ENZYME TECHNOLOGY

Overview Enzyme definition Enzyme classification & nomenclature The nature of enzymes Enzyme preparation & source of commercial food enzyme Enzyme applications in food industry Enzyme immobilization

 Biocatalysts include any enzymaticallycatalyzed reaction, whether the enzyme has been purified or is part of a whole (microbial) cell
Amylases, glucanases, proteinases widely used in the brewing process Bakers yeast (Saccharomyces cerevisiae) to convert sugars to CO2 to make leavened bread

What is an enzyme?
Enzymes are usually proteins of high molecular weight (15 000<MW<several million daltons) that catalyze biologically important reactions

Enzyme classification & nomenclature

The accepted system for classification & nomenclature of enzymes embodies 3 general principles: Enzyme names, ending in -ase should be used only for single enzymes i.e. single catalytic entities Enzyme is named & classified according to the reaction it catalyzed i.e the observed chemical changes produced by the enzyme Based on the type of reaction catalysed, and together with the name(s) of the substrate(s) this provides a basis for naming individual enzymes. It is also the basis for classification and code numbers.

1.

2.

3.

EC 1.1.3.4,
glucose oxidase trivial/working systematic name, -D-glucose:oxygen 1oxidoreductase

 These code numbers contain four elements separated by points, with the following meaning: (i) the first number shows to which of the six main divisions (classes) the enzyme belongs, (ii) the second figure indicates the subclass, (iii) the third figure gives the sub-subclass, (iv) the fourth figure is the serial number of the enzyme in its sub-subclass.

 EC 2.1.1 Methyltransferases EC 2.1.2 Hydroxymethyl-, Formyl- and Related Transferases EC 2.1.3 Carboxyl- and Carbamoyltransferases EC 2.1.4 Amidinotransferases

 A given enzyme often has 2 names: 1. Systematic

formed in accordance with definite rules, showed the reaction it catalyzed, thus identifying the enzyme precisely.

2. Working or trivial
sufficiently short for general use, but not systematic

Enzyme classes
1. Oxidoreductases oxidation-reduction reactions
Acting on the CH-OH group of donors Acting on the aldehyde or oxo group of donors One C-groups Aldehyde or ketonic groups

2. Transferases transfer of functional groups

3. Hydrolases (hydrolysis reactions)

Esters Glycosidic bonds Peptide bonds

4. Lyases (addition of double bonds)

Carbon-carbon lyases Carbon-oxygen

Racemases & epimerases Cis-trans isomerases

5. Isomerases (isomerization reactions)

6. Ligases (formation of bonds with ATP cleavage)

Forming carbon-oxygen bonds Forming carbon-sulfur bonds

CLASSES 1 Oxidoreductases

E.G. INDUSTRIAL ENZYMES Peroxidases Catalases Glucose oxidases Fructosyl-transferases Glucosyl-transferases

Transferases

3
4 5 6

Hydrolases
Lyases Isomerases Ligases

Amylases, Lipases, Pectinases Cellulase, Proteases

Pectate lyases -acetolactate decarboxylases Glucose isomerase DNA ligase (Not use at presentexclusive for biological purposes)

The nature of enzymes

1. The enzyme remains unaltered at the end of reaction

S+E
Substrate Enzyme

SE

P+E
Enzyme

Enzyme Product substrate complex

Enzyme is not used up in the reaction but can be used over & over again. Implication: small amount of enzyme can catalyze the bioconversion of a large amount of substrate, thus save cost

2. Chemical reactions take place under mild conditions Chemical catalysts often require organic solvents, high temperature, extreme pH & high pressure Most enzyme operate in aqueous solution, at mild temperature & pH, and at atmospheric pressure.
Lower energy & materials cost

E.g. Maltose is usually synthesised by hydrolysis of starch Non-enzymatic Maltose + H2O boil, HCl 2 Glucose Enzymatic
Maltose + H2O
maltase, 25oC

2 Glucose

3. Highly specific action Requirement for complementarily in the configuration of substrate and enzyme explains the specificity of most enzymes.

Analogy that a substrate molecule binds to the enzyme is like a key in a lock.

4.

Fast reaction rates

Enzymes reduce the activation energy - less energy for each molecule of substrate converted to product. More molecules of substrate would be converted when the enzyme is present than when it is absent The reaction is faster in a given period of time.

Advantages of enzymes
Enzyme 1. Higher product quality
Produce consistent quality products Improve the conversion of raw material to its constituent parts e.g. hydrolysis of starch to glucose Acid hydrolysis gives limited conversion whereas enzyme can improve yield

2. Lower manufacturing cost

Enzyme can be reused Better use of raw materials

3. Less waste
Effluent from enzyme hydrolysis is less toxic, therefore cheaper in term of waste disposal environmental benefit

4. Reduced energy consumption

Chemical treatment
Generally non-specific Not always easily controlled May create harsh conditions Produce undesirable side effects waste disposal problems

Enzyme preparations used in food processing

Definition consist of biologically active proteins, at times combined with metals, carbohydrates and/or lipids. They are obtained from animal, plant or microbial sources and may consist of whole cells, parts of cells, or cell-free extracts May contain one or more active components as well as carriers, solvents, preservatives, antioxidants and other substances consistent with good manufacturing practice. They may be liquid, semi liquid, dry or in an immobilized form (immobilized enzyme preparations are preparations which have been made insoluble in their intended food matrix by physical and/or chemical means). Their colour may vary from virtually colourless to dark brown.

Material used in the production of enzyme preparations: Plant - must consist of components that leave no residues harmful to health in the processed finished food under normal conditions of use. Animal tissues - must comply with meat inspection requirements and be handled in accordance with good hygienic practice. Microbial sources - may be native or variants strains of microorganisms, or by the processes of selective serial culture or genetic modification.

Source of commercial food enzyme

3 primary sources: 1. Plants Malt amylase (malted barley), papain (papaya), bromelain (pineapple), ficin (figs) 2. Animal tissue Pepsin & rennet (stomach mucosa), catalase (liver), proteases, amylase, lipases (pancreas) 3. Microorganisms Yeast (Aspergillus), mold, bacteria (Bacillus)

Selection of microorganisms
Strain able to give high yield of enzyme at short fermentation time Produce extracellular enzyme for easier isolation A food-grade m/o (GRAS) that does not produce any toxic substances Able to grow on inexpensive medium containing cheap substrate Low amount of interfering by-products (i.e. pigments, slime, proteases)

Advantages of enzyme production from microbes

1. Fast & ease of growth
Large volumes can be produced with a uniform quality and high purity Supply of ingredients extracted from animals/plants/humans is limited by the availability of a potentially unsuitable range of raw materials that can vary in quality

2. Can be easily controlled during growth

can be made consistently to recognised quality standards

3. Produce enzymes that are easy to extract esp. extracellular enzymes

Safety of microbial enzyme preparations used in food

Primary consideration in evaluating safety of a production strain: 1. Toxigenic potential Possible synthesis of toxins that are active 2. Pathogenic potential Not usually an area of concern for consumer safety but important to worker safety Enzyme preparation rarely contain contain viable organisms

Production of enzymes
Naturally occurring enzymes are quite often not readily available in sufficient quantities for food applications or industrial use Isolating microbial strains that produce the desired enzyme & optimizing the conditions for growth obtain commercial quantities

Production of enzymes
1. Cultivate the organisms producing the desired enzyme via fermentation
Surface cultures Submerged cultures

2. Cell separated from the media filtration, centrifugation

Extracellular enzyme fermentation broth Intracellular enzyme cells biomass
Recovery involves disruption of cells & removal of cell debris & nucleic acid

Enzyme in starch industry

Starch polysaccharide of plants Commercial source the seed of cereal grains (rice, corn, wheat, sorghum), roots (tapioca) and tubers (potato) Most starch contain 2 types of glucose polymer
Amylose- linear polymer with -1,4 linkage Amylopectin- branched polymer at -1,6 branch point

Representative partial structure of amylose

Representative partial structure of amylopectin

 Starches from different sources differ from each other in terms of

Length of the chains Degree of branching

the amounts & combinations of enzymes used to hydrolyze starch are different in different operations

 Traditionally, starch was, and still is, hydrolyzed to low-molecular-weight dextrins and glucose using acid, but enzymes have several advantages.
First, the specificity of enzymes allows the production of sugar syrups with well-defined physical and chemical properties. Second, the milder enzymatic hydrolysis results in few side reactions and less browning.

 Production of High Fructose Corn Syrup (HFCS)

Closely mimicked the sweetness of sucrose (table sugar). Converts large quantities of corn & other botanical starches to HFSC & other useful sweeteners High sweetening property can be used to replace sucrose syrups in foods & beverages Sweeteners soft drinks, candies, baking, jams, jellies, etc.

Major steps in production of sweeteners

Starch Slurry preparation
-amylase
Glucoamylase/pullulanase

Liquefaction Saccharification Purification

Maltodextrins

Maltose syrups Glucose syrups Mixed syrups

Glucose isomerase

Isomeration Refining
Frutose syrups

1.

Hydrolysis with -amylase (liquefaction)

Starch is gelatinized at temperature 105-110oC & liquefied to reduce viscosity using thermostable -amylase, pH7
To make starch chains shorter To make more chain ends

2.

Hydrolysis with glucoamylase (saccharification)

Glucoamylase or -amylase enzymes are used to produce glucose & maltose syrups from the dextrin respectively, pH 3.55.0 & 4.8-6.5 respectively; 60oC Pullulanase added along with glucoamylase to improve enzyme efficiency to evolve glucose syrups containing 95-96% glucose in shorter periods of time Glucose isomerase converts glucose to fructose by isomerization Since glucose & fructose have a roughly equimolar equilibrium, the product is a mixture of about 50-53% glucose, 42-45% fructose & 5% other products Finishing steps ion exchange, decolourization & evaporation to give HFCS 42, or enrich it to increase its fructose content

3.

Isomerization with glucose isomerase

 3 major types of amylase: 1. -amylase (starch liquefying enzyme; endoenzyme)

breaks -1,4 glycosidic bonds randomly on the amylose chain & solubilizes amylose to give maltose & short oligosaccharides hydrolyzed -1,4 glycosidic bonds on the non-reducing ends of amylose to yield maltose capable of cleaving both -1,4 and -1,6 glucose linkages, which releases glucose Pullulanase hydrolyzes -1,6 glycosidic linkages in branched polysaccharides e.g. amylopectin

2. -amylase (saccharifying enzyme, exo-enzyme)

3. Glucoamylase (saccharifying enzyme)

Enzyme in brewing industry

Beer production involve 2 biological process malting & fermentation Starch present in cereal grain (usually barley) is broken down Yeasts cannot metabolise starch & in order to yield alcohol (brewing), the starch needs to be hydrolyzed

 Bacterial -amylase in mashing

Splits insoluble & soluble starch into shorter chains -amylase: starch maltose & dextrins glucoamylase: dextrins glucose

-glucanase in mashing
Assist in mashing of grits, reduce wort viscosity & improve beer finability & filterability reduce haze formation

Glucoamylase in mashing
Cleavage of terminal -1,6 glycosidic bonds of oligosaccharides, thus an additional amount of fermentable glucose in the wort.

 Fungal -amylase in fermentation

Increase fermentable CHO in the wort extract produce more alcohol

Proteases
Hydrolyzed high molecular weight proteins into simpler peptides reduce haze & better foam stability

Special brewing process

Low calorie beer (diet/light lagers) highly carbonated fermentation products with almost complete conversion of all CHO into alcohol & CO2 assisted by bacterial & fungal enzymes e.g. glucoamylase

Enzyme in fruit juice processing

The fruit cell wall The most important characteristic affecting the extraction of juice The composition of the cell wall can vary significantly for different types of fruit, but mainly consists of pectin, hemicellulose, cellulose, lignin and other components. 1st application of enzymes in the fruit juice industry. 1930s pectinases for fruit juice clarification Juices extracted from ripe fruit contain significant amount of pectin Pectins contribute to fruit juice viscosity & turbidity

Fruit juice extraction process

 Mixture of pectinases & amylases is used to clarify fruit juices Degrade pectin & starch during clarification stage prevents post-bottling haze formation Decreases filtration time up to 50% Pectinases in combination with other enzymes (hemicellulases, arabinases, cellulases & xylanases) Lowering the viscosity of pulp Preventing araban haze formation after concentration of the juice Increase the pressing efficiency of the fruits for fruit extraction increase juice yield Better colour extraction

Enzyme in dairy industry

Milk itself contains a large number of enzymes, some of which are important in milk processing:
naturally occuring proteases - contribute for the flavour characteristics of the cheese naturally occuring lipases - frequently lead to the development of rancidity in products containing milk fat.

 Main category of enzyme involved in dairy industry:

1.Lactase hydrolyse lactose in milk & whey
-galactosidase/lactase: lactose glucose + galactose

Potential use of hydrolysed lactose:

Nutritional sweet syrup dairy, confectionery, baking, beverage An accelerating fermentation medium in yogurt, cheese Lactose free products for lactose intolerance ppl

2. Milk clotting enzymes Initially, unpurified crude preparations extracted from the stomachs of ruminants were used to coagulate milk a. Rennet milk clotting enzymes derived from animal source
Bovine rennet consists of 2 main proteinases, the milk clotting enzyme chymosin & pepsin

b. Coagulants milk clotting enzymes derived from microbes

Derived from fungal sources Produced by fermentation where the proenzyme is converted to its active form during production by the slightly acidic environment

c. Fermentation produced chymosin milk clotting enzymes produced using genetic engineering
Produced by fermentation brought about by an organism genetically modified with a gene for chymosin

4. Lipase Milk fat consists mainly of triglycerides

Lipase: triacylglycerols di- and mono-acylglycerols, free fatty acids & glycerol

Contribute to distinctive flavor development during ripening stage of cheese production

Enzyme in baking industry

Introduction
Ancient Egyptians made use of enzymes present endogenously in the flour 20th century enzymes used as flour improvers 1st application in baked goods- supplementation of -amylase by addition of malt to correct the concentration of endogenous -amylase in the flour Malt substituted by microbial -amylase more suitable thermostability for baking

Main component of wheat flour is starch, gluten , non-starch polysaccharides, lipids & trace amount of minerals
Amylase: degrade starch & produce small dextrins for the yeast to act upon

Gluten combination of proteins which forms a large network during dough formation
Xylanases (hemicellulases), lipases & oxidases directly or indirectly improve the strength of the gluten network improve the quality of the finished bread

Enzyme Amylase

Effect

Maximizes the fermentation process to obtain an even crumb structure & a high loaf volume Oxidizes free sulphydryl Glucose oxidase groups in gluten to make weak doughs stronger & more elastic Lipase Dough conditioning by producing more uniform, smaller crumb cells & a silkier texture & whiter crumb colour

Enzyme Lipoxygenase Xylanase

Protease

Effect Bleaching & strengthening dough Dough conditioning. Easier dough handling & improved crumb structure Weakens the gluten to provide the plastic properties required in doughs for biscuits

Enzyme immobilisation
Definitions Immobilized enzymes have been defined as enzymes that are physically confined or localized, with retention of their catalytic activity, and which can be used repeatedly and continuously
Applicable to enzymes, cellular organelles, microbial cells, plant cells & animal cells, that is, to all types of biocatalysts

Advantages of immobilisation
1. 2. Reusability Easily recovered & separated from the product Suitable for continuous processes Lowering processing costs Stability Increase if the carrier provides a micro environment capable of stabilizing the enzyme

3. Product is not contaminated with the enzyme Enzyme can be readily removed from the reaction mixture Useful especially in the food and pharmaceutical industries 4. Lower reaction time Due to higher enzyme to substrate ratios 5. Reduce effluent problems Downstream processing is easier

Disadvantages
1. Diffusion of substrates & products may be hampered by partitioning of the enzyme in immobilised layer 2. The enzyme may have a more constrained conformation in the immobilised state, giving it a lower catalytic activity 3. High initial investment compared to free enzyme
Cost of supports & reagents

Type of support - 3 categories: 1. Hydrophilic biopolymers based on natural polysaccharides such as agarose, dextran and cellulose 2. Lipophilic synthetic organic polymers such as polyacrylamide, polystyrene and nylon 3. Inorganic materials such as controlled pore glass and iron oxide.

 Selection of support material

The binding capacity
Charge density, functional groups, porosity & hydrophobicity of the support surface

Stability & retention of enzymatic activity

Functional groups on support material & microenvironment conditions If immobilization causes some conformational changes on the enzyme, or if reactive groups on the active site of the enzyme involved in binding, a loss in enzyme activity can take place upon immobilization

Desirable enzyme carrier possesses: large surface area permeable insolubility chemical, mechanical and thermal stability high rigidity suitable shape and particle size resistance to microbial attack regenerability

Immobilisation techniques
2 major methods of immobilization
Surface immobilization
Adsorption Covalent bonding Cross-linking Matrix entrapped Membrane entrapped

Entrapment

1. Adsorption Attachment of enzymes on the surfaces of support particles by weak physical forces i.e. van der Waals, hydrogen bonding, hydrophobic interaction, or combined action Support materials

Inorganic alumina, silica, porous glass, ceramics, diatomaceous earth, clay, bentonite Organic cellulose (CMC, DEAE-cellulose), starch, activated carbon, ion-exchange resins (Amberlite, Sephadex, Dowex)

Common problem desorption of enzymes esp. in the presence of strong hydrodynamic forces
Stabilized by cross-linking with glutaraldehyde

2. Covalent binding Retention of enzymes on support surfaces by covalent bond formation Bind to support material via certain functional groups i.e. amino, carboxyl, hydroxyl & sulfhydryl groups Functional groups on support material usually activated using chemical reagents i.e. cyanogen bromide, carboiimide & glutaraldehyde

3. Cross-linking Intermolecular cross-linking of enzyme molecules using bi- and multifunctional compounds reagents i.e. glutaraldehyde, bis-diazobenzidine & 2,2-disulfonic acid Cause changes in the active site of enzymes, severe diffusion limitations

4. Entrapment Physical enclosure of enzyme in a small space Methods

Matrix entrapment Membrane entrapment macroscopic membrane, microencapsulation

Matrices used polymeric materials e.g. Ca-alginate, agar, -carragenan, polyacrylmide, collagen; solid matrices e.g. activated carbon, porous ceramic, diatomaceous earth

 Membrane used nylon, cellulose, polysulfone, polyacrylate Disadvantages

Enzyme leakage into solution Diffusional limitations e.g. substrates of high molecular mass Lack of control of microenvironment conditions reduced enzyme activity & stability

QUIZ 1
1. Name 3 main vectors used in recombinant DNA? (3 marks) 2. Name 5 basic media composition for the fermentation process. (5 marks)

3. What is the purpose of downstream processing? (2 marks)

 Plasmid, bacteriophage, cosmids Water, carbon, nitrogen, vitamin & mineral To obtain the product with requisite concentration & purity