SUPEROXIDE DISMUTASE

Superoxide dismutases  are a class of enzymes that catalyze the dismutation of superoxide into oxygen and hydrogen peroxide. As such, they are an important antioxidant defense in nearly all cells exposed to oxygen. One of the exceedingly rare exceptions is Lactobacillus plantarum and related lactobacilli, which use a different mechanism

General

Discovered by Irwin Fridovich and Joe McCord, SOD enzymes were previously thought to be several metalloproteins with unknown function (for example, CuZnSOD was known as erythrocuprein). Several common forms of SOD exist: they are proteins cofactored with copper and zinc, or manganese, iron, or nickel. For example, Brewer (1967) identified a protein that became known as superoxide dismutase as an indophenol oxidase by protein analysis of starch gels using the phenazine-tetrazolium technique.

There are three major families of superoxide dismutase, depending on the metal cofactor: Cu/Zn (which binds both copper and zinc), Fe and Mn types (which bind either iron or manganese), and finally the Ni type, which binds nickel.

Copper and zinc – most commonly used by eukaryotes. The cytosols of virtually all eukaryotic cells contain an SOD enzyme with copper and zinc (Cu-Zn-SOD). (For example, Cu-Zn-SOD available commercially is normally purified from the bovine erythrocytes: The Cu-Zn enzyme is a homodimer of molecular weight 32,500. The two subunits are joined primarily by hydrophobic and electrostatic interactions. The ligands of copper and zinc are histidine side-chains.

Iron or manganese – used by prokaryotes and protists

Iron – E. coli and many other bacteria also contain a form of the enzyme with iron (Fe-SOD); some bacteria contain Fe-SOD, others Mn-SOD, and some contain both. (For the E. coli Fe-SOD: . Fe-SOD can be found in the plastids of plants. The active sites of Mn and Fe superoxide dismutases contain the same type of amino acid side-chains.

Manganese – Chicken liver (and nearly all other) mitochondria, and many bacteria (such as E. coli) contain a form with manganese (Mn-SOD). (For example, the Mn-SOD found in a human mitochondrion: The ligands of the manganese ions are 3 histidine side-chains, an aspartate side-chain and a water molecule or hydroxy ligand, depending on the Mn oxidation state (respectively II and III).

nickel – prokaryotic. A hexameric structure built from right-handed 4-helix bundles, each containing N-terminal hooks that chelate a Ni ion. The Ni-hook contains the motif His-Cys-X-X-Pro-Cys-Gly-X-Tyr; it provides most of the interactions critical for metal binding and catalysis and is, therefore, a likely diagnostic of NiSODs.

DNase

A deoxyribonuclease (DNase, for short) is any enzyme that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone. Deoxyribonucleases are thus one type of nuclease. A wide variety of deoxyribonucleases are known, which differ in their substrate specificities, chemical mechanisms, and biological functions.
Modes of action
Some DNases cleave only residues at the ends of DNA molecules (exodeoxyribonucleases, a type of exonuclease). Others cleave anywhere along the chain (endodeoxyribonucleases, a subset of endonucleases).
Some are fairly indiscriminate about the DNA sequence at which they cut, while others, including restriction enzymes, are very sequence-specific.Some cleave only double-stranded DNA, others are specific for single-stranded molecules, and still others are active toward both.DNase enzymes can be inhaled using a nebuliser by cystic fibrosis sufferers. DNase enzymes help because white blood cells accumulate in the mucus and when they breakdown they release DNA which adds to the 'stickiness' of the mucus. DNase enzymes breakdown the DNA and the mucus is much easier to clear from the lungs.
Types of deoxyribonucleases


The two main types of DNase found in metazoans are known as deoxyribonuclease I and deoxyribonuclease II.
Other types of DNase include Micrococcal nuclease

ASPARAGINASE ENZYME

Asparaginase is an enzyme that catalyzes the hydrolysis of asparagine to aspartic acid. It is marketed under the brand name Elspar, to treat acute lymphoblastic leukemia (ALL) and is also used in some mast cell tumor protocols. [1] Unlike other chemotherapy agents, it can be given as an intramuscular, subcutaneous, or intravenous injection without fear of tissue irritation

Mechanism of action

The rationale behind asparaginase is that it takes advantage of the fact that ALL leukemic cells are unable to synthesize the non-essential amino acid asparagine, whereas normal cells are able to make their own asparagine; thus leukemic cells require high amount of asparagine. These leukemic cells depend on circulating asparagine. Asparaginase, however, catalyzes the conversion of L-asparagine to aspartic acid and ammonia. This deprives the leukemic cell of circulating asparagine.

Side effects

The main side-effect is an allergic or hypersensitivity reaction. Asparaginase has also been associated with pancreatitis. Additionally, it can also be associated with a coagulopathy as it decreases protein synthesis, including synthesis of coagulation factors (eg progressive isolated decrease of fibrinogen) and anticoagulant factor (generally antithrombin III; sometimes protein C & S as well), leading to bleeding or thrombotic events such as stroke

IS ENZYMES USED IN FRUIT JUICE

Uses of Enzymes in Fruit Juice Wine Brwing Diltilling Industries
One of the major problems in the preparation of fruit juices and wine is cloudiness due primarily to the presence of pectins. These consist primarily of a-1, 4-anhydrogalacturonic acid polymers, with varying degrees of methyl esterification. They are associated with other plant polymers and, after homogenization, with the cell debris. The cloudiness that they cause is difficult to remove except by enzymic hydrolysis. Such treatment also has the additional benefits of reducing the solution viscosity, increasing the volume of juice produce (e.g. the yield of juice from white grapes can be raised by 15%), subtle but generally beneficial changes in the flavour and, in the case of wine-making, shorter fermentation times. The enzymes used in brewing are needed for saccharification of starch (bacterial and fungal a-amylases), breakdown of barley a-1.4- and a-1,3-linked glucan (b-glucanase) and hydrolysis of protein (neutral protease) t increase the (later) fermentation rate, particularly in the production of high-gravity beer, where extra protein is added. Cellulases are also occasionally used, particularly where wheat is used as adjunct to help break down the barley is b-glucans

.Due to the extreme heat stability of the B. amyloliquefaciens a-amylase where this is used, the wort must be boiled for a much longer period (e.g. 30 minutes) to inactivate it prior to fermentation. Papain is used in the later post-fermentation stages of beer-making to prevent the occurrence of protein-and tannin- containing ‘chill-haze’ otherwise formed on cooling the beer.Recently, ‘light’ beers, of lower calorific content, have become more popular. These require a higher degree of saccharification at lower starch concentrations to reduce the alcohol and total solid content of the beer. This may be achieved by the use of glucoamylase and/or fungal a-amylase during the fermentation.

A new enzyme preparation of fungal pectin lyase (EC 4.2.2.10) was shown to be useful for the production of cranberry juice and clarification of apple juice in the food industry (Semenova et al., 2006). A comparative study showed that the preparation of pectin lyase is competitive with commercial pectinase products. The molecular weight of homogeneous pectin lyase was 38 kDa. Properties of the homogenous enzyme were studies. This enzyme was most efficient in removing highly esterified pectin.
The zygomycete microfungus R. microsporus var. microsporus produced a 1,3-1,4-beta-D-glucan 4-glucanhydrolase (EC 3.21.73) which was able to hydrolyse beta-D-glucan that contains both the 1,3-and 1,4-bonds (barley beta-glucans) (Celestino et al., 2006). Its molecular mass was 33.7 kDa. Maximum activity was detected at pH values in the range of 4-5, and temperatures in the range of 50-60ºC. The enzyme was able to reduce both the viscosity of the brewermash and the filtration time, indicating its potential value for the brewing industry
Landbo et al, (2006) examined the clarification and haze-diminishing effects of alternative clarification strategies on black current juice including centrifugation and addition of acidic protease and pectinolytic enzyme preparation and gallic acid.

USE OF ENZYMES IN INDUSTRY

Enzymes Used in Industry
Lactose is hydrolysed to its constituent monosaccharides, glucose and galactose, by lactase. Glucose itself can be removed from some foodstuff, for example, prior to drying where glucose would cause discoloration, by fungal glucose oxidase.
Enzyme
   

Uses

Bacterial glucose isomerase                        Glucose ----> Invert sugar (i.e., fructose formation)

Bacterial a-Amylase Fungal amyloglucosidase              Starch -----> Glucose

Fungal a-Amylas                  Partial degradation of starch in supplementation of amylase-deficient flour for bread making

Microbial rennets                  k-casein -----> para-casein (in milk curdling for cheese manufacture) 

Bacterial protease                Removal of protein-based stains and laundering (in biological washing powders)

Papain (from papaya melon)  Several protease applications including meat tenderization and dehazing of beer

Cellulose                              Cellulose -----> Glucose

Fungal pectinase         Pectin degradation(In fruits and vegetable processing)
   
Glucose isomerase (immobilized)        Production of invert sugar and of high fructose syrups from glucose

Penicillin acylase (immobilized)            Hydrolysis of penicillin-G to make 6-aminopenicillanic acid for production of new penicillins


Aminoacylase (immobilized)                 Resolution of DL-amino acids to produce L-amino acids for food supplementation

IS PROTEASE IS USED IN FOOD INDUSTRY?

Uses of Proteases in Food Industry
Certain proteases have been used in food processing for centuries and any record of the discovery of their activity has been lost in the midst of time. Rennet (mainly chymosin), obtained from the fourth stomach (abomasum) of unweaned calves has been used traditionally in the production of cheese. Similarly, papain from the leaves and unripe fruit of the pawpaw (Carica papaya) has been used to tenderize meats.

These ancient discoveries have led to the development of various food applications for a wide range of available proteases from many sources, usually microbial.Proteases may be used at various pH values, and they may be highly specific in their choice of cleavable peptide links or quite non-specific. Proteolysis generally increases the solubility of proteins at their isoelectric points.The action of rennet in cheese making is an example of the hydrolysis of a specific peptide linkage, between phenylalanine and methionine residues (-Phe105Met106-) in the k-casein protein present in milk. Calf rennet, consisting of mainly chymosin with a small but variable proportion of pepsin, is a relatively expensive enzyme and various attempts have been made to find cheaper alternatives from microbial sources

These have ultimately proved to be successful and microbial rennets are used for about 70% of US cheese and 33% of cheese production worldwide. The development of unwanted bitterness in ripening cheese is an example of the role of proteases in flavour production in foodstuff. The action of endogenous proteases in meat after slaughter is complex but "hanging" meat allows flavour to develop, in addition to tenderizing it. It has been found that peptides with terminal acidic amino acid residues give meaty, appetizing flavours akin to that of monosodium glutamate.The presence of proteases during the ripening of cheese is not totally undesirable and a protease from Bacillus amyloliquefaciens may be used to promote flavour production in Cheddar cheese. Lipases from Mucor miehei or Aspergillus niger are sometimes used to give stronger flavours in Italian cheese by a modest lipolysis, increasing the amount of free butyric acid

Meat tenderization by the endogenous proteases in the muscle after slaughter is a complex process which varies with the nutritional, physiological and even psychological (i.e., frightened or not) state of the animal at the time of slaughter.Meat of older animals remains tough but can be tenderized by injecting inactive papain into the jugular vein of the live animals shortly before slaughter. Proteases are also used in the baking industry.

TYPES OF ENZYMES

Categories or Types of Enzymes
All enzymes contain a protein backbone. In some enzymes this is the only component in the structure. However there are 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 affect 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 noncovalent or covalent forces. Enzymes are classified according to the report of a Nomenclature Committee appointed by the International Union of Biochemistry (1984).

APPLICATION OF ENZYMES IN BT

Applications of Enzymes in Biotechnology
For thousands of years processes such as brewing, bread-making and the production of cheese have involved the unrecognized use of enzymes. In the West the industrial understanding of enzymes revolved around yeast and malt, where traditional baking and brewing industries were rapidly expanding. Much of the early development of biochemistry was centred on yeast fermentations and processes for conversion of starch to sugar. Several enzymes, especially those used in starch processing, high-fructose syrup manufacture, textile desizing and detergent formulation, are now traded as commodity products in the world market. Relatively few enzymes, notably those in detergents, meat tenderizers and garden composting agents, are sold directly to the public.

Most are used by Industry to produce improved or novel products, to bypass long and involved chemical synthetic pathways or for use in the separation and purification of isomeric mixtures. Many of the most useful, but least-understood, uses of free enzymes are in the food industry.
There is now a raid proliferation of uses and potential uses for more highly purified enzyme preparation sin industrial processing, clinical medicine and laboratory practice. The range of pure enzymes now available commercially is rapidly increasing. Most of the enzymes used on an industrial scale are extracellular enzymes, i.e., enzymes that are normally excreted by the microorganisms to act upon their substrate in an external environment, and are analogous to the digestive enzymes of human beings and animals. Thus, when microorganisms produce enzymes to split large external molecules into an assimilable form, the enzymes are usually excreted into the fermentation media. In this way the fermentation broth form the cultivation of certain microorganisms, e.g. bacteria, yeasts or filamentous fungi, then becomes a major source of proteases, amylases and (to a lesser extent) cellulases, lipases, etc. Some intracellular enzymes are now being produced industrially and include glucose oxidase for food preservation, asparaginase for cancer therapy and penicillin acylase for antibiotic conversion. Since most cellular enzymes are by nature intracellular, more advances can be expected in this area.

Enzymes in soluble form have been used in the food industry for many years.This is especially so in the baking and brewing industries, the latter being the best example of traditional biotechnology.