Enzymes for pharmaceutical and therapeutic applications

Enzymes are highly efficient and selective biocatalysts, present in the living beings. They exist in enormous varieties in terms of the types of reactions catalyzed by them for instance oxidation–reduction, group transfers within the molecules or between the molecules, hydrolysis, isomerization, ligation, bond cleavage, and bond formation. Besides, enzyme based catalyses are performed with much higher fidelity, under mild reaction conditions and are highly efficient in terms of number of steps, giving them an edge over their chemical counter parts. The unique characteristics of enzymes makes them highly applicable fora number of chemical transformation reactions in pharmaceutical industries, such as group protection and deprotection, selective acylation and deacylation, selective hydrolysis, deracemization, kinetic resolution of racemic mixtures, esterification, transesterification, and many others. In this review, an overview of the enzymes, their production and their applications in pharmaceutical syntheses and enzyme therapies are presented with diagrams, reaction schemes and table for easy understanding of the readers.


Introduction
Enzymes are the functional proteins or nucleic acids (Ribozymes), also known as biocatalysts that facilitate the execution of biochemical reactions at the rates which are suitable for the normal functioning, growth, and proliferation of any living system, including unicellular or multicellular plants as well as animals [1][2][3]. The ability of enzymes to remain viable and perform catalytic activities even outside their source organism, that is, under in situ conditions [4] allows them to be exploited for carrying out a number of industrial processes that rely on chemical transformations of substrates to their corresponding products. The reactions catalyzed by enzymes are highly efficient, that is, they occur under ambient environmental conditions, that is, temperature, pH, and pressure (the conditions depend on the physiological conditions of the source organisms and its environmental conditions). For instance, an enzyme obtained from a mesophilic organism (optimal growth temperature is 37 • C and growth temperature range from 20 to 40 • C), inhabiting neutral environments, shall work efficiently at mild temperatures, neutral pH, and atmospheric pressure. Enzymes may be used as isolated free forms or immobilized forms (as whole cells or isolated enzyme immobilized on a suitable support) [5,6]. As enzymes are functional outside the cells (or organisms), they are used in a number of industrial applications such as synthesis of pharmaceuticals such as drugs, process grain juices into lager and wine, leaven dough for bread production, production of agrochemicals, artificial flavors, biopolymers, waste remediation, and many others [7].
Majority of the industrial enzymes come from microorganisms as they are the most convenient sources, which gives the propensity of faster production, easy scale up, recovery and purification, strain manipulation for over-expression, enzyme activity, specificity modulations, and so on [8]. At present, 1. Enzymes are highly selective biocatalysts, catalyzing varieties of reactions such oxidation-reduction, group transfers within the molecules or between the molecules, hydrolysis, isomerization, ligation, bond cleavage, and bond formation. 2. Enzymes are applicable in pharmaceutical industries, for group protection and deprotection, selective acylation and deacylation, selective hydrolysis, deracemization, kinetic resolution of racemic mixtures, esterification, transesterification, and many other reactions. 3. In this review, an overview of the enzymes' production and applications in pharmaceutical syntheses and enzyme therapies has been discussed. boosting the immune system, preventing inflammatory bowel diseases, and curing skin burns and stomach ulcers. However, other sectors like animal feed segment, where protease are used for improving the nutritional or digestive properties of fodder and upkeep of animal gut health shall hold a great share of protease market. Carbohydrases crossed USD 2.5 billion in 2016 and are expected to show a good increase of more than 1/3 over the existing figure by 2024. The prominent application sectors shall be in food, beverage, and pharmaceutical industry.
Lipases are expected to achieve the increase in sale by around 6.8% by 2024. The main area of lipase market shall be in the field of healthcare for the treatment of obesity, which is becoming an emerging issue in developed countries. As lipase break down fats into glycerol and fatty acids under natural conditions, its demand is expected to rise in healthcare industry as an aid for weight control and management in obese people [13]. Several drugs or pharmaceutical formulations are comprised of active pharmaceutical ingredients (APIs) that are synthesized using enzymes as important components of the manufacturing process [2,14]. The usage of biocatalysts for pharmaceuticals' production as well as rising interest in the production of chiral intermediates and green synthetic processes has substantiated the interest in the applications of biocatalysts in these fields [15]. These have been possible due to improvements in the technologies for biocatalyst selection, screening, improvement, production, and supply. Although the applications of enzymes in pharmaceuticals may be vast, the aim of this review is to focus on the prospects of biocatalysts in the manufacturing of APIs, as aids for the production of health supplements, and as enzyme therapy for treatment of a number of diseases.

Production of Pharmaceutical Enzymes
Most of the industrial enzymes including those used in pharmaceutical industries are produced through fermentation of suitable microbial strains mainly belonging to bacteria and fungi due to their easy handling, fast growth rates, and convenient scale up in large vessels (fermenters) [16][17][18]. Bacteria like Escherichia coli, Bacillus subtilis, lactic acid bacteria, and the filamentous fungi such as Aspergillus oryzae, Aspergillus niger, Trichoderma atroviride, and Yeasts, for example, Saccharomyces cerevisiae, Pichia pastoris, and so on are the most exploited microorganisms for enzyme production by the biotechnology industries over the world [8,18]. As the strains have been improved through genetic engineering, they produce enzymes in very high yields [8,19]. Selection of suitable microbial strains for the production of various industrial enzymes is a very important aspect for their successful industrial applications. Ideally, the enzymes produced should be secreted out in the fermentation medium by the producing microbial strain as it makes the downstream processing more convenient and economically feasible; however, this may not be the case with most of the industrial strains. Pharmaceutical enzymes are produced by employing the fermentation technology, mainly using the microorganisms (bacteria and fungi), which comes under the Generally Recognized as Safe category [8,19]. This production is mainly carried out employing two processes, that is, submerged fermentation (SmF) and solid-state fermentation (SSF) [20]. Both the processes have their own benefits and limitations. Most industries have adopted SmF process for enzyme production; but there has been a renewed interest in SSF for certain specific industries [18,21].

Enzyme Production Through SmF
Submerged fermentation, as the name suggests, is carried out in a liquid medium, wherein the nutrients and other medium components are either dissolved or remain suspended in an aqueous medium. The microorganisms also grow and proliferate in the liquid fermentation medium in the suspended state. Generally, aerobic microorganisms are grown in submerged cultures in a stirred-tank reactor for industrial production processes for extracellular enzymes and even for the intracellularly overexpressed enzymes as in the case of recombinant E. coli and other bacterial and yeast strains [8]. The fermenters for SmF applications have been developed successfully and are being used for the production of enzymes and other biotechnological products [22,23]. The main reason behind the success of SmF is feasibility of process scale up from pilot scale fermenters (ß100 L) to production scale fermenters having capacities of millions of liters (vessel volumes) with well-developed offline and/or online control over several parameters such as, temperature, dissolved oxygen, pH, and foam formation. Further, there is no problem of mass transfer and heat dissipation; however, it is generally achieved at the cost of higher energy consumption [18,23]. Submerged fermentation for industrial production processes are manly carried out in four ways, namely batch culture, continuous culture, perfusion culture, and fed batch culture. In batch culture, the production strains are inoculated in a fixed volume of medium and thereafter no further addition of medium is carried out during the entire fermentation process (except the addition of acid, alkali, and antifoam). In continuous culture, the fermentation process is initiated in the batch mode. However, after certain time period fresh medium or nutrient concentrate is added into the fermenter at a rate which approximately matches the growth rate of the microorganism used and simultaneously the medium containing the product and biomass is continuously withdrawn from the overflow line of the vessel [24,25]. A perfusion culture is somehow similar to a continuous culture process, in that it also involves the constant feeding of fresh media and removal of spent media and product. However, it differs from the previous in retention of high numbers of viable cells by using alternating tangential-flow and standard tangential-flow filtration or by binding the cells to a substrate (capillary fibers, membranes, microcarriers in fixed bed, and so on) in the fermenter [26]. In the case of fed batch culture, the concentrated components of the nutrients are added to the batch culture in small lots at regular time points or based on the requirement of the process [24,25].

Enzyme Production Through Solid-State Fermentation
Solid-state fermentation has a great potential for enzyme production [18]. This process offers several advantages over the SmF process, such as high product titer, lesser effluent generation, use of simple fermentation equipment, less trained labor, and so on [27]. However, SSF is in general more suitable for those processes where the crude fermented products themselves are used as the final product rather than the isolated enzymes. Agroindustrial residues are commonly used substrates for the SSF processes, including those used to produce enzymes. A variety of substrates have been used for the cultivation of enzyme producing microorganisms. Examples of the substrates used are wheat bran, rice bran, sugar cane bagasse, wheat straw, rice straw, saw dust, corncobs, banana waste, cassava waste, palm oil mill waste, oil cakes, and so on [18,28].

Synthesis of 6-amino penicillanic acid by penicillin acylases
Penicillin acylases are a group of enzymes that cleave the acyl chain of penicillins to yield 6-amino penicillanic acid (6-APA) and the corresponding organic acid [29]. Screening of penicillin acylase-producing microorganisms has shown that these enzymes are produced by bacteria, actinomycetes, yeasts, and fungi [30][31][32].
Based on their substrate specificity, penicillin acylases have been classified into three groups: penicillin G acylases, penicillin

SCHEME 1
Penicillin acylase catalyzed synthesis of some important semi-synthetic β-lactam antibiotics (adopted from Ref. [43]) V acylases, and ampicillin acylases. Penicillin acylase-catalyzed synthesis of 6-APA has replaced the traditional chemical processes used for its synthesis. The conventional chemical method of 6-APA synthesis, started in 1970s, was dependent on a one-pot diacylation reaction of penicillin G (produced through fermentation), using hazardous reagents and solvents. This process of production continued for next 15-20 years and thereafter it was replaced substantially by penicillin G acylase-based method, as the enzymatic method allowed the production of 6-APA in good yields. Some researchers proposed an alternative process for the synthesis of 6-APA, based on penicillin V acylase and penicillin V [33,34]. The advantage of this process relied on the fact that the penicillin V exhibited higher stability at lower pH during the extraction process from the fermented broth, which led to higher yield of 6-APA. In addition, penicillin V acylases-based processes afforded 6-APA production even at higher substrate concentrations compared with penicillin G acylases. Further, their wider optimal pH range alleviated the need of pH regulation during hydrolysis [32,35]. Enzyme-based production of 6-APA is economically viable with immobilized forms of the enzymes. It results in great cost savings in terms of easy separation of the enzyme from the product, reuse of the enzyme and enhanced stability. Penicillin acylases from Bacillus megaterium, E. coli, and Alcaligenes faecalis have been immobilized for efficient application [36][37][38][39][40][41].

Synthesis of semisynthetic penicillins by penicillin acylases
Semisynthetic penicillins have been shown to have better properties than penicillin G and V, such as higher stability, easier absorption and lesser side effects and are also better candidates against the adaptive microbial resistance to antibiotics [42]. Large-scale production of semisynthetic antibiotics that are derived from penicillin are based on the condensation of the β-lactam nucleus with appropriate D-amino acid catalyzed by penicillin acylases (Scheme 1) [43][44][45][46]. Another application of penicillin G acylase is kinetic enantioselective acylation of the racemic azetidinone intermediate for the synthesis of the carbacephalosporin antibiotic Loracarbef, an analogue of Cefaclor and anti-platelet agent Xemilofiban [47,48]. Penicillin G acylase has also been reported for enantioselective acylation of the l-enantiomers of methyl esters of phenylglycine and 4-hydroxyphenylglycine in organic solvents (Scheme 2) [49]. The process resulted in easy isolation of the enantiomerically pure d-enantiomer, which is of practical use in the preparation of β-lactam antibiotics.

Synthesis of β-lactam antibiotic key intermediate, 7-aminocephalosporanic acid
The cephalosporin-derived antibiotics are regarded as one of the most efficient and key drugs in the treatment of bacterial infections/diseases and presently a broad range of drugs have been derived from hetero-cyclic key moiety of the cephalosporins [50,51]. Several cephalosporin-based semisynthetic β-lactam antibiotics have been commercialized such as cefotaxime, ceftriaxone, cefuroxime, and cefdinir. The lately developed ceftobiprol and ceftaroline fosamil are capable of treating the severe infections caused by MRSA (methicillin resistant Staphylococcus aureus). Cephalosporin C is the most feasible starting material for the synthesis of 7-aminocephalosporanic acid (7-ACA), as it is available in the desirable quantities at affordable cost, obtained through microbial fermentation. Additionally, cephalosporin C is a direct precursor leading to 7-ACA by amide bond cleavage [52]. Enzymatic synthesis of 7-ACA involves a combined enzymatic and spontaneous reaction cascade, which consists of a d-amino acid oxidase-catalyzed oxidation of the amino acid side chain in to the corresponding α-keto acid. This is followed by a

Enzymes for the DKR of drugs
Enzymes are exceptional in performing enantioselective reactions because of their high chemoselectivity. Enantioselective enzymes act one of the enantiomers (either R or S form) and selectively transform it into the corresponding product, whereas the other enantiomer remains untouched. At this point, the product can readily be separated from the other enantiomeric form of the substrate. However, the conventional method for resolution of racemates (i.e., the enzymatic resolution of a racemic mixture of a compound like amino acids or any synthetic or natural biomolecule) results in 50% yield of the required form of the isomers [53]. To overcome this barrier, the dynamic kinetic resolution (DKR) principle has been used recently in a variety of enzymatic reactions. Yasukawa et al. [54] reported the production of optically pure α-amino acids via enzyme catalyzed DKR of α-aminonitriles. This process is performed in three sequential steps: (i) nonselective hydrolysis of racemic α-aminonitrile by a Nitrile Hydratase (NHase), (ii) breakdown of the resulting amide by a stereoselective amino acid amide hydrolase and (iii) simultaneous racemization of the α-amino acid amide by α-amino-ε-caprolactam racemase (Scheme 4). Many compounds were hydrolyzed using the combinations of these enzymes; the results were promising wherein both (R)-and (S)-amino acids were obtained in excellent yields and ee's (>99%). An investigation with Burkholderiacepacia lipase to catalyze the DKR of racemic α-aminonitrile, resulted in enantioselective production of acetylated amine with substantial yield of 87%-90% and ee's of 85%-88% [55] (Scheme 5).
In another study DKR of racemic mandelic acid, was catalyzed sequentially using three enzymes, that is, mandelate racemase, d-mandelate dehydrogenase, and l-amino acid dehydrogenase to yield the corresponding enantiopure αamino acid l-phenylglycine, with high conversion and ee in excess of 97% [56] (Scheme 6).

Enzymes for the synthesis of amino acids
Amino acids are building blocks of life and thus are in dispensable for proper health and nutrition of both humans and animals [57][58][59]. In addition, due to their chirality, they are biochemically very significant and are very useful in chemical synthesis [60,61]. The nine essential amino acids, that is, l-leucine, l-isoleucine, l-valine, l-lysine, l-histidine, l-methionine, l-phenylalanine, l-threonine, and l-tryptophan are not synthesized in animals and humans and therefore must be ingested with food. Enzymes and whole cell biocatalysts have been important in the production of proteinogenic and nonproteinogenic d-and l-amino acids, and enantiomerically pure amino acid derivatives, which are used as important building blocks for active ingredients that are used in the manufacture of pharmaceuticals, cosmetics, and agrochemicals [62]. The rapid expansion in the amino acid market from 1980s is mainly due to cost effective production and isolation of amino acid products [58]. Out of different methods of amino acids' production the two methods, that is, the fermentation, and enzymatic catalysis are predominant, owing to their cost effective and ecofriendly nature [63].

Enzymatic production of proteinogenic amino acids
Enzymes have been exploited from the last 40 years for the production of l-amino acids in Japan (Scheme 7) [58,64,65]. The production of l-methionine, which is used in special diets, has been carried out using enzymatic resolution with acylase of Aspergillus oryzae [66]. Several hundred tons of l-methionine and l-valine are now produced each year using enzyme membrane reactor technology to minimize the enzyme loss [66]. Another amino acid that is preferably obtained through enzyme catalysis is l-aspartic acid. The enzyme aspartase catalyzes the addition of amino group from ammonia to fumaric

SCHEME 3
Enzymatic transformation of cephalosporin C into 7-ACA catalyzed by D-amino acid oxidase and glutaryl-7-ACA acylase (redrawn from Ref. [52]) acid to directly produce the l-aspartate, which is used on industrial scale to produce the artificial sweetener l-aspartame. l-Alanine is produced from l-aspartate using aspartate βdecarboxylase enzyme [67,68]. l-Cysteine has been produced from l-cystine mainly through electrochemical reduction process. However, now its industrial production is feasible through enzymatic hydrolysis and recemization of the substrate dl-2amino-2-thiazoline-4-carboxylic acid (ATC) carried out by three enzymes, that is, l-ATC hydrolase, S-carbamoyl-l-cysteine hydrolase, and ATC racemase [69]. Recently, substantial production of l-cysteine has been achieved through fermentation of an E. coli strain with disrupted l-cysteine regulon, that is, the yciW gene [70].

Enzymatic production of nonproteinogenic amino acids
Enzymatic production of d-amino acids and nonproteinogenic l-amino acids are becoming a more acceptable and environmental-friendly method. d-Amino acids can be obtained as the byproducts through enzymatic resolution of racemic mixtures of dl-amino acids used to produce l-amino acids [65].
However, it is also possible to produce d-amino acids directly, for example, using a d-specific acylase, from racemic acetyl amino acids [66]. Still another method of industrial importance is the production of building blocks for the semisynthetic antibiotics ampicillin and amoxycillin, that is, the d-phenylglycine and p-hydroxy-d-phenylglycine, which are produced using the enzyme system hydantoinase/carbamoylase. The recent advances in modern molecular biological methods (directed evolution), has made it possible to switch the d-specificity of hydantoinases to l-specificity [71]. Further, it is now possible to coexpress racemases with d-or l-selective hydantoinases and carbamoylases in highly potent recombinant whole-cell systems capable of producing a variety of d-and l-amino acids (Scheme 8) [72]. Recently, a more cost-effective enzymatic method for d-amino acid production has been reported involving dynamic kinetic resolution of N-succinyl amino acids carried out by two enzymes namely the d-succinylase and N-succinyl amino acid racemase, which enantioselectively hydrolyzed the N-succinyl-d-amino acids to their corresponding d-amino acids [73].

SCHEME 6
Dynamic kinetic resolution of mandelic acid (adopted from Ref. [

Enzymatic synthesis of l-tert-leucine
l-Tert-leucine is the basic chiral building block for many pharmaceutically active compounds such as atazanavir, boceprevir, and telaprevir, which are potent protease inhibitors for human immunodeficiency virus (HIV) protease, hepatitis C virus genotype 1 protease, and hepatitis C NS3-4A serine protease, respectively [83,84]. Li et al. [83] has reported the successful coexpression of Exiguobacterium sibiricum leucine dehydrogenase (LeuDH) with Bacillus megaterium glucose dehydrogenase (GDH) in E. coli BL21 for the production of l-tert-leucine. Here, LeuDH was used to convert the substrate trimethylpyruvic acid to l-tert-leucine and GDH was used to regenerate the cofactor NADH from NAD + , which was required to continue the synthesis reaction. By using the coexpressed whole cells, they were able to prepare decagram of l-tertleucine at 1 L scale from 0.6 M (78.1 g L −1 ) of substrate with 99% conversion after 5.5 H, resulting in 80.1% yield and >99% ee (Scheme 12). In another report, recombinant LeuDH from Thermoactinomyces intermedius and recombinant format dehydrogenase (FDH) from Pichia pastoris were used to catalyze the synthesis of L-tert-leucine from trimethylpyruvic acid and NADH regeneration, respectively (Scheme 13). Using this system, a reaction yield of ˃95% with an ee of ˃99.5% was obtained at 100 g/L substrate input [84].

Enzymatic synthesis of antiplatelet agent, Xemilofiban
A racemic mixture of ethyl 3-amino-5-(trimethylsilyl)-4pentynoate was resolved through an enantioselective acylation catalyzed by penicillin G amidohydrolase (penicillin G acylase) from E. coli to yield the S-isomer (Scheme 16), which can be used as a chiral synthon for the synthesis of the antiplatelet agent Xemilofiban [91]. The first step is the synthesis of desilylated phenylacetamide racemic ethyl Biotechnology and Applied Biochemistry SCHEME 7 L-Amino acid/D-amino acid production using acylases (adopted from Ref. [66]) 3-amino-5-(trimethylsilyl)-4-pentynoate, which is a nonenzymatic step. In the second step, phenylacetamide is enzymatically hydrolyzed to produce (R)-amide in a quantitative yield and (S)-amine with yield of 90%.

Enzyme Therapy
Enzyme therapy refers to the applications of enzymes for treating enzyme deficiencies and other medical conditions in human beings. In humans, enzymes assist in food digestion, body detoxification, strengthening of the immune system, muscle contraction, reduction of stress on the vital organs like pancreas and others. In this regard, enzyme therapy has numerous possible medical applications, for example, treatment of pancreatic insufficiency and cystic fibrosis (CF), metabolic disorders, lactose intolerance, removal of dead tissues, cancers or tumors, and so on. The therapy may be systemic or nonsystemic, and may be administered via multiple routes of administration, most often orally, topically or intravenously [92]. A summary of enzymes used as therapeutic agents is presented in Table 1.

Adenosine deaminase
Adenosine deaminase (adenosine aminohydrolase, or ADA) is an enzyme involved in metabolism of purine bases. It performs the breakdown of adenosine obtained through food and from the turnover of nucleic acids in tissues. Its primary function in humans is the development and maintenance of the immune system [101]. This enzyme has emerged as therapeutic agent for the treatment of immunological disorders. Adenosinedeaminase (Adagen: pegadamase bovine) is used for the treatment of Severe Combined Immunodeficiency Disease (SCID) [102]. It is the first successful use of an enzyme for the treatment of an inherited immunological disorder [103]. ADA plays an important role in the metabolism of excess adenosine present in the circulation of these patients and decreases the toxicity to the immune system of the raised adenosine levels. The success of the treatment depends upon the modification of ADA with PEG [104]. PEG enhances the half-life of the enzyme (originally less than 30 Min) and reduces the possibility of immunological reactions due to the bovine origin of the drug [105,106]. The PEGylated forms of enzyme are commercially available as pegadamase bovine with the brand name Adagen.  [107,108]. Mutations in the glucocerebrosidase gene are also associated with Parkinson's SCHEME 8 D-Amino acid/L-amino acid production using hydantoinases/carbamoylases (adopted from Ref. [67])

SCHEME 9
Enzymatic kinetic resolution of racemic 3-cyano-5-methylhexanoic acid esters using Arthrobacter sp. esterase (adopted from Ref. [79]) disease [108,109]. This enzyme was approved for use in enzyme replacement therapy of Gaucher disease in which an exogenous modified placental glucocerebrosidase was targeted to its correct compartment within the body [108]. The commercial formulations of the enzyme were available as alglucerase injection with the brand name Ceredase.

Sacrosidase
Sacrosidase (β-fructofuranoside fructohydrolase) is another enzyme that is used for the treatment of congenital sucraseisomaltase deficiency (CSID). CSID patients cannot digest the disaccharide sucrose. The sacrosidase-based drug hydrolyses sucrose, helping in its digestion allowing the consumption of regular food items having added sucrose or otherwise sucrose-containing fruits [110]. A sacrosidase from S. cerevisiae that can be taken orally has been used for the treatment [111].

Enzymes used as digestive aids
A number of enzymes are employed to cure the digestive problems induced by some sugars. The α-galactosidase enzyme is taken as a digestive aid for people who develop the symptoms of bloating, gas, and diarrhea upon ingesting foods like beans and Brassica vegetables such as, cabbage, broccoli, Brussels, and so on [112]. The hydrolase α-galactosidase assists in breakdown of terminal α-galactosidic residues of sugar substrates

SCHEME 11
The chemo-enzymatic route for the synthesis of Boc-(R)-3-amino-4-(2,4,5-trifluorophenyl)-butyric acid (adopted from Ref. [82]) present in these foods, which when remain undigested cause the discomfort, probably due to the bacterial fermentation of the undigested sugars [113]. Now-a-days α-galactosidase and lactase are available as a number of ready to use supplements. Lactose intolerance is a condition in which the patient is unable to produce sufficient quantities of lactase enzyme that is responsible for the digestion of the milk sugar lactose [114]. The lactose intolerant people cannot digest lactosecontaining foods and therefore, suffer from stomach upset when they consume lactose containing food such as milk and milk products containing lactose [115]. For such people, lactase supplements such as milk fortified with lactase, lactase powder, and so on are available. These supplements help to break down the lactose into its monomers, that is, glucose and galactose and alleviate the symptoms of lactose intolerance such as bloating, gas, and diarrhea [116,117].
Another disease (genetic disorder) is phenylketonuria (PKU), an inheritable disorder, requires strict compliance with a specialized diet [118]. PKU occurs due to little or absence of phenylalanine hydroxylase enzyme. This enzyme helps in converting phenylalanine to tyrosine, thereby maintaining its normal levels in the body [118]. An oral treatment for the disorder has been developed, which is composed of a plant phenylalanine ammonia lyase (PAL) overexpressed and obtained from a recombinant yeast, available in market with the trade name Phenylase TM . PAL has been demonstrated to hydrolyze phenylalanine in the gastrointestinal tract [119].
As a general digestive aid for the benefit of people with weak immunity a cocktail of pancreatic enzymes, including amylases proteases, and lipases, has been developed. The cocktail has been shown to be effective in the alleviating the problem of fat malabsorption in patients suffering from HIV [120,121]. This enzyme cocktail is also beneficial in treating the pancreatic insufficiency, which is common in patients of CF [122]. A cocktail of pancreatic enzymes with the trade name "TheraCLEC Total TM " is available commercially.

Enzymes for the treatment of infectious diseases
Lysozyme a bactericidal enzyme produced naturally in human body is added in many food products. It has been discovered that this enzyme has activity against HIV, similar to RNase A and urinary RNase U, it selectively degrades viral RNA [125] and therefore is a promising candidate for the treatment of  [127][128]. Thus, the application of lytic bacteriophages as a treatment for bacterial infections is also under development and is assumed tobe effective against antibiotic resistant bacterial strains.

Enzymes for the treatment of cancer
Application of therapeutic enzymes in cancer treatment is an emerging field of research. Lately, it has been discovered that PEG immobilized arginine deaminase (an arginine-degrading enzyme) can inhibit human hepatocellular carcinomas and skin cancers which are deficient in arginine due to lack of arginosuccinate synthetase activity [129]. A better enzyme therapy has been developed using PEGylated l-asparaginase with the name Oncaspar (pegaspargase). It has shown better results for the treatment of acute lymphoblastic leukemia, acute myeloid leukemia, and non-Hodgkin's lymphoma [130]. In other words, the normal cells, that is, the noncancerous cells can synthesize asparagine, whereas the cancer cells cannot, and die in the presence of enzymes which degrade asparagine. Although the cost of the PEG-asparaginase formulations is higher than the native enzyme the overall cost of the treatment is very similar in both cases [131]. In fact, asparaginase and PEG-asparaginase are better alternatives to standard chemotherapy.

Future Prospects of Enzymes in Pharmaceuticals
As enzymes have successfully replaced many conventional chemical catalysts-based pharmaceutical manufacturing processes, the future of enzymes in this sector is very promising. There are some enzymes that have been shown to have cellular detoxification function such as superoxide dismutase and catalase. Both these enzymes work in conjunction to detoxify the body. The superoxide dismutase transforms the highly toxic superoxide anion to moderately toxic hydrogen peroxide [132]. The catalase then converts hydrogen peroxide to harmless water and oxygen. However, both these enzymes could not be used for these desired activities even in their PEGylated forms [133]. Nevertheless, these enzymes have been reported for prolonging the life of the nematode, Caenorhabditis elegans and it is assumed that they can be used for prolonging the human life as well [134]. Improvement in the structure and functional properties of these enzymes shall be helpful in avoiding organ damage in hemorrhagic shock [135]. A serum detoxifying enzyme as an example the human butyrylcholinesterasehas been shown to be effective in the treatment of cocaine overdose [136]. Structural engineering of this enzyme has been shown to increase its detoxifying activity toward cocaine [137]. In this respect, directed evolution is proposed to provide more efficient forms of the therapeutic enzymes and other enzymes that will catalyze the pharmaceutical biotransformations with great efficiencies [138].

Conclusions
Enzymatic processes have replaced the conventional chemicalbased methods for the synthesis of many APIs such as the production of different semi-synthetic antibiotics, active enantiomers of drugs through kinetic resolution, synthesis of enantiomerically pure forms of amino acids (d-and l-amino acids) and others. Enzymes have also been used for therapeutic purposes, for example, as enzyme therapy for the treatment of various metabolic disorders and genetic diseases such as SCID, Gaucher's disease, Parkinson's disease, PKU, and so on. More recent uses of enzymes are in cancer treatment and treatment of infectious diseases, where antibiotics are no longer useful (due to appearance of resistance in bacteria against the antibiotics). They are also used as digestive aids, for example, proteases, lipases, and amylases, and have been used sepa-rately or as cocktails for the treatment of digestive problems. However, despite the current progress, there is still ample scope for enzymes in the pharmaceutical sector. In future, the established enzymes will be tried for novel applications and new enzymes, discovered from natural sources or developed through enzyme engineering or directed evolution, shall be used to catalyze still unexploited reactions.