Enzymatic Synthesis of Я-Lactams: Constraints and Control

Indian Journal of Biotechnology Vol 11, January 2012, pp 7-15

Enzymatic synthesis of ß-lactams: Constraints and control

Timir B Samanta Department of Biotechnology, West Bengal University of Technology, Salt Lake City, Kolkata 700 064, India

Received 20 July 2010; revised 28 February 2011; accepted 12 April 2011

The constraint in synthesis of ß-lactams by penicillin acylase is the hydrolysis of the activated acyl donor and the synthetic product. The thermodynamically controlled synthesis of ß-lactams deals with direct acylation of a nucleophile (6- APA, 7-ACA or 7-ADCA) by free acids in acidic pH and high concentration of solvent to achieve good yield. On the other hand, the kinetically controlled synthesis with esters is much faster. However, success and yield of kinetically controlled synthesis depend on combined application of an insolubilized catalyst, optimum pH of the reaction, addition of suitable solvent and high concentration of actvated acyl donor and ß-lactam nucleus. Water miscible solvents play an important role in the synthesis. The choice of solvent is guided by its inhibitory effect on enzyme activity and deleterious effect on enzyme stability. Synthetic yield of cefazolin in presence of water-miscible solvent is reduced, while the reaction carried in water- immiscible solvent markedly improves the yield of cefazolin, possibly due to reduction in hydrolysis of acyl donor and the product. Ethylene glycol inhibits more the hydrolysis of ampicillin than the hydrolysis of PGME and the synthetase/amidase ratio varies depending on the concentration of the solvent used. Hydrolytic vs synthetic activity of penicillin acylase is also influenced by water activity of the insolubilized catalyst. Shuffling of PA-encoding genes has been shown to improve the ß- lactam synthesis. Delineation of enzyme-solvent interaction needs close scrutiny to ensure further success in biocatalysis and synthesis of ß-lactams in particular.

Keywords: Immobilized penicillin acylase, ß-lactams, solvents, synthesis, water activity

Introduction hydrolyze penicillin F, penicillindihydro F and Penicillin acylases or penicillin amidases (PAs) penicillin K. Although it has been described as a (EC 3.5.1.11) are enzymes which cleave the acyl side penicillin V acylase4, the results indicate that this chain of ß-lactams to furnish 6-aminopenicillanic acid enzyme should be considered as penicillin K acylase. (6-APA) and an acyl residue11 (Fig. 1). Penicillin acylases (PAs) are produced by bacteria, actinomycetes, yeasts and fungi2. Penicillin acylases are classified as Type-I (penicillin V acylases), Type II (penicillin G acylases) and Type III (ampicillin acylases). Type I acylases are composed of four identical subunits, each of a mass of 35 kDa. Type II acylases are heterodimers composed of a small subunit (16-26 kDa) and large ß-subunit (54-66 kDa). Again, Type II acylases are subdivided into Type IIA and Type IIB. Type IIA comprise of penicillin G acylases and Type IIB acylases are glutaryl acylases. Type-III acylases (ampicillin acylases) are reported to be homodimers consisting of identical subunits with a molecular mass of 72 kDa. However, substrate specificity study of penicillin acylase from Streptomyces lavendulae3 has revealed that it can ______Author for correspondence: Tel: +91-33-23341014; Fax: +91-33-23341030 Mobile: +91-9830281585 Fig. 1—Penicillin acylase catalysed hydrolysis of penicillins E-mail: [email protected] to 6-APA 8 INDIAN J BIOTECHNOL, JANUARY 2012

The availability of chromogenic analogs of treated with acridine orange. The resultant mutants penicillindihydro F and penicillin K could allow exhibited enhanced production of PGA and screening of novel penicillin acylase-producing inactivation of ß-lactamase7. The mutant BDCS-N- microorganisms and lead to a more detailed M36 showed very negligible expression of classification of this group of enzymes5. ß-lactamase activity and 2-fold increase in PGA activity. Isolation and characterization of a natural Modified Penicillin Acylases β-lactamase-free penicillin acylase from Alcaligenes Enzymes with improved properties can be sp. (Fig. 2) had been reported for the first time from developed either by classical screening, or by the author’s laboratory8,9. chemical modification of the existing proteins, or by The PA from Alcaligenes sp. shares properties of using modern genetic and protein-engineering both type II and type III PA, although it is of type II techniques. Chemical methods used for modification origin. This enzyme is quite stable and it has been of proteins are not specific. They modify all shown that the enzyme stability is not due to the accessible residues with common side chains or they presence of disulphide bond like PA from A. are dependent on suitable amino acids to be modified, faecalis10. and often they are unable to modify amino acid difficult to reach, unless the enzyme molecule is Structure of Penicillin Acylase unfolded. Moreover, chemical modification requires The mature E. coli PGA is a periplasmic 86 kDa additional processing steps and chemicals to prepare heterodimer. The two monomer chains referred to as the enzyme. Enzyme modification through A and B contain 209 and 557 amino acid residues, mutagenesis of the encoding gene does not suffer respectively. The enzyme is synthesized as a from the problems mentioned above. Desired precursor protein, from which an active enzyme is properties may include altered specificity, altered pH generated by proteolytic processing. The PGA dependence or altered stability. Mutagenesis can be precursor polypeptide consists of four segments: the achieved either by random or by site-directed signal sequence, the A-subunit (A1-A209), the spacer technique. peptide (residues 210-263) and the B-subunit Random mutagenesis is usually done by treating (B1-B557). The signal sequence directs the transport whole microorganism with chemical mutagens or of PGA precursor into the periplasmic space, while mutagenizing radiation. High probability of isolating the 54 amino acid spacer between A and B chains desired mutant enzymes by random mutagenesis helps to ensure the correct folding of the polypeptide involves cloning of the encoding enzyme, before cleavage to generate the mature enzyme. The mutagenizing in vitro or in vivo and expressing the N-terminal serine of the B chain reacts with phenyl encoded enzyme by recloning of the mutated gene in methyl sulphonyl fluoride, which is consistent with a a suitable host. Also in this case suitable biological catalytic role for the serine hydroxyl group. protocols should be available in order to select the Modification of serine to a cysteine inactivates the desired mutant enzymes. Site-directed mutagenesis enzyme, whereas threonine, arginine or glycine (SDM) is the most suitable way of obtaining modified substitution prevents in vivo processing of the enzymes, enabling specific substitution of one or more amino acids by any other desired amino acid. Recombinant production and export of Bacillus megaterium ATCC 14945 penicillin G acylase into the growth medium has been reported for use in retro- synthesis of ß-lactam antibiotics6.

ß-Lactamase-free Penicillin Acylase Penicillin acylases produced by microorganisms are very often associated with ß-lactamase. They are made ß-lactamase-free by induced mutation. Three wild Escherichia coli strains, BDCS-N-FMu10,

BDCS-N-S21 and BDCS-N-W50, producing both penicillin G acylase (PGA) and ß-lactamase were Fig. 2—Transmission electron micrograph of Alcaligenes sp. SAMANTA: ENZYMATIC SYNTHESIS OF ß-LACTAMS 9

enzyme, indicating that this must be an important recognition site for cleavage. The crystal structure of penicillin acylase from E. coli has been determined at 1.9 Ao resolution11and more recently crystal structure of PGA from Providencia rettgeri has been determined at 2.5 Ao12. Analysis of the structure shows that two chains of the molecule are closely intertwined and form a pyramidal structure that contains a deep cone-shaped depression, at the bottom of which is the active site.

Catalytic Mechanism of Penicillin Acylases Penicillin acylases are members of the N-terminal nucleophile (Ntn) hydrolase superfamily. These enzymes are characterized by a distinct fold and a catalytic residue in the N-terminal position13. Even Fig. 3—Catalytic pocket of penicillin V acylase showing active site residues Cys 1, Asp20, Asn175, Arg228,Tyr82 and Arg17 though the nucleophilic residue differs (serine for penicillin G acylase and cysteine for penicillin V The thermodynamically controlled synthesis deals acylase), both these acylases share a striking similar with direct acylation of a nucleophile (6-APA or 7- arrangement of their catalytic environment. The ACA or 7-ADCA) by free acids in low pH–a typical fold of Ntn-hydrolase superfamily consists of condition under which most of the substrate remains a 4-layered catalytically active αßßα-core structure14. in the non-dissociated form. High solvent This core is formed by two anti-parallel ß-sheets concentration has been used to achieve a substantial packed against each other, and these ß-sheets are decrease in the ionized form of the substrate covered by a layer of α-helices on one side (Fig. 3). ultimately to favour the synthesis. In addition, the pK Like all Ntn-hydrolases, penicillin acylases go value of the substrate and products may be increased through a post-translational process15 that leads to an to ensure increase in solubility of non-dissociated autocatalytically activated enzyme. form of the acids18. Penicillin acylases from E. coli

and Kluvera citrophila have been successfully used in Synthesis of ß-Lactams by Penicillin Acylase the synthesis of antibiotics in medium containing 50% Semi-synthetic penicillins are superior to penicillin organic solvent19. The enzyme remains active in this G and V in respect of stability, absorption and side environment due to high stabilization secured by effects. They also represent a practical solution to the multipoint covalent immobilization and efforts made problem of adaptive microbial resistance to to increase the stability of the immobilized enzymes antibiotics. The semi-synthetic penicillins can be in hostile medium. Artificial microenvironment prepared by chemical and biological means. The around the immobilized enzymes has been observed enzymatic method is preferred because: i) the reaction to increase the stabilizing effect in the presence of is stereospecific, ii) reactants do not require side chain high concentration of organic solvents with minimal protection, iii) reduces environmental problem loss in catalytic activity20. The combination of because of no need for organic solvent, iv) unlike thermodynamic controlled synthesis in presence of chemical route less steps are required, vi) there is water-miscible solvents, enzyme stabilization through least amount of byproduct, and vii) extreme immobilization, and in situ product removal seems to conditions of pressure and temperature are not be a very favourable condition for the synthesis of ß- required. lactam antibiotics by penicillin acylase. Semi-synthetic penicillins and cephalosporins are Kinetically controlled synthesis involves an acyl produced by condensation of appropriate D-amino group transfer where activated acids and esters are acid derivative with ß-lactam nucleus catalyzed by used as acylating agent. The kinetically controlled penicillin acylase16. This type of condensation can be synthesis and its yield depend on the rate of done by either thermodynamically or kinetically 3 different reactions catalyzed by the enzyme, i.e., controlled synthesis17. a) the synthesis of the ß-lactamic compound, 10 INDIAN J BIOTECHNOL, JANUARY 2012

b) hydrolysis of the activated acyl donor, and c) hydrolysis of the synthesized product (Fig. 4). Obviously, design of synthesis would be to favour the synthetic process and to reduce hydrolysis. This can be achieved by combined application of an insolubilized catalyst, optimum pH for the reaction, addition of suitable solvent and high concentration of activated acyl donor and ß-lactam nucleus21. Taking these into considerations, kinetically controlled synthesis proved to be equally successful for the synthesis of different semi-synthetic ß-lactams22. Fig. 4—Enzymatic synthesis of ampicillin Advantages of performing penicillin acylase- catalyzed synthesis of new penicillins and nucleus during ampicillin synthesis. Similar cephalosporins by enzymatic acyl transfer to the improvements in acyl transfer were obtained for the ß-lactam antibiotic nuclei in the supersaturated synthesis of amoxicillin, cephalexin and cefadroxil, solutions of substrates have been demonstrated23. It making the new hybrid enzymes interesting has been shown that the effective nucleophile candidates for the biocatalytic synthesis of several β- reactivity of 6-aminopenicillanic (6-APA) and lactam antibiotics. 7-aminodesacetoxycephalosporanic (7-ADCA) acids Enhanced synthesis of different ß-lactams in ice in their supersaturated solutions continue to grow at –20°C catalyzed by penicillin G acylase has been 25 proportionally to the nucleophile concentration. As a reported by Sheldon et al . The initial ratio between result, synthesis/hydrolysis ratio in the enzymatic aminolysis and hydrolysis of the acyl-enzyme synthesis can be significantly increased due to the complex in the synthesis of cephalexin increased from nucleophile supersaturation. In the antibiotic nuclei 1.3 at 20°C to 25 at –20°C. The free penicillin-G conversion to the target antibiotic, remarkable acylase could perform transformations in frozen improvement (up to 14%) has been achieved. media with predominant hydrolytic activity under Methods of obtaining relatively stable supersaturated above conditions. Relative rates of hydrolysis and solutions of 6-APA, 7-ADCA, and synthesis of ß-lactams can be modified by site- D-p-hydroxyphenylglycine amide (D-HPGA) have directed mutagenesis of the enzyme. In fact, mutation been developed and syntheses of ampicillin, of phenylalanine at position 24 in the ß-subunit to amoxicillin, and cephalexin starting from the alanine of penicllin G acylase from E. coli yielded a supersaturated solutions of substrates were performed. mutant that showed a higher synthesis-hydrolysis ratio. Higher synthetic efficiency and increased productivity The transformation by enzyme has been found to of these reactions compared to the heterogeneous be affected by various factors, viz., reaction medium, aqueous solution-precipitate systems were observed. pH, ionic strength, temperature, organic solvent, It has been suggested that this approach seems to be water activity, stability of the catalyst with respect to an effective solution for the aqueous synthesis of the reaction conditions and reactant and product stability. broad spectrum ß-lactam antibiotics, i.e., amoxicillin, Methyl esters have been observed to be better acyl cephalexin, cephadroxil and cephaclor. donor than free acid. The equilibrium of the reaction can be shifted in favour of acylation by changing the Improvement in synthesis of ß-lactam antibiotics water activity of the catalyst with organic solvents, has also been reported by shuffling of PA-encoding e.g., polyethylene glycol, methanol, ethanol, genes of E. coli, K. cryocrescens and Providencia propanol, butanol and acetone. rettgeri24 .Out of these three, the PA from E. coli possessed the best properties for the synthesis of ampicillin. Three clones with improved synthetic Synthesis of ß-Lactams by Immobilized Penicillin properties were selected and sequence analysis Acylase showed that the shuffled genes were hybrids of the The immobilized enzyme is superior to its free PA-encoding genes from E. coli and K. crocrescens. counterpart because of easy separation of enzyme The hybrid enzymes displayed a 40-90% increase in from the product, reuse of the enzyme and enhanced the relative rate of acyl transfer to the β-lactam stability. Immobilization methods can be divided into SAMANTA: ENZYMATIC SYNTHESIS OF ß-LACTAMS 11

3 types: binding to a carrier, encapsulation in an through three consecutive transformations catalyzed inorganic or organic polymeric gel, or by cross- by D-amino acid oxidase, glutaryl acylase and PGA32. linking of the protein molecules by a bifunctional All these techniques are oriented to reduce cross-linking agent26. The hydrophilic resin, such as, manufacturing costs and mass transfer limitations in glyoxyl-agarose (activated with aldehyde groups) has order to increase their competitiveness for industrial been shown to be superior to hydrophobic acrylic- applications. At present, worldwide demand of 9000 epoxy supports like Eupergrit C during N-acylation of tons 6-APA is served by enzymatic means33. 7-aminocephalosporanic acid by Arthrobacter Enzymatic production of 6-APA is only economically viscocus27 .The use of cross-linked enzyme crystals viable when immobilized biocatalysts are used. (CLECs) as industrial biocatalyst had been introduced Research is now focused on the preparation of very in the early 1990s for commercial purpose. CLECs robust biocatalysts that may improve enzyme stability proved significantly more stable to denaturation by over a broad range of temperature, pH and solvents. heat, organic solvents and proteolysis than the Recently, whole cell immobilized penicillin G acylase corresponding soluble enzyme or lyophilized powder. have been reported to compete with biocatalysts Later cross-linked aggregates (CLEAs) were preferred prepared with isolated enzymes. In fact, whole cell of to CLECs because of productivity. In fact, penicillin several strains of E. coli and its mutants have been G acylase CLEAs, prepared by precipitation with entrapped within polymethacrylamide beads34. The ammonium sulphate, proved to be the effective immobilized preparations were effective in the catalyst for the synthesis of ampicillin28. Penicillin hydrolysis of penicillin G. Similarly, synthesis of ß- acylase from E. coli, B. megaterium or A. faecalis lactam antibiotics has been affected by reacting an have been immobilized by different methods that amino-ß-lactam component with a corresponding range from binding to prefabricated carrier materials amino group containing acylating side-chain to packaging in enzyme crystals or powders. In fact, component in presence of penicillin acylase from E. covalent binding of penicillin V acylase from coli covalently immobilized on support particles. The Streptomyces lavendulae to epoxy-activated acrylic resulting ß-lactam antibiotic is solubilized by adding beads (Eupergit C) have shown improved production an acid, such as, sulfuric acid, to lower the pH to 1.0 of 6-APA and enhanced pH and thermal stability29 . at a temperature in the range of 0° to +5°C. The However, inspite of stabilization due to enzyme immobilized penicillin acylase is substantially attachment to the support, immobilized penicillin G inactivated by the acid. After separating the product acylase on Eupergit C exhibited lower turnover rates (ß-lactam antibiotic), the immobilized enzyme can be in penicillin G hydrolysis than the enzyme in solution. reactivated for reuse in antibiotic synthesis by The hydrophobic acrylic epoxy-supports (Eupergrit treatment with a buffer having a neutral pH. C) also acted as a poor matrix and showed low Antibiotics, viz., ampicillin, amoxicillin, cephalexin, turnover rate in kinetically controlled N-acylation of cefaclor and cefadroxil, can be produced in this way. 7-amino cephalosporanic acid catalyzed by penicillin Enzymatic synthesis of ampicillin, in particular, acylase from A. viscosus. This might be due to can be carried out by thermodynamically or diffusion limitations of substrate and product kinetically controlled route. However, thermodynamic inhibition. Mateo et al30 reported a new epoxy-coated studies showed that synthesis is highly unfavourable support called Sepabeads-EP, which showed excellent for ampicillin due to low chemical energy of results as immobilizing agent. Later Phadtare et al31 zwitterionic phenylglycine35. Penicillin G acylase developed an enzyme (penicillin acylase)-fatty lipid from E. coli requires that the PG carboxyl should be biocomposite for more efficient immobilization. The protonated, while at the same time, the amino group modulation of penicillin G acylase (PGA) properties of 6-amino penicillanic acid should be neutral, via immobilization techniques has been performed available for neucleophilic interactions. The studying the acylation of 7-aminocephalosporanic kinetically controlled synthesis of ampicillin can be acid with R-mandelic acid methyl ester. PGA from E. achieved by the use of activated substrates like phenyl coli, immobilized onto agarose activated with glycidol glycine methyl ester (PGME) at pH values that are (glyoxyl-agarose), has been used for the design of a optimal for the enzyme. PGA from E. coli novel one-pot synthesis of cephamandole in aqueous immobilized on an agarose-glyoxyl derivative or medium and without isolation of intermediates, chitosan has been used for synthesis of ampicillin. 12 INDIAN J BIOTECHNOL, JANUARY 2012

The presence and magnitude of interactions between markedly increased (95%) compared to those reaction variables were examined. The best synthetic obtained in fully aqueous medium under yield (56.9%) was at 4oC and pH 6.5. The highest compromising reaction conditions37. The kinetically productivity was achieved at 25oC and pH 6.536. Also, controlled synthesis with esters is much faster, since by controlling the pH, it is possible to inhibit the two Gibb’s energy of activated side chain is improved and undesirable hydrolysis reactions and increase the more alkaline pH values may be applied, where the yield of synthesis. acylase is more active. The higher Gibb’s energy is also the reason for the yields better than Synthesis in Water Miscible Solvents thermodynamic equilibrium. These condensations Synthesis of semi-synthetic penicillins by penicillin have to be terminated in time, since the formation of acylase is possible both by thermodynamically and antibiotic reaches a maximum. This is because of kinetically controlled synthesis. While the former is hydrolysis of the activated side chain followed by slow, the latter is fast and in both the modes, i.e., synthetic ß-lactam hydrolysis. At the maximum yield, water soluble solvents, play an important role on the rate of synthesis is equal to the rate of hydrolysis of yield of synthesis. The enzyme activity and stability the condensed product. Very often these effects act account very much for overall yield of the product in controversial, e.g., a higher pH, higher temperature or synthetic/hydrolytic reaction. more enzyme input increases the activity for In the thermodynamically controlled synthesis free hydrolysis and synthesis. For enzyme input, a acids act as acyl donor. The reaction is slow because compromise between reaction time and yield needs to of low pH and PGA is not so active, thereby affecting be made. Also high substrate concentration will the yield. At the end, thermodynamically equilibrium enhance the synthesis followed by faster hydrolysis, is reached, which is convenient to stop the reaction. but better yields will be achieved at higher load. This approach works only with amidases, not with α- Water soluble solvent and ionic strength of the amino acid esterases, since these enzymes catalyse an medium will have the same effect on the yield of the α-amino acyl transfer. The quilibrium constant product as mentioned above. However, the overall follows the charge state of the side chain, which performance of the enzyme in the synthetic mode is a depends on the Ks of the acid and pH value. The combination of two effects (i) the inhibitory effect of dissociation constant can be shifted to more the solvent on the enzyme and (ii) the desirable favourable values by water soluble solvents as ions increase in the non-ionized form of the substrate in are less well hydrated. In contrast, the Ks is reduced the presence of solvent. The ratio of the enzyme by high ionic strength as ions are better stabilized. activity in its synthetic mode to its hydrolytic mode Water soluble solvents have also a direct influence on seems to be a useful guide for the selection of a the equilibrium, since they reduce the water activity suitable solvent38. and therefore water, which is a by-product of synthesis, is apparently removed and drives the Synthesis in Biphasic Solvent reaction forward. Also, increase in synthetic yield can The utility of synthetic route of penicillin acylase is be achieved by increase in concentration of ß-lactic often limited by the necessity of using organic nucleus and side chain. Temperature and enzyme solvents, which have an inhibitory effect on the input have got influence on reaction time, but less on enzyme activity and also a deleterious effect on the yields. Increase in yield is possible when the reaction enzyme stability. The first limitation can be overcome is run in water miscible solvent at low substrate by careful selection of the organic solvent so as to concentration. In contrast, at high substrate ensure that high synthetic yields are obtained without concentration, yields are reduced in synthesis run in excessively reducing the activity of the enzyme water soluble solvents. Synthesis of cephalothin from derivative, and the second by using enzymes thienylacetic acid and 7-aminocephalosporanic acid previously stabilized. Activation and stabilization of has been carried out in the presence of high the enzyme can also be ensured by the addition of concentration of organic cosolvents, e.g., 50% N,N- polyols, viz., glycerol and glycol. In fact, the effects dimethyl formamide (DMF), under a variety of of organic solvents on penicillin acylase catalyzed experimental conditions of pH and temperature by kinetically controlled synthesis of cefazolin ((CEZ) using penicillin G acylase. Synthetic yields are have been examined in various water-solvent SAMANTA: ENZYMATIC SYNTHESIS OF ß-LACTAMS 13

mixtures39. In the presence of water-miscible solvents, of available water, which differs considerably the initial rate and maximum yield of CEZ synthesis depending on solutes. The water activity of the were found to be reduced. The extent of inhibition biocatalyst is usually estimated by isopiestic method was increased with increasing hydrophobicity of the devised by Bull and Bresse41. The penicillin acylase solvent in the reaction mixture. It is interesting to note activity (hydrolytic and synthetic) of immobilized that the reaction when carried in water immiscible cells and periplasm tested under reduced water solvent, e.g., ethylacetate or carbon tetrachloride activity showed that, depending on water activity, the (two-phase system), the yield of CEZ was markedly catalyst can be used for hydrolysis or synthesis of improved. It was suggested that enhanced effect of penicillins42. EtOAc or CCl4 on synthetic yield was possibly due to reduction of hydrolysis of acyl donor and product in Conclusion and Future Trends the two-phase system rather than extraction of the Penicillin acylase is now an important alternative product into the solvent phase. Similarly the effects of for the cost-effective synthesis of β-lactam antibiotics. organic cosolvents on the synthesis of ampicillin from Immobilized penicillin acylase is preferred to free phenylglycine methyl ester (PGME) and cells because it can be re-used and product separation 6-aminopenicillanic acid (6-APA) using immobilized from the reaction mixture is easy. Again, synthesis is B. megaterium penicillin G acylase have been favoured over hydrolysis during kinetically controlled examined. Several cosolvents were tested for their synthesis of ß-lactams by the immobilized catalyst. influence on the enzyme in terms of enzyme stability Water activity of the immobilized catalyst is an and hydrophobicity. Among the cosolvents tested, important determinant during synthesis of ß-lactams. ethylene glycol was found to increase the yield of Enzyme engineering, immobilized enzyme, non- ampicillin by 39-50%. The effect of ethylene glycol aqueous biocatalysis, delineation of enzyme-solvent on the pKa of PGME, the hydrolysis of ampicillin and interaction are the areas which need to be defined PGME, and synthetase/amidase and esterase/amidase precisely to ensure further success in biocatalysis and ratios was also been studied. In fact, ethylene glycol synthesis of ß-lactams. inhibited more the hydrolysis of the ampicillin than the hydrolysis of the PGME and the Acknowledgement synthetase/amidase ratio varied from 0.2 to 0.88 when The present work was supported by Council of the concentration (v/v) of the cosolvent increased Scientific and Industrial Research (CSIR) and the from 0 to 40%. The enhancement of the synthetic Department of Biotechnology (DBT), New Delhi. The yield was mainly due to reduction in the hydrolysis of author wishes to thank Anjan Pal, Shuvendu Das, acyl donor (PGME) and product (ampicillin) in the Jiaur R Gayen and Sutapa Biswas Majee who were water-cosolvent system40. associated with this work. He also thanks the Director, Bose Institute, Kolkata for encouragements. Synthesis under Reduced Water Activity The real challenge in biocatalysis is the alteration References of substrate specificity of the enzyme. Although the 1 Shewale J G & Siva Raman H, Penicillin acylase: Enzyme stereo-chemical outcome of a given enzyme-substrate production and its application in manufacture of 6-APA, Process Biochem, 24 (1989) 146-154. reaction is predetermined, it got two options to alter 2 Sudhakaran V K & Borkar P S, Microbial transformation of the result of the reaction, viz., change of reactants or β-lactam antibiotics: Enzymes from bacteria, sources and change of reaction conditions. With the former, study—A sum up, Hindustan Antibiot Bull, 27 (1985a) redesigning of enzyme by site-directed mutagenesis is 63-119; Idem, ibid, 27 (1985b) 44-62. a powerful tool towards the goal; while in latter, 3 Torres-Guzman R, de la Mata I, Torres-Bacete J, Arroyo M, Castillon M P et al, Substrate specificity of penicillin acylase reports are available on water activity (aw = P/P0, from Streptomyces lavendulae, Biochem Biophys Res where P and P0 are vapour pressures over a sample Commun, 291 (2002) 593-597. and pure water, respectively) which has been used to 4 Batchelor F R, Chain E B, Richards M & Rolinson GN, 6- explain the results in biocatalysis. APA VI: Formation of 6-APA from penicillin by enzymatic hydrolysis, Proc R Soc Lond (B) Biol Sci, 154 (1961) Water activity is a much better choice than water 522-531. content since microorganisms do not recognize the 5 Arroya M, Torres-Guzman R, Torres-Bacete J, de la Mata I, water content of a particular material but the amount Castillon MP et al, Chromogenic analogs of penicillin 14 INDIAN J BIOTECHNOL, JANUARY 2012

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