DEFINICIÓN DEL NEGOCIO

Enzymes are vital for life, as they catalyze spe-cifi c reactions as well as enhance the rate of reac-tions, thus forming the basis of metabolism. They

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also offer tremendous opportunities in industry to carry out biocatalytic conversions, thereby making the process effi cient and economical.

Microbial enzymes can be used as free enzymes or selectively induced intracellularly in microbial cells. Unlike many chemical processes in con-ventional synthetic chemistry, enzymes require non-toxic and non-corrosive conditions to carry out catalysis.

Extremozymes are enzymes produced by extremophilic microbes and are increasingly fi nding applications in industry, replacing the conventional catalysts as they can withstand harsh conditions. This has revolutionized the arena of synthesis of active pharmaceutical ingre-dients (APIs), thereby enhancing manufacturing operations. Moreover, chirally pure compounds/

drugs can be synthesized (enantioselective syn-thesis) using microbial enzymes to avert such disasters as the thalidomide cases.

The screening of microbes that produce novel enzymes for process development is the key step. It is based on (1) selecting a process and a particular type of enzymatic activity; (2) deciding on the types of microorganisms to be selected and screened; and fi nally (3) develop-ing an appropriate, sensitive, and convenient assay that would facilitate the screening. D-p- hydroxyphenylglycine and its derivatives serve as important side chain precursors for the semi- synthetic penicillins and cephalosporins.

Industrial production of D -amino acids involves the synthesis of hydantoin substrates. Stereo- specifi c hydrolysis of hydantoins was catalyzed by D-hydantoinase (microbial origin) and decarbamoylation. Chemical decarbamoylation is carried out by treating the amino acid inter-mediate with an equimolar amount of nitrile under aerobic conditions. A novel enzyme

D -carbamoylase has been found and engineered for use in the decarbamoylation process, thereby making the process cleaner and sim-pler. The recombinant mutant enzyme has been used for the large-scale production of D-p-hydroxyphenylglycine (~2,000 tons/year) with simultaneous use of D -hydantoinase (Ogawa and Shimizu 2000 ).

9.4.1 Cell-Free Biocatalysis

Trans-4- L -hydroxyproline is an important chiral precursor for the synthesis of antiphlogistics, car-bapenem antibiotics, and angiotensin- converting enzyme (ACE) inhibitors. This requires stereo-selective and region-stereo-selective hydroxylation of L -proline to yield hydroxy-l - proline isomers, leading to the discovery of specifi c proline hydroxylases: 4-hydroxylase and 3-hydroxylase in Dactylosporangium sp. RH1 and Streptomyces sp. TH1, respectively. 4-hydroxylase specifi cally produces trans-4- hydroxy- l -proline, whereas 3- hydroxylase produces cis -3-hydroxy- L -proline.

These were subsequently cloned in E. coli . Kyowa Hakko Kogyo Co. started the production of trans-4- hydroxy- l -proline using this method.

L -DOPA (l-3,4 dihydroxyphenylalanine) is a medicine for Parkinson’s disease. Tyrosine phe-nol lyase (TPL) is a pyridoxal phosphate (PLP) enzyme isolated from Erwinia herbicola , Citrobacter freundii that catalyzes the revers-ible transformation of tyrosine into phenol, pyruvate, and ammonia (Yamada and Kumagai 1975 ). Pyrocatechol was used as a substrate for this enzyme for the production of L -DOPA. E.

herbicola cells accumulated 110 g/l of L -DOPA, and in 1993, Ajinomoto Co. started commercial production using this system and currently pro-duces half of the worldwide supply of L -DOPA, which is around 250 tons/year.

Lipases have been used as biocatalysts for the synthesis of chiral compounds through kinetic

Biocatalysis is the use of free (extracellu-lar) enzymes to perform chemical transfor-mation of organic compounds.

Biotransformation is the chemical transfor-mation of organic compounds carried out by intracellular enzymes and is often referred to as whole cell biocatalysis.

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resolution of racemic mixtures or enantioselec-tive enzymatic desymmetrization of prochiral compounds. Candida rugosa lipase is one of the employed serine hydrolases in organic synthesis.

Pfi zer has prepared a procedure of (S)-ibuprofen through enantioselective hydrolysis of the corre-sponding methoxyethyl ester catalyzed by C.

rugosa lipase in an immobilized bioreactor.

Similarly, lipases from Serratia and Pseudomonas have been used for the enantiomeric/chiral syn-thesis of organic compounds (Table 9.4 ).

Penicillin G acylase has been isolated from many microorganisms and is currently being used for the selective hydrolysis of penicillin G to afford 6-APA.

9.4.2 Whole Cell Biocatalysis

F. oxysporum has been responsible for the pro-duction of an enzyme that catalyzes the revers-ible hydrolysis of aldonate lactones and butyrolactones. The hydrolysis is stereospecifi c by recognizing the hydroxyl group confi guration at the two position of carbon (Haworth projec-tion). If the hydroxylation is below the carbon atom then hydrolysis takes place. A racemic mix-ture of pantolactone has been resolved into

D -pantoic acid and L -pantolactone. D -pantoic acid is the chiral building block for the commercial

production of the vitamin, D -pantothenic acid (vitamin B 5 ). The mycelium of F. oxysporum is entrapped in calcium alginate and incubated with the racemic mixture pantolactone for 21 h at 30

°C at pH 7; approximately 90 % of D -isomer was hydrolyzed with high optical purity between 93 and 98 %. This process has been used by Daiichi Fine Chemicals for the commercial production of

D-pantolactone (~3,000 tons/year calcium D -

PANTOTHENATE ) (Kataoka et al. 1995 ).

Redox reactions play a signifi cant role in the sustainable and productive synthesis of drugs.

Oxidoreductases are employed using whole cells due to their dependence on cofactors that need to be regenerated. Whole cells of Gluconobacter sub-oxydans are responsible for the regio- selective oxi-dation of N-protected-1-amino- D -sorbitol. The reaction product is (3S, 4R, 5S)-1, 3, 4, 5- tetrahydroxy - 6 - (alkylamino) hexan-2-one; a sorbose derivative is a key intermediate in the syn-thesis of oral α-glucosidase inhibitors (Landis et al.

2002 ). Similarly, Streptomyces species has been used by Bristol-Myers Squibb to carry out bio-transformation of compactin to pravastin (sodium (3R,5R)-3,5-dihydroxy-7-((1S, 2S, 6S, 8S, 8aR)-6- hydroxyl-2-methyl-8-((S)-2- methylbutanoyloxy) -1, 2, 6, 7, 8, 8a- hexahydronaphthalen-1-yl) heptanoate).

Whole cell reductions have also been carried out industrially. Whole cells of Rhodococcus

Table 9.4 Lipases in chiral synthesis of drugs

Immobilized Candida antarctica lipase B (CALB)

Pseudomonas cepacia lipase PS30 (Amano)

Paclitaxel (anti-tumor agent)

Serratia marcescens lipase

Diltiazem (calcium canal blocker)

Tanabe Pharmaceutical (3S,3aR,6aR)-3-hydroxy-

3,3a,4,6a-tetrahydro- 2Hcyclopenta[b]furan-2-one

Pseudomonas fl uorescens lipase

Carbovir (antiviral agent) Celltech Group 9 Microbial Enzymes and Their Industrial Applications

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erythropolis SC13854 reduced (1S)-[3-chloro-2- oxol-(phenylmethyl)propyl] carbamic acid, leading to the formation of tert-butyl [(2S,3R)-4-chloro-3-hydroxy-1-phenylbutan-2-yl] carba-mate, an alcohol intermediate for an HIV endopeptidase inhibitor atazanavir (Patel et al.

2003 ).

Aryl aryl ketones have also been reduced by whole cell biocatalysis. ((S,E)-methyl 2-(2-(3-(3-(2-(7-chloroquinolin-2-yl)vinyl) p h e n y l ) - 3 - h y d r o x y p r o p y l ) p h e n y l ) - 2 - methylpropanoate) has been produced from the corresponding ketone in the synthesis of the anti- asthma drug montelukast, produced at Merck & Co. Inc., Rahway, USA, by bio-reduc-tion catalyzed by whole cells of Microbacterium campoquemadoensis . The industry-relevant examples of whole cell biocatalysis are given in Table 9.5 .

9.4.3 Phytochemical Extraction with Microbial Enzymes

Recently, microbial enzymes have also been used in the isolation of phytochemicals as they improve yield and protect the phytochemical from degra-dation. Lutein, phenolics, and lignans have been extracted using enzyme-assisted extraction meth-ods. Lutein is a xanthophyll that has been extracted from fl ower petals of marigold ( Tagetes erecta ) (Tekwani and DeMello 2010 ). Lutein is a reactive oxygen species (ROS) scavenger and is well used in nutrition and in the cosmetic and pharmaceutical industries. Lutein is also impli-cated in vision improvement, protection of skin from ultraviolet (UV)-induced damage, and reduction in risk of CVDs. Conventional methods of lutein extraction from marigold fl owers results in partial degradation and substantial loss of

Table 9.5 Whole cell biocatalysis in synthesis of drugs

Product Biocatalyst Drug Company

6-(alkylamino) hexan-2-one

Gluconobacter suboxydans α- glucosidase inhibitors Bayer, Germany Sodium (3R,5R)-3,5-dihydroxy-

7-((1S,2S,

Rhodococcus erythropolis SC13854

Zygosaccharomyces rouxii Talampanel™ Eli-Lilly and Company

Montelukast (anti-asthma) Merck & Co.

(S)-ethyl

4-chloro-3-hydroxybutanoate

Escherichia coli Atorvastatin (lateral chain) Kaneka Co.

(S)-methyl

4-chloro-3-hydroxybutanoate

Geotrichum candidum SC 5469

Atorvastatin (lateral chain) Bristol–Myers Squibb (S)-2-amino-5-(1,3-dioxolan-2-

yl)-pentanoic acid

E. coli cells over-expressing phenylalanine dehydrogenase from Sporosarcina

Omapatrilat (anti-hypertensive)

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carotenoids. Thus, enzyme treatment has been proposed as an alternate stage to solvent extrac-tion processes to improve the yield and prevent partial degradation. A 10 % improvement in yield of lutein was observed when the extraction was carried out using cellulases and pectinases.

Solvent use was also reduced, making the pro-cess more economical and greener.

Phenolic extraction from agricultural and industrial wastes has gained much attention recently as cheap and safe sources of strong anti-oxidants. Bioactive phytochemicals are present as soluble, suspended, or colloidal forms in com-plexes with the cell wall components in the plants after the cell wall is ruptured. Thus cell-wall- hydrolyzing enzymes such as cellulase, hemicel-lulase, and pectinase hydrolyze the plant materials, and have often been proposed as tools for extraction. Phenolics have been extracted from apple peel using cellulases apart from the recovery of sugars.

Lignans are diphenolic compounds resulting from the dimerization of two coniferyl alcohol moieties. Some lignan derivatives have been found to possess chemopreventive properties against breast and prostatic tumors after being transformed by gut fl ora in humans. Main fl ax seed ( Linum usitatissimum ) lignan secoisolarici-resinol is converted after ingestion by human intestinal microbiota into enterodiol, which has been shown to reduce the development of mam-mary and prostatic tumors. Onuzuka RS ( T. reesei cellulase by Merck & Co.) was found to give a better yield of secoisolariciresinol from seed hulls and whole seeds of fl ax as compared with β-glucosidase.