Microbial Enzymes: Production, Purification, and Isolation

Volume 2, Issue 2 119

MICROBIAL ENZYMES: PRODUCTION, PURIFICATION, A N D ISOLATION

Authors: B. Volesky Department of Chemical Engineering McGill University Montreal, Quebec, Canada

John H. T. Luong Biotechnology Research Institute National Research Council of Canada Montreal, Quebec, Canada

Referee: Knud Aunstrup Department of Enzyme Microbiology Novo Industries A/S Bagsvaerd, Denmark

I . INTRODUCTION

All forms of life live by enzymes and also produce enzymes. As a result, enzymes can be obtained from three different sources: plants, animals, and microorganisms.

Some commercial enzymes such as papain, bromelain (bromelin) ficin, and malt diastase are derived from plant sources. In the animal body, the highest accumulation of enzymes is in the glands. Stomach, spleen, pancreas, and stomach mucus are rich in several important enzymes. Some widely used enzymes such as pepsin, trypsin, a-chymotrypsin, lipase, catalase, rennin, and pancreatic enzymes are currently obtained from animal sources.

Microorganisms are becoming a favored source of industrial enzymes since the number of enzymes which can be recovered economically from plants and animals is limited. Animal enzymes, e.g., are supplied by the meat-packing industry and the size of the slaughter limits the total enzyme supply. Plants may be grown in any desired quantity if the enzyme market is precisely estimated well ahead of time. However, except in a few special cases, plants are generally utilized for other purposes rather than for enzyme production, and their growth is dependent upon geography, soil conditions, and climate. Some plant and animal enzymes of industrial importance are listed in Table 1.'

While certain plant enzymes (e.g., papain) and animal enzymes (pepsin, trypsin) will continue to have important applications, microbial enzyme production based on im- proving biotechnology is making significant advances.

11. SOURCES OF MICROBIAL ENZYMES

The development of fermentation methods for the production of microbial enzymes has assured an unlimited supply. For the production of industrial enzymes, microbial cells are selected from the groups of fungi, bacteria, or yeasts. Four enzymes are now produced on a large scale: protease, glucamylase, alpha-amylase, and glucose isomerase.

Microorganisms used in the production of industrial enzymes must have good biological activity. Most of microorganisms used for industrial enzyme production are aerobic strains, i .e., they require oxygen for their metabolism. The number of known

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120 CRC Critical Reviews in Biotechnology

Table 1 SOME PLANT AND ANIMAL ENZYMES OF INDUSTRIAL

IMPORTANCE'

Name

Plant enzymes Papain

Brornelain. ficin

Diastase

Rennet Pepsin Pancreas protease

Animal enzymes

Trypsin

u-Chymotrypsin Lipase

Source

Papaya

Pineapple, fig tree

Malt

Calf stomach Animal stomach Animal pancreas

Animal pancreas

Animal stomach Hog pancreas

Application

Digestive aid; medical uses; beer haze re-

Digestive aid; medical uses; beer haze re-

Digestive aid; supplement to bread; syrup

moval; meat tenderizer

moval; meat tenderizer

Cheese manufacture Digestive aid; meat tenderizer Digestive aid; cleaning; leather bating; dehairing; feed improvement

Medical uses; meat tenderizers; beer haze removal

Medical uses Cheese production; digestive aid; laun- dering cotton and linen; feed supple- mentation for mink; poultry and swine feeds

individual enzymes is around 750, of which about 100 have been isolated in crystalline form. Daviesl presents a very comprehensive list with names of the production microbial strains, the enzymes they produce, and some properties of these enzymes. Some of these microorganisms are employed for commercial production of industrial enzymes; many of them have a market potential in the near future. Fewer than 50 microbial enzymes are of industrial importance today. Most of the important enzymes can be obtained from strains of Aspergillus oryzae, Aspergillus niger, and Bacillus subtilis groups.

Among bacteria, Bacillus licheniformis is used for the production of protease, pen- icillinase, and heat-stable alpha-amylase, with other important strains being B. amyloliquefaciens and B. subtilis. Among fungal organisms, strains of A. oryzae and A . flavus are employed in the production of proteases, amyloglucosidase, and other amylases. Strains of A. niger are mainly used for the production of amylases, amyloglucosidase, glucose oxidase, catalase, lipase, and petic enzymes. Table z3 presents some microbial enzymes of industrial interest and their sources.

It is important to note that the multiplicity of enzymes produced by a single microorganism is sometimes a major disadvantage. In general, microbial cells have the capacity to produce at least 2500 different enzymes. Therefore, if an industrial process requires only a specific enzymatic conversion, the presence of contaminating enzymes may cause undesirable reactions. In such cases, it is necessary to remove the undesirable enzymes. This step may be technically very difficult and costly. Fortunately, many enzyme purification methods have been developed which enable enzyme manufacturers to provide commercial enzyme products with the necessary performance characteristics.

Fermentation technology know-how developed in the large-scale production of an- tibiotics plus a recent understanding of microbial genetics and protein biosynthesis are undoubtedly making microorganisms a major and a most attractive source of enzymes.

111. PRODUCTION OF MICROBIAL ENZYMES A new enzyme product becomes a commercial success only if a market exists and if

it can be produced economically. Ideally, the following requirements should be fulfilled for the successful development of a commercial enzyme process:

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Table 2 SOME ENZYMES OF INDUSTRIAL INTEREST3

Enzymes Microorganisms

Proteolytic enzymes Bacillus proteases Streptomyces proteases Aspergillus proteases

Yeast proteases

Yeast proteases

Microbial Rennett Other proteases

15 species of the genus Bacillus Streptomyces rectus, S. hygroscopicus Aspergillus oryzae, A. candidus, A. sojae, A.

Candida lipolytica, Rhodotorula glutinins, Torula

Candida lipolytica, Rhodotorula glutinis, Torufa

Mucor miehei, M. pusillus, Enclothia parasitica Tritirachium album, Malbranchea pulchella, Acre- monium kiliense, Fusarium, Giberelle sp., Penicil- lium dupontii. Armellaria mellea, Fusarium semi- tectum

ocheaceus, A. niger

thermophila

thermophila

Carbohydrases Pectinase A. niger Lactase S. fragilis. S. lactis, Aspergillus sp. (0-Galactosidase)

u-Galactosidase

Invertase Hemicellulase xylanase P-Glucanase Dextranases

Cellulase

Amylolytic enzymes Bacillus alpha-amylase

Bacillus saccharifying amylase

Fungal alpha-amylase

Am yloglucosidase

Alpha-glucosidase Maltogenic and debranching amylases

Penicillium cyclopium, Sclerotium tuliparum, Cor- ticium rolfsi, Mucor pusillus, Bacillus stearothermophilus, Bacilhs coagulans, Thermus aquaticus, Streptomyces coelicolor

Lac to b a cill i , Penicillium dupon tii, Aspergillus a wa - mori. Monascus sp., Streptomyces, sp., Absidia sp., Mortierella vinacea

Saccharomyces cerevisiae Malbranchea pulchella B. subtilis, B. pumilus, A. niger Penicillium luteum, Penicillium funiculosum, P. lil- acin urn, Aspergillus carneus, Fusarium rn on oli- forme, Streptococcus mutans, Flavobacterium sp., Bacillus sp., Arthrobacter globigiformis. Brevi- bacterium fuscum, Cladosporium resinae

Trichoderma reesei Sporotrichum pulverulenturn, Sporotrichum thermophile, Chaetomium thermophile. Thermomonospora curva ta, Actinomycetes sp., Irpex lacteus, Aerobactersp..

B. amyloliquefaciens, 8. Iicheniformis B. caldolyti-

B. subtilis marburg, B. natto, B. amylosacchariti-

Aspergillus oryzae. A. niger, Paecilomyces subglo-

A. foetidus, A. niger, Rhizopus, Endomyces spp.,

Mucor javanicus, B. cereus Klebsiella pneumoniae, Aerobacter aerogenes,

cus, B. acidocaldarius

cus

bosurn, Penicillium expansum

Trichoderma viride

Pseudomonas deramosa, B. polymyxa. B. cereus, B. amyloliquefaciens, B. megatherium, B. circu- lans, Pseudomonas spp.. Streptomyces spp.

Other amylases Cyclodextrin transferase Exoa- Bacillussp. Pseudomonas stutzeri, Klebsiella pneu- mylase

Other enzymes Glucose isomerase

moniae

Streptomyces sp., Actinoplanes missouriensis, Ba- cillus coagulans, B. stearothermophilus, Arthro- bacter sp.,

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1 .

2.

3 .

4.

5 . 6.

7.

Table 2 (continued) SOME ENZYMES OF INDUSTRIAL INTEREST3

Enzymes Microorganisms

Lipase

Penicillin acylase

Geotrichum candidurn. Mucor javanicus, Aspergil- lus niger, Rhizopus delernar, Penicillium cyclopium. Candida cylindrace

Bovista plumbea, Phialomyces macrosporum, Lep- tosphaerulina australis, Robillarda SQ., Esche- richia coli, Kluyvera citrophila, Erwinia aroidea. Gumnaoascus polypaecilum

The microorganisms should be able to grow on an inexpensive medium at a rapid rate. The organism should produce the enzyme with high yields at a high concentration. Undesirable enzyme contaminants and the content of metabolites in the fermentation broth should be minimal. In order to increase the fermentor enzyme productivity, the microorganism should be able to grow on a concentrated medium in a dense culture. Recovery of the enzyme should be technically feasible and inexpensive. In terms of biohazards, the production process and its product must be safe to the personnel involved and to the consumer. The treatment of the effluent frvm the production process should be technically and economically feasible.

The fulfillment of these objectives requires a combined optimization of the strain properties and process parameters. Optimization of microbial strain properties is very attractive since it offers an inexpensive and permanent solution.

A. Screening Methods Various levels of different individual enzymes are produced by microbial cells. This

characteristic is dependent upon the particular purpose of each enzyme. In general, most enzymes are produced in the quantities required by the cell since the enzyme formation is regulated by the dominant genes. However, certain types of microbial cells stand out in the excess production of one or a few specific enzymes.

In many cases, the type of microorganism employed in the production of a commercial enzyme is common knowledge and while some enzyme activity levels are docu- mented in the literature, data on commercial processes are often confidential and not readily available.

For the development of a new enzyme, the best source of this particular enzyme must be sought among appropriate organisms by suitable screening methods. Cultures for screening can be obtained from culture collections and from new isolates from natural habitats, particularly those rich in the substrate upon which the desired enzyme must act. Once the most outstanding strain from a production point of view is selected (highest enzyme activity, good cell yields, easy propagation condition, lower sensitivity to infection, inexpensive nutrient requirements, etc.), the medium composition and growth conditions have to be established for maximizing the production of the desired enzyme.

The development of a new enzyme usually requires a search for a high-producer microorganism. The screening on laboratory scales is very tedious and time consuming. Its successful result, however, is of great economic value to the enzyme industry.

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B. Mutants It is not unusual that mutant strains can produce many times the yield of enzyme

than obtainable from the wild parent culture. In other cases, mutants may produce fewer undesirable enzyme contaminants and/or other metabolites, thus facilitating enzyme purification steps.

Mutants for enzyme production are obtained by subjecting cultures to mutagenic agents (chemicals, heat, UV, etc.) to such a degree that most of the microbial cells are killed. The survival cells are then screened for superior desirable characteristics as compared to their parental strain. The mutagens used for enzyme overproducing strains are those commonly applied in microbiology. A detailed discussion of microbial genetics, including mutation applied to industrial microbiology, is reported el~ewhere.~

It is important to note that sites at which genetic control is exerted during enzyme production by microorganisms include induction (direct), end product repression (in- direct through enzyme function), and catabolite repression (direct). Today, geneticists can cite a number of methods for eliminating any one of these mechanisms when it is being used by the cell to prevent production of large amounts of a given e n z ~ r n e . ~ However, these mechanisms may not always exist in the given strain.

The industrial production of enzymes is expected to significantly grow in the next decade as recombinant DNA techniques are applied to the microbial production of enzymes. Because an enzyme itself is a direct product of a gene, the yield can be improved by introducing multiple copies of the gene into the DNA of the microorganism. The yield can also be improved by maximizing the expression of the gene through insertion of regulatory sites in the DNA called promoters, and/or by facilitating the secretion of the enzyme from the cell. Genetic engineering has already proved feasible with certain microorganism^.^

C. Strain Maintenance The highly developed production strains must be protected against the risk of degen-

eration, contamination, and loss of viability. One of the most common methods is to store the strains lyophilized or at the temperature of liquid nitrogen.

The master culture is usually kept alive on an agar slant and stored in the refrigera- tor. The agar slant culture must be transferred at regular intervals which represents a certain degree of risk. Such culture has to be periodically checked for its level of activity before it is used as starter for the preparation of inoculum for an industrial batch.

D. General Methods of Cultivation Similar to other fermentation processes, for enzyme production the microorganisms

are cultivated by inoculating pure cultures into a sterile medium. There are two methods of cultivation: surface culture (semisolid or koji method) and submerged culture. Traditionally, all commercial microbial enzymes were produced by surface culture methods until the Second World War. Some amylase production methods employed cultures grown in a film on liquid medium in trays up until the 1950s. Submerged culture has since come into extensive use.

E. Surface Culture This method has still been employed for the production of a few products, primarily

of fungal origin such as Aspergillus amylase, proteases from Aspergillus and Mucor species, pectinases from Penicillium and Aspergillus species, and also cellulases. In this method, the cultures are cultivated on the surface of a solid substrate where wheat bran is often the main constituent of the medium. Carbohydrates, nitrogen compounds, minerals, and other chemicals are then added to the medium. The mixture is moistened with water and sterilized with steam at 30 psi for 2 hr, sometimes directly in

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the propagating equipment. It is more economical, however, to separate the two operations with an aseptic transfer of sterilized mixture.

The moist bran can be incubated either in thin layers on perforated trays or in a slowly rotating horizontal drum. Originally, cultures grown in trays were handled man- ually, but mechanical systems for cleaning, filling, and emptying the trays are now available. An interesting system uses enclosed sterile plastic bags. Cold and moist air is circulated over the bran, and parts of it are constantly replaced with fresh humidified sterile air to maintain proper temperature and to remove CO,. Occasionally, spraying of water over the culture is needed to keep the proper moisture level. The pH is controlled by inclusion of buffers in the medium or addition of acid and alkali to the spray water.

After the fermentation is completed, which may vary from 1 to 7 days, the enzyme systems may be extracted from the “moldy bran” with water. Subsequent recovery operations are then similar to those employed for the submerged fermentation process broth.

The surface culture process has advantages in simplicity of operation, low power requirements, and a low level of contamination. Whereby contamination is a serious problem with surface culture processes, it is of a different nature. The low levels of contamination often present do not necessarily affect the process in a significant way. Contamination in the submerged culture process often means it is rapid and widespread resulting in a complete waste of a whole batch. Surface culture, however, requires excessive space and costly manual labor. Submerged culture methods are now dominating the production of enzymes because handling costs and the risk of infection are greatly reduced. Furthermore, the submerged method often results in higher yields on the substrate than the surface culture. Some advantages and disadvantages of the surface culture and the submerged culture are summarized in Table 3.* The production methods discussed below refer to the submerged culture method. A detailed discussion of the surface culture method can be found elsewhere.’

F. Submerged Culture Fermentation Process Since the advent of penicillin, considerable advances have been made in the tech-

nique of growing fungi in highly aerated submerged culture. This technique offers considerable advantages with respect to ease of control of pH, temperature, medium composition, agitation, and aeration. Of still greater interest is a shorter fermentation time. Lower operation costs are the most important factor.

With many significant improvements in sterilization procedures, instrumentation, and controls, submerged culture methods are progressively displacing surface culture techniques.

I . Preparation of Plant-Scale Inoculum A pure culture of a selected strain of the microorganism is grown in laboratory shake

flasks. These flasks are used to inoculate seed tanks which in turn are used to inoculate production vessels. The growth rate of microbial cells may differ noticeably from a shake flask to an aerated bioreactor. Using an inoculum in the logarithmic phase nor- mally does not produce any significant lag or delay period in the fermentor. The inoculum, however, must contain a sufficient amount of cells to serve as a starter for a final batch on production-scale propagation. The optimal size of inoculum must be determined by practical experience and usually ranges from 2 to 10% of the total volume of production medium. There are exceptions, however, where the size of inoculum for a 500-1 culture may be as small as 200 m l (0.04%) or as large as 80 I (16%). In general, the inoculum must be large enough to give a speedy start, to decrease the chance of contamination spoilage, and to result in the best yield under the processing conditions.

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Table 3 COMPARISON O F SURFACE AND SUBMERGED PROCESS6

Surface culture Submerged culture

Requires much space for trays Requires much hand labor Uses low-pressure air blower Little power requirement

Minimal control necessary Little contamination problem Recovery involves extraction with aqueous solution,

filtration, or centrifugation, or perhaps evaporation and/or precipitation

Uses compact closed fermentors Requires minimum of labor Requires high-pressure air Needs considerable power for air, compressors, and

Requires careful control Contamination frequently a serious problem Recovery involves filtration or centrifugation, and

perhaps evaporation and/or precipitation

agitators

From an economic point of view, it is always desirable to keep the amount of inoculum as low as possible since the cost of nutrients and the equipment per volume (or weight unit) are more costly for the inoculum than for the final batch. However, it should be kept in mind that the success of the enzyme production depends principally on the quality of the inoculum. The cost of an inoculum amounting to 1/1000 or less of the final batch is usually acceptable.

The inoculum may contain vegetative cells or spores (either sexual spores or coni- diospores) which corresponds to the nature of the final batch and to the characteristics of the culture used.

2. Medium Composition The medium must meet the nutritional requirements of the microorganism. It basi-

cally consists of a fermentable carbon energy source, a nitrogen source, and some growth requirements such as essential amino acids and vitamins. Sometimes, special nutrients must frequently be added to stimulate enzyme production. Pectin compounds, e.g., must be provided if certain pectinases are desired. Minerals are added as supplementary sources of nitrogen, phosphorous, sulfur, and calcium. Tap water and/ or raw materials usually contain adequate amounts of trace metals required for enzyme fermentations. Trace metals such as Ca, Mg, Mn, Fe, Au, Cu, Co, and Mo are required for growth, some of them being essential constituents of coenzymes. Among these elements, Ca is often the most essential one for high activity and stability of many extracellular enzymes.

For most microorganisms, a metabolizable carbohydrate is usually supplied as a carbon and energy source. Occasionally, the carbohydrate source may fulfill the role of inducer for inducible enzyme production, e.g., dextran for dextranase production, maltose for maltase formation, and starch for amylase production. Although most bacteria can utilize amino acids as a carbon source, a small quantity of carbohydrate is usually added to the medium. For the production of extracellular enzymes by Clos- tridia and Streptococci,* 0.25% glucose is usually added even though these bacteria can grow quite well in carbohydrate-free media. The presence of carbohydrate may also stimulate the minimal growth and quantity of enzyme formation. For example the addition of 0.45% glucose to a 3 .3% Evans peptone-1.5% beef extract medium stimulated both growth and total extracellular hyaluronidase and lecithinase-C by C. welchii? Stimulation of the rate of formation of the inducible galactose-fermenting enzymes in Saccharomyces cerevisiae under anaerobic conditions by a small amount of fermentable sugar (0.1 g/P) was also r e p ~ r t e d . ~ In some cases, the presence of a carbohydrate source is mandatory when the energy source in a medium is one to which the inoculum is not adapted. Without the addition of 0.20% glucose to the medium,

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Table 4 TYPICAL MEDIUM COMPONENTS FOR ENZYME FERMENTATION”

Carbohydrates Molasses, corn, barley, wheat, starch hydrolyzate, cereal starch, lactose, beet pulp, citrus pulp, apple po-

Nitrogen Sources Corn steep liquor, yeast hydrolyzate, gluten, whey, soybean meal, cottonseed meal, peanut peptone, gelatin,

casein Minerals Ammonium phosphate, sodium chloride, magnesium sulfate, dipotassium phosphate, monopotassium phos-

mzce

phate, iron, ammonium citrate, calcium carbonate, manganous chloride

C. flarum and C. laniganiicannot grow in a peptone, yeast extract, and sodium pectate medium.1°

The growth and enzyme formations of many organisms are stimulated by supplying complex forms of nitrogen such as amino acid mixtures, protein digests, nucleic acid digests, etc. Sometimes, the presence of complex nitrogenous substances only stimu- lates the extracellular enzyme formation, but not growth and these nitrogenous compounds are not even required for growth. The stimulation of extracellular enzyme production by complex nitrogenous materials can be explained by the fact that there is an extra supply of growth factors, trace elements, or perhaps an inducer function of some peptides (in the case of proteinase). However, in some cases, complex nitrogen sources are unsuitable for extracellular enzyme formation, even though growth is satisfactory. For cellulolytic enzyme formation by Trichoderma reesei,” ammonium sulfate and ammonium nitrates are the best nitrogen sources, while organic nitrogen sources result in lower yields through microbial growth is in fact stimulated.

The enzyme formation also often depends upon the presence of an inducer in the medium. According to current knowledge, enzyme production is controlled by three factors: (1) a gene determining the structure and properties of the enzyme, (2) a repressor substance which prevents the gene functioning, and (3) an inducer which releases the gene from inhibition by the repressDr. The ability of the microorganism to synthesize repressors and inducers is determined by the presence of appropriate genes. If the organism cannot synthesize the repressor, the enzyme may become constitutive. Con- versely, if the inducer cannot be synthesized because of a gene deficiency, an external supply of inducer must be provided for enzyme synthesis to occur and the enzyme becomes inducible. Cell autolysis occurring during prolonged incubation can supply inducers. Complex nitrogenous compounds such as corn-steep liquor, yeast extract, peptone, meat extracts, or protein hydrolyzates may supply inducer for proteinase synthesis, while amino acids or low peptides will prove inhibitory.

Oxygen is another essential fermentation constituent for aerobic fermentations. En- riched air (>21% oxygen) and the cryogenic fractionation of air into its component gases may be used to reduce the cost of pumping large volumes of air into the medium.

The raw materials must be available in large quantities and relatively inexpensive since they could contribute up to 60 to 80% of the variable costs of an enzyme fermentation process.12 Furthermore, the raw materials which have some adverse effects on product quality, enzyme recovery and purification, or wastewater treatment must be avoided in enzyme fermentations. Some typical constituents of industrial media for enzyme fermentation are listed in Table 4.”

The medium fermentations developed by the individual manufacturers for enzyme productions are kept confidential and very little information about them is published. Although mechanisms of end-product inhibition and restriction of enzyme formation by gene-controlled repressions are fairly well understood, it is still not possible to pre-

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dict the optimal conditions for the production of a particular enzyme by a given microorganism. The appropriate fermentation medium for maximum enzyme production is empirically determined for each individual fermentation process. In general, the following requirements must be fulfilled when formulating the optimal conditions for the production of a specific enzyme:

1. 2. 3. 4.

5 .

Adequate aeration for aerobic organisms The requirement of a suitable inducer for inducible enzymes An adequate supply of trace metals An adequate supply of substances which the microorganism cannot synthesize i.e., growth factors, amino acids, etc. Avoidance of high concentrations of certain carbon and energy sources which inhibit the formation of enzymes.

However, a medium formulated for good growth will not necessarily result in a good desirable enzyme quantity. The enzyme production may be repressed by one of the medium constituents.

3. Medium Sterilization Most media are heat sterilized either batchwise in the fermentor, or by passing

through separate continuous sterilizers. A method similar to that used in calculating equivalent “thermal denaturation” processes can be used to determine the effects of sterilization processes.

The large-scale long heating cycle is effective, however, it can cause change or de- struction of essential nutrients as well as reactions between medium components. In this case, certain medium components must be sterilized separately from the main batch and then added aseptically at fermentation temperature to minimize undesirable changes of the fermentation medium. The sterilization of glucose, amino acids, and trace elements should follow this procedure. Heat-sensitive materials in the medium such as vitamins may even be filter sterilized. Filter sterilization, however, is not effective for eliminating viruses and phage.

In order to avoid undesirable heat-caused changes in fermentation media, continuous high-temperature, short-time sterilization methods are gaining wider application. External (plate) heat exchangers are employed to heat medium by high pressure steam to higher temperatures maintained for only short periods of time. The heat exchanger acts as a heat economizer, and recovery of heat is an additional advantage. In some cases, the direct injection of steam into the medium and subsequent “flashing off” under vacuum of excess condensed water is used.

4. Fermentation After temperature, pH, and agitation rate of the sterilized medium are adjusted to

the optima, the inoculum is transferred into the solution under aseptic conditions. The formation of a desired enzyme may occur during any phase of microbial growth

except the lag phase. For example, while production of bacterial amylases occurs mostly during the stationary phase, certain bacterial proteinases are almost entirely synthesized during the exponential phase. Most commercial enzymes are formed during the stationary phase.

Except for a few enzymes such as glucose isomerase and oxidase, yeast lactase, invertase, and catalase, most enzymes of industrial interest are extracellular, i.e., excreted from the microbial cells into the broth. However, it is not clear whether an extracellular enzyme is excreted by the organism as a normal physiological process or whether it appears in the liquid due to cell autolysis or as a result of cell damage.

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From a practical point of view, this mechanism is not important since the main practical concern is to determine the conditions which will give the best yield of enzyme. Some commercial enzymes are intracellular. They are either located within the cell cytoplasmic membranes (truly intracellular enzymes) or are bound to the surface structures of the cell. Intracellular enzymes are probably produced mainly during the exponential phase.

The maximum level of the desired enzyme may remain constant for a significant period of culture time or it may decline slowly or rapidly following its peak accumulation.

Most microorganisms produce more than one type of extracellular enzyme. The formations of extracellular enzymes are not necessarily simultaneous during the fermentation. Therefore, it may be advisable to sacrifice yield of a desired enzyme by termi- nating the fermentation before some undesirable contaminants are excreted into the broth.

The typical yield of a useful enzyme may vary between 1 to 5% of the initial medium dry substance. The cellular yield may be 2 to 10% on a similar basis. About 5 to 10% of metabolites and residual substrates remains unused in the broth at the end of a fermentation.12

a. Some Factors Affecting Enzyme Fermentation The optimal culture conditions such as pH, temperature, aeration-agitation rates,

and nutrient compositions for enzyme production must be rather empirically determined for each individual fermentation process. The optimal conditions for growth are not necessarily applicable for maximum enzyme production.

Effect of temperature - In many cases, the formation of extracellular enzyme is greater when the microorganism is grown at temperatures lower than the optimal growth temperature.z For example, it is reported that procedures which restrict the growth rates of Pestaloptiopsis, Westerdijkii, Trichoderma reesei, and a Basidiomycete SP., especially lowering the temperature, appreciably increased production of cellulolytic enzymes." Table 5 lists the enzymes and organisms for which the optimum growth temperature is noticeably different from the optimum temperature for enzyme synthe- s k 2 The effect of growth temperature on the properties of an enzyme may aIso be important. Some controversy exists concerning an interesting finding that @-amylase produced by the thermophilic Bacillus coagulans formed during growth at 55°C retains 88 to 90% of its activity after 60 min at 9O"C, whereas the enzyme formed during growth at 35OC retains only 6 to 10% of its activity.15 Many Streptococcal strains which have no typical Mantigen when grown at 37°C possess this antigen when grown at 220C.16

Effect of pH - Most microbial extracellular enzymes are produced in greatest yield at a growth pH somewhere near the pH for maximum enzyme activity. However, there are many cases where the growth pH for maximum yield deviates significantly from the optimal pH for activity.* Table 6 presents some enzymes and organisms for which the optimum growth pH is noticeably different from the optimum pH for enzyme production and activity.

Effect of aeration - Most aerobic enzyme fermentations have a high oxygen de- mand. In general, they require aeration and agitation rates similar to those used in antibiotic fermentations. However, the optimum level of aeration for the production of some extracellular enzymes by aerobic microorganism is somewhat difficult to pre- dict. Some aeration is necessary but the degree of aeration required appears to depend on the microorganism and/or the enzyme species. Some microorganisms require vigorous aeration, while the production of some extracellular enzyme is inhibited by aeration.2 Table 7 lists the degree of aeration on the optimum production of some extracellular enzymes.

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Table 5 TEMPERATURE FOR ENZYME BIOSYNTHESIS'

Optimum temperature ("C)

Enzyme

Pectin esterase Polygalacturonase Dextransucrase fi-Glucuronidase Proteinast

Proteinase Lysozyme-like enzyme

Enzymes hydrolyz- ing the pentosans of wheat flour

Acid proteinase Alkaline proteinase

Microorganism

Botrytis cinera Aspergillus niger Leuconostoc rnesen teroides Escherichia cofi Micrococcus lysodeikticus Proreus x 19 Bacillus megaterium Coc-

Pseudomonas fluorescens Bacillus so btilis

cus P (Sarcina flava)

Bacillus pumilus

Aspergillus oryzae Aspergillus oryzae

For enzyme For growth production

- 12 12

37 23 37 25

-

37 26

20 0 37 30

40 26

- 22 - 20

Table 6 EFFECT O F pH VALUE ON ACTIVITY AND PRODUCTION OF

MICROBIAL EXTRACELLULAR ENZYMES

Microorganism

Bacillus macerans Lactobacillus bifidus Leuconostoc mesen teroides Escherichia coli Bacillus subtilis Bacillus subtilis Bacillus subtilis Erwinia carotovora Erwinia atroseptica Penicillium lilacinurn Penicillium funicufosurn

pH Optimum for enzyme

Enzyme Activity Production

Amylase Endo-dextranse Dextransucrase p-Glucuronidase Alkaline proteinase Subtilisin Endo-polygalacturonase Endo-poly-galacturonase Endo-poly-galacturonase Endo-dextranase Endo-dextranase

5-6 5.4-6.5

5.5 6.2

9.5-10.5 10-1 1

8.5 6.5 6.5

5.9-6.1 4-5.5

Not below 6.6 7 .2

6.7 2 0.1 7.3

6.5-6.8 6.0-7.5

7-9 7-9

Close to 4 Near pH 8

7-a

Harvesting time - Depending upon the organism and enzyme produced, the propagation time may take I to 5 days. From a microbial point of view, when laboratory tests indicate that maximum enzyme production has been reached, it is the harvesting time. However, from an economic point of view, the optimal harvesting time is determined by relative costs of raw materials, utilities, recovery steps, and utilization of plant capacity. In many cases, the process is terminated before or carried on beyond the maximum enzyme productivity point in order to obtain a broth with properties which facilitate product recovery, e.g., containing less other contaminants.

G . Pretreatment of the Fementation Broth After completion of the fermentation, the liquid broth is rapidly cooled from the

fermentation temperature to about 5°C. Strict hygenic measures in the plant are very crucial during this stage since the liquid broth is especially vulnerable to microbial contamination. The addition of some preservatives such as toluene, organic acids and

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Table 7 AERATION REQUIREMENTS FOR ENZYME BIOSYNTHESIS

Enzyme

Alkaline proteinase o-Amylase Proteinase

Neutral proteinase Gelatinase Pentosanases Polygalacturonase

Deoxyribonuclease Invertase

Pectin esterase and polygalactu. ronase

Microorganism

Bacillus subtilis Pseudornonas saccharophila Many microorganisms

Bacillus stearotherornophilus Pseudomonas rnyxogenes Aspergillus niger Botrytis cinera and some species of Aspergillus, Rhizopus, Mu- cor. and Penicillium

Strphylococcus aureus Saccharornyces uvarium and some unisexual strans of Saccharomy- ces kluyveri

persici Fusariurn oxysporurn var. lyco-

Degree of aeration for enzyme production

Vigorous Vigorous

Aeration is generally not favora-

Inhibited by aeration 25-fold increased by aeration High aeration is unnecessary Aeration is not favorable

ble

Vigorous Increased by aeration

20-fold increased by aeration (to be compared in cultures on solid bran)

their salts, phenolic compounds, and sodium fluoride to the liquid broth may be help- ful to prevent possible microbial spoilage. However, if the enzyme is involved in food preparation, the quantity of preservatives cannot exceed 10 ppm in the final enzyme preparation or 0.05 ppm in the food. Some preservatives have also been found to adversely affect the subsequent biological treatment of the wastewater. I 3

Broth containing yeasts or myceIiaI organisms is ready to process; it requires no special treatment. Broth of bacterial fermentation is very difficult to process. Usually, the broth contains large amounts of colloidal particles which present some technical difficulties in liquid-solid separation and to the wastewater treatment plant. In this case, the addition of a flocculating or coagulating agent is needed to aid precipitation. Inorganic salts and synthetic polyelectrolytes are good flocculating agents. Recovery of the flocculent from microbial cells is often not feasible and flocculent should therefore be inexpensive. The flocculent should also be nontoxic if the microbial cells are to be used as animal feeds or if the enzyme produced is a food additive. Gelatin and other flocculents with positive nutritional value have an advantage for this application.

H. Separation of Solids After the pretreatment step of fermentation broth, the next task in recovery of an

extra- or intracellular enzyme is to separate microbial cells and other suspended solids from the liquid medium. On a large scale, the problems of solid-liquid separation are very complex and diverse. The preliminary step may be a screening to remove coarse suspended solids. Then centrifugation, filtration, or settling follows which depends on whether yeast, bacteria, mycelia, spores, or viruses are to be removed. The recovery of yeasts is usually accomplished by centrifugation. The separation of mycelial organisms from the liquid broth can be achieved by filtration. Since mycelial organisms may create considerable resistance to filtration, the filter is frequently precoated with a filtration aid such as diatomaceous earth or expanded perlite to prevent filter binding and to increase filtration rates. The filtration aid may also be added directly to the liquid medium.

Bacteria are much smaller than yeast and mycelial organisms and usually create extremely high filtration resistance. They are more difficult to centrifuge because of their

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lower density and smaller diameter. If an efficient method of flocculating bacteria is developed, the solid-liquid separation can be achieved by sedimentation on classifica- tion. Heat generated during centrifugation may create problems to the stability of the enzyme produced. In this case, a centrifuge equipped with a chilled-water cooling system must be used.

In practical terms, continuous centrifuges must be used to accommodate the large volumes to be processed. During the past decade, there has been little development in centrifugation equipment.” Three types of continuous centrifuges are available: disc- type centrifuges, hollow bowl centrifuges, and high speed ultracentrifuges.

The use of vacuum drum filters precoated with suitable filtration aid is particularly widespread in the industry. However, the filtration technique is rarely employed for separation of the microbial debris from the suspension resulting from a cell disruption step (intracellular enzyme process).

I. Disintegration of Microbial Cells In the case of intracellular enzymes, the supernatant liquid is discarded, and the

microbial biomass is slurried in water containing appropriate buffer and/or salts. To harvest the cells, centrifugation is usually preferred to filtration to avoid mixing the microbial cells with the filtration aid.

In many industrial applications, the cells are used directly without extraction of the enzyme. If the enzymes must be extracted, either physical or chemical methods are used for the disintegration of the cells. On an industrial scale, physical methods are always preferred.

Hughes et al.” presented one of the few reviews specifically dedicated to the chemical and physical disintegration of microorganisms. Considerable coverage is also presented by Melling and P h i l l i p ~ , ~ ’ . ~ ~ Wang et al.,” and Scawen et a1.2’ The following extraction methods have found application in large-scale enzyme technology:

1.

2 .

Physical methods: solid shear, agitation with abrasives, liquid shear, freeze-thawing Chemical methods: detergents, enzyme treatment, osmotic shock and alkali treatment

A recent survey of 24 large-scale intracellular isolation procedures indicates that liquid shear is the most popular meth0d.l’ However, the choice of this method may well have been influenced by availability of equipment. In some cases, the enzymes may be partially inactivated when subjected to a shear force, however.

Some advantages and disadvantages of chemical and physical methods for cell disintegration are summarized in Table 8. When the cells are broken, the enzyme can be removed from the resulting solution and purified the same way as an extracellular enzyme. The process is usually more difficult due to the presence of nucleic acids and cell debris from the broken cells.

Full details are not readily available on commercial processes for producing an intracellular enzyme of industrial importance. A schematic diagram for invertase production (from baker’s or brewer’s yeast) is presented in Figure 1. A full detail of this process is available

IV. VOLUME REDUCTION AND CONCENTRATION OF ENZYME- CONTAINING SOLUTIONS

After the solid-liquid separation step, a fairly clear enzyme-containing solution is obtained. This aqueous solution may often be highly diluted. If the process is well

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Physical methods Agitation with abra-

sives (glass beads)

Solid sheaf (freeze- pressing)

Liquid shear

Freeze-thawing

Chemical methods Enzyme treatment

Detergent treatment

Alkali treatment

Table 8 DISRUPTION OF MICROBIAL CELLS

Methods of operation

Rapid mixing with glass beads

Using for brewer's yeast Saccharomyces cerevisiae

Pressure extension of frozen material through a narrow hole

Disruption of the cells is probably caused by deformation of organisms embedded in the ice

Passage of a cell suspension of (10-17 '7% w/v) through a restricted orifice at pressures of up to 200 MPa

Freeze-thawing the suspension at -20 to -3S"C

Ice crystals are respon- sible for breaking the cell membrane

Lysis by egg-white lysozyme

Large-scale lysis: Mi- crococcus lysodeikti- CUS

Without a strong cell wall, the resulting pro- toplast o r spheroplast lyzes because of the high osmotic pressure of the cell contents (5-25 Kg/cm*)

Ionic detergents: sodium dodecyl sulfate and cetyl trimethyl ammonium bromide

Nonionic detergents: Tween or Triton x 100

pH: (11.5-12.5) for 20-30 min

To extract the therapeu- tic enzyme L-asparagi- nase from Erwinia chrysanthemi

Advantages

Effective against tough cell wall

Enzyme activity com- pares favorably with that obtained by other methods

Suitable for Iarge-scale enzyme production

Particularly advantageous when enzymes are sensitive to heat

High enzyme yields are obtained

High percentage of the cells are lysed

Effective for bacterial cells and to a lesser extent fungal cells

Very popular Suitable for large-scale enzyme production

Gentle and selective

Effective for the commercial production of cholesterol oxidase

Inexpensive

Disadvantages

The removal of glass beads from the dis- rupted cells

Absence of commercially available large- scale equipment

Freezing of cells prior to disintegration

Heat generation Single pass operations usually insufficient

Irregular particle size (filament or pellets) reduces the efficiency of cell breakage

Most intracellular enzymes defy release

Most cells are resistant to disruption

Slow and inefficient

Possible disvuction of enzyme by cellular proteases

Expensive Enzymes are mixed with

the whole cell lysate which adversely affects the purification steps

May cause enzyme inac- tivity

The presence of detergents affects the purification step

May cause enzyme inac-

Reduces pyrogen tivation

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Table 8 (continued) DISKUPTION OF MICROBIAL CELLS

Osmotic shock Rapid change in salt Only low levels o f solu- Tough cell walls are less concentration ble protein are re- susceptible to this

leased, facilitating the method subsequent purifica- Cells from solid-liquid tion recovery steps often

contain much occluded salt

optimized, the enzyme should be a dominant protein. The recovery and purification of enzymes may be technically difficult and costly if an enzyme species exists in a very low concentration in the liquid along with thousands of others. For instance, a 15,000- I culture of Srrepromyces RCJ only produces a 3 g of D.D carboxypeptidase-transpep- tidase.

There are also some technical problems associated with enzyme recovery, Enzymes are relatively fragile molecules sensitive to certain chemicals. They also retain their biological activity only within relatively narrow ranges of pH and temperature.

The recovery and purification of enzymes may be more or less extensive, depending on the intended use of the product. Many useful industrial enzyme preparations are not highly purified. On the other hand, for analytical, pharmaceutical, and some special industrial applications, it is desirable to obtain the enzyme in a highly purified form. From a commercial point of view, the desired enzyme purity depends on the potential market, quality and quantity considerations, production costs, and available technology.

The enzyme recovery and purification procedure consists of two basic type of operations: concentration (volume reduction) and purification. A general schematic diagram of extracellular enzyme recovery processes is illustrated in Figure 2.

Microbial enzymes cannot be stored conveniently as crude supernatant solutions, since they are often unstable and susceptible to microbial attack in this state. An initial concentration step is necessary to remove a large volume of water before any standard purification method can be applied. The enzyme solution is usually concentrated by one of the following methods: vacuum evaporation, drying, ultrafiltration, and/or reverse osmosis.

A. Vacuum Evaporation Different types of vacuum evaporators (rotary, shell and tube, falling or climbing

film) are often used for concentrating the enzyme solution. The operating temperature is usually kept below 40°C to minimize heat denaturation. During the vacuum concentration, the enzyme is protected by maintaining the pH at the optimum for stability. Gelatin or protein hydrolyzates are often added to the solution as stabilizers, however, foaming problems then may be encountered. The tendency of enzyme solution to foam causes considerable difficulties and fouling of equipment. The viscosity of the concen- trate becomes very high if there is a significant residual sugar in the enzyme solution. It should be noted that increased concentrations of other solutes in the concentrates may deactivate the enzyme.

B. Drying Freeze drying and spray drying are occasionally used for concentration of microbial

enzymes. Freeze drying can provide a stable product which can be stored easily if

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further processing (final purification) must occur on a staggered time basis. This technique, however, is expensive and relatively slow if it is considered for initial microbial enzyme concentration. The application of freeze drying technique for biological materials is limited. However, it is a very convenient method for high purity preparations and is widely used when applicable. The salt concentration in the solution must be reduced significantly in order to avoid the formation of eutectic mixtures resulting in complete drying and protein denaturation.

Although spray drying can be used for concentration of enzyme solutions, it is usually employed in many industrial enzyme production processes to obtain a dry-powered final product.

C. Ultrafiltration and Reverse Osmosis Ultrafiltration and reverse osmosis, comparatively new membrane techniques, have

been successfully developed for large-scale applications. The term “ultra filtration” describes a process in which molecules or particles of much greater size than the solute are retained when a solution is hydraulically forced through a membrane of a very small pore size. Where even finer pore-size membranes are used and the necessary pressure attains the value of the appropriate osmotic pressure, the procedure is called “reverse osmosis” which passes the water molecules through the small membrane po- res.

In comparison with vacuum evaporation, ultrafiltration has the advantage of remov- ing smaller molecules (molecules of a molecular weight below 10,000 form the per- meate). As a consequence, it is possible to obtain a more concentrated enzyme solution by ultrafiltration than by vacuum evaporation. Ultrafiltration is commonly used to remove salts and low molecular weight species in the final purification step.

The application of ultrafiltration for concentration of microbial enzymes, however, also possesses some drawbacks. One of these drawbacks is that the membranes are frequently clogged by precipitates which may form during the concentration of the enzyme solution. Microbial contamination of the solution may also cause problems.

V. PURIFICATION

Where microbial enzymes of a high degree of purity are desired, a purification step is required to separate the specific enzyme protein from the nonenzyme protein and from the nonenzyme components present in one solution. Since microbial enzymes have their distinct physical properties (solubilities, binding at an interface), it is quite possible to separate a specific enzyme from a multicomponent mixture containing other enzymes and other biochemicals.

According to the enzyme properties, the following methods are usually employed to purify an enzyme solution:

1.

2. 3.

Differences in ionic properties: precipitation, electrophoresis, and ion-exchange chromatography Differences as absorbates: adsorption and affinity chromatography Differences in size: molecular sieve chromatography, dialysis, ultrafiltration, and ultracentrifugation

A. Enzyme Fractionation in Aqueous Salt Solution The solubility of protein macromolecules is dependent on electrolyte concentration.

This fact is being exploited in enzyme recovery processes. Electrolytes can have one of two effects on protein in solution. In dilute solution, the solubility of these ampholytes is enhanced as a result of increased interactions of small ions of neutral salts with polar

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groups of the protein side chains decreasing the activity coefficient of the protein.2s.16 This is known as “salting in”. As the concentration of the electrolyte is increased, a point of maximum solubility of the protein is reached, after which the protein begins to precipitate out of solution. This is known as “salting out”. The effect is caused by increased competition between the polar groups of the protein and ions of the salt for the polar water molecules. Thus, as the ionic strength increases, by increasing the salt concentration, the number of water molecules for the solvation of the protein decreases, increasing the intermolecular protein interaction, and allowing the protein molecules to associate in a solid

I . p H Effect Changing the pH of the solution changes the number of charged groups on the

protein molecules. When a protein is in its isoelectric state, repulsive forces between solute molecules are at a minimum and attractive forces at a maximum, easily forming a crystal lattice, and making the protein less soluble. Therefore, the isoelectric point of the protein is the most effective region of “salting out” and should be taken advantage of in isolated procedures. On either side of the isoelectric point, these amphoteric macromolecules exist predominantly as anions or cations repelling one another and inter- acting to a greater extent with water molecules surrounding them.z7

2. Temperature Effect One of the best methods of crystallizing a protein from solution is to slowly change

the temperature. In dilute electrolyte solution, proteins are more soluble above its freezing point. In concentrated solutions, it is found that different proteins exhibit different solubilities at different temperatures. Also, the saturation point of a salt solution is dependent on temperature, and changing that parameter will change the concentration of the electrolyte, affecting the salting out process. Caution must be exer- cised during the fractionation of very large volumes of proteins which have been stored in the cold. The whole extract must be warmed uniformly to ensure that the entire batch will be affected by the same factors.Z6 Applications of the temperature-change enzyme precipitation are obviously limited.

3. Protein Concentration It was shown by Dixon and Webb26 that in order to obtain good separation and

consistently reproducible results, the concentration of the protein must be high and constant in each purification attempt. Dilute protein solutions require greater salt concentration, making the process more expensive, especially when employed in large- scale operations. Successive fractionation under the same conditions is of limited value. At high salt concentrations, a mixture of proteins will precipitate independently when subjected to fractional salting out.

Another method used in separating a particular protein from a mixture depends on the observation that each type of protein has a different degree of stability with respect to factors such as heat or pH. Thus, removal of the unwanted labile proteins can be accomplished as an initial step in the purification process by denaturation and centrifugation. Stability of most enzymes is increased in the presence of their substrates or inhibitor^.^^.^^

An important factor in the salting out process is the type of electrolyte used. Uni- valent salts such as NaC1 are not as effective as divalent and trivalent ones. The higher the valence the higher the ionic strength produced in solution, and the more effective the electrolyte as a precipitating agent. The salting out constant varies with the type of salt used. Examples of some of the salts employed in this process are KHIP04, KzHP04, Na2S04, Na,C6Hs0,, (NH,)SO,, MgSO,, and sodium phosphate. The salt

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may be added as a solid, a saturated solution, or it may be dialized through a semipermeable membrane. In each case, care must be taken to ensure that the electrolyte spreads uniformly through the protein extract without forming pockets of high concentration. The most common and practical method of changing the concentration of a salt in solution with a protein extract is by the addition of a predetermined weight of the salt. The concentration units traditionally have been used as percent saturation of the electrolyte. Many homograms have been used as percent saturation of the electrolyte. Many homograms have been prepared to expedite the preparation of appropriate concentrations.

The most frequently used salt is ammonium sulfate as it is very soluble, reasonably inexpensive, and does not harm most enzymes. Often, it has a stabilizing effect on enzymes and prevents bacterial growth in protein extracts during storage. Disadvan- tages are few and can be minimized. The salt is slightly acidic and proper control of the pH must be maintained. Good quality (NH4),S04 should be used to reduce the presence of toxic impurities and free acid. For better separation, it is advisable to wait at least 15 min before centrifugation. Finally, the salt has to be removed, usually by dialysis. The presence of the salt affects the choice of the analytical protein estimation method. The major disadvantage of using ammonium sulfate is in sensitivity of certain enzymes to the salt and in costs associated with wastewater treatment.

Of the other salts mentioned earlier, phosphates also work well. Phosphate salts act as buffers, give good separation, and do not interfere with nitrogen estimation. How- ever, these are less soluble and of limited US^.^'^" Application of the salting out process is relatively cheap and does not denature enzymes. Frequently, it is used as one of the effective initial concentration steps, often following vacuum evaporation, which is another concentration technique.,’ A disadvantage in the use of the salting out technique for use in industrial continuous systems is that it takes a long time - up to several hours - for equilibration of the solution to O C C U ~ . ’ ~

B. Fractionation with Organic Solvents The method of fractional precipitation using organic solvents is quite versatile. The

solubility of proteins in aqueous solution can be altered by the addition of certain organic solvents, provided the solution is of low and constant ionic strength and constant pH. Increasing the solvent concentration tends to decrease protein solubility by decreasing the dielectric constant of the medium and perhaps by attracting the protein bound water, as a result, dehydrating and precipitating the enzyme.’’

Because organic solvents tend to denature proteins, this procedure is often carried out at temperatures below 0°C. The low temperatures not only reduce denaturation, but also stabilize a variety of enzymes. Organic solvents that are used must be mixable with water, depressing the freezing point of the solution and enabling work to be carried out at temperatures ranging from -1 to -50°C.’0 Ethanol is the most widely used solvent because of its acceptance by the pharmaceutical industry. However, other solvents, such as methanol or acetone, may be more efficient or have a lower denaturing e f f e ~ t . ’ ~ , ~ ~ The use of solvents, however, may result in increased production expenses reflecting the solvent recovery costs. Versatility of this method is demonstrated by adjusting one of the many variables that affects precipitation.

Lowering the temperature in successive steps addition of a solvent at below 0°C will precipitate certain proteins at each new decrease. Solubility of proteins in aqueous organic solvent solution decreases with temperature. The cost of chilling the solution needs to be considered in selecting the most feasible process sequence.

As in salt fractionation, pH has a great effect on the solubility of proteins when this method is applied. Again, proteins are least soluble a t their isoelectric point. Keeping the pH of the medium near one end of the pH scale will minimize protein-protein

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interactions and will allow fractional precipitation by changing the pH in one direction. Addition of a buffer (0.01 to 0.05 M 17) will facilitate keeping the pH constant when another parameter is varied.30

Neutral salts increase protein solubility in organic solvents; therefore, high concentrations of these salts should be avoided in order to minimize the volume of the solvent used axid to prevent the precipitation of the salt.30

Multivalent cations such as Zn’+ and Ca2+ combine with certain proteins and render them less soluble in both water and organic solvents. This property is of value in iso- lating very soluble proteins. Care must be taken to avoid buffers such as phosphates which will combine with the ion and precipitate out of solution. A detailed procedure for separating proteins using ethanol is discussed in some detail e l ~ e w h e r e . ~ ~

Organic solvents, those mentioned earlier, as well as isopropanol and diethyl ether, have been used in large-scale enzyme preparations. However, problems exist such as maintaining low temperatures, decreasing “adverse side effects on many enzymes” in enzyme activity, high equipment cost (explosion proof motors and switches), and the necessity of pollution control as well as of the removal of highly toxic and explosive vaporsz2 generated during the process operation. In many cases, the possibility of solvent reuse makes up for other expenses.

Using charged polymers such as polyethyleneimine, dextran sulfate, or DEAE-dextran for large-scale protein precipitation was found to be too costly, even though these agents produced good and quick precipitation of proteins reducing centrifugation time in the process.”

C. Recovery by Chromatography In recent years, improvements in chromatography have made this method one of the

most important techniques for the separation of proteins. The method depends on the distribution of a mixture of proteins between a solid phase and a liquid phase. The solid phase, made up of certain polymers, can either be packed in a column or left in a large container. In a column, during elution, proteins are retarded by the stationary phase. The method of retardation depends on the type of material used in the solid phase which specifies the type of chromatography being employed. The most common types of chromatographic methods are gel, ion exchange, and affinity. In the latter two methods, selective adsorption can be carried out in one of two ways, known as negative or positive, depending on whether the required enzyme is eluted first from the stationary phase. Improvements in the basic batch mode of chromatographic operations made them very popular and competitive.

1. Gel Chromatography Since the early 195Os, gel chromatography has undergone a number of changes, not

only technical improvements but also changes in name. Among eight or so names given to this procedure, some of the more common ones are gel filtration, exclusion chromatography, restricted diffusion chromatography, molecular sieve chromatography, and molecular sieve filtration.

Cross-linked dextrans were introduced into column chromatography in the late 1950s. Since then, the technique has become one of the major ones in use of the purification and characterization of proteins. The cross-linked dextrans became known commercially as Sephadex (Pharmacia, Uppsala, Sweden). The introduction of agarose and polyacrylamide gel has increased the range and flexibility of this method.

Cross-linked dextrans form porous, stable, bead-like structures. The process depends on the partitioning of a mixture of macromolecules between the solvent inside and outside the beads. The inside volume determines the size of molecules to be trapped and retarded. Smaller molecules will percolate through the column and diffuse

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into the stationary aqueous phase. Molecules too large to enter the beads are readily eluted at the beginning of the process. The size of the macromolecules trapped depends on the degree of hydration, which in turn depends on the amount of cross linking. Larger molecules are fractionated by gels with greater degree of hydration and smaller degree of cross linking per unit of dry weight of the gel.’O Sephadex G-25@ , e.g., has a greater degree of cross linking than Sephadex G-200@ .33

A detailed analysis of gel chromatography of proteins was covered by Ackers in 1 970.3 One of the widely used chromatography materials is a polyacrylamide-agarose combination prepared in the form of beads containing different amounts of the former. It provides a variety of effective fractionation ranges with extremely narrow size- distribution results in sharp zones of separated components. The beads are rigid and preswollen at an optimum size of 60 to 40 pm, small enough to give high resolution, yet allow good flow rates at reasonable pressures. This material can be used for high molecular weight biomolecules, as well as in affinity chromatography and preparation of immunosorbents.

Hydroxyapatite-agarose materials for gel chromatography commonly contain 40% of the former and are useful for separation of proteins, nucleic acids, and low molecular weight compounds such as nucleotides.

2. Adsorption Chromatography Adsorption chromatography is an effectual technique for enzyme isolation, as par-

tial purification and concentration can be accomplished in one step due to specific interactions between the enzyme and the matrix. This technique has been used successfully on a large scale in the isolation of high cost enzymes destined for research or analytical purposes.22

3. Zon-Exchange Chromatography Fractionation of proteins on ion-exchange media depends on the establishment of

attractions between the stationary phase or mattrix and the sample. Interactions between the adsorbent and the solute are mainly caused by weak, reversible, intermolecular forces such as Van der Waals, electrostatic, hydrophobic, and hydrogen bonds. At low-solute concentrations, the amount of solute that attaches to the adsorbent is directly proportional to its concentration. At very high-solute concentrations, there is an upper limit, known as the capacity, which is the maximum amount that can be adsorbed by the packing material.21 The number of the intermolecular interactions between the matrix and the solute will influence the concentration of ions in the eluting solvent necessary to displace the charged groups of the proteins from the adsorbent. The electrostatic bonds can be broken by changing the pH of the solvent or by changing its concentration, or both. Protein affinity to the ion-exchange medium will depend not only on the number of charged sites, but also on the charge distribution of both components, as well as pH and salt c~ncentration.~~~’~~~~*~~

Of the various types of materials used for packing ion-exchange columns, derivatized celluloses, cross-linked dextrans (Sephadex), or agarose gels give better results than ion-exchange resins. The former, being unstable in highly acidic and basic soh- tions and being less substituted than ion-exchange resins, has a lower density charge and can separate proteins under mild conditions, whereas resins (dowex, amberlite), because of their high density charge, can denature many enzymes. For enzymes that are not denatured, resins provide a quick and effective method of separation, as they have good settling and flow properties.32

Isolation of any given enzyme can be done in one of two ways. The enzyme can be either eluted first or last from the ion-exchange column. In either case, optimum pH range, isoelectric point, and electrophoretic mobility must be known in order to

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Volume 2 , Issue 2 141

achieve the most efficient separation. An enzyme can be eluted either on a cation or anion exchanger depending a n whether the majority of the charged groups on the protein are positively o r negatively charged at the chosen pH. The p H of the medium should be chosen so that the required protein will retain its stability and will have a n overall charge. In order to elute the desired enzyme first, a column containing the same kind of charges as those found on the enzyme must be used, provided the contaminating proteins are less basic or less acidic.

Adsorption can be accomplished by a column or a batch method. For large-scale processing, the batch method is more useful than column chromatography. Pumping large volumes through a column is time consuming and impractical. Packing material can compact, thereby reducing and eventually blocking flow. To minimize the decrease in flow rate, smaller particle-size gels and resins should be avoided and the packing material should be washed extensively before use to remove excessively fine material, commonly referred to as d fine^".^' The adsorbents should be stored at 4°C as moist solids or aqueous suspensions. Addition of 0.03% toluene or 1% butanol to ion-exchangers and 0.02% sodium azide to dextran gels prevents bacterial

Batch adsorption method is advantageous for very large volumes as it saves a great deal of time in achieving a high concentration factor; however, a lower resolution is obtained than in column chromatography. In batch chromatography, the matrix is first suspended in the solution to be separated, then it is removed by filtration, after being equilibrated. Elution of proteins adsorbed can be accomplished in one of two ways, either by packing a column with the “loaded” matrix and then using the gradient elution technique, o r by the batch method elution. Solutions containing particulate matter should be separated by batch adsorption. Also, adsorption should not be at- tempted on a previously packed column. Both of these actions may clog the system, decreasing the flow rate and increasing the chances for d e n a t ~ r a t i o n . ~ ~

Gel filtration column chromatography systems can be scaled up for larger operations without altering the elution profile by keeping the ratio of the length of the column to its diameter and the product of column diameter and flow rate constant. However, increasing flow rates too high increases the risk of inferior separation. Charm and matte^^^ give sample calculations comparing data for small and large columns.

Most column chromatography methods are discontinuous or at best cyclic. A continuous chromatography system has been used by the team of Fox3’.’” who employed a rotating annular column where components of a sample separated and moved down by different spiral pathways. Each component left the column at a different place, depending on the elution time and direction of movement. The rotating annular column can be adapted fo r large-scale operations. While ion-exchange resins find only a limited use in enzyme purifications, the most wideIy used a re the following derivatized celluloses: 16.28.36

. Most widely used ion-exchangers.

Cation exchangers Carboxyrnethyl cellulose (CM-). Phosphorylated cellulose (P-) Sulfothyl cellulose (SE-) Sulfomethyl cellulose (SM-)

pArninobenzyl cellulose (PAB-) Triethylarninoethyl cellulose (TEAE-) EACTEOLA cellulose (mixed arnines) Guanido ethyl cellulose (GE-) Diethylaminoethyl cellulose (DEAE-)’

Anion exchangers

DEAE, CM, SE. QAE cross-linked dextrans - are also being used as Sephadex

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142 CRC Critical Reviews in Biotecbnology

4. Affinity Chromatography This chromatographic method offers extremely high resolution as it is based on very

specific and reversible biological interactions between the desired protein and the adsorbent. The system is composed of a stationary insoluble matrix such as derivatives of cellulose, polyacrylamide, polystyrene, beaded agarose to which a ligand molecule is covalently bound.39 The ligand molecule must have specific affinity for the enzyme being isolated. Two categories of ligands exist:

1.

2.

The specific ligand will isolate only the desired enzyme, e.g., the substrate, substrate analogue or inhibitor for that enzyme. The general ligand, on the other hand, is specific for a certain class of enzymes such as coenzymes 5’ AMP, 2’, 5‘ ADP, or NADZ2 or will attract a specific side chain of the desired molecule, i.e., a hydrocarbon chain.39 Molecules without any attraction towards the ligand will pass through unaffected. Molecules attracted to the ligand will be adsorbed. Elution of the enzyme from the ligand can be achieved by changing the ionic strength or the pH or by applying a specific co- factor or substrate that will compete with the ligand for the active site of the enzyme.

It was found that certain enzymes would be adsorbed by the ligand in limited quantities, or not at all, because of steric hindrance. The distance between the solid support and the ligand is critical in this purification process.39 Molecules that increase the distance between the ligand and the matrix by being covalently bonded to both are known as spacers or spacer arms. The spacers can be added to the system by attaching them to the ligand first and then to the insoluble support, or by attaching them to the solid support and then to the ligand.*O Proteins and other macromolecules as well as hydrocarbon chains were used as Hydrocarbons best suited for this purpose consisted of six to eight carbon

Some methods used to bond spacer arms to the solid matrix create unwanted hydro- philic and hydrophobic groups in addition to the required ligands; thus, the substituted resin may also act as an ion e~changer.’~.‘’ There are synthetic processes available which minimize the formation of these unwanted free amino and carboxyl groups on the matrix. The severity of the ion-exchange problem also can be reduced by acylation or esterification of these groups.39

Another problem that can develop in derivatized agarose matrix is that of leakage. Solubilization of agarose and bond cleavage between the ligand and the adsorbent are possible explanations. Leakage reduces the efficiency of the column as the protein to be purified will preferentially adsorb to the free ligand, especially when the affinity of the two is very high. Extensive washing of the substituted resin with buffers of high ionic strength and wide pH range will minimize the problem. The use of polyvalent spacers, such as polylysine, forming a number of bonds, will cross link the agarose and lower leakage caused by bond breakage.39

Chromatographic columns packed with agarose polyhydrazide derivatives overcome the aforementioned problems as these derivatives are stable during coupling and elution, lack charged groups, and yet can be easily modified. In addition, properties of agarose that make it ideal for affinity chromatography are unchanged. Nonspecific interactions are minimal, the flow rate is good, and the resin is porous enough to allow the passage of macromo1ecules.39

Mono- and dichlorosubstituted triazinyl dyes, such as cibrachron dyes (Ciba-Geigy Ltd.) and procion dyes (Imperial Chemical Industries Ltd.), can be immobilized easily on gels for the purpose of affinity chromatography.” Because they are produced commercially for the textile industry, these dyes are commercially available and inexpen-

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Volume 2 , Issue 2 143

A detailed discussion of the preparations of a number of ligand-resin derivatives is given by Wilchek and H e ~ t e r . ~ ~

Affinity chromatography has an excellent possibility of becoming a large-scale recovery technique. Laboratory trials with 3HB dehydrogenase from Rhodopseudomo- nas spheroides produced yields of 99% .22 Trial runs with other enzymes proved interesting. Very few steps are required for the purification of macromolecules using this technique. Cuatrecasas et al. recovered staphylococcal nuclease in a single step.43

D. Dialysis Dialysis is used to remove low molecular weight contaminants, counterions, or sol-

vent components from a sample of macromolecules being purified. This method involves the movement of undesirable small particles across a semipermeable membrane which, due to its specific pore size, prevents the escape of the desired macromolecules. Materials used as dialysis membranes are chemically inert polymers in the form of very thin films, stored dry and flat. In the presence of a liquid, the film swells and becomes a molecular sieve.

Examples of some of the materials in use are animal membranes, collodion, polyeth- ylene, Kel-F, and cellophane. The most widely used is cellophane as it has fixed pore dimensions, reproducible porosity, and is commercially available."

Dialysis membranes very often contain a certain amount of chemical substances which are used as preservatives and/or plasticizers. For best results, these impurities need to be removed prior to the use by washing the membrane with distilled water or dilute (0.01 N) acetic acid. For some applications, a more thorough washing process is sometimes required.44

Preservation of the membrane is very important. Decrease in porosity and formation of pin holes may result if the membrane is allowed to dry out. Microbial degradation of the membrane is prevented by addition of benzoic acid or formaldehyde into the storage aqueous solution.

Large-volume dialysis is carried out with different types of commercial apparati often utilizing the countercurrent mode of

E. Electrophoresis Proteins, being arnpholytes, react to surrounding charges: ionic strength, pH, or

electric current. In a system where a stable pH gradient is established and a direct current is applied, a protein will migrate between the two electrodes, the anode and the cathode, until it reaches the pH gradient that equals its isoelectric point, PI, a condition in which the overall charge of the protein is zero. A protein applied to a position in the system where the pH equals its isoelectric point will not move. Each protein has its own specific and characteristic isoelectric point which is useful tool in its identification and recovery.

The acceleration of charged particles in an electric field is opposed by the friction created by the surrounding medium, causing the ampholytes to travel at a constant rate which is proportional to their charge. The rate of migration will also vary with temperature, applied current, molecular shape, and size."

A number of electrophoretic techniques have been developed through the years, but very few can be scaled up for industrial preparation. There are a number of factors generated by the process that have adverse effects on biologically active material and on the stability of the system used. Passage of electric current through the medium generates heat. Changes in temperature affect a number of other parameters: (1) ther- molabile proteins can denature: (2) convection currents may be created as a result of changes in density: (3) lower resolution may result from the decrease in viscosity and increase in diffusion: and (4) increase in evaporation will affect ionic strength and pH". The effect of heat was reviewed by Bier.45

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Production of convection currents is more pronounced in free solutions and thus techniques classed as “free electrophore~is” ,~~ those without a supporting medium, do not lend themselves well to scaling up. Even some techniques using stabilized media may produce uneven migration of boundaries. Therefore, there is a small number of electrophoretic techniques that can be utilized for large-scale preparation of proteins. Besides the heat-related disadvantages, many techniques require expensive equipment and take a long time for separation; Ko1i1-1‘~ used a sucrose density gradient in a liquid column to prevent convection currents.

VI. CONCLUSIONS

The early investigations of enzymes were chiefly concerned with the isolation of known enzymes from their substances and in demonstrating their activity under conditions controlled only to a certain limited degree. Gradually, we became aware that enzymes could be harnessed to perform a variety of industrial tasks. The complex structure of the protein molecule makes possible the isolation of enzymes in virtually limitless variations, each with its own substrate. The feasibility of producing enzymes in commercial quantities has already been demonstrated and new enzyme-catalyzed reactions and conversions are being utilized at an escalating rate.

The practice, summarized in this review, shows that all techniques used in protein isolation and purification in general are quite applicable in separation of a specific enzyme from other biochemicals present in the solution or fermentation broth. Each enzyme requires a specific purification technique or sequence of its own, and no general recovery procedures can be quoted as such. The requirements of the enzyme application determine the degree of purification and techniques employed. In some cases, a combination of different fractionation techniques, (repetition of individual steps whole sequences) is required to obtain a purified enzyme product from a mixture of other enzymes and/or proteins of perhaps very similar chemical and physical properties.

A newly developing technique which is highly specific and offers a great promise is based on purification by immunoadsorption. The ability to derive antibodies to a single compound of a heterogeneous mixture opens up a new approach to the purification of natural products. To separate a single component from a mixture, one merely has to contact in a column the mixture with a monoclonal antibody specific only to the desired component. The antibody-antigen complex can then be separated, leaving the pure component as the product. The advantages of this procedure relative to conven- tional methods are obvious. First, a one-step purification is involved as opposed to a tedious, many-step process, and second, yields are greater.

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Documents

Microbial Enzymes: Production, Purification, and Isolation