Overview of Microbial Therapeutic Enzymes

Int.J.Curr.Microbiol.App.Sci (2015) 4(4): 17-26

ISSN: 2319-7706 Volume 4 Number 4 (2015) pp. 17-26 http://www.ijcmas.com

Review Article Overview of Microbial Therapeutic Enzymes

Prajakta Mane and Vidya Tale*

Department of Microbial Biotechnology, Rajiv Gandhi Institute of IT & Biotechnology, Bharati Vidyapeeth Deemed University, Pune, India *Corresponding author

A B S T R A C T

Enzymes possess specificity, greater affinity, and high catalytic efficiency. They are required for many chemical inter conversions that support life and speed up all the metabolic processes. These entire characteristics discern them from all other K e y w o r d s types of drugs. Due to these, enzymes are widely been used for different therapeutic purposes and enzyme therapies are acquiring much attention. Both Enzymes, digestive and metabolic enzymes can be used either separately or in combination Therapeutic, with other therapies for the treatment of several diseases such as leukemia, skin Digestive, ulcers, Pompe s disease, cardiovascular diseases, celiac disease, Parkinson s Metabolic, disease, Fabry s disease, inflammation, digestive disorders, pancreatic disorders Drugs etc. They are also employed in diagnosis, biochemical investigation and monitoring of many alarming diseases. Medically important enzymes produced by microorganisms have advantage of being economically feasible and consistent. They have high yield and are easy for product modification and optimization. The present review compiles the information on the sources and application of medically important enzymes produced by microorganisms and future prospects of these enzymes as drugs.

Introduction

The enzyme technology is applied to Regular consumption of enzymes and pharmaceutical research, development and enzyme-rich foods contributes to vibrant manufacturing and is a growing field. health, prevention of disease, and anti- Therapeutic enzymes have been in use for ageing process. Each cell in our body needs around at least 40 years. For example, a enzymes for its biochemical functions, and a therapeutic enzyme was described as a part deficiency of these enzymes will accelerate of replacement therapies for genetic the aging process. Some of the important deficiencies in 1960s by de Duve (Vellard, functions of enzymes are regulation of the 2013). Attempts are made to capitalize on growth of the body from a single cell to a the advantages of enzymes as drugs at every mature organism, conversion of food to pharmaceutical research center in the world energy to fulfill the body s needs, and break (Gonzalez and Isaacs, 1999). down or buildup of certain substances within the cell (Kaur and Sekhon, 2012). 17

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Enzymes were largely ignored as drugs ready purification. Medically important other than digestion aids. In the later years enzymes are usually marketed as lyophilized of 19th century, crude proteolytic enzymes pure preparations with biocompatible were used to treat gastrointestinal disorders buffering salts and mannitol diluent. The only e.g. pepsin for dyspepsia. Later, cost of these enzymes is high but do not researchers observed that an extra cellular exceed or are comparable to those of secretion i.e. nuclease (enzymatically therapeutic agents or treatments (Gurung et degrades nucleic acid) of Bacillus al., 2013). pyocyaneus kills anthrax bacilli and protects mice from otherwise lethal bacterial Different types of therapeutic enzymes inoculum. This became a milestone in the use of parental enzyme in the treatment of Medically important enzymes (digestive and infections, cancers and finally diverse metabolic) can be used either alone or in spectrum of diseases. Supplements of combination with other therapies for treating enzymes are available in pills, capsules and a variety of diseases safely. These enzymes powder form and often consist of a have two important features, a) they often combination of different enzymes (Gonzalez bind and act on their targets with a high and Isaacs, 1999). Enzymes have chiral affinity and specificity; b) they have selectivity property which is employed to catalytic property and convert multiple prepare enantiomerically pure target molecules to the desired products. pharmaceuticals (Underkofler et al., 1957). These two features are exploited to make enzymes specific and potent drugs for a Majority of medically important enzymes numerous disorders (Cooney and are obtained from a limited number of fungi, Rosenbluth, 1975). yeast and bacteria. These organisms are also considered when a new enzyme is required Medically important enzymes produced by (Teal and Wymer, 1991). Medically microorganisms find their application in important enzymes are required in very less removal of cytotoxic substances within the quantity as compared to the industrially blood circulation, treatment of life important enzymes. But they should have a threatening disorders as oncolytics, high degree of purity and specificity. The thrombolytics, anti-coagulants and as replacements for metabolic deficiencies (Kaur and Sekhon, 2012). There is very less kinetics of these enzymes are low and information about the utilization of microbial enzymes for therapeutic purposes except for some anticancer enzymes and the enzymes active against cystic fibrosis (Sabu, 2003). high max, therefore it has maximum There major application is in the treatment of cancer (prodrug activator enzymes and efficiency even at low concentrations of antineoplastic enzymes) and various other enzymes and substrates. The sources of diseases as genetic diseases including these kinds of enzymes should be selected Gaucher, Fabry, MPS I, Pompe, MPS VI, with great care and precautions to prevent SCID, CF and PKU & infectious diseases any possibility of undesirable contamination caused by protozoa, fungi or bacteria. They by incompatible material and also to enable

Int.J.Curr.Microbiol.App.Sci (2015) 4(4): 17-26 can also be used to aid digestion where they individual s body s natural healing process are used to supplement lipase, protease and (Ostlie et al., 2012). amylase in lactose intolerant people who require lactose as their body is unable to Lipase is used as digestive aids. It is also produce it. used in the treatment of malignant tumors as they have the ability to activate tumor Asparginase is employed for the treatment necrosis factor. Lipases were used in the of acute lymphocytic leukemia. Tumor cells treatment of dyspepsia, gastrointestinal lack aspartate-ammonia ligase activity, disturbances, cutaneous manifestations of which stops the synthesis of nonessential digestive allergies, and many more such amino acid L-asparagine. The activity of infections in the past. Lipase from Candida asparginase is based on this fact. The rugosa synthesizes lovastatin, a drug that asparginase does not affect the normal cells has the ability to lower serum level of which have the capability to synthesize L- cholesterol. The hydrolysis of 3- asparagine for their own need, but they phenylglycidic acid ester, which is cause a decline in the free exogenous asymmetric, is a key intermediate in the concentration, which causes a state of fatal synthesis of diltiazem hydrochloride. It is a starvation in the tumor cells. The enzyme widely used coronary vasodilator and is can be administered intravenously and is synthesized using S. marcescens lipase effective only when the asparagine levels (Matsumae et al., 1993). within the bloodstream are extremely low (Gurung et al., 2013). Nattokinase is a serine proteinase obtained from Bacillus subtilis. It can reduce some Chitinase has antimicrobial property. Chitin factors of blood clotting and lipids that are is the component of cell wall of many associated with an increased risk for pathogenic organisms, including fungi, cardiovascular disease (CVD). Oral protozoa, and helminthes and is a good administration of nattokinase could be target for antimicrobials (Fusetti et al., considered as a CVD neutraceutical. It 2002). The cell walls of Streptococcus decreases the plasma levels of fibrinogen, pneumonia, Bacillus anthracis, and factor VII, and factor VIII (Hsia et al., Clostridium perfringens are targeted using 2009). Nattokinase shows prolonged action lytic enzyme derived from bacteriophage of preventing coagulation of blood and (Zimmer et al., 2002). These lytic enzymes dissolving existing thrombus (Milner, 2008). derived from bacteriophages can be used for the treatment of several infections and also Serratiopeptidase is useful in the treatment shows activity against new drug-resistant of pain and inflammation. It has three bacterial strains. Proteolytic enzymes have mechanisms to reduce inflammation. It anti-inflammatory actions. Huge number of breaks down fibrin, the insoluble protein these proteolytic enzymes of bacterial origin byproducts of blood coagulation and thins can also be employed in the removal of dead the fluids formed from inflammation and skin of burns (Gurung et al., 2013). injury. It also facilitates their drainage which increases the speed of the tissue repair Collagenase helps in the healing of burns process. It also alleviates pain as it inhibits and skin ulcers. It helps to break up and the release of bradykinin, a specific pain remove dead skin and tissue and thus help in inducing peptide (Rothschild, 1991; Esch repair mechanism. This in turn helps and Fabian, 1989). antibiotics to work better and speed up an 19

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Thrombolytic drugs (Fibrinolytics) are created a great impact in the medicinal, used to lyse thrombi/clot to regain the industrial, dairy, and agricultural sectors. normal flow of occluded blood vessels. Many chemical processes were developed Instead of being prophylactic, they are by a number of scientists to meet this raising curative. Their action involves the activation demand. But in the later years many harmful of the natural fibrinolytic system and thus effects were observed due to the use of these the activation of plasminogen. Some chemical catalysts to fast up the processes. plasminogen activators are Streptokinase, The drawbacks of chemical transformation Urokinase, etc. Streptokinase is inactive but processes were both commercial and when it combines with the circulating environmental. Poor product yield may be plasminogen, it forms an activator complex obtained due to nonspecific reactions. High which then results in limited proteolysis of temperatures and/or high pressures required other plasminogen molecules to plasmin. It for reactions and large volumes of cooling is antigenic, has ability to cause water downstream, increases the cost of the hypersensitivity reactions and anaphylaxis. process. High capital investment and Urokinase directly activates plasminogen specially designed equipment and control and it is non-antigenic (Banerjee et al., systems are needed for harsh and hazardous 2004; Olson et al., 2011). processes involving high pressures, temperatures, alkalinity or acidity. The L-amino acid -ligase obtained from reaction may result in unwanted and harmful Empedobacter brevis catalyzes the ligation by-products which can induce negative of two amino acids L-alanine and L- impact on the environment, as well as the glutamine. This dipeptide i.e. Ala-Gln is chemicals and energy consumption. easily digested in human body and hence is used in nutritional therapy. In multiple- Hence there is a need of environment trauma patients, parenteral supplementation friendly process/biocatalyst, for which a of alanyl-glutamine dipeptide was associated biotechnology came into picture, where with better insulin sensitivity. It also different live organisms were utilized to prevents muscle wasting and increases obtain desirable products in an ecofriendly synthesis of protein in muscle (Bohumil and way. Industrial enzymes are obtained from Frantisek, 2006). In Bacillus subtilis, a novel biological systems hence effectively enzyme coded by a gene YwfE was contribute to sustainable development as identified, which catalyzed this dipeptide they are isolated from microorganisms formation from unprotected amino acids in which are fermented using primarily an ATP-dependent manner. This novel renewable resources. Both solid and liquid enzyme was classified into the category of enzyme preparations take up very little L-amino acid -ligase (LAL) (Kazuhiko and storage space as only small amounts of Shin-ichi, 2005). enzymes are needed. Uncomplicated and widely available equipment can be used as Industrial production of therapeutic the operating conditions are mild. It also microbial enzymes reduces the impact of manufacturing on the environment by reducing the utilization of Industrial expansion in the 19th up till mid- energy, chemicals, and water, and the 20th century, well known as industrial subsequent waste production (Gram et al., revolution, resulted in a great increase in 2001; Raven, 2002; Clark and Dickson, population and its demand for survival. This 2003). In pharmaceutical industry, manufacture or processing of enzymes for 20

Int.J.Curr.Microbiol.App.Sci (2015) 4(4): 17-26 use as drugs is an important aspect. The transferred into a selected microbial advantages of enzymes as drugs, is now production strain. This strain has all the been capitalized at virtually every desired features such as safety, high pharmaceutical research center in the world expression levels and for which the growth (Cassileth, 1998). medium is optimum. Hence the individual Medically important enzymes are broadly enzyme producing strains need not be distributed in animal tissues, plants, and optimized (Kaul, 2008). The principle microorganisms including bacteria, yeast, underlying rDNA technology is cloning of and fungi. The use of microbial enzymes in cDNA protein, insertion of cloned cDNA medical field is limited, although they have into expression vector, transformation of E. a good potential, because they are coli it s over expression and finally incompatible with the human body. But the purification (Sabu, 2003). Genetic use of microbial enzymes is still increasing engineering techniques allows enzyme because of their economic feasibility. manufacturer to produce sufficient amounts Various methods involving fermentation of enzyme from any microbial source. technology are available for the production Protein engineering facilitates enzyme of microbial enzymes (Sabu et al., 2000). manufacturer to adjust the properties of These include solid statefermentation, enzymes prior to production (Kaul, 2008). submerged fermentation, and immobilization, etc. These methods are Future Prospects utilized for bulk production of therapeutic enzymes on commercial scale than liquid The use of microbial enzymes in various cultures in huge bioreactors (Lozano et al., fields such as industrial and pharmaceutical 2012). Various production techniques and have increased greatly during the past few downstream processing of large-scale years. More biopharmaceuticals are entering production of microbial therapeutic enzymes the drug discovery and development have been reported (Sabu et al., 2005). pipelines in the recent times (John, 2009). Enzymes are already been used in clinical Recombinant DNA technology in the test reagents and further development in this production of therapeutic microbial field can be expected. Development in the enzymes field of clinical application of enzymes is also seen. Proteolytic enzymes are used for The production of large amount of debridement of wounds. Injection of certain therapeutic enzymes is made feasible due to enzymes such as streptokinase also promises the development of recombinant DNA positive clinical results. The use of small technology. This enhances the activity and molecular pharmaceuticals may increase, stability of an enzyme prior to its production once the diseases are better understood at (Kaul, 2008). It also reduces the cost and molecular level. Crystalline and extremely facilitates easy production. The modification purified enzymes will be necessary for in protein activity by rDNA can be clinical and therapeutic uses. Rapid overcome by site directed mutagenesis and advances may be expected in the availability shuffling functional domains. This modifies of high purity enzymes on an industrial activity and regulation of enzyme and avoids scale. Various industries including enzyme unwanted side effects (Sabu, 2003). In manufacturers are carrying out enzyme rDNA technology, the gene coding for the research currently to find new and improved enzyme with required characteristics is methods for using enzymes, to improve

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yields of industrial microbial enzymes, oncolytics, thrombolytics, fibrinolytics, &find new enzymes for industrial and mucolytics, anti-inflammatories, medical purposes. This research will help in antimicrobial and digestive aids. Diseases continuous usage of old and new enzymes which are resurging after acquiring (Underkofler et al., 1957). resistance to antibodies can also be treated There is a great and growing market for using microbial enzymes. Combinations of therapeutic enzymes. Many diseases have enzymes and drugs also have the ability to increased the demand of enzymes as induce synergistic effects and can treat therapeutic agents. Presently, therapeutic various diseases by counteracting their side enzymes are available as pills, capsules, effects. Hence, it can be concluded that there powders and food supplements. Studies are is an indeed need of research in the near been conducted to utilize the varied future in these biomolecules which will later microbial resources including both marine on prove beneficial to mankind in their and terrestrial microorganisms. Medically relevance. important enzymes produced by microorganisms are used as anticoagulants,

Table.1 Microbial therapeutic enzymes and their application

S. Enzyme Source Use No. Acid protease (Kaur and Sekhon, Aspergillis niger & Aspergillus 1 Stomach disorders 2012) oryzae Alkaline protease (Vishalakshi et al., Bio cleaning agent for 2 Streptomyces gulbargensis 2009) washing surgical instruments 3 Amylase (Kaur and Sekhon, 2012) Aspergillus sp. Easy digestion 4 Arginase (Kaur and Sekhon, 2012) Bacillus subtilis & E. coli Antitumor 5 Asparginase (Jain et al., 2012) E. coli Leukemia 6 Bacilysinsynthetase (Torsten, 2005) Bacillus subtilis Antibiotic Bacitracin synthetase (Pfaender et 7 Bacillus licheniformis Antibiotic al., 1973; Dirk and Andrea, 1997) 8 Collagenase (Dolynchuk et al., 2000) Clostridium perfringens Skin ulcers Glucose oxidase (Bankar et al., Aspergillus, Penicillium, & 9 Biosensors, Antimicrobial 2009) Saccharomyces sp. Glucosidase (Kaur and Sekhon, 10 Aspergillus niger Antitumor 2012) Glutaminase (Spiers and Wade, 11 E. coli SFL-1 Leukemia 1976) Gramicidin synthetase (carboxyl- 12 activating synthetases) (Edward and Bacillus brevis Antibiotic Arnold, 1988) L-amino acid -ligase (Makoto and Empedobacterbrevis& Bacillus 13 Patient infusion Shin-ichi, 2008; Kazuhiko and Shin- subtilis

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ichi, 2005) Aspergillus oryzae, Candida Digest lipids, Treatment of 14 Lipase (Kaur and Sekhon, 2012) lipolytica, & Candida rugosa disorders of the pancreas 15 Maltase (Kaur and Sekhon, 2012) Aspergillis oryzae Therapy for Pompe s disease 16 Nattokinase (Hsia et al., 2009) Bacillus subtilis Cardiovascular disease Nonribosomal peptide synthetase Inhibitor of microtubule 17 Aspergillus fumigatus (Deirdre and Claire, 2007) assembly hence anti-tumor Penicillin production/broad Penicillin acylase (Erickson and 18 Penicillium sp. spectrum antibiotic Bennett, 1965) production Celiac disease, Clot Bacillus polymyxa, &Beauveria 19 Peptidase (Kaur and Sekhon, 2012) formation, Inflammation, bassiana and Repair Phenylalanine racemase (Edward 20 Bacillus brevis Antibiotic and Arnold, 1988) Protease (Izrael- ivkovi et al., 21 Pseudomonas aeruginosa Antibacterial 2010) 22 Rhodanase (Kaur and Sekhon, 2012) Sulfobacillus sibiricus Cyanide poisoning 23 Ribonuclease (Lin et al., 2013) Sacchromyces sp. Antiviral RNase P ribozyme (Kaur and 24 Bacillus subtilis Antiviral Sekhon, 2012) Sacrosidase (Kaur and Sekhon, Congenital sucrase- 25 Saccharomyces cerevisiae 2012) isomaltase deficiency Serratiopeptidase (Kaur and Sekhon, 26 Serratiamarcescens Anti inflammatory 2012) Staphylokinase (Kaur and Sekhon, Staphylococcus aureus, 27 Thrombolytic agent 2012) &Streptococci sp. 28 Streptokinase (Banerjee et al., 2004] Streptococci sp. Anticoagulant Superoxide dismutase (Kaur and Mycobacterium sp. & Nocardia Anti-oxidant, Anti- 29 Sekhon, 2012) sp. inflammatory Tyrosinase (Kaur and Sekhon, 2012; Streptomyces glausescens, Antitumor, Treatment of 30 Para et al., 1984) &Erwinia herbicola Parkinson s disease Urease (Banerjee andAggarwal, Lactobacillus sp., & Klebsiella Nitrogen metabolism of 31 2013) aerogenes ruminants 32 Uricase (Terkeltaub, 2009) Aspergillus flavus Gout 33 Urokinase (Zaitsev et al., 2010) Bacillus subtilis Blood clots 34 VibrilaseTM (Ozcan et al., 2002) Vibrio proteolyticus Treatment of damaged tissue Fabry s disease, Prevention Aspergillussp. &Streptomyces 35 -Galactosidase (Anisha et al., 2008) of xenorejection, Blood griseoloalbus group transformation Ochrobactrum anthropi, Aminopeptidase (Jan and Nicolai, 36 &Sphingosinicellaxeno- Anti-oxidant 2009) peptidilytica Removal of lactose from 37 -Galactosidase (Husain, 2010) Aspergillus sp. milk 23

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