Biotechnology - Yu.O. Sazykin 2006

Applied Biotechnology
Pharmaceutical products obtained by biotechnological methods
Enzymes

Ferments or Enzymes (from fermentum meaning "leaven" and enzyme meaning "in Yeast") are high-molecular-weight protein compounds that act as catalysts for biochemical reactions both inside and outside living organisms. To date, only a single non-protein enzymatic reaction has been discovered, in which an RNA molecule catalyzes its own self-Cleavage.

A living Cell contains a multitude of diverse compounds, yet the reactions between them are far from random; rather, they form strictly defined metabolic pathways characteristic of that specific cell. A cell's individuality is largely determined by the unique Complement of enzymes it is genetically programmed to produce. The absence of even a single enzyme—or any structural defect within it—can lead to severe adverse consequences for the Organism.

All enzymes are Globular Proteins, with each enzyme performing a specific function tied to its characteristic globular Structure. However, The activity of many high-molecular-weight enzymes—ranging from 10 kDa to 1 MDa or more (for comparison: the Molecular Weight of glucose is 180, carbon dioxide 44, and Amino Acids range from 75 to 204)—depends on non-protein, low-molecular-weight compounds known as Cofactors.

The molecular complex formed by the protein moiety (apoenzyme) and the cofactor is called a holoenzyme. Metal Ions (Zn²⁺, Mg²⁺, Mn²⁺, Fe²⁺, Cu²⁺, K⁺, Na⁺) or complex Organic compounds can act as cofactors. Organic cofactors are commonly referred to as Coenzymes, some of which are vitamin derivatives. The bonds between an enzyme and its cofactor vary in nature: sometimes they exist independently and bind only during the course of a reaction, while in other cases the cofactor and enzyme are permanently bound, occasionally via strong covalent bonds (in which case the non-protein portion is termed a prosthetic group). Vitamin derivatives are among the most critical coenzymes, whereas minerals (macro- and microelements) serve as cofactors. A deficiency in Vitamins AND MINERALS leads to a sharp decline in enzymatic activity, which underlies many pathological processes within the body.

Enzymes are classified into simple and complex types. Simple enzymes consist exclusively of amino acids, whereas complex enzymes (which comprise the majority) incorporate a non-protein group (a cofactor or coenzyme) In addition to the protein component.

A defining feature of enzymes is their exceptionally high catalytic activity, accelerating reactions by orders of magnitude. A single enzyme molecule can catalyze The conversion of anywhere from a thousand to a million substrate molecules per minute under normal temperatures—a reaction velocity unattainable by non-biological catalysts. Another equally vital property of enzymes is their Specificity (selectivity) regarding substrate structure, reaction type, and operating conditions.

Possessing high specificity, enzymes steer chemical transformations along strictly defined pathways. They catalyze reactions under mild conditions—namely, at normal atmospheric pressure, moderate temperatures, and near-neutral pH values—while remaining exceptionally sensitive to shifts in ambient pH and Temperature. Because maximum enzyme activity relies on the optimal conformation of the enzyme molecule as a whole and its Active Site in particular, even minor environmental alterations that affect substrate binding or the Tertiary Protein Structure will correspondingly impact The rate of the enzymatic reaction.

The optimal pH for a given enzyme ensures that its ionization state achieves the highest degree of complementarity. Temperature variations produce a dual effect: on the one hand, raising the temperature up to 37–40 °C increases the reaction rate, as is typical for catalysis; on the other hand, temperatures exceeding 50 °C trigger enzyme Denaturation. Enzymatic processes run without Side Reactions, characteristically yielding a 100% output of the target product.

Enzymes are subject to regulation, meaning they can alter their activity in response to various factors, which in turn varies the yield of the target product. This regulatory mechanism ensures the precise temporal coordination of all metabolic processes.

The rate of an enzymatic reaction is directly proportional to The amount of enzyme present. Consequently, an enzyme deficiency within the organism results in a sluggish conversion rate for specific compounds. Conversely, one of the ways an organism adapts to environmental changes is by upregulating The production of the required enzyme.

Over 6,000 enzymes have been identified, many of which catalyze solely a single reaction; 150 of these have been isolated in crystalline form.

Enzymes are classified not as individual chemical substances, but rather as catalysts of specific chemical transformations or groups of transformations, and are accordingly divided into six major classes:

✵ Oxidoreductases — catalysts of oxidation-reduction reactions;

✵ Transferases — Reactions Involving the transfer of specific chemical groups from one molecule to another;

✵ Hydrolases — hydrolytic cleavage of chemical bonds (utilizing Water);

✵ Lyases (synthases) — reactions resulting in the joining or splitting of molecules (via the addition or removal of water, ammonia, carbon dioxide, etc.);

✵ Isomerases — interconversion of isomers (structural rearrangement within a single molecule);

✵ Ligases (synthetases) — bond formation driven by the Condensation of two distinct compounds coupled with ATP energy.

The recognition of the pivotal role enzymes play in all cellular processes has led to their widespread application across various industries, including pharmaceutical manufacturing, as well as in medical practice as therapeutic and diagnostic agents.

Currently, three primary directions of research in medical enzymology have emerged: enzymopathology, enzymodiagnostics, and enzymotherapy.

Enzymopathology. As is well known, among more than two thousand hereditary human diseases, the molecular developmental mechanism has been elucidated for only a few dozen. Most frequently, the onset of pathology is directly linked to a hereditary deficiency or the complete absence of the synthesis of a single specific enzyme in the patient's body. A classic example of such a link between a disease and the lack of hepatic synthesis of a specific enzyme is Phenylpyruvic Oligophrenia (phenylketonuria), a hereditary disorder leading to death in early childhood or severe mental retardation. The enzyme phenylalanine 4-hydroxylase, which catalyzes the Conversion of the essential amino acid phenylalanine into Tyrosine, fails to be synthesized in Liver Cells. Treatment largely boils down to eliminating The amino acid phenylalanine from the child's diet (including breast milk). Similarly, The Development of another severe hereditary condition, galactosemia (an intolerance to milk sugar), stems from the absence of liver cell enzymes that catalyze the conversion of galactose into glucose. The consequence of this anomaly is the accumulation of galactose in Tissues, leading to early-onset cataracts and damage to liver and Brain tissues, frequently resulting in the child's death. In this case, treatment consists of excluding milk sugar from the diet.

Beyond hereditary disorders, enzymopathology successfully addresses the Pathogenesis of somatic diseases, aiming to uncover the molecular foundations of conditions such as malignant cell proliferation, atherosclerosis, and rheumatoid Arthritis.

Enzymodiagnostics. The extent of organ damage, cellular Biomembranes, and subcellular structures—as well as the severity of a pathological process—can be assessed by the appearance (or a sharp surge in the levels) of organ-specific enzymes and Isoenzymes in the Blood serum of patients, which forms the Subject Matter of diagnostic enzymology.

Presently, over 30 enzymes are known whose blood levels rise during cellular damage to various Organs in the acute and chronic phases of disease. Determining the activity of most of these serum enzymes is utilized for diagnosing and monitoring the treatment of numerous pathologies. For each such enzyme, baseline reference values (activity levels) and normal fluctuation ranges have been established for both blood serum and the respective organ.

As an example, consider the assay of transaminase activities—specifically aspartate aminotransferase and Alanine aminotransferase. Under normal conditions, the serum activity of these enzymes ranges from 5 to 40 IU. In Heart Failure and coronary artery disease, serum levels of both transaminases experience a modest elevation; however, upon the onset of a myocardial infarction, within a mere 20 minutes the serum activity of both transaminases sharply surges, exceeding the baseline levels of a healthy individual by tens or hundreds of times.

In addition to serum transaminases, Lactate dehydrogenase and creatine phosphokinase tests are highly informative diagnostic enzyme assays for myocardial infarction, which also fall under the category of necrotic enzyme Methods. When a portion of The Heart Muscle is damaged and disintegrates due to a coronary artery occlusion by a thrombus, breakdown products, including enzymes, are washed out of the ischemic zone into the bloodstream. In favorable cases, serum enzyme levels return to normal by the second or third day post-infarction. However, in recurrent myocardial infarction, which typically occurs During the first week of the illness, the Electrocardiogram often fails to detect it, whereas enzyme tests react with a repeated and sharp increase in their serum levels.

The diagnostic value of enzymes increased significantly after the Introduction of isoenzyme Determination Methods into clinical practice. In this regard, It is worth noting the Diagnostic significance of two enzymes whose isoenzyme profile assays have been adopted in almost all clinical laboratories worldwide.

The first of these is lactate dehydrogenase (LDH), which catalyzes the reversible conversion of pyruvic acid into lactic acid. It should be emphasized that LDH is a key enzyme of anaerobic Carbohydrate METABOLISM in All living organisms, determining the rate of energy production in the form of adenosine triphosphate (ATP). The widely distributed LDH enzyme is synthesized in almost all cells of The Human Body. There are two types of LDH: the so-called cardiac H-type (from English heart) and the muscle M-type (from English muscle); each consists of four subunits. In organic heart muscle damage, such as myocardial infarction, the level of total lactate dehydrogenase in blood serum rises sharply, which is primarily due to isoenzymes 1 and 2 (this is crucial for diagnostic accuracy).

In Skeletal Muscle damage, inflammatory processes (hepatitis), viral liver tissue damage, and finally, carbon tetrachloride or other poisonings where the liver is primarily affected causing tissue necrosis, the level of LDH isoenzymes 3 and 4 sharply increases while the levels of isoenzymes 1 and 2 remain almost unchanged. Naturally, treatment methods will differ significantly; the H- or M-type LDH isoenzyme profile plays a considerable role in choosing the appropriate approach.

The second enzyme, whose diagnostic value is even higher—especially in myocardial infarction—is creatine phosphokinase (CPK), which catalyzes The Biosynthesis of creatine phosphate from creatine and ATP. Creatine phosphokinase is a key enzyme in the biosynthesis of the high-energy substrate creatine phosphate, which, alongside ATP, plays a prominent role in the Bioenergetics of the heart muscle and the entire body. The CPK molecule is also composed of Two Types of subunits: muscle (M) and brain (B) (from English brain); three CPK isoenzymes have been isolated and characterized.

Diagnostic enzymology has achieved significant success in diagnosing diseases not only of the aforementioned organs, but also of others, particularly the Kidneys, Pancreas, Stomach, intestines, and Lungs.

One of the primary tests for liver damage is the determination of alkaline phosphatase activity—an enzyme that catalyzes the cleavage of a phosphate group from organic compounds, namely phosphoric acid esters. The activity of this enzyme increases significantly in Primary and secondary liver neoplasms, hepatic cholestasis, and liver cirrhosis. Clinical practice also widely utilizes, for instance, determining serum transamidinase—an enzyme discovered exclusively in Kidney and pancreatic tissue—or measuring the activity of histidase, an enzyme found only in liver cells and Skin epidermis. In organic lesions of these organs, inflammatory processes, trauma, and surgical interventions, these enzymes appear in the patients' blood serum, normally being absent from it.

Enzyme therapy. Currently, over 40 enzyme preparations of animal, plant, and microbial origin are used for pharmaceutical purposes. A number of proteolytic agents have been obtained (Trypsin, Chymotrypsin, etc.); special fibrinolytic agents (fibrinolysin, streptokinase, etc.); preparations that reduce the viscosity of hyaluronic acid (hyaluronidase, ronidase), and others. These medications are frequently employed in treating conditions accompanied by purulent-necrotic processes, thromboses and thromboembolisms, digestive disorders, and more. Enzyme preparations are also utilized in oncological treatment (asparaginase). The development of novel drug delivery systems—immobilized enzymes (streptodecase)—opens up new Prospects for the successful application of enzymatic agents.

Simultaneously, the range of medications whose action is associated with Enzyme inactivation has begun to expand. These include proteolytic Enzyme Inhibitors (pantripyn, ingitril, etc.), widely used in the treatment of acute pancreatitis and other conditions; selectively acting Fibrinolysis inhibitors (aminocaproic acid, etc.) used as antihemorrhagic agents; a large group of anticholinesterase drugs; monoamine oxidase inhibitors used as psychotropic agents; and Carbonic anhydrase inhibitors employed as Diuretics. The efficacy of allopurinol in hyperuricemia is related to its inhibition of the enzyme xanthine oxidase, while the action of disulfiram (teturam) in treating alcoholism is due to its suppression of acetaldehyde oxidase.

An important group of medicinal substances consists of enzyme reactivators that restore inactivated enzyme function (cholinesterase reactivators).

The following enzyme preparations are obtained for medical purposes via microbiological synthesis:

✵ solizym — a fat-hydrolyzing lipolytic enzyme used in chronic gastrointestinal diseases;

✵ α-amylase — a starch-hydrolyzing saccharolytic enzyme, used as part of the therapeutic drug «festal» in case of exocrine pancreatic insufficiency;

✵ terrilytin — a proteolytic enzyme recommended for the treatment of purulent burn wounds and trophic ulcers;

✵ streptokinase — a fibrinolytic enzyme effective in the treatment of thrombosis;

✵ galactosidase — a saccharolytic enzyme that has proven effective in the treatment of lactase deficiency.

Traditional biotechnologies based on the Processing of animal tissues supply medical practice with:

✵ trypsin, chymotrypsin — Proteolytic Enzymes used to resolve scars and adhesion processes;

✵ urokinase — a proteolytic enzyme intended for the treatment of thrombosis;

✵ Pepsin — a proteolytic enzyme indispensable for digestive disorders.

Those same traditional biotechnologies, but based on the processing of plant raw Materials, yield proteases such as bromelain, Papain, and ficin, which are used in enzyme replacement therapy for digestive disorders and in serological Diagnostics to detect the Rhesus factor.

Enzyme-based pharmaceutical formulations are characterized by great diversity, including tablets, capsules, ointments, and aerosols.

In our country and abroad, proteolytic enzymes—the most thoroughly studied among all known enzymes—rank first in terms of production volume. Proteases were among the first proteins to be obtained in a highly purified crystalline state. The Primary Structure of such enzymes as chymotrypsin, trypsin, papain, and subtilisin has been elucidated. To date, four classes of proteases are known: Serine, carboxy, Cysteine, and metalloproteases. This Classification is based on the specific STRUCTURE OF THE active centers of these enzymes, which represent a unique combination of specific amino acid residues located at various points along the polypeptide chain. Specific inhibitors are used to identify particular reactive groups.

The Class of serine or alkaline proteases encompasses a group of proteolytic enzymes containing the triad asparagine — serine — Histidine in their active center. Specific inhibitors of these enzymes, whose maximum activity is exhibited in the pH range of 7.5–12.5, are phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate.

Alkaline proteases include enzymes of animal origin: trypsin and chymotrypsin, which are widely used in medical practice for both local and parenteral application. They are also administered as aerosols for inflammatory respiratory tract conditions—tracheitis, Pneumonia—as well as via intramuscular injections for Osteomyelitis, thrombophlebitis, and Various Forms of periodontal disease. These preparations are inactive and safe regarding healthy tissues due to the presence of trypsin inhibitors in those tissues.

Extracellular alkaline proteinases have been isolated and thoroughly studied from various strains of Bacillus subtilis microorganisms. Subtilisins are among the few proteins whose three-dimensional structure has been determined and molecular model constructed. Due to their broad action specificity, these enzymes hydrolyze up to 80 peptide bonds in proteins. They exhibit both esterase and amidase activities; they are stable in the pH range of 5–10, with an optimal action range at pH 9–10. In terms of MECHANISM OF ACTION and substrate specificity, subtilisins are closely related to trypsin and chymotrypsin, despite the complete lack of structural similarity between them. In the biotechnological industry, alkaline proteases from bacterial cultures are utilized for Protein Hydrolysis, with subsequent application of the hydrolysates as components of nutrient media supplied to clinical and bacteriological laboratories.

The primary Representatives of the carboxyl or acid protease class include pepsin, chymosin, and fungal proteases. The primary structure of pepsin has been deciphered and comprises 300 amino acid residues. Pepsin is produced by the gastric mucosa cells as a zymogen—an inactive enzyme form that is activated by another activating enzyme that cleaves off an amino acid from the N-terminus of the zymogen molecule. The active center of the zymogen contains two carboxyl groups of aspartic acid. A specific inhibitor of this class of proteases is the natural peptide pepstatin. Acid proteases share several common features. They are stable in an acidic environment (pH 2.0–5.0); their optimal activity occurs at pH 1.5–5.0, and they are rapidly inactivated at neutral pH values. Pepsin and chymosin lack alpha-helices but are characterized by the presence of an antiparallel beta-structure. Japanese researchers suggest that the stability of acid proteases at low pH values is specifically associated with this feature of their Secondary structure.

Thiol-dependent or cysteine proteases relevant to medical practice include papain from the melon tree, ficin from plant latex, bromelain from pineapple fruit, and a number of enzymes of microbial origin.

Cysteine proteases contain an amino acid triad in their active center: cysteine-histidine-aspartic acid. The activity of thiol proteases is inhibited by specific sulfhydryl group blockers (iodoacetate, iodoacetamide, p-chloromercuribenzoate, heavy metal ions). Their maximum activity is observed at pH 6.0–9.5. Papain and chymopapain are temperature-resistant. A distinctive feature of these enzymes is their high resistance to Denaturing Agents; for example, their activity remains unchanged in 6–8 M urea or 70% methyl alcohol.

Studies on their hydrolytic activity have demonstrated that these enzymes cleave proteins more extensively compared to enzymes of animal and bacterial origin. Thus, papain is capable of hydrolyzing virtually any peptide bond. In addition to peptide bonds, thiol-dependent enzymes hydrolyze amide and ester bonds.

Bromelain, papain, and ficin are used in medicine to lyse non-viable necrotic tissues in the treatment of purulent-inflammatory processes. As a result of enzymatic wound debridement, healing times are reduced by 1.5–2.0 times. Bromelain, papain, and ficin are employed in replacement therapy for digestive disorders, as well as in obstetric practice for identifying and preventing Rhesus incompatibility situations.

The class of neutral or metalloproteases consists of enzymes primarily of microbial origin. Neutral proteases contain Ca²⁺ and Zn²⁺ ions in their active center. Effective at neutral pH values, metalloproteases exhibit strictly endopeptidase activity and do not cleave peptide bonds formed by amino acid residues with free NH₂ and COOH groups. Neutral proteases hydrolyze casein, gelatin, and egg albumin, and unlike subtilisins, they do not possess esterase activity. In medical practice, terrilytin from Aspergillus terricola is widely used to treat acute purulent diseases and trophic ulcers. Inflammatory processes in the lungs are treated with terrilytin inhalations. Another neutral protease of microbial origin, collagenase, is used in eye surgery to dissolve scar tissue.

Despite the fact that the laboratory synthesis of a number of enzymes—such as Ribonuclease, Lysozyme, ferredoxin, and cytochrome c—has already been accomplished, it is unlikely that synthetic enzyme production will become widespread in the coming decades due to its complexity and high cost. Therefore, isolation from biological sources remains the only viable method for obtaining enzymes. Enzymes are isolated similarly to other proteins, although certain techniques are applied primarily to enzymes. Among these, glycerol extraction is noteworthy, as it preserves the native Properties of Enzymes, as well as the acetone powder method, which involves precipitation and rapid dehydration (at temperatures not exceeding 10 °C) of tissues or tissue extracts containing enzymes.

Such methods also include the production of enzymes via adsorption followed by elution (desorption) from the adsorbent. The adsorption method for enzyme Isolation and Purification has been thoroughly developed. One of its modifications is Affinity Chromatography, where the adsorbent is a substance with which the enzyme interacts selectively. As a result, only this enzyme is retained on the Column, while all accompanying substances are eluted with the developer flow. By changing The Nature of the developer, the desired enzyme is eluted from the column. This method achieves Enzyme Purification by several thousand times using a single-step (affinity sorption–elution) isolation scheme.

Along with this, Ion-exchange chromatography, molecular sieving, Electrophoresis, and especially isoelectric focusing are widely used.

For the successful extraction of enzymes from cellular contents, very fine grinding of the initial material is required, up to the disruption of subcellular structures (specifically Lysosomes, Mitochondria, and nuclei) that house many individual enzymes. Special attention is paid during enzyme isolation to performing all operations under conditions that exclude Protein Denaturation, which leads to the loss of enzymatic activity. Therefore, these technological operations are carried out in the presence of protective additives, specifically SH-containing compounds (cysteine, Glutathione, mercaptoethanol, cysteamine, etc.). It is crucial to maintain a low temperature at all stages of enzyme isolation, as some enzymes lose activity even at 80 °C. Standard Methods of Protein chemistry are employed to assess the homogeneity of the enzyme preparation. The purity of an enzyme preparation is generally judged by its biological activity: if the activity does not increase upon further purification, the preparation can be considered homogeneous.

As an example, brief technological schemes for obtaining three Enzymes can be cited: terrilytin, streptokinase, and lipase.

The proteolytic enzyme preparation terrilytin is obtained by cultivating a strain of Aspergillus terricola followed by Separation from the mycelium. The resulting native solution is then freed from pigments by passage through an anion exchanger (a polycondensation product of formaldehyde phenoxy derivatives at neutral pH). Next, the enzyme solution is desalted and concentrated via ultrafiltration; the resulting concentrate is lyophilized. To shorten the process duration and increase the yield of the target product, the depigmented native solution is additionally purified from high-molecular-weight ballast substances.

Streptokinase is obtained by CONTINUOUS CULTIVATION OF the producer strain Streptococcus equisimilis on a nutrient medium comprising meat-peptone broth containing beef heart infusion, glucose, and potassium bicarbonate. The supply of the nutrient medium is regulated depending on Changes in the pH of the culture suspension.

Subsequently, the target product is isolated and purified. To increase the yield of the target product, the nutrient medium is heated to a specified pH prior to cultivation.

Lipase is obtained from cotton seeds, which are irradiated for a specific duration with pulsed concentrated light. The seeds are then germinated in the dark. The germinated seeds are crushed and treated with acetone, after which lipase is isolated from the acetone extract by sequential precipitation with ammonium sulfate solution and acetone. To increase the yield of the target product and enhance its activity, the seeds are pre-soaked prior to irradiation.

Review Questions

1. What chemical modifications of the steroid molecule are carried out using biotransformation?

2. What serves as the primary source of raw materials for the production of steroid drugs, and why?

3. What is the basis for selecting microorganisms capable of transforming Steroids?

4. What forms The basis of the vitamin classification?

5. Which methods dominate in vitamin production?

6. Which stage of ascorbic acid production is a biotechnological method?

7. What amino acid-based medicinal drugs exist?

8. What is the difference between Threonine biosynthesis and Lysine biosynthesis?

9. What are the causes of dysbiosis?

10. What requirements must the production strain of a symbiont microorganism meet?

11. What are The main classes of enzymes?

12. What are the Specific features of enzyme purification and Isolation Methods?