Enzymes (Essentials of Biochemistry and Enzymology)
Enzymes
Enzymes are biological molecules that act as catalysts in living organisms. They are protein in nature. They speed up chemical reactions by lowering the activation energy required for a reaction to occur. Enzymes are highly specific, meaning each enzyme catalyzes a particular reaction or a group of similar reactions. They are crucial for various biological processes in the body, such as digestion, metabolism, and cell signaling.
* Most enzymes lose their activity when heated, a process known as denaturation. This is due to the disruption of their protein structure.
Types of Enzymes
Cofactor:
The non-protein part is called a cofactor and is necessary for the catalytic function of the enzymes.
* Cofactors are generally stable to heat, meaning they can withstand high temperatures without losing their activity.
Types of Cofactors:
Prosthetic Group: A cofactor that is firmly bound to the apoenzyme. It is considered an integral part of the enzyme structure.
Coenzyme: A cofactor that is loosely bound to the apoenzyme. It can dissociate from the enzyme and function independently.
Apoenzyme:
The protein component of an enzyme without its cofactor, which is inactive.
Holoenzyme:
A biochemically active compound formed by the combination of an enzyme with a coenzyme.
Proenzymes or Zymogens:
These are inactive forms of enzymes that are secreted or synthesized in the body.
They are activated under specific conditions, often through a process known as proteolytic cleavage, where a portion of the protein is removed to reveal the active site.
Examples:
Pepsinogen: this is the inactive form of pepsin, an enzyme involved in protein digestion in the stomach. Pepsinogen is activated by hydrochloric acid and pepsin itself in a position feedback loop.
Trypsinogen: An inactive form of trypsin, an enzyme involved in protein digestion in the small intestine. Trypsinogen is activated by enterokinase, an enzyme secreted by the intestinal mucosa.
Properties of Enzymes
Enzymes are protein in nature.
They are generally larger than the molecules they act upon (substrates).
The substrate binds to a specific region of the enzyme called the active site or active center.
The active site has a unique shape and chemical properties that are complementary to the structure of the substrate, allowing for a precise fit between them.
The active site is made up of 2 main components:
Binding Site: This part is responsible for recognizing and binding the substrate molecule.
Catalytic Site: Contains the amino acid residues that participate in the chemical reaction catalyzed by the enzyme.
Only a few amino acids in the active site are directly involved in the catalytic mechanism.
Remaining amino acids contribute to the specificity of the enzyme, determining which substrate it can bind to and catalyze.
The amino acids in the active site often have reactive side-chain groups, such as cysteine, histidine, and serine. (These groups play a crucial role in the catalytic mechanism, facilitating the chemical reaction that transforms the substrate into the product.)
The enzyme specificity depends upon the particular atomic structure and configuration of both substrate and enzyme.
Rate of enzyme catalyzed reaction is rapid.
Enzymes promote reactions under mild conditions, such as at relatively low temperature, compared to non-enzymatic reactions.
Enzymes also work optimally at neutral pH values, which is similar to the pH of most biological fluids.
The synthesis and regulation of enzymes are controlled by the direction of genes and the factor that influences these genes. This ensures that the right enzymes are produced at the right time and in the right amounts.
A distinctive feature of enzyme-catalyzed reactions is the phenomenon of saturation. This occurs when the enzyme is working at its maximum capacity, and increasing the concentration of the substrate no longer significantly increases the reaction rate.
Nomenclature & Classification of Enzymes
The international Union of Biochemistry established this commission to standardize the naming and classification of enzymes.
Enzymes are divided into major classes and subclasses based on the type of reaction they catalyze.
There are 3 ways to describe an enzyme:
Trivial Name: A short and commonly used name.
Systematic Name: A more detailed name that identifies the specific reaction catalyzed.
EC Number: A unique numerical code assigned by the Enzyme Commission for precise and unambiguous identification.
There are 6 major classes of enzymes:
* Oxido-reductases: Catalyze oxidation-reduction reactions by transferring hydrogen or electrons. Example: catalase.
* Transferases: Catalyze the transfer of functional groups from one molecule to another. Example: glucokinase.
* Hydrolases: Catalyze hydrolysis reactions, breaking down molecules by adding water. Example: alkaline phosphatase.
* Lyases: Catalyze the cleavage of carbon-carbon, carbon-oxygen, carbon-nitrogen, and sulfur-sulfur bonds by elimination (not hydrolysis), leaving double bonds or adding groups to double bonds. Example: fumarate hydratase.
* Isomerases: Catalyze isomerization reactions within a single molecule. Example: mutase.
* Ligases: Catalyze the formation of bonds between two molecules, often involving the cleavage of ATP. Involved in the biosynthesis of compounds.
Alkaline phosphatase
Trivial name : alkaline phosphatase
Systematic name : orthophosphoric monoester phosphohydrolase
Reaction catalyzed : Orthophosphoric monoester + H2O « An alcohol + H3PO4
Classification number : EC 3.1.3.1, where EC stands for Enzyme Commission
The first digit (3) for the class name (hydrolases: enzymes that break chemical bonds by adding water).
The second digit (1) for the subclass (acting on ester bonds)
The third digit (3) for the sub-subclass (phosphoric monoester)
The fourth digit (1) designates alkaline phosphatase
Lipase
Recommended name : lipase
Systematic name : glycerol ester hydrolase
Reaction catalyzed : A triglyceride + H2O « A diglyceride + a fatty acid
Classification number : EC 3.1.1.3, where EC stands for Enzyme Commission
The first digit (3) for the class name (hydrolases)
The second digit (1) for the subclass (acting on ester bonds)
The third digit (1) for the sub-subclass (carboxylic ester)
The fourth digit (3) designates lipase
Hydrolases
These enzymes are widely used in the food industry.
Types of Hydrolases Enzymes
Amylases:
* A type of hydrolase that specifically targets α-1,4 glycosidic bonds found in starch.
* There are two main types: α-amylase and β-amylase.
α-Amylase:
* Cleaves α-1,4 bonds in amylose and amylopectin in a random manner.
* Produces small units with free non-reducing end groups, resulting in low molecular weight dextrins.
β-Amylase:
* Cleaves α-1,4 bonds in amylose and amylopectin in an orderly fashion, starting from the non-reducing end.
* Produces maltose units.
α-Amylase and β-Amylase:
* Both enzymes cannot cleave α-1,6 linkages present in amylopectin.
Uses of Amylase:
During Fermentation:
α-amylase catalyzes the dextrinization of damaged starch granules. Dextrins are further hydrolyzed by β-amylase to produce maltose. Maltose provides fermentable sugar for yeast cells to utilize.
During Baking:
* α-amylase activity is destroyed due to the high oven temperature.
* The presence of amylases during fermentation contributes to:
Greater bread volume
Deeper crust color
Softer crumb
Improved grain and texture
Fungal amylase enzymes(α-, β- and amylo-1,6-glucosidase) are used to produce a well flavored, low viscous syrup consisting of dextrose, maltose, and dextrin.
2.β-D-Fructofuranosidase (Invertase)
Invertase is also known as Sucrase
This enzyme is important in the confectionary industry. It's used in the production of sweets and candies.
Involves in the hydrolysis of sucrose. (Invertase catalyzes the breakdown of sucrose into its constituents monosaccharides, glucose and fructose. This process is called Hydrolysis.)
The products of sucrose hydrolysis, glucose and fructose, are collectively known as invert sugar. Invert sugar has a sweeter taste compared to sucrose.
3. Pectinolytic Enzymes
Enzymes that act on pectic substances, which are components of plant cell walls.
Types of pectinolytic enzymes include polygalacturonase, pectin methyl esterase, and pectate lyases.(These enzymes break down pectic substances)
Polygalacturonase: Breaks the 𝛼-1,4- glycosidic bond between the anhydro galacturonic units in pectic substances.
Pectin methyl esterase: Breaks the methyl ester bond of pectin, producing pectic acid and methanol.
Pectic acid, a product of pectin methyl esterase activity can flocculate (form clumps) in the presence of calcium ions(Ca2+).
Pectinolytic Enzymes: Are used in the clarification of fruit and vegetable juices because they can break down the pectic substances that cause cloudiness.
4. Glucoamylase
Glucoamylase is produced by bacterial and fungal cultures.
It cleaves β-D-glucose units from the non-reducing end of 1,4-α-D-glucan.
The 𝛼-1,6-branching bond is cleaved about 30 times slower than α-1,4-linkages in straight chains.
The swelling, gelatinization, and liquefaction of starch can occur in a single step using heat-stable bacterial α-amylase.
Amylases yield starch syrup, which is a mixture of glucose, maltose, and dextrins.
5. 𝛃-D-Galactosidase (Lactase)
Lactase breaks down lactose, a sugar found in milk and dairy products, into glucose and galactose.
Lactase can be produced by various organisms, including fungi like aspergillus niger and yeast.
Uses:
Lactase is used in the dairy industry to hydrolyze lactose, making dairy products suitable for people with lactose intolerance.
Immobilized lactase enzymes can be used to produce lactose-free milk, which is beneficial for individuals who cannot digest lactose.
6. Proteases
Proteases catalyze the breakdown of peptide bonds in proteins.
Endopeptides,a type of protease, is used in the food industry for various purposes.
Proteases can be obtained from different sources, including animal organs, higher plants, and genetically engineered microorganisms.
Utilization:
In the dairy industry, proteases like chymosin or rennin are used to form casein curd, a crucial step in cheese making.
Rennin is particularly suitable for cheesemaking because it is relatively free of other undesirable proteinases.
Proteases like papain, pepsin, ficin, or bromelain, and microbial proteases can help prevent haze formation in beer by breaking down large polypeptides into smaller ones.
These proteases (papain, pepsin, ficin, bromelain also known as sulfhydryl proteases) are characterized by the presence of a sulfhydryl group (-SH) in their active site, which is essential for their catalytic activity.
Proteases in wheat flour modify rheological properties of dough and firmness of the end product.
By adding proteases to wheat flour, it's possible to modify the dough's rheological properties (how it flows and handles) and the firmness of the final baked product.
During dough treatment, the hard wheat gluten is partially hydrolyzed to a soft-type gluten.
Proteases can break down the gluten proteins in wheat flour, resulting in a softer dough texture.
Proteases are used for tenderization of meat.
Enzymes like trypsin, papain, bromelain, and ficin can tenderize meat by breaking down muscle tissue components.
7. Lipases
Lipases play a major role in cheese manufacturing.
Lipases from microbial sources are used in cheese ripening to develop specific flavors and textures.
Lipases are responsible for hydrolytic rancidity in dairy products.
Lipases can break down the fatty acids in dairy products, leading to a rancid flavor.
Lipases catalyze the hydrolysis of the ester bond in glycerides, releasing fatty acids.
Lipases can be used to improve the shelf life of bakery products by releasing mono- and diacylglycerols, which acts as emulsifiers and retard staling.
Lipases can be used to defat bones, which is a necessary step in the production of gelatin.
Oxidoreductases :
involved in oxidation-reduction reactions.
oxidize or reduce substrates by transfer of hydrogen or electrons or by oxygen.
Glucose Oxidase
It is produced by fungi such as Penicillium notatum and Aspergillus niger.
Uses :
It removes traces of glucose and oxygen from food products, such as fruit juices, mayonnaise, beer, wine ,etc.
It acts as an analytical reagent for the specific determination of glucose.
It oxidizes glucose into gluconic acid in presence of oxygen and hydrogen peroxide.
Catalase
Catalase breaks down hydrogen peroxide (H2O2) into water and oxygen.
Plants produce h2o2 as a byproduct of their metabolic processes. Catalase helps to remove excess H2O2, which can be harmful to the plant cells.
Uses H2O2 in oxidation of phenols, alcohols and other hydrogen donors.
Catalase is often used in combination with glucose oxidase to rapidly convert glucose into non-fermentable gluconic acid.
Basic Mechanism of Catalase
The basic mechanism of the working of this enzyme involves the breakdown and subsequent breakdown of the reactive oxygen specie i.e. hydrogen peroxide (H2O2) into oxygen and water thus relieving the oxidative stress caused by this substrate.
Catalase in production of cheese
The enzyme Catalase has been identified to be of use in one particular area of cheese production. Hydrogen peroxide (H2O2) is a potent oxidizer and toxic to cells.
It is used instead of pasteurization, when making certain cheeses such as Swiss, in order to preserve the natural milk enzymes that are beneficial to the end product i.e. cheese and subsequent flavor development in the processed cheese.
Ascorbic Acid Oxidase
* Catalyzes the following reaction:
L-Ascorbic acid + ½ O2 → dehydroascorbic acid + H2O
Ascorbic acid oxidase converts ascorbic acid to dehydroascorbic acid, releasing water.
Significance in Fruits and Vegetables:
* This reaction is significant in fruits and vegetables.
* When fruits and vegetables like apples, bananas, potatoes, and brinjal are cut, they undergo enzymatic browning, which is a discoloration caused by the action of enzymes.
* Ascorbic acid oxidase is responsible for initiating this browning reaction and for the eventual loss of all vitamin C activity.
This process is known as Enzymatic Browning.
Mechanism of enzymatic browning
When the tissue is injured or cut and the cut surface is exposed to air, phenol oxidase enzymes are released at the surface. These acts with the polyphenols present in the fruits and oxidizes them to ortho quinones, which gives the brown color to cut tissues.
Browning can be prevented by the following methods
1. Inactivation of polyphenol oxidase by applying heat.
2. Elimination of oxygen by vacuum packing.
3. Change of pH to prevent enzyme action.
4. Dipping of fruits and vegetables in brine and sugar solutions.
5. Use of antioxidants such as ascorbic acid to retard oxidation.
4. Lipoxygenase
It is used in the bleaching of flour.
It can be used in the improvement of the rheological properties of dough.
5. Peroxidase
Common plant peroxidases contain iron.
Peroxidases found in animal tissues and milk (lactoperoxidase) are flavoprotein peroxidases.
The peroxidase test is used as an indicator of satisfactory blanching of fruits and vegetables. (Blanching is a process used to preserve fruits and vegetables by inactivating enzymes. The peroxidase test can be used to determine if the blanching process has been effective.)
* Peroxidase catalyzes the following reaction:
H2O2 + AH2 → 2H2O + A
H2O2 is hydrogen peroxide.
AH2 is an oxidizable substrate, which can be a variety of compounds, including phenols, alcohols, and amines.
6. Phenolases :
They are also known as polyphenol oxidases or polyphenolases.
They are present in potatoes, apples, peaches, bananas, tea leaves, coffee beans etc.
They have the ability to oxidize phenolic compounds to o-quinones.
They are involved in enzymatic browning.
They are also desirable in the processing of tea and coffee.
Various factors 🡪 rate of enzyme catalyzed reactions :
Substrate concentration,
Enzyme concentration,
Temperature,
pH,
Specific activators,
inhibitors.
Pectin?
Pectin is a soluble fiber (polysaccharide) found in fruits and vegetables.
It is used as a thickener in cooking and baking.
It is also sometimes used to make medicine.
Pectin binds substances in the intestines and adds bulk to the stools.
It might also reduce how much cholesterol the body absorbs from foods.
What is pectin made of?
Pectin is typically made from apples (apple pectin) or citrus peels (citrus pectin), and often requires heat and sugar to set.
Though fruit pectin is primarily used in jam production.
It's also used in place of gelatin in other food products, like gummy candies.
What is pectin used for?
The main use for pectin is as a gelling agent, thickening agent and stabilizer in food.
The classical application is giving the jelly-like consistency to jams or marmalades, which would otherwise be sweet juices.
Pectin also reduces syneresis in jams and marmalades and increases the gel strength of low-calorie jams.
Is pectin the same as gelatin?
The main difference between gelatin and pectin is where the ingredients come from.
Gelatin is derived from collagen that originates in animals, whereas pectin is extracted from citrus fruit peels.
Companies will make the switch from gelatin to pectin when they want to make their products vegan friendly or animal free.
4 Common Types of Pectin
There are also different types of pectin that can be used for different things. There are four primary types.
HM pectin: High Methoxyl (HM) Pectin: Most common type of pectin, extracted from citrus fruit peels.
Comes in two types: Rapid-set and Slow-set.
Rapid-set Pectin:
* Requires higher temperature and less time to set.
*Ideal for recipes needing suspension, like jams and preserves (helps fruit morsels remain suspended in the jam).
Slow-set Pectin:
*Requires lower temperature and more time to set.
*Best for smooth jellies, without any suspended particles.
HM Pectin Requirements:
Needs sugar and specific acid levels to firm up.
Commonly used for making fruit preserves, jams, and jellies.
2. LM pectin Low methoxyl pectin (LM) also comes from citrus peels.
It’s often used for low-calorie jams and jellies since it relies on calcium instead of sugar to solidify.
It’s great for dairy-based recipes that don’t need sugar, too.
LM pectin gets increasingly firmer as calcium is added until it hits a saturation point. At that time, the process reverses and it becomes less firm.
3. Apple pectin. Apple pectin is pectin that is derived from apples and it’s usually sold as a powder.
It can be used as a gelling and thickening agent, as well as a food stabilizer.
4. Pectin NH. Pectin NH is an apple pectin that’s usually used for fruit glazes and fruit fillings.
It’s a type of modified LM pectin.
Pectin NH needs calcium to gel, like any other type of LM pectin, but it less of it.
It’s also thermally reversible, which means that it can be melted, set, remelted, and then reset again.
Structure of Pectin
Pectin is the methylated ester of polygalacturonic acid, which consists of chains of 300 to 1000 galacturonic acid units joined with 1α→4 linkages.
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