Enzyme Engineering & Technology

Enzyme Engineering & Technology Lecturer Dr. Kamal E. M. Elkahlout Assistant Prof. of Biotechnology

CHAPTER 1Fundamentals of Enzymes

Fundamentals of enzymes • Why enzymes? • Enzyme nomenclature • Enzyme units • Sources of enzymes • Screening for novel enzymes • Media for enzyme production • Preparation of enzymes

Why Enzymes • Catalysts increase the rate of reactions without any net change in their structure. • Concept of catalysis is developed as early as the 19thcentury. • The biocatalysts were called enzymes and were later found to be proteins. • They mediate all synthetic and degradation reactions carried out by living organisms. • They are very efficient catalysts. • Superior to conventional chemical catalysts.

They are being employed increasingly in today's high-technological society, as a highly significant part of biotechnological expansion. • Their utilization has created a billion dollar business including a wide diversity of industrial processes, consumer products, and the burgeoning field of biosensors. • Further applications are being discovered constantly. • Advantages of using enzymes: • Specificity and selectivity not only for particular reactions but also in their discrimination between similar parts of molecules (regiospecificity) or optical isomers (stereospecificity).

Catalyze only the reactions of very narrow ranges of reactants (substrates). • Substrates consist of a small number of closely related classes of compounds (e.g. trypsin catalyses the hydrolysis of some peptides and esters in addition to most proteins). • Substrates of a single class of compounds (e.g. hexokinase catalyses the transfer of a phosphate group from ATP to several hexoses), or a single compound (e.g. glucose oxidaseoxidizes only glucose amongst the naturally occurring sugars). • This means that the chosen reaction can be catalyzed to the exclusion of side-reactions, eliminating undesirable by-products. • Thus, higher productivities may be achieved, reducing material costs.

The product is generated in an uncontaminated state so reducing purification costs and the downstream environmental burden. • Often a smaller number of steps may be required to produce the desired end-product. • Certain stereospecific reactions (e.g. the conversion of glucose into fructose) cannot be achieved by classical chemical methods without a large expenditure of time and effort. • Enzymes work under generally mild processing conditions of temperature, pressure and pH. • This decreases the energy requirements, reduces the capital costs due to corrosion-resistant process equipment and further reduces unwanted side-reactions.

The high reaction velocities and straightforward catalytic regulation achieved in enzyme-catalyzed reactions allow an increase in productivity with reduced manufacturing costs due to wages and overheads. • Some disadvantages: • The high cost of enzyme isolation and purification still discourages their use, especially in areas which currently have an established alternative procedure. • The generally unstable nature of enzymes, when removed from their natural environment, is also a major drawback to their more extensive use.

Enzyme nomenclature • All enzymes contain a protein backbone. • In some enzymes this is the only component in the structure. • Some enzymes have additional non-protein moieties usually present which may or may not participate in the catalytic activity of the enzyme. • Covalently attached carbohydrate groups are commonly encountered structural features which often have no direct bearing on the catalytic activity, although they may well effect an enzyme's stability and solubility.

Other factors often found are metal ions (cofactors) and low molecular weight organic molecules (coenzymes). • These may be loosely or tightly bound by non-covalent or covalent forces. • They are often important constituents contributing to both the activity and stability of the enzymes.

Trivial name • Gives no idea of source, function or reaction catalyzed by the enzyme. • Example: trypsin, thrombin, pepsin.

Classes of Enzyme Specificity • Absolute: enzyme reacts with only one substrate • Group: enzyme catalyzes reaction involving any molecules with the same functional group • Linkage: enzyme catalyzes the formation or break up of only certain category or type of bond • Stereochemical: enzyme recognizes only one of two enantiomers 19.5 Specificity of the Enzyme-Substrate Complex

Nomenclature of Enzymes • In most cases, enzyme names end in –ase • The common name for a hydrolase is derived from the substrate • Urea: remove -a, replace with -ase = urease • Lactose: remove -ose, replace with -ase = lactase • Other enzymes are named for the substrate and the reaction catalyzed • Lactate dehydrogenase • Pyruvatedecarboxylase • Some names are historical - no direct relationship to substrate or reaction type • Catalase • Pepsin • Chymotrypsin • Trypsin 19.1 Nomenclature and Classification

Systematic Name • According to the International union Of Biochemistry an enzyme name has two parts: -First part is the name of the substrates for the enzyme. -Second part is the type of reaction catalyzed by the enzyme.This part ends with the suffix “ase”. Example: Lactate dehydrogenase

EC number Enzymes are classified into six different groups according to the reaction being catalyzed. The nomenclature was determined by the Enzyme Commission in 1961 (with the latest update having occurred in 1992), hence all enzymes are assigned an “EC” number. The classification does not take into account amino acid sequence (ie, homology), protein structure, or chemical mechanism.

EC numbers • EC numbers are four digits, for example a.b.c.d, where “a” is the class, “b” is the subclass, “c” is the sub-subclass, and “d” is the sub-sub-subclass. The “b” and “c” digits describe the reaction, while the “d” digit is used to distinguish between different enzymes of the same function based on the actual substrate in the reaction. • Example: for Alcohol:NAD+oxidoreductase EC number is 1.1.1.1

The Six Classes • EC 1. Oxidoreductases • EC 2. Transferases • EC 3. Hydrolases • EC 4. Lyases • EC 5. Isomerases • EC 6. Ligases • A list of the subclasses for each class is given below. Additional information on the sub-subclasses and sub-sub-subclasses (ie, full enzyme classification and names) can be found at the referenced web link. • From the Web version, http://www.chem.qmul.ac.uk/iubmb/enzyme/index.html

EC 1. Oxidoreductases • EC 1. Oxidoreductases :catalyze the transfer of hydrogen or oxygen atoms or electrons from one substrate to another, also called oxidases, dehydrogenases, or reductases. Note that since these are ‘redox’ reactions, an electron donor/acceptor is also required to complete the reaction.

Oxidoreductases catalyze redox reactions • Reductases • Oxidases

EC 2. Transferases • EC 2. Transferases – catalyze group transfer reactions, excluding oxidoreductases (which transfer hydrogen or oxygen and are EC 1). These are of the general form: • A-X + B ↔ BX + A

Transferases transfer a group from one molecule to another • Transaminases catalyze transfer of an amino group • Kinases transfer a phosphate group 19.1 Nomenclature and Classification

EC 3. Hydrolases • EC 3. Hydrolases – catalyze hydrolytic reactions. Includes lipases, esterases, nitrilases, peptidases/proteases. These are of the general form: • A-X + H2O ↔ X-OH + HA

Hydrolases cleave bonds by adding water • Phosphatases • Peptidases • Lipases 19.1 Nomenclature and Classification

EC 4. Lyases • EC 4. Lyases – catalyze non-hydrolytic (covered in EC 3) removal of functional groups from substrates, often creating a double bond in the product; or the reverse reaction, ie, addition of function groups across a double bond. • A-B → A=B + X-Y X Y • Includes decarboxylases and aldolases in the removal direction, and synthases in the addition direction.

Lyases catalyze removal of groups to form double bonds or the reverse break double bonds • Decarboxylases • Synthases

EC 5. Isomerases • EC 5. Isomerases – catalyzes isomerization reactions, including racemizations and cis-tran isomerizations.

Isomerases catalyze intramolecular rearrangements • Epimerases • Mutases 19.1 Nomenclature and Classification

EC 6. Ligases • EC 6. Ligases -- catalyzes the synthesis of various (mostly C-X) bonds, coupled with the breakdown of energy-containing substrates, usually ATP

Ligases catalyze a reaction in which a C-C, C-S, C-O, or C-N bond is made or broken

Enzyme units • The amount of enzyme present or used in a process is difficult to determine in absolute terms (e.g. grams). • Its purity is often low and a proportion may be in an inactive, or partially active, state. • More relevant parameters are the activity of the enzyme preparation and the activities of any contaminating enzymes. • These activities are usually measured in terms of the activity unit (U) which is defined as: • Amount which will catalyze the transformation of 1 μmoleof the substrate per minute under standard conditions. • This is 10-6 - 10-11 Kg for pure enzymes and 10-4 - 10-7 Kg for industrial enzyme preparations.

Another unit of enzyme activity katal (kat) which is defined as the amount which will catalyze the transformation of one mole of substance per second (1 kat = 60 000 000 U). • It is an impracticable unit and has not yet received widespread acceptance. • Sometimes non-standard activity units are used, such as Soxhet, Anson and Kilo Novo units, which are based on physical changes such as lowering viscosity and supposedly better understood by industry.

Rightfully, such units are gradually falling into disuse. • The activity is a measure of enzyme content that is clearly of major interest when the enzyme is to be used in a process. • For this reason, enzymes are usually marketed in terms of activity rather than weight. • The specific activity (e.g. U Kg-1) is a parameter of interest, some utility as an index of purity but lesser importance.

There is a major problem with these definitions of activity; the rather vague notion of "standard conditions". • These are meant to refer to optimal conditions, especially with regard to pH, ionic strength, temperature, substrate concentration and the presence and concentration of cofactors and coenzymes. • However, these so-termed optimal conditions vary both between laboratories and between suppliers. • They also depend on the particular application in which the enzyme is to be used.

Additionally, preparations of the same notional specific activity may differ with respect to stability and be capable of very different total catalytic productivity (this is the total substrate converted to product during the lifetime of the catalyst, under specified conditions). • Conditions for maximum initial activity are not necessarily those for maximum stability. Great care has to be taken over the consideration of these factors when the most efficient catalyst for a particular purpose is to be chosen

Sources of enzymes • Biologically active enzymes may be extracted from any living organism. • A very wide range of sources are used for commercial enzyme production from Actinoplanes to Zymomonas, from spinach to snake venom. • Of the hundred or so enzymes being used industrially, over a half are from fungi and yeast and over a third are from bacteria with the remainder divided between animal (8%) and plant (4%) sources (Table 2.1). • A very much larger number of enzymes find use in chemical analysis and clinical diagnosis. • Non-microbial sources provide a larger proportion of these, at the present time.

Microbes are preferred to plants and animals as sources of enzymes because: • 1) they are generally cheaper to produce. • 2) their enzyme contents are more predictable and controllable, • 3) reliable supplies of raw material of constant composition are more easily arranged, and • 4) plant and animal tissues contain more potentially harmful materials than microbes, including phenolic compounds (from plants), endogenous enzyme inhibitors and proteases. • Attempts are being made to overcome some of these difficulties by the use of animal and plant cell culture.

a The names in common usage are given. As most industrial enzymes consist of mixtures of enzymes, these names may vary from the recommended names of their principal component. Where appropriate, the recommended names of this principal component is given below. • b The EC number of the principal component. • c I - intracellular enzyme; E - extracellular enzyme. • d +++ > 100 ton year-1; ++ > 10 ton year-1; + > 1 ton year-1; - < 1 ton year-1. • etriacylglycerol lipase; • fchymosin; • g Endo-1,3(4)-β-glucanase; • hxyloseisomerase; • isubtilisin; • jα-dextrin endo-1,6- α -glucosidase; • kglucan1,4- α -glucosidase; • lβ -galactosidase; • m microbial aspartic proteinase; • npolygalacturonase; • oα -galactosidase; • pβ -fructofuranosidase.

The majority of microbial enzymes come from a very limited number of genera. • Aspergillus,Bacillus&Kluyveromyces (also called Saccharomyces) species predominate. • Most of the strains have employed by the food industry for many years. • or have been derived from such strains by mutation and selection. • Very few examples of the industrial use of enzymes having been developed for one task. • Such developments are the production of high fructose syrup using glucose isomerase and the use of pullulanase in starch hydrolysis.

Producers of industrial enzymes and their customers will share the common aims of economy, effectiveness and safety. • They will wish to have high-yielding strains of microbes which make the enzyme constitutively and secrete it into their growth medium (extracellular enzymes). • If the enzyme is not produced constitutively, induction must be rapid and inexpensive. • Producers will aim to use strains of microbe that are known to be generally safe.

Users will pay little regard to the way in which the enzyme is produced but will insist on having preparations that have a known activity and keep that activity for extended periods, stored at room temperature or with routine refrigeration. • They will pay little attention to the purity of the enzyme preparation provided that it does not contain materials (enzymes or not) that interfere with their process. • Both producers and users will wish to have the enzymes in forms that present minimal hazard to those handling them or consuming their product.

The development of commercial enzymes is a specialized business which is usually undertaken by a handful of companies which have high skills in • 1) screening for new and improved enzymes, • 2) fermentation for enzyme production, • 3) large scale enzyme purifications, • 4) formulation of enzymes for sale, • 5) customer contact, and • 6) dealing with the regulatory authorities

Screening for novel enzymes • Natural samples, usually soil or compost material found near high concentrations of likely substrates, are used as sources of cultures. • It is not unusual at international congresses of enzyme technologists to see representatives of enzyme companies collecting samples of soil to be screened later when they return to their laboratories.

The first stage of the screening procedure for commercial enzymes is to screen ideas, • Determine the potential commercial need for a new enzyme. • Estimate the size of the market and to decide, approximately, how much potential users of the enzyme will be able to afford to pay for it. • In some cases, the determination of the potential value of an enzyme is not easy, for instance when it might be used to produce an entirely novel substance. • In others, for instance when the novel enzyme would be used to improve an existing process, its potential value can be evaluated very accurately.

In either case, • A cumulative cash flow must be estimated. • Balancing the initial screening and investment capital costs including interest, tax liability and decline, against the expected long term profits. • Full account must be taken of inflation, projected variation in feedstock price and source, publicity and other costs. • In addition, the probability of potential market competition and changes in political or legal factors must be considered. • Usually the sensitivity of the project to changes in all of these factors must be estimated, by informed guesswork, in order to assess the risk factor involved. • Financial re-appraisal must be frequently carried out during the development process to check that it still constitutes an efficient use of resources.

If agreement is reached, probably after discussions with potential users, that experimental work would be commercially justifiable. • The next stage involves the location of a source of the required enzyme. • Laboratory work is expensive in manpower so clearly it is worthwhile using all available databases to search for mention of the enzyme in the academic and patents literature. • Cultures may then be sought from any sources so revealed. • Some preparations of commercial enzymes are quite rich sources of enzymes other than the enzyme which is being offered for sale, revealing such preparations as potential inexpensive sources which are worth investigating.

If these first searches are unsuccessful, it is probably necessary to screen for new microbial strains capable of performing the transformation required. • This should not be a 'blind' screen: there will usually be some source of microbes that could have been exposed for countless generations to the conditions that the new enzyme should withstand or to chemicals which it is required to modify. • Hence, thermophiles are sought in hot springs, osmophiles in sugar factories, organisms capable of metabolizing wood preservatives in timber yards and so on. • A classic example of the detection of an enzyme by intelligent screening was the discovery of a commercially useful cyanide-degrading enzyme in the microbial pathogens of plants that contain cyanogenic glycosides.

Enzyme Engineering & Technology