hand side of the equation- The first term represents e due to the mRNA carried over from the growing phase and the second term is the change of e due to the turnover synthesis and degradation of the mRNA.
6. Cultivation Techniques of Microorganisms:
Solid Substrate Cultivation:
This method plays an important role in commercial enzyme production from fungal sources, especially in Japan. Advantages and disadvantages of this method, as compared with the submerged culture technique, have often been analyzed.
Considering modern deep bed processes, the following advantages can be stated as a matter of fact- (1) enzyme yield per unit volume of incubator is high; (2) power requirement is low; (3) minimum control is necessary; (4) extraction yields highly concentrated enzyme solutions; (5) only small equipment for enzyme recovery is required due to the small amounts of extracts obtained; (6) scaling-up is easy.
Problems which can be solved, if need be, may be listed as follows- (1) continuous operation is possible; (2) feeding substrates during cultivation is possible; (3) defined media can be applied by using suitable inert carriers. Caused by the nature of the complex media used, the extracts contain considerable amounts of fungal pigments, the removal of which is difficult and costly.
The methods of solid substrate cultivation can be divided into two groups- thin layer and deep bed processes.
The thin layer techniques, also called tray processes, work with substrate layers of 2 to 4 cm height spread on wooden or metallic trays. These are incubated in air-conditioned rooms or cabinets (hence cabinet method). The process is described in detail by under kofler and Hickey (1954).
Jeffreys (1948) has tried to mechanize this process, but it does not seem that manufacturers have been very successful in fully mechanized tray methods. Usually the heat produced by the growing culture is removed by moistened cool air which passes over the surfaces of the trays or is pressed through the bran mass. Kalashnikov et al. (1960) recommended a water-cooling, system for the trays.
The deep bed process, developed by Terui and co-workers (1958, 1959A.B) in order to meet the enormous demand for enzymes needed for the traditional soybean fermentations, uses substrate layers usually as deep as 0.6 m (2 ft), but beds as deep as 1.5-1.8 m (5-6 ft) have also been reported. The dimension of rectangular beds is of the order of 5.5 x 61 m (18 x 200 ft).
Circular beds are also operated. In general, the equipment used in deep bed processes is quite similar to that known in the malting industry. The deep bed plants are fully automated. Continuously operated deep bed techniques may, for example, guide the culture mass through air-conditioned tunnels by means of conveyor belts, as claimed by Christensen (1940).
The use of rotating drums, as recommended by Takamine (1913) for the production of mold bran on an industrial scale, was only of temporary importance. With this method manufacturers experienced many difficulties and the desired results were not always obtained. Some workers attributed this to damage of the mold mycelium by mechanical action, but it is not really clear why this method does not work.
Media used in solid substrate fermentations are mainly based on wheat bran. This material is particularly suitable because of its high content of nutrients and its large surface. Other basic substrates are rice bran and soybean or sweet potato flakes, as well as grains or soybeans, etc. Kernels should be selected for optimal size or cracked to give particles of the desired size.
On bran, superficial growth of the fungus is sufficient for utilization of nutrients. Kernels, however, must permit the inoculum to penetrate. This is not difficult with polished rice, but corn or soybeans must be cracked or freed of the hulls. The amount of water needed for moistening the substrates is in the range of 40 to 70%. In the case of grains and soybeans the optimum content of moisture is about 30%.
Pressure sterilization of large masses of wet bran in bulk presents serious problems. A convenient method to ensure thorough sterilization is by direct steam injection into the bran. During this process the mass is agitated so that each particle of the moist bran is in constant .direct contact with the steam.
Experience, however, has shown that when acidic solutions are employed in place of water for moistening the bran it was quite sufficient to sterilize the bran at 95°C for 15 to 30 min. In some cases decontamination of the bran was achieved by means of bactericides, e.g., formaldehyde or β-propiolactone.
Inoculation of the sterilized medium is carried out by use of spores in a dry or suspended form. The amount of inoculum varies from process to process depending on a number of factors. The actual amount must be determined empirically. Under kofler et al. (1947) found that even as low an inoculation ratio as 0.04% of dry spore culture was satisfactory in fungal amylase production.
Attempts had been undertaken to use an inoculum in the mycelial form which can be easily produced in large quantities by submerged culture. However, this method is not convenient, resulting in non-uniform growth of the fungus throughout the bran mass.
Conidia can be produced in large quantities in special cultures, whereby the method of fermentation is quite similar to that applied in enzyme production. In order to promote spore formation it may be beneficial to add a balanced solution of trace elements (Fe3+, Zn++, C+ +, Mn++).
Among the conditions of incubation, moisture and temperature present the most serious problems. It was soon recognized that these two factors are dependent variables. Growing cultures produce heat which tends to evaporate the water of the substrate. This effect is intensified by warming the air when it passes the culture and thereby enlarging its capacity to dissolve water vapor.
These processes inevitably cause a drying out of the culture. However, the water lost is partially replaced by water that is formed by the metabolic activity of the organism. The difference must be made up by application of sprayed water (this method, of course, is unfit for non-agitated cultures). The microbiological approach to this problem is the selection of slowly growing strains with high productivity. Short-time fermentations may also be helpful.
Heat production is, indeed, considerable. Its magnitude can be readily appraised by the loss in dry weight of the culture. It has been demonstrated that almost all this loss is due to the oxidation of organic compounds to CO2. In many cases it was found that approximately half of the dry weight of the bran disappears.
Kalashnikov et al. (1960) observed with Aspergillus niger that under industrial conditions up to 380 J.h-1.kg-1 of fermented bran were liberated at maximum heat production. Removal of this amount of heat required 20 m3 airs with 20°-28°C temperature and 100% moisture.
The problem of heat removal is complicated by the heat-insulating properties of the bran. Attempts to minimize this problem by diluting the bran through incorporation of inert materials, such as grain husks, only cause other problems, e.g., a greater requirement of space.
Regarding the moisture content of the medium, it has been found in many cultures that careful observation of the tolerated limits has a decisive influence on the production of the enzyme.
Initial pH and the course of the pH during the development of the culture also play a great role. Several means are known to influence the pH development. For instance, this can be done by incorporation of suitable inorganic or organic salts into the medium.
Submerged Cultivation Techniques:
Treatment of this subject may be limited to some considerations of enzyme production. The fermentation equipment used for the large-scale production of enzymes is the same as that used in the production of other microbial metabolites. As far as is known, processes are batch-operated; however, attempts have been made to introduce continuous processes to enzyme production.
Continuous fermentation may be employed if the optimal conditions for the process are known. For inducible enzymes, however, optimal conditions for cell growth are often different from those required for induction and synthesis of the enzymes. In such cases a 2-stage, continuous- culture system can be used efficiently for production of the enzyme. By using this method, the growth stage can be operated with an optimal set of culture conditions.
It may be useful to insert a washing of the cells between the first and the second stage in order to decrease the requirement of inducer and to produce less impure preparations. Two-stage, continuous- culture processes can also be use when reduction or elimination of catabolite repression due to a high medium concentration is desired.
Addition of a substrate to a batch process under controlled conditions is a well-known technique in fermentation technology for prolonging growth and increasing product accumulation. Edwards et al. (1970) have called this method “extended culture.”
Since it permits prolonged maintenance of a constant environment with respect to the added substrate, the system is more similar to a continuous process than to batch fermentation. In many cases drastic increases in enzyme production result from the use of extended culture.
Quite similar to solid substrate fermentations, the pH also plays an important role in submerged cultivations. Generally, the culture starts with a certain pH which depends on the strain employed and the enzyme desired. The course of pH during the fermentation is often manipulated by addition of suitable agents. In some processes the pH is “fixed” at a certain level, within limits.
This is the case in the production of amyloglucosidase from Aerobacter aerogenes. In a number of batch processes it is convenient to change the pH because of differences in optimum pH for growth and enzyme formation. For example- in the case of neutral metalloproteinase from a Bacillus species, the pH is allowed to change “naturally.” The course of pH may, however, be programmed. As an example- the acid stable α-amylase of Aspergillus niger may be mentioned.