Enzyme Engineering

Summary

Catalysts accelerate the rate of chemical reactions without being essentially changed, and enzymes are biological catalysts that accelerate the rate of reactions that occur in living systems. Enzyme engineering identifies enzymes that have potentially useful catalytic activities and chemically or structurally modifies them to increase their activity, change their substrate specificity, change the types of reactions they catalyze, or change the properties of enzymes and the manner in which they are regulated. Engineered enzymes can generate novel molecules or new, improved ways to synthesize useful molecules.

Definition and Basic Principles

Enzymes are widely used as catalysts in several industrial ventures, ranging from food to synthetic chemistry to many other industrial processes. However, enzymes often show insufficient substrate selectivity, poor stability, and catalytic activities that are not robust enough for industrial use. To remedy this shortcoming, enzyme engineering builds new enzymes or modifies existing enzymes to give them novel, useful properties, or the ability to catalyze valuable chemical reactions. Engineered enzymes come in several forms. Semisynthetic enzymes (synzymes) have specific amino acids that have been chemically modified. These modifications can significantly alter the activity, specificity, or properties of the enzyme. Directed evolution subjects the gene that encodes the enzyme to multiple rounds of mutation. The variant enzymes generated by these mutagenic genes are then sieved by some kind of selection scheme that identifies those mutant forms that display the desired characteristics or activities.

A cheaper strategy is rational design. Rational design uses detailed knowledge of the structure of proteins to identify those regions that are essential for its function and properties. By changing only those amino acids thought to be necessary for the modification of that function, the enzyme is potentially tailored for a new function with little investment.

Enzyme engineers use catalytic antibodies or abzymes. Antibodies are Y-shaped proteins made by vertebrate immune systems that bind to specific chemicals. Abzymes bind to chemicals and force them into the transition state of a chemical reaction, which accelerates the formation of the product from the reactants.

Background and History

Enzyme engineering arose only after advances in several other fields made it possible to determine the primary amino acid sequences and three-dimensional structure of enzymes and directly manipulate them at the molecular level. Swedish biochemist Pehr Victor Edman gave birth to protein sequencing in 1950, when he designed the Edman degradation reactions that can determine the primary amino acid sequence of proteins. In 1958, English biochemist John Cowdery Kendrew used X-ray crystallography to solve the three-dimensional structure of the muscle oxygen-storing protein myoglobin. In the 1970s, American biochemist Herbert Wayne Boyer and American geneticist Stanley Norman Cohen pioneered molecular cloning techniques that gave scientists the means to clone genes and insert them into bacteria for propagation.

The first studies in enzyme engineering examined the effects of mutations on enzyme active sites. Beta-lactamase, the enzyme used by bacteria to degrade beta-lactam antibiotic (penicillin, ampicillin, and amoxicillin) was one of the first enzymes examined by enzyme engineering. In 1978, Canadian chemistMichael Smith and his colleagues invented site-directed mutagenesis, which gave biochemists a much better way to place targeted mutations into the genes that encode enzymes and thereby change their primary amino acid sequence. In 1986, the laboratories of Peter Schultz (University of California, Berkeley) and Richard Lerner (Research Institute of Scripps Clinic) made the first catalytic antibodies that could split ester bonds.

How It Works

Semisynthetic Enzymes. Enzymes that are modified by chemical means are known as semisynthetic enzymes. There are two main ways to produce semisynthetic enzymesatom replacement or group attachment.

Atom replacement exchanges one atom within an enzyme for a different atom. Such replacements can modify enzyme activity or change the substrate specificity of the enzyme. Group attachment involves the use of particular chemical reagents to attach particular molecules to enzymes. Attaching additional molecules to enzymes can also markedly change enzyme activity and substrate specificity.

Directed Evolution. Directed evolution randomly changes amino acids in a protein without prior knowledge of the exact function of each amino acid. The first step, diversification, takes the gene that encodes the enzyme of interest and replicates it many times while using a copying machinery that is inherently error-prone. This introduces random mutations into the gene and creates a large collection of gene variants that are usually grown in bacteria. The second step, selection, tests or screens these enzyme variants for a desired property. Once the desired variants are identified, they undergo the third step, amplification, which replicates the identified variants and sequences them to determine which mutations produced the desired properties. Collectively, these three steps constitute one round of directed evolution, and the vast majority of such experiments require multiple rounds. The goal is to find those variant enzymes that show the most desired characteristics to the greatest extent. Directed-evolution studies suffer from the need to make huge numbers of mutants that produce no discernible effect since up to 90 percent of all mutants made are uninformative.

Semirational Design. This enzyme engineering strategy employs sophisticated computer programs that assemble all the available structural information of the enzyme under study and predict how the mutations introduced into different locations within the enzyme might affect its activity. The enzyme engineer then notes the predicted changes that will potentially generate the desired property changes and uses this information to conduct targeted mutagenesis experiments. Targeted mutagenesis experiments introduce mutations into specific locations of a protein. Once these mutations are made, the variant enzyme with the engineered changes is tested to determine if it has the specific properties the enzyme engineer was hoping to produce in the enzyme. These approaches combine structural information with rational design. Two computer programs that make such predictions include Protein Sequence-Activity Relationship or ProSAR and Combinatorial Active-Site Saturation Test, otherwise known as CASTing.

Rational Design. If a great deal of structural information about the enzyme in question is available, then that structural information informs which amino acids should be changed. Many rational design attempts have not succeeded because of uncertainties regarding protein structure.

De Novo Design. A computer builds an enzyme around the transition state of a reaction from scratch. The computer begins by designing the active site by placing specific amino acids in strategic positions so that they efficiently bind the transition state of the chemical reaction and stabilize it. The program then constructs a protein backbone that supports and properly positions the active-site amino acids but still provides a coherent protein structure that is predictably stable under the desired conditions.

This particular strategy suffers from gaps in the ability to predict protein structure accurately and correlate this ideal structure with enzymatic activity. For example, two enzymes (retro-aldol enzyme and a Kemp elimination catalyst) were built completely from scratch by using computer programs. However, both enzymes required further optimization by directed evolution to achieve maximum activity.

Catalytic Antibodies. The immune system of some vertebrates makes Y-shaped proteins that specifically bind to and neutralize foreign substances that invade the body. Immunizing laboratory animals with stable analogs of the transition states of various reactions directs the immune systems of those animals to synthesize antibodies that cannot only bind particular chemical reactants but force them into the transition state of the reaction, which subsequently forms the product.

Applications and Products

Pharmaceutical Production. Beta-lactam and cephalosporin antibiotics are commonly prescribed to combat various illnesses. Both of these drugs kill bacteria by inhibiting the synthesis of the bacterial cell wall. Beta-lactam antibiotics include such widely recognized drugs as penicillin, ampicillin, and amoxicillin, whereas cephalosporin antibiotics include such popularly used antibiotics as Ceftin (cefuroxime), Kephlex (cephalexin), and Ceclor (cefaclor). Unfortunately, with repeated use, bacteria can become resistant to commonly used antibiotics, and making new, improved antibiotics is essential to treat some of the more recent and aggressive infectious diseases. To make new cephalosporin antibiotics, enzyme engineers have used enzymes called acylases to convert simple starting chemicals into various versions of these drugs. By engineering these acylase enzymes, pharmaceutical companies have been able to make new cephalosporin and beta-lactam antibiotics that have novel properties and can kill bacteria that are resistant to older drugs.

Enzymes as Medicines. When a person is cut, blood oozes from the damaged tissue. Fortunately, blood clotting (also known as coagulation) eventually stanches this blood flow. Blood clotting is an essential part of wound healing, but it is also a very highly regulated event. The formation of blood clots inside undamaged blood vessels clogs those vessels and leads to heart attacks if clots form inside the vessels that surround the heart, or a stroke, if they occur within vessels that surround the brain. The human body has ways to destroy unnecessary clots. An enzyme called tissue plasminogen activating factor (TPA) activates other enzymes in the body that degrade harmful clots. Commercially available, native TPA is called Alteplase, which has a half-life in the bloodstream of four to six minutes. Engineered forms of TPA are also clinically available. Reteplase, a shortened version of TPA (consists of 357 of the 527 amino acids of Alteplase), has a longer half-life (thirteen to sixteen minutes). Tenecteplase, which has two amino acid changes (substitutes asparagine¹⁰³ with a threonine and asparagine¹¹⁴ with glutamine), has an even longer half-life of twenty to twenty-four minutes.

Engineered enzymes are also used in enzyme-replacement therapies. Several genetic diseases, known as lysosomal storage diseases, result from the inability to make functional versions of enzymes that degrade various biological molecules. The accumulation of these molecules kills brain cells and causes the death of the patient. Engineered enzymes used in enzyme-replacement therapies include Cerezyme (imiglucerase, used to treat Gaucher's disease), Naglazyme (galsulfase, used to treat mucopolysaccharidosis VI), Myozyme (alglucosidase alfa, used to treat Pompe disease), and Aldurazyme (laronidase, used to treat mucopolysaccharidosis I).

Enzymes for Food Production. Enzymes are used in many areas of food production, particularly in dairy farming for such purposes as milk coagulation, cheese ripening, flavor modification, hypoallergenic dairy production, lactose reduction, and sterilization, among others. Enzyme engineers have worked to develop helpful enzymes for these purposes. For example, cheese coagulation is traditionally accomplished using calf rennet, a combination of the enzymes rennin and pepsin extracted from the stomachs of calves. However, much cheese is now made with a vegetarian rennet extracted from genetically modified yeast and fungi that provide the necessary enzymes.

Enzymes in Manufacturing. Enzymes are also used in the production of paper, to remove adhesives introduced to the pulp during recycling of paper, and in textiles, to reduce impurities and prepare fabrics for dyeing, among other uses. Natural enzymes are only effective within limited temperature and pH ranges and may be inhibited by other chemicals present. Enzyme engineers are always working to find new enzymes or modify existing enzymes that will be more broadly useful. The search for enzymes that are effective within a wider temperature range is also important in the manufacturing of laundry detergents, which need to be effective in both hot and cold washes.

Enzyme Immobilization. By attaching enzymes to surfaces, embedding them in gel matrices, hollow fibers, or cross-linking them to each other, enzymes are immobilized on insoluble surfaces. This increases their stability, simplifies their recycling, and increases the tolerance of enzymes to high levels of substrate and products. Detergent enzyme preparations, such as Alcalase, immobilize the protease subtilisin by attaching it to insoluble particles. Attaching the enzyme to inert material increases its reuse as it degrades proteinaceous matter.

Making Enzymes Soluble in Organic Solvents. Enzymes usually work in water, but many reactions between organic chemicals occur in organic solvents. Although Russian chemist Alexander Klibanov showed that several enzymes are active in organic solvents, many enzymes are neither soluble in organic solvents nor work properly in such environments. Attaching a molecule called polyethylene glycol (PEG) to some enzymes makes them soluble and active in organic solvents. It allows them to make things such as polyester, peptides (small proteins), esters (sweet-smelling things found in foods), and amides (nitrogen-containing compounds). Such modified enzymes also have clinical uses. For example, the enzyme asparaginase can kill cancer cells but is toxic, unstable, and some patients have severe allergies to it. PEG-treated asparaginase is not as toxic as the native enzyme, is much more stable, and does not cause allergy. PEG-asparaginase is used to treat tumors in humans.

Catalytic Antibodies. A notable variety of reactions are catalyzed by catalytic antibodies that range from forming or breaking carbon-carbon bonds, rearrangements, hydrolysis of various bonds, transfer of chemical groups, and even an industrial reaction called the Diels-Alder reaction. However, abzymes are expensive and tedious to make, and their catalytic activity is well below that of typical enzymes. However, their long half-life and high serum concentration may compensate for their lower reaction rates.

Recombinant antibodies, like catalytic antibodies, are unpredictable, which makes research and clinical trials more difficult. However, because of the potential that catalytic antibodies present in advancing treatments of immunity and infection-based diseases, scientists continue to develop the field. The catalytic antibodies 3D8 scFv treat COVID-19 using a nucleic acid-hydrolyzing activity. Other applications include the treatment of triple-negative breast cancer, multiple sclerosis, avian influenza virus (H1N1), and Alzheimer's disease. Methods of expressing catalytic antibodies include yeast, mammalian cells, and prokaryotic expression systems.

Careers and Course Work

Anyone who wishes to study enzyme engineering must have a good understanding of general, organic, and physical chemistry and biochemistry. Knowledge of calculus is also essential, as is mastery of computers since many structural studies of enzymes use somewhat sophisticated computer programs. Enzyme engineers also must have some mastery of the tools of molecular biology, gene cloning, and microbiology. Enzyme engineers will also need graduate training since many of the tools used in enzyme engineering are simply beyond the typical undergraduate laboratory curriculum. A bachelor's degree in chemistry or biochemistry is necessary to work as a technician in the enzyme engineering field. A master's degree will also be sufficient for a technician, but to run an enzyme engineering lab, a Ph.D. in chemistry or biochemistry is required.

Enzyme engineers work either in academia or industry. In academia, they will run their own lab and train graduate students. Most of the research in academic settings is not applied but theoretical. Academic enzyme engineers usually try to develop new technologies for enzyme engineering or use enzyme engineering techniques to study various enzymes. In industry, enzyme engineering research is much more applied since the goal is to optimize an enzyme for a specific synthetic process that saves time, money, and resources.

Enzyme engineering is almost certainly the next frontier in biochemistry. Many industries make copious use of enzymes already, and the need to tailor these enzymes to fit the needs of industrial uses is pressing. Enzyme engineers need to be good collaborators, visionary, and very patient since most experiments require extensive trials before something interesting is discovered.

Social Context and Future Prospects

Because modified enzymes can make certain products more cheaply, the public response to modified enzymes is generally positive. However, the genetically modified organisms (GMOs) used to produce these enzymes give many people pause since the introduction of GMOs into the environment may have long-term consequences that are presently unrecognized. Strict government regulation that forbids the release of GMOs into the environment without approval alleviates most of these concerns. However, some people are still troubled by the use of GMOs to make products they eat or use.

Enzyme engineering is one of the up-and-coming fields in chemistry and biochemistry. Since the 1990s, the use of enzymes in industrial and academic chemistry has greatly increased. There are many advantages to using enzymes. They can act outside cells and under mild conditions, which minimizes side effects. Additionally, they are environmentally innocuous, compatible with other enzymes, and efficient, though highly selective, catalysts. The largest drawback of using enzymes is that a suitable enzyme is sometimes unavailable to catalyze the desired reaction. Enzyme engineering can eliminate this significant drawback.

Furthermore, as biochemists achieve a more profound understanding of protein structure, cheaper and faster enzyme engineering methods become more successful and practical. This will shorten the time required for enzyme engineering experiments and reduce costs. Many companies have invested in enzyme engineering research and development, recognizing the field's potential.

The coronavirus disease 2019 (COVID-19) global pandemic showed the importance of developing ways to combat viruses. Enzyme engineers hope to eventually create an enzymic cascade that can accelerate the distribution of oral anti-viral therapeutics. Researchers also see promise in using a catalase, a naturally occurring enzyme, to treat virus symptoms and stop of the replication of viruses and infections in the body.

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