Like all catalysts, enzymes increase the rate of a reaction by lowering its activation energy. Some enzymes can make their conversion of substrate to product occur many millions of times faster. For example, the reaction catalyzed by orotidine 5'-phosphate decarboxylase will consume half of its substrate in 78 million years if no enzyme is present. When decarboxylase is added, the same process takes just 25 milliseconds. Chemically, enzymes are like any catalyst and are not consumed in chemical reactions, nor do they alter the equilibrium of a reaction. Enzymes differ from most other catalysts by being much more specific. Enzyme activity can be affected by other molecules. Inhibitors are molecules that decrease enzyme activity, and activators are molecules that increase activity. Drugs and poisons are often enzyme inhibitors. Enzymes are also affected by features of their environment, such as temperature, pressure, and pH.
Some enzymes are used commercially, for example, in the synthesis of antibiotics. Some household products use enzymes to speed up chemical reactions: enzymes in biological washing powders break down protein or fat stains on clothes, and enzymes in meat tenderizer break down proteins into smaller molecules, making the meat easier to chew.
By the late 17th and early 18th centuries, the digestion of meat by stomach secretions and the conversion of starch to sugars by plant extracts and saliva were known but the mechanisms by which these occurred had not been identified.
French chemist Anselme Payen discovered the first enzyme, diastase, in 1833. A few decades later, when studying the fermentation of sugar to alcohol by yeast, Louis Pasteur concluded that this fermentation was caused by a vital force contained within the yeast cells called "ferments", which were thought to function only within living organisms. He wrote that "alcoholic fermentation is an act correlated with the life and organization of the yeast cells, not with the death or putrefaction of the cells."
In 1877, German physiologist Wilhelm Kühne (1837–1900) first used the term enzyme, which comes from Greek ἔνζυμον, "leavened", to describe this process. The word enzyme was used later to refer to nonliving substances such as pepsin, and the word ferment was used to refer to chemical activity produced by living organisms.
Eduard Buchner submitted his first paper on the study of yeast extracts in 1897. In a series of experiments at the University of Berlin, he found that sugar was fermented by yeast extracts even when there were no living yeast cells in the mixture. He named the enzyme that brought about the fermentation of sucrose "zymase". In 1907, he received the Nobel Prize in Chemistry for "his discovery of cell-free fermentation". Following Buchner's example, enzymes are usually named according to the reaction they carry out: the suffix -ase is added to the name of the substrate (e.g., lactase is the enzyme that cleaves lactose) or to the type of reaction (e.g., DNA polymerase forms DNA polymers).
The biochemical nature of enzymes was at this point still unknown. Many scientists observed that enzymatic activity was associated with proteins, but others (such as Nobel laureate Richard Willstätter) argued that proteins were merely carriers for the true enzymes and that proteins per se were incapable of catalysis. In 1926, James B. Sumner showed that the enzyme urease was a pure protein and crystallized it; he did likewise for the enzyme catalase in 1937. The conclusion that pure proteins can be enzymes was definitively demonstrated by John Howard Northrop and Wendell Meredith Stanley, who worked on the digestive enzymes pepsin (1930), trypsin and chymotrypsin. These three scientists were awarded the 1946 Nobel Prize in Chemistry.
The discovery that enzymes could be crystallized eventually allowed their structures to be solved by x-ray crystallography. This was first done for lysozyme, an enzyme found in tears, saliva and egg whites that digests the coating of some bacteria; the structure was solved by a group led by David Chilton Phillips and published in 1965. This high-resolution structure of lysozyme marked the beginning of the field of structural biology and the effort to understand how enzymes work at an atomic level of detail.
Enzymes are generally globular proteins, acting alone or in larger complexes. Like all proteins, enzymes are linear chains of amino acids that fold to produce a three-dimensional structure. The sequence of the amino acids specifies the structure which in turn determines the catalytic activity of the enzyme. Although structure determines function, a novel enzyme's activity cannot yet be predicted from its structure alone. Enzyme structures unfold (denature) when heated or exposed to chemical denaturants and this disruption to the structure typically causes a loss of activity.
Enzyme are usually much larger than their substrates and sizes range from just 62 amino acid residues, for the monomer of 4-oxalocrotonate tautomerase, to over 2,500 residues in the animal fatty acid synthase. Only a small portion of their structure (around 2–4 amino acids) is directly involved in catalysis (catalytic site). This catalytic site is located next to one or more binding sites where residues orient the substrates and together these comprise the enzyme's active site The remaining majority of the enzyme structure serves to maintain the precise orientation and dynamics of the actives site..
In some enzymes, no amino acids are directly involved in catalysis, instead, the enzyme contains sites to bind and orient catalytic cofactors. Enzymes may also contain allosteric sites where the the binding of a small molecule causes a conformational change that increases or decreases activity.
A small number of RNA-based biological catalysts called ribozymes exist which again can act alone or in complex with proteins. The most common of these is the ribosome.:2.2
Some of the enzymes showing the highest specificity and accuracy are involved in the copying and expression of the genome. These enzymes have "proof-reading" mechanisms. Here, an enzyme such as DNA polymerase catalyzes a reaction in a first step and then checks that the product is correct in a second step. This two-step process results in average error rates of less than 1 error in 100 million reactions in high-fidelity mammalian polymerases.:5.3.1 Similar proofreading mechanisms are also found in RNA polymerase,aminoacyl tRNA synthetases and ribosomes.
Whereas some enzymes have broad-specificity, as they can act on a relatively broad range of different physiologically relevant substrates, many enzymes possess small side activities which arose fortuitously (i.e. neutrally), which may be the starting point for the evolutionary selection of a new function; this phenomenon is known as enzyme promiscuity.
Enzyme changes shape by induced fit upon substrate binding to form enzyme-substrate complex. Hexokinase has a large induced fit motion that closes over the substrates adenosine triphosphate and xylose. Shown with binding sites in blue, substrates in black and Mg2+ cofactor in yellow (PDBs:2E2N,2E2Q).
"Lock and key" model
Enzymes are very specific, and it was suggested by Emil Fischer in 1894 that this was because both the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another. This is often referred to as "the lock and key" model.:8.3.2 This model explains enzyme specificity, but fails to explain the stabilization of the transition state that enzymes achieve.
Induced fit model
In 1958, Daniel Koshland suggested a modification to the lock and key model: since enzymes are rather flexible structures, the active site is continuously reshaped by interactions with the substrate as the substrate interacts with the enzyme. As a result, the substrate does not simply bind to a rigid active site; the amino acid side-chains that make up the active site are molded into the precise positions that enable the enzyme to perform its catalytic function. In some cases, such as glycosidases, the substrate molecule also changes shape slightly as it enters the active site. The active site continues to change until the substrate is completely bound, at which point the final shape and charge is determined. Induced fit may enhance the fidelity of molecular recognition in the presence of competition and noise via the conformational proofreading mechanism.
Distorting the bound substrate(s) into their transition state form, thereby reducing the amount of energy required to complete the reaction.
Creating an environment with a charge distribution complementary to that of the transition state.
By providing an alternative pathway; for example, temporarily reacting with the substrate to form an intermediate ES complex, which would be impossible in the absence of the enzyme.
By reducing the reaction entropy change through productive orientation of substrate molecules. This entropic effect involves destabilization of the ground state, and its contribution to catalysis is relatively small.
The internal dynamics of enzymes are important for their catalytic function. Internal dynamics are the movement of parts of the enzyme's structure, such as individual amino acid residues, a group of amino acids, or even an entire protein domain. These movements occur at various time-scales ranging from femtoseconds to seconds and can be studied using biophysical techniques such as nuclear magnetic resonance spectroscopy or time resolved crystallography. Networks of protein residues throughout an enzyme's structure can contribute to its function through collective dynamic motions. This behavior can be modeled by extension of the Michaelis-Menten kinetic model to multiple reaction pathways. Protein dynamics are important for binding and releasing substrates and products, and for interacting with other proteins involved in regulating an enzyme's activity, but the role of dynamics in catalysis itself is controversial.
Allosteric sites are pockets on the enzyme that bind to molecules in the cellular environment. The sites form weak, noncovalent bonds with these molecules, causing a change in the conformation of the enzyme. This change in conformation translates to the active site, which then affects the reaction rate of the enzyme. In this way, allosteric interactions can either inhibit or activate enzymes. Allosteric interactions with metabolites upstream or downstream in an enzymes metabolic pathway causes feedback regulation, matching the activity of the enzyme to the flux through the rest of the pathway.
An example of an enzyme that contains a cofactor is carbonic anhydrase, which is shown in the ribbon diagram above with a zinc cofactor bound as part of its active site. These tightly bound molecules are usually found in the active site and are involved in catalysis.:8.1.1 For example, flavin and heme cofactors are often involved in redox reactions.:17
Enzymes that require a cofactor but do not have one bound are called apoenzymes or apoproteins. An enzyme together with the cofactor(s) required for activity is called a holoenzyme (or haloenzyme). The term holoenzyme can also be applied to enzymes that contain multiple protein subunits, such as the DNA polymerases; here the holoenzyme is the complete complex containing all the subunits needed for activity.
Since coenzymes are chemically changed as a consequence of enzyme action, it is useful to consider coenzymes to be a special class of substrates, or second substrates, which are common to many different enzymes. For example, about 1000 enzymes are known to use the coenzyme NADH.
Coenzymes are usually continuously regenerated and their concentrations maintained at a steady level inside the cell: for example, NADPH is regenerated through the pentose phosphate pathway and S-adenosylmethionine by methionine adenosyltransferase. This continuous regeneration means that even small amounts of coenzymes are used very intensively. For example, the human body turns over its own weight in ATP each day.
The energies of the stages of a chemical reaction. Uncatalysed (dashed line), substrates need a lot of activation energy to reach a transition state, which then decays into lower-energy products. When enzyme catalysed (solid line), the enzyme binds the substrates (ES), then stabilizes the transition state (ES‡) to reduce the activation energy required to produce products (EP) which are finally released.
As with all catalysts, enzymes do not alter the position of the chemical equilibrium of the reaction. In the presence of an enzyme, the reaction runs in the same direction as it would without the enzyme, just more quickly.:8.2.3 For example, carbonic anhydrase catalyzes its reaction in either direction depending on the concentration of its reactants.
The rate of a reaction is dependent on the activation energy needed to form the transition state which then decays into products. Enzymes increase reaction rates by lowering the energy of the transition state. First, binding forms a low energy enzyme-substrate complex (ES). Secondly the enzyme stabilises the transition state such that it requires less energy to achieve compared to the unanalysed reaction (ES‡). Finally the enzyme-product complex (EP) dissociates to release the products.
Enzymes can couple two or more reactions, so that a thermodynamically favorable reaction can be used to "drive" a thermodynamically unfavourable one so that the combined energy of the products is lower than the substrates. For example, the hydrolysis of ATP is often used to drive other chemical reactions.
Enzyme kinetics is the investigation of how enzymes bind substrates and turn them into products. The rate data used in kinetic analyses are commonly obtained from enzyme assays. In 1913 Leonor Michaelis and Maud Leonora Menten proposed a quantitative theory of enzyme kinetics, which is referred to as Michaelis-Menten kinetics. The major contribution of Michaelis and Menten was to think of enzyme reactions in two stages. In the first, the substrate binds reversibly to the enzyme, forming the enzyme-substrate complex. This is sometimes called the Michaelis-Menten complex in their honor. The enzyme then catalyzes the chemical step in the reaction and releases the product. This work was further developed by G. E. Briggs and J. B. S. Haldane, who derived kinetic equations that are still widely used today.
Enzyme rates depend on solution conditions and substrate concentration. To find the maximum speed of an enzymatic reaction, the substrate concentration is increased until a constant rate of product formation is seen. This is shown in the saturation curve on the right. Saturation happens because, as substrate concentration increases, more and more of the free enzyme is converted into the substrate-bound ES complex. At the maximum reaction rate (Vmax) of the enzyme, all the enzyme active sites are bound to substrate, and the amount of ES complex is the same as the total amount of enzyme.:8.4
Vmax is only one of several important kinetic parameters. The amount of substrate needed to achieve a given rate of reaction is also important. This is given by the Michaelis-Menten constant (Km), which is the substrate concentration required for an enzyme to reach one-half its maximum reaction rate; generally, each enzyme has a characteristic Km for a given substrate. Another useful constant is kcat, also called the turnover number, which is the number of substrate molecules handled by one active site per second.:8.4
The efficiency of an enzyme can be expressed in terms of kcat/Km. This is also called the specificity constant and incorporates the rate constants for all steps in the reaction up to and including the first irreversible step. Because the specificity constant reflects both affinity and catalytic ability, it is useful for comparing different enzymes against each other, or the same enzyme with different substrates. The theoretical maximum for the specificity constant is called the diffusion limit and is about 108 to 109 (M−1 s−1). At this point every collision of the enzyme with its substrate will result in catalysis, and the rate of product formation is not limited by the reaction rate but by the diffusion rate. Enzymes with this property are called catalytically perfect or kinetically perfect. Example of such enzymes are triose-phosphate isomerase, carbonic anhydrase, acetylcholinesterase, catalase, fumarase, β-lactamase, and superoxide dismutase.:8.4.2 The turnover of such enzymes can reach several million reactions per second.:9.2
Michaelis-Menten kinetics relies on the law of mass action, which is derived from the assumptions of free diffusion and thermodynamically driven random collision. Many biochemical or cellular processes deviate significantly from these conditions, because of macromolecular crowding and constrained molecular movement. More recent, complex extensions of the model attempt to correct for these effects.
Competitive inhibitors bind reversibly to the enzyme, preventing the binding of substrate. On the other hand, binding of substrate prevents binding of the inhibitor. Substrate and inhibitor compete for the enzyme.
The coenzyme folic acid (top) and the anti-cancer drug methotrexate (bottom) are very similar in structure. As a result, methotrexate is a competitive inhibitor of many enzymes that use folates.
A competitive inhibitor and substrate cannot bind to the enzyme at the same time. Often competitive inhibitors strongly resemble the real substrate of the enzyme. For example, methotrexate is a competitive inhibitor of the enzyme dihydrofolate reductase, which catalyzes the reduction of dihydrofolate to tetrahydrofolate. The similarity between the structures of folic acid and this drug are shown in the figure to the right. This type of inhibition can be overcome with high substrate concentration. In some cases, the inhibitor can bind to a site other than the binding-site of the usual substrate and exert an allosteric effect to change the shape of the usual binding-site.
A non-competitive inhibitor binds to a site other than where the substrate binds. The substrate still binds with its usual affinity and hence Km remains the same. However the inhibitor reduces the catalytic efficiency of the enzyme so that Vmax is reduced. In contrast to competitive inhibition, non-competitive inhibition cannot be overcome with high substrate concentration.:76–78
An uncompetitive inhibitor cannot bind to the free enzyme, only to the enzyme-substrate complex, hence these types of inhibitors are most effective at high substrate concentration. In the presence of the inhibitor, the enzyme-substrate complex is inactive.:78 This type of inhibition is rare.
A mixed inhibitor binds to an allosteric site and the binding of the substrate and the inhibitor affect each other. The enzyme's function is reduced but not eliminated when bound to the inhibitor. This type of inhibitor does not follow the Michaelis-Menten equation.:76–78
In many organisms, inhibitors may act as part of a feedback mechanism. If an enzyme produces too much of one substance in the organism, that substance may act as an inhibitor for the enzyme at the beginning of the pathway that produces it, causing production of the substance to slow down or stop when there is sufficient amount. This is a form of negative feedback. Major metabolic pathways such as the citric acid cycle make use of this mechanism.:17.2.2
An important function of enzymes is in the digestive systems of animals. Enzymes such as amylases and proteases break down large molecules (starch or proteins, respectively) into smaller ones, so they can be absorbed by the intestines. Starch molecules, for example, are too large to be absorbed from the intestine, but enzymes hydrolyze the starch chains into smaller molecules such as maltose and eventually glucose, which can then be absorbed. Different enzymes digest different food substances. In ruminants, which have herbivorous diets, microorganisms in the gut produce another enzyme, cellulase, to break down the cellulose cell walls of plant fiber.
Several enzymes can work together in a specific order, creating metabolic pathways.:30.1 In a metabolic pathway, one enzyme takes the product of another enzyme as a substrate. After the catalytic reaction, the product is then passed on to another enzyme. Sometimes more than one enzyme can catalyze the same reaction in parallel; this can allow more complex regulation: with, for example, a low constant activity provided by one enzyme but an inducible high activity from a second enzyme.
Enzymes determine what steps occur in these pathways. Without enzymes, metabolism would neither progress through the same steps and could not be regulated to serve the needs of the cell. Most central metabolic pathways are regulated at a few key steps, typically through enzymes whose activity involves the hydrolysis of ATP. Because this reaction releases so much energy, other reactions that are thermodynamically unfavorable can be coupled to ATP hydrolysis, driving the overall series of linked metabolic reactions.:30.1
Control of activity
There are five main ways that enzyme activity is controlled in the cell.:30.1.1
Enzymes can be either activated or inhibited by other molecules. For example, the end product(s) of a metabolic pathway are often inhibitors for one of the first enzymes of the pathway (usually the first irreversible step, called committed step), thus regulating the amount of end product made by the pathways. Such a regulatory mechanism is called a negative feedback mechanism, because the amount of the end product produced is regulated by its own concentration.:141–48 Negative feedback mechanism can effectively adjust the rate of synthesis of intermediate metabolites according to the demands of the cells. This helps with effective allocations of materials and energy economy, and it prevents the excess manufacture of end products. Like other homeostatic devices, the control of enzymatic action helps to maintain a stable internal environment in living organisms.
Since the tight control of enzyme activity is essential for homeostasis, any malfunction (mutation, overproduction, underproduction or deletion) of a single critical enzyme can lead to a genetic disease. The malfunction of just one type of enzyme out of the thousands of types present in the human body can be fatal. An example of a fatal genetic disease due to enzyme insufficiency is Tay-Sachs disease, in which patients lack the enzyme hexosaminidase.
An enzyme's name is often derived from its substrate or the chemical reaction it catalyzes, with the word ending in -ase.:8.1.3 Examples are lactase, alcohol dehydrogenase and DNA polymerase. Different enzymes that catalyze the same chemical reaction are called isozymes.
These sections are subdivided by other features such as the substrate, products, and chemical mechanism. An enzyme is fully specified by four numerical designations. For example, hexokinase (EC 188.8.131.52) is a transferase (EC 2) that adds a phosphate group (EC 2.7) to a hexose sugar, a molecule containing an alcohol group (EC 2.7.1).
Enzymes are used in the chemical industry and other industrial applications when extremely specific catalysts are required. Enzymes in general are limited in the number of reactions they have evolved to catalyze and also by their lack of stability in organic solvents and at high temperatures. As a consequence, protein engineering is an active area of research and involves attempts to create new enzymes with novel properties, either through rational design or in vitro evolution. These efforts have begun to be successful, and a few enzymes have now been designed "from scratch" to catalyze reactions that do not occur in nature.
^Schomburg I, Chang A, Placzek S, Söhngen C, Rother M, Lang M et al. (Jan 2013). "BRENDA in 2013: integrated reactions, kinetic data, enzyme function data, improved disease classification: new options and contents in BRENDA". Nucleic Acids Research41 (Database issue): D764–72. doi:10.1093/nar/gks1049. PMID23203881.
^Radzicka A, Wolfenden R (Jan 1995). "A proficient enzyme". Science267 (5194): 90–931. PMID7809611.
^de Réaumur R (1752). "Observations sur la digestion des oiseaux". Histoire de l'academie royale des sciences1752: 266, 461.
^Kühne coined the word "enzyme" in: Kühne W (1877). "Über das Verhalten verschiedener organisirter und sog. ungeformter Fermente" [On the behavior of various organized and so-called unformed ferments]. Verhandlungen des naturhistorisch-medicinischen Vereins zu Heidelberg. new series (in German) 1 (3): 190–193. The relevant passage occurs on page 190: "Um Missverständnissen vorzubeugen und lästige Umschreibungen zu vermeiden schlägt Vortragender vor, die ungeformten oder nicht organisirten Fermente, deren Wirkung ohne Anwesenheit von Organismen und ausserhalb derselben erfolgen kann, als Enzyme zu bezeichnen." (Translation: In order to obviate misunderstandings and avoid cumbersome periphrases, [the author, a university lecturer] suggests designating as "enzymes" the unformed or not organized ferments, whose action can occur without the presence of organisms and outside of the same.)
^Holmes FL (2003). "Enzymes". In Heilbron JL. The Oxford Companion to the History of Modern Science. Oxford: Oxford University Press. p. 270.
^"Eduard Buchner". Nobel Laureate Biography. Nobelprize.org. Retrieved 23 February 2015.
^Blake C, Koenig D, Mair G, North A, Phillips D, Sarma V (May 1965). "Structure of hen egg-white lysozyme. A three-dimensional Fourier synthesis at 2 Ångström resolution". Nature206 (4986): 757–61. doi:10.1038/206757a0. PMID5891407.
^Chen L, Kenyon G, Curtin F, Harayama S, Bembenek M, Hajipour G et al. (Sep 1992). "4-Oxalocrotonate tautomerase, an enzyme composed of 62 amino acid residues per monomer". The Journal of Biological Chemistry267 (25): 17716–21. PMID1339435.
^Smith S (Dec 1994). "The animal fatty acid synthase: one gene, one polypeptide, seven enzymes". FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology8 (15): 1248–59. PMID8001737.
^Boyer R (2002). "Chapter 6: Enzymes I, Reactions, Kinetics, and Inhibition". Concepts in Biochemistry (2nd ed.). New York, Chichester, Weinheim, Brisbane, Singapore, Toronto.: John Wiley & Sons, Inc. pp. 137–8. ISBN0-470-00379-0. OCLC51720783.
^English B, Min W, van Oijen A, Lee K, Luo G, Sun H et al. (Feb 2006). "Ever-fluctuating single enzyme molecules: Michaelis-Menten equation revisited". Nature Chemical Biology2 (2): 87–94. doi:10.1038/nchembio759. PMID16415859.
^Olsson M, Parson W, Warshel A (May 2006). "Dynamical contributions to enzyme catalysis: critical tests of a popular hypothesis". Chemical Reviews106 (5): 1737–56. doi:10.1021/cr040427e. PMID16683752.
^Chapman-Smith A, Cronan J (1999). "The enzymatic biotinylation of proteins: a post-translational modification of exceptional specificity". Trends Biochem. Sci.24 (9): 359–63. PMID10470036.
^Fisher Z, Hernandez Prada J, Tu C, Duda D, Yoshioka C, An H et al. (Feb 2005). "Structural and kinetic characterization of active-site histidine as a proton shuttle in catalysis by human carbonic anhydrase II". Biochemistry44 (4): 1097–115. doi:10.1021/bi0480279. PMID15667203.
^Yoshikawa S, Caughey W (May 1990). "Infrared evidence of cyanide binding to iron and copper sites in bovine heart cytochrome c oxidase. Implications regarding oxygen reduction". The Journal of Biological Chemistry265 (14): 7945–58. PMID2159465.
^Skett P, Gibson GG (2001). "Chapter 3: Induction and Inhibition of Drug Metabolism". Introduction to Drug Metabolism (3 ed.). Cheltenham, UK: Nelson Thornes Publishers. pp. 87–118. ISBN978-0748760114.
^Suzuki H (2015). "Chapter 4: Effect of pH, Temperature, and High Pressure on Enzymatic Activity". How Enzymes Work: From Structure to Function. Boca Raton, FL: CRC Press. pp. 53–74. ISBN978-981-4463-92-8.
^Kamata K, Mitsuya M, Nishimura T, Eiki J, Nagata Y (Mar 2004). "Structural basis for allosteric regulation of the monomeric allosteric enzyme human glucokinase". Structure12 (3): 429–38. doi:10.1016/j.str.2004.02.005. PMID15016359.
^Froguel P, Zouali H, Vionnet N, Velho G, Vaxillaire M, Sun F et al. (Mar 1993). "Familial hyperglycemia due to mutations in glucokinase. Definition of a subtype of diabetes mellitus". The New England Journal of Medicine328 (10): 697–702. doi:10.1056/NEJM199303113281005. PMID8433729.
^Okada S, O'Brien J (Aug 1969). "Tay-Sachs disease: generalized absence of a beta-D-N-acetylhexosaminidase component". Science165 (3894): 698–700. PMID5793973.
^Fieker A, Philpott J, Armand M (2011). "Enzyme replacement therapy for pancreatic insufficiency: present and future". Clinical and Experimental Gastroenterology4: 55–73. doi:10.2147/CEG.S17634. PMID21753892.
^Misselwitz B, Pohl D, Frühauf H, Fried M, Vavricka S, Fox M (Jun 2013). "Lactose malabsorption and intolerance: pathogenesis, diagnosis and treatment". United European Gastroenterology Journal1 (3): 151–9. doi:10.1177/2050640613484463. PMID24917953.
^Dulieu C, Moll M, Boudrant J, Poncelet D. "Improved performances and control of beer fermentation using encapsulated alpha-acetolactate decarboxylase and modeling". Biotechnology Progress16 (6): 958–65. doi:10.1021/bp000128k. PMID11101321.
^Tarté R (2008). Ingredients in Meat Products Properties, Functionality and Applications. New York: Springer. p. 177. ISBN978-0-387-71327-4.
^Molimard P, Spinnler H (Feb 1996). "Review: Compounds Involved in the Flavor of Surface Mold-Ripened Cheeses: Origins and Properties". Journal of Dairy Science79 (2): 169–184. doi:10.3168/jds.S0022-0302(96)76348-8.
^Guzmán-Maldonado H, Paredes-López O (Sep 1995). "Amylolytic enzymes and products derived from starch: a review". Critical Reviews in Food Science and Nutrition35 (5): 373–403. doi:10.1080/10408399509527706. PMID8573280.