Enzymes PDF - University of the Philippines Los Baños

ENZYMES  ENZYMES Lowers the activation energy of chemical reactions https://www.kosmotime.com /  ENZYMES catalyzes both forward and reverse reactions not consumed with high degree of specificity for a certain s...

ENZYMES  ENZYMES Lowers the activation energy of chemical reactions https://www.kosmotime.com /  ENZYMES catalyzes both forward and reverse reactions not consumed with high degree of specificity for a certain substrate or group of substrates  ENZYMES APPLICATIONS: 1. upgrade product quality (e.g., clarification of juice) 2. preparation of synthetic food 3. improvement of flavor 4. improve processing techniques & procedures 5. utilization of by-products  ENZYMES SOURCES: 1. Plants 2. Animals 3. Microorganisms versatile and produces large amount of enzyme easily modified by genetic engineering enzymes produced are easily recovered (extracellular)  ENZYMES GUIDELINES IN SELECTING RIGHT MCG: 1. Mcg should be non-pathogenic 2. Mcg should be non-toxigenic 3. Should not produce antibiotics 4. Stable and consistent 5. High yield and activity  ENZYMES ADVANTAGES: 1. natural and non-toxic 2. non-toxic products 3. minimal side reactions 4. mild operating conditions 5. controlled rate of reaction 6. easily inactivated  ENZYMES DISADVANTAGE: Limited substrate range - Enzymes can act only on one substrate or on a family of structurally similar substrate ENZYMES  ENZYMES COMPONENTS: HOLOENZYME ⚬ protein portion + non-protein portion; active enzyme APOENZYME ⚬ Protein part of the enzyme; inactive COFACTOR ⚬ Non-protein portion ⚬ Helps in catalyzing biochemical reactions  ENZYMES THREE TYPES: Co-enzymes - vitamin derivative (vitamin itself or derived from it) ⚬ usually, water soluble Metal ions - metals (Na, Fe, Mn, Mg, Cu, K, Zn) Ligands - forms complexes with biomolecules  ENZYMES TERMINOLOGY: 1.Enzymology study of enzymes 2. Isozymes multiple forms of enzymes w/c catalyzes the same reactions but differ in protein structure (1o AA sequence) ⚬ Ex. Hexokinase and glucokinase 3. Active site (AS) part where the chemical reactions occurs  ENZYMES TERMINOLOGY: 4. Allosteric Factors (AF) activators or inhibitors of enzymatic activity that alters kinetic properties of the enzyme 5. Regulatory site (RS) part where allosteric factor binds binding of AF at the RS can modulate the enzyme's activity  ENZYMES TERMINOLOGY: 6. Enzyme kinetics study of reaction of enzyme catalysis deals with the rate/ speed of catalyzed reaction 7. Transition State an activated complex formed between enzyme and substrate where the substrate can either return to the original state or form new products  ENZYMES TERMINOLOGY: 8. Activation energy (Ea) catalysis of reaction amount of energy to bring 1 mole of reactant or substrate to transition state 9. Catalysis process by which enzymes facilitate chemical reactions  ENZYMES MECHANISM: https://www.youtube.com/watch?v=ZQzzBCf5wck https://www.youtube.com/watch?v=qgVFkRn8f10  ENZYMES ENZYME CLASSIFICATION & NOMENCLATURE (by IUBMB)  ENZYMES ENZYME CLASSIFICATION 1. Oxidoreductase oxidize or reduce substrates by transfer of hydrogens or electrons or by use of oxygen the systematic name is formed as “donor : acceptor + oxidoreductase” ⚬ Example: H2O2 + H2O2 → O2 + 2H2O hydrogen peroxide:hydrogen peroxide oxidoreductase (catalase, EC 1.11.1.6)  ENZYMES ENZYME CLASSIFICATION 2. Transferase remove groups (not including H) from substrates and transfer them to acceptor molecules (not including water) the systematic name is formed as “donor : acceptor + group transferred + transferase” ⚬ Example: ATP + D-glucose →ADP + D-glucose 6-phosphate ATP: D-glucose 6-phosphotransferase (glucokinase, EC 2.7.1.2)  ENZYMES ENZYME CLASSIFICATION 3. Hydrolase hydrolytic or cleavage reaction water participates in the breakage of covalent bonds of the substrate the systematic name is formed as “substrate hydrolase” when the enzyme specificity is limited to removal of a single group, the group is named as a prefix a. Example: Triacylglycerol + H2O →diacylglycerol + a fatty acid anion Triacylglycerol acylhydrolase (triacylglycerol lipase, EC 3.1.1.3)  ENZYMES ENZYME CLASSIFICATION 4. Lyases remove groups from their substrates (not by hydrolysis) to leave a double bond or which conversely add groups to double bonds the systematic name is formed as “substrate prefix-lyase” Prefixes indicate the type of reaction ⚬ Example: Malate → fumarate + H2O malate hydro-lyase (fumarate hydratase, EC 4.2.1.2; formerly known as fumarase)  ENZYMES ENZYME CLASSIFICATION 5. Isomerase isomerization reaction or intramolecular rearrangement the systematic name is formed as “substrate prefix- isomerase” the prefix indicates the types of isomerization involved ⚬ cis-trans- isomerase ⚬ keto-enol- isomerase ⚬ mutase (intramolecular transfer of a group) ⚬ racemases or epimerases (inversions of asymmetric groups) ￭ Example: L-alanine →D-alanine Alanine racemase (alanine recemase, EC 5.1.1.1)  ENZYMES ENZYME CLASSIFICATION 6. Ligases/ Synthetase catalyze the covalent linking of two molecules, coupled with the breaking of a pyrophosphate bond as in ATP the systematic name is formed as “X:Y ligase (Z),” where X and Y are the two molecules to be joined together and Z is the product formed ⚬ Example: ATP + L-aspartate + NH3 →AMP + pyrophosphate + L- asparagine L-aspartate:ammonia ligase (AMP-forming) (aspartate-ammonia ligase, EC 6.3.1.1).  ENZYMES DEGREE OF SPECIFICITY - Most enzyme relatively catalyzes small number of reactions 1. Stereochemical specificity enzyme is only specific to a single type of isomer (D or L) Sugars- D form, amino acids- L-form e.g., Lactic acid dehydrogenase specific for L- lactic acid to pyruvic acid  ENZYMES DEGREE OF SPECIFICITY 2. Low specificity enzyme shows specificity only to the type of linkage or bond to be split e.g., Lipase- specific to a linkage between an alcohol and an acid (glycerol and FA)  ENZYMES DEGREE OF SPECIFICITY 3. Group specificity enzyme acts upon the substrate in w/c there is a specific chemical linkage and a specific group on one side of the linkage e.g., Chymotrypsin- peptide bond cleavage of aromatic AA ⚬ Trypsin- basic AA in peptide bond  ENZYMES DEGREE OF SPECIFICITY 4. Absolute specificity most abundant an exclusive type of specificity for one substrate and one reaction only ⚬ e.g., Urease- urea to CO2 and NH3 ⚬ Maltase- maltose to 2 glucose  ENZYMES CATALYSIS MODELS 1. Lock & Key Hypothesis proposed by E. Fischer substrate is the key and enzyme is the lock the substrate should be in complement  ENZYMES CATALYSIS MODELS 2. Induced Fit Model proposed by D. Koshland substrate is initially not complementary with the enzyme conformation of enzyme is changed by the binding of the substrate only the substrate can induce a change in the tertiary structure to the active form of the enzyme as substrate approaches the enzyme surface, the AA residues of the enzyme conforms to the shape of the substrate  ENZYMES CATALYSIS MODELS 2. Induced Fit Model  ENZYMES CATALYSIS MODELS 2. Induced Fit Model  ENZYMES FACTORS AFFECTING ENZYME ACTIVITY: 1. pH Extreme pH inactivates enzymes Due to protein unfolding (apoenzyme) Optimum pH for enzymes: pH 4.5–8.0 ⚬ Exemptions: example pepsin (1.5–2.0), arginase (pH 10)  ENZYMES FACTORS AFFECTING ENZYME ACTIVITY: 2. Temperature Optimum at 30–40˚C Exemption: DNA polymerase- opt. at 72˚C, stable up to 94–95˚C Begins to inactivate at 45˚C At subfreezing temp, some enzymes show minimal activity, but most remain significantly active  ENZYMES FACTORS AFFECTING ENZYME ACTIVITY: 3. Substrate concentration as substrate concentration increases, the rate of enzyme-catalyzed reactions also increases at the saturation point, the enzyme is working at its maximum capacity  ENZYMES FACTORS AFFECTING ENZYME ACTIVITY: 4. Enzyme concentration as enzyme concentration increases, the rate of enzyme-catalyzed reactions also increases there are more active sites available for substrate binding, leading to more product formation  ENZYMES FACTORS AFFECTING ENZYME ACTIVITY: 5. Water activity Enzyme activities usually occur in aqueous media Low water activity can reduce enzyme activity due to decreased mobility and interactions between the enzyme and substrate molecules. High water activity can lead to dilution effects, reducing the concentration of reactants and thus affecting enzymatic activity negatively. ENZYME ENZYMES REACTION BEING CATALYZED SOURCES α-AMYLASE starch/ glycogen to glucose barley (with traces of maltose and Aspergillus niger maltooligosaccharides) Aspergillus oryzae β- AMYLASE starch/glycogen to maltose barley Bacillus cereus Bacillus polymyxa CELLULASE Cellulose to β- dextrin Aspergillus niger INVERTASE sucrose to fruc and glu Saccharomyces sp. LACTASE lactose to galac and glu Aspergillus niger Aspergillus oryzae PECTINASE demethylates pectin, Aspergillus niger hydrolyzes pectin Rhizopus oryzae  ENZYMES ENZYME REACTION BEING CATALYZED SOURCES BROMELAIN/ proteins to low MW peptides pineapple PAPAIN papaya RENNET coagulates milk in cheese 4th stomach ruminants making, flavor development LIPASES TAGs to DAGs, MAGs, glycerol 4th stomach ruminants and fatty acids CATALASE hydrogen peroxide to oxygen Aspergillus niger and water  ENZYMES ENZYME REACTION BEING CATALYZED SOURCES GLUCOSE glucose to gluconic acid and Aspergillus niger OXIDASE hydrogen peroxide LIPOXYGENASE PUFA to hydroperoxide Soybean flour GLUCOSE glucose to fructose Bacillus coagulans ISOMERASE  ENZYMES IMMOBILIZED ENZYMES: Enzymes fixed to a carrier Movement of enzymes is restricted through physical/chemical means Thus, it can be used repeatedly in batch operations  ENZYMES IMMOBILIZED ENZYMES: ADVANTAGES: 1. High bio-catalytic activity 2. High yield 3. Continuous process 4. Reusability 5. Economical 6. Product purity  ENZYMES IMMOBILIZED ENZYMES: DISADVANTAGE: Enzymes are sensitive to external factors such as pH, temperature, contaminants, etc.  ENZYMES IMMOBILIZED ENZYMES: WAYS OF IMMOBILIZING A. IMMOBILIZATION BY BINDING 1. Adsorption 1.1 Biophysical force - immobilization onto a H2O-insoluble carrier - by H-bond or hydrophobic interaction 1.2 Electrostatic interaction/ ionic bonding/ heteropolar binding - opposite charge group of carrier material and enzyme  ENZYMES IMMOBILIZED ENZYMES: WAYS OF IMMOBILIZING A. IMMOBILIZATION BY BINDING 2. Covalent bond/ homopolar binding - true bond between carrier and enzyme - linking by shared electron  ENZYMES IMMOBILIZED ENZYMES: WAYS OF IMMOBILIZING A. IMMOBILIZATION BY BINDING 3. Cross linking/ Co-crosslinking * Cross linking- enzymes are linked with each other by bi- or multi- functional reagents to produce high MW & insoluble aggregates * Co-cross linking- enzyme links with each other and another inactive compounds of high MW  ENZYMES IMMOBILIZED ENZYMES: WAYS OF IMMOBILIZING A. IMMOBILIZATION BY BINDING 3. Cross linking/ Co-crosslinking  ENZYMES IMMOBILIZED ENZYMES: WAYS OF IMMOBILIZING B. IMMOBILIZATION BY PHYSICAL RETENTION 1. Matrix Entrapment - Enzymes are embedded in natural or synthetic polymers a. in form of beads e.g., calcium alginate b. in form of network e.g., cellulose fiber  ENZYMES IMMOBILIZED ENZYMES: WAYS OF IMMOBILIZING B. IMMOBILIZATION BY PHYSICAL RETENTION 2. Membrane Enclosure a. by encapsulation, e.g., dialysis bag - small pores membrane retard high MW enzymes b. by membrane reactor - contained within a specialized membrane system for specific reactions  ENZYMES IMMOBILIZED ENZYMES: WAYS OF IMMOBILIZING  ENZYMES MODIFICATION OF FOODS BY ENDOGENOUS ENZYMES 1.Pectin Esterase enzymes that react with pectin and cleaves them pectin are naturally occurring in plants and responsible for formation of gel  ENZYMES MODIFICATION OF FOODS BY ENDOGENOUS ENZYMES 2. Amylases starch hydrolysis 𝛼-amylase hydrolyzes starch in random manner and results in rapid decrease on the viscosity of the solution β-amylase attacks only the end units of starch chains that results in the increase sweetness of the solution  ENZYMES MODIFICATION OF FOODS BY ENDOGENOUS ENZYMES 3. Lipase hydrolysis of ester linkage TAG causes of desirable and undesirable flavors from the hydrolyzed FA  ENZYMES MODIFICATION OF FOODS BY ENDOGENOUS ENZYMES 4. Phenolase hasten oxidative browning exposure of enzyme to air causes rapid oxidation of phenolic compounds to orthoquinones which can polymerize to form melanin pigment  ENZYMES MODIFICATION OF FOODS BY ENDOGENOUS ENZYMES 5. Peroxidase enzymes which contains a heme prosthetic group leads to destruction of Vitamin C, bleaching of carotenoids, peroxidation of FA indicator of heat treatment effectiveness  ENZYMES MODIFICATION OF FOODS BY ENDOGENOUS ENZYMES 6. Ascorbic acid oxidase copper containing enzyme that catalyzes oxidation of ascorbic acid lowers Vit. C content of citrus fruit juices END OF PRESENTATION