The realm of penicillin G acylase in β-lactam antibiotics

The realm of penicillin G acylase in β-lactam antibiotics

Available online at www.sciencedirect.com Enzyme and Microbial Technology 42 (2008) 199–207 Review The realm of penicillin G acylase in ␤-lactam an...

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Available online at www.sciencedirect.com

Enzyme and Microbial Technology 42 (2008) 199–207

Review

The realm of penicillin G acylase in ␤-lactam antibiotics Anuj K. Chandel a,b , L. Venkateswar Rao b , M. Lakshmi Narasu a , Om V. Singh c,∗ a

Department of Biotechnology, Jawaharlal Nehru Technological University, Hyderabad 500007, India b Department of Microbiology, Osmania University, Hyderabad 500007, India c Department of Pediatrics, The Johns Hopkins School of Medicine, Baltimore, MD-21287, USA

Received 1 September 2007; received in revised form 10 November 2007; accepted 19 November 2007

Abstract Penicillin G acylase (PGA; EC 3.5.1.11) is a hydrolytic enzyme that acts on the side chains of penicillin G, cephalosporin G and related antibiotics to produce the ␤-lactam antibiotic intermediates 6-amino penicillanic acid (6-APA) and 7-amino des-acetoxy cephalosporanic acid (7-ADCA), with phenyl acetic acid (PAA) as a common by-product. These antibiotic intermediates are among the potential building blocks of semi-synthetic antibiotics, such as ampicillin, amoxicillin, cloxacillin, cephalexin, and cefatoxime. Currently, ␤-lactam antibiotics have annual sales of ∼$15 billion and make up 65% of the total antibiotics market; the annual consumption of PGA is estimated to be in the range of 10–30 million tons. The high demand for PGA is being met through a submerged fermentation process that uses genetically manipulated Escherichia coli and Bacillus megaterium microorganisms. Advancements in biotechnology such as screening of microorganisms, manipulation of novel PGAencoding traits, site-specific mutagenesis, immobilization techniques, and modifications to the fermentation process could enhance the production of PGA. Commercially, cheaper sources of carbohydrates and modified fermentation conditions could lead to more cost-effective production of PGA. These methodologies would open new markets and create new applications of PGA. This article describes the advancements made in PGA biotechnology and advocates its simulation for production of ␤-lactam antibiotics. © 2007 Elsevier Inc. All rights reserved. Keywords: Penicillin G acylase; ␤-Lactam antibiotics; Fermentation; Immobilization; Down stream recovery

Contents 1. 2.

3. 4. 5. 6. 7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microorganisms: an asset in PGA production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Molecular cloning and PGA production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Optimization of fermentation media for PGA production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Fermentation types for commercial significance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Product recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immobilization and biocatalytic stability of PGA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Commercial significance of PGA immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Penicillin G acylase and biotransformation of ␤-lactam antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

199 201 201 202 203 203 204 204 204 205 205 205

1. Introduction ∗

Corresponding author at: 600 N Wolfe Street, CMSC 3-106, The Johns Hopkins School of Medicine, Baltimore, MD-21287, USA. Tel.: +1 410 614 1804; fax: +1 410 955 1030. E-mail addresses: [email protected], [email protected] (O.V. Singh). 0141-0229/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2007.11.013

Due to the rising interest in sustainable development and environmentally friendly practices, microbial enzyme transformation processes are generally preferred over the conventional chemical conversion process. The former have multiple advan-

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tages, including less chemical load on the environment, higher efficiency, and the ability to dilute multiple downstream transformation attempts while maintaining product yield and recovery. The enzyme penicillin G acylase (PGA; EC 3.5.1.11) is a heterodimeric protein consisting of a small ␣ subunit and a large subunit, which are formed by the processing of a single polypeptide precursor (Fig. 1) [1]. PGA belongs to the structural superfamily of N-terminal nucleophile hydrolases that share a common fold around the active site bearing a catalytic serine, cysteine, or threonine at the N-terminal position [1,2]. Functionally, PGA acts on the side chains of penicillin G, cephalosporin G, and other related antibiotics to produce antibiotic intermediates such as 6-amino penicillanic acid (6-APA) and 7-amino desacetoxy cephalosporanic acid (7-ADCA), leaving behind phenyl acetic acid (PAA) as a common by-product (Fig. 2) [3,4]. These antibiotic intermediates are the building blocks of semi-synthetic penicillins (ampicillin, amoxicillin, cloxacillin, salbactum) and cephalosporins (cephadroxil, cefalexins, etc.) [3,5]. ␤-Lactam antibiotics, in particular penicillins and cephalosporins, represent one of the world’s major biotechnology markets. With annual sales of ∼$15 billion, they make up ∼65% of the total antibiotics market [5]. ␤-Lactam antibiotics alone constitute most of the world’s antibiotic sales: 3 × 107 kg/year out of a total 5 × 107 kg/year produced worldwide [6]. Therefore, the annual consumption of PGA is estimated to be in the range of 10–30 million tons [6]. To meet the requirements for the bulk production of ␤-lactam antibiotics, significant growth has occurred in the past two decades. Industries that produce ␤-lactam antibiotics have introduced PGA biocatalysis by replacing multistep conversion processes with cheap and promising enzymatic conversion, which has an efficiency of ∼80–90% [5]. PGA-mediated conversion of ␤-lactam antibiotics provides a novel direction

Fig. 1. PGA dimeric structure showing beta-subunit—magenta, A-subunit— blue ribbon. The polypeptide regions trimmed from the N terminus of the Asubunit and from the C terminus of the beta-subunit are indicated in green. The amino acid residues to be connected with the four amino acids linker are labeled. In red, at the center of the molecule, the catalytic serine residue is indicated (Source: Flores et al., 2004, with permission and courtesy of “Protein Science”). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

for antibiotics industries and promotes a safer and cleaner environment [3,5]. Apart from ␤-lactam hydrolysis, recent developments have resulted in multiple applications of PGA, including peptide synthesis, resolution of racemic mixture,

Fig. 2. Enzymatic conversion of penicillin G and cephalosporin G into 6-APA and 7-ADCA leaving phenyl acetic acid as common side product.

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Table 1 Profound applications through implemented mechanisms of penicillin G acylase Mechanism

Mechanistic applications

References

Peptide-synthesis (fortification of amino groups)

Production of d-phenyl dipeptides, esters applied in food additives, chitinase inhibitors, production of antifungal, anti allergic and antiviral compounds Chiral compounds (amino acids) removal Loracarbef, Carba cephalosporin antibiotic synthesis Xemilofiban, anti-platlet synthesis ␤-Amino acids synthesis

[8]

Cephalothin production from 7-ADCA and amide derivatives of 2-thienylacetic acid (2-TA) using PGA Hydrolysis of racemic iso-propylamide of mandelic acid. Derivatives were used for the preparation of cefamandole and cefonicid Synthesis of a series of structurally related compounds-2-aryloxy-2-aryl acetic acids together with a thioisostere derivatives Synthesis of +(−) methyl penylmalonate

[13]

Resolution of racemic mixture

Cephalothion synthesis Enantioselectivity (modulation of enantioselectivity of immobilized PGA) Enantioselective acylation (enantioselective recognition) Chiral hydrolysis (hydrolysis of prochiral diethyl and dimethyl phenylmalonate)

enantioselective acylation, etc. [7]. Table 1 summarizes the specified applications of PGA. The surplus demand for PGA can be met by exploring cheaper methods of PGA fermentation. In the past, PGA has primarily been manufactured by fed-batch fermentation, followed by batch and intermittent batch processes with recombinant Escherichia coli, Bacillus megaterium, and Arthrobacter viscous, utilizing sucrose and glucose as potential carbohydrate substrates [4]. This process was simple, but was found to be uneconomical because of the system-specific requirements and the high cost of carbohydrate sources for fermentation. In addition, the downstream recovery systems contributed substantially to the high price of PGA. One way to overcome such impediments may be to intervene at the molecular level via cloning and expression of PGA genes in a variety of hosts [4,17–20]. The process development has also been verified by changing fermentation-related parameters [21,22] and altering the production medium [23–27]. The key question is whether it is possible to exploit the availability of microbial sources and cheaper carbohydrate sources to develop an industrially feasible PGA production technology, despite the fact that the indigenous demand for PGA is met using pure forms of glucose and sucrose substrates. Rather than summarizing all of the existing literature on PGA production, the present article deals with the crucial parameters for developing an indigenous PGA fermentation technology. We will review various fermentation medium parameters, downstream recovery methods, and immobilization technology for biocatalytic stability and explore the optimization of enzymecatalyzed hydrolysis of ␤-lactam antibiotics. 2. Microorganisms: an asset in PGA production Microorganisms have excelled at producing primary and secondary metabolites from a variety of raw carbohydrates for billions of years under varying cultivation processes. Today, the results of studying the giant “microbial libraries” currently in vogue for microbial conversion of alternative carbohydrates into

[9] [10] [11] [12]

[14]

[15]

[16]

value-added products can be applied to PGA production. In addition, timely interventions, such as strain improvement through mutagenesis, gene cloning and expression, and optimization of potential fermentation parameters can enhance the production of selective metabolites. 2.1. Mutagenesis A suitable microorganism must be able to survive over a wide range of complex substrates at higher levels of carbohydrate with little or no toxicity of the formed product. Therefore, modifying the microbial strains for fed-batch fermentation of PGA appears to be a reasonable approach to economizing the overall PGA fermentation process. In past, multiple studies have shown progress toward strain improvement by physical means, such as UV irradiation, [28] and certain chemicals, such as N-methyl N-nitro-N-nitrosoguanidine (MNNG), 6-bromoheximide, and acridine orange modification agents [29–31]. The PGA activity (231 IU/mg) observed from a MNNG mutagenized strain of E. coli (PCS 1R-102) was more than three times greater than the activity observed in a wild-type strain (77 IU/mg) [31]. Site-directed mutagenesis was helpful for tracking the molecular changes in the pga gene in lieu of one base with an altered amino acid sequence, resulting in a cloned pga gene [32]. This approach was used to modify the rate of enzymatic hydrolysis and the synthesis of ␤-lactam antibiotics [3,33]. Site-directed mutagenesis of phenylalanine to alanine at position 24 in the ␤-subunit of E. coli produced a protein with a higher synthesis/hydrolysis ratio, increased acylase activity, and more resistance to inhibition by PAA [34]. Several studies have been performed to evaluate the mutagenic effect on pga-encoding genes and their subsequent effect on translated proteins [30,35,36]. Table 2 summarizes the studies that have used site-directed mutagenesis. The ultimate goal is to achieve a stable formed product over several generations of a modified microorganism. This process is crucial for the prolonged sur-

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Table 2 Site directed mutagenesis and modulated production of penicillin G acylase Organisms

Mutagenic sites

Modulated PGA production

References

E. coli

Ser 290

Mutants show improved PGA activity (92%) by T289C followed by T289S (85%) and T289G (20%). 20% decrease in mutated protein. 100-fold increased Kcat/Km toward glutaryl-l-leucine.

[37]

Two mutants (K299S, K299Q) showed no processing; one mutant (K299H) showed very slow processing and overall loss of activity (90%). Higher accumulation of pro PAC precursor by mutant (Arg B263) in periplasm while mutant Asn B241 stabilizes the tetrahedral intermediate. Changes in the transferase/hydrolase ratios, 40-fold decrease for ␣F146Y and ␣F146W to a 3-fold increase for ␣F146L and ␤F24A using 6-APA as the nucleophile mutants. 90 and 50% of activity loss in PGA activity on Pen. G and phenylacetyl-l-leucine respectively. Mutant showed accumulation of precursor in periplasm. Catalytic activity (kcat) decreased by a factor of more than 10. Bro 1 mutants strain exhibited altered substrate specificity consistent with the ability of the mutant to process 6-bromohexanamide. Mutant bK427A showed a great stability to organic solvents, bK430A, bK430A/bK427A showed a good stability to solvent and thermostable. Half lives of b K427A and bK430A improved by 60% and 166%, respectively, in comparison with the parent PGA.

[39]

Ser290 substituted by Cys or Secys. Replaced the B-chain residue PheB71 with either Cys or Leu. Lys (K299) residue in the active site

Invariant Arg B263, Asn B241 residues

Mutation of ␣ F146, ␤ F24 and ␤ F57 to Tyr, Trp, Ala or Leu

K. citrophila

Transversion mutation of thymine to guanine at position 1163 Gly 21 Serine residue of putative active-site

P. rettgeri

Met ␣ 140

B. megaterium

Lys at ␤ chain 427 and 430

vival of any fermentation industry that needs additional effort to establish itself. 2.2. Molecular cloning and PGA production A strong “degeneration” in selective mutant strains can occur upon the storage of conidial material; therefore, molecularbiology-based modern genetics and protein engineering have put forth new avenues to create genetically engineered microorganisms (GEMs) that can function as “booster biocatalysts.” Past studies revealed that manipulated forms of the pga gene in E. coli, A. viscous, P. rettgeri, K. citrophila, B. megaterium Achromobacter xylosoxidans and A. faecalis have been successfully cloned and expressed [20,44–47]. A bacterium harboring a plasmid containing the K. citrophilla acylase sequence gene produced >30 times PGA than the parent strain [17]. In another study, Ohashi et al. [48] cloned the pga gene from Arthrobacter viscosus and expressed it in both E. coli and Bacillus subtilis. The authors reported >72-fold increase in PGA activity intracellularly from E. coli and extracellularly from B. subtilis. Another group doubled the PGA production by growing Bacillus sp. in a process that used two consecutive fermentation media [25]. Mart´ın et al. [49] cloned a pga gene encoding for PGA from B. megaterium ATCC 14945 into E. coli HB101. This selective gene was designed to produce enhanced levels of intracellular PGA in E. coli. An expression system that gave an even higher yield of PGA was constructed by employing E. coli RE3 as a host for a recombinant plasmid pKA 18 [46]. The latter was

[32] [38]

[40]

[34]

[35] [41] [42] [29]

[43]

constructed by cloning the chromosomal pga gene in the E. coli strain RE3 on multicopy vector pK 19. This system gave a total PGA activity of about 4500 U/L along with specific activity of about 1000 U/g of cellular dry weight [46] and was found to be commercially useful. The pga gene was expressed with its own promoter in different E. coli host strains and a maximum recombinant PAC (1820 U/L) was obtained in E. coli DH5␣ [50]. It is now feasible to clone and express the PGA gene into a stable vector using cloning and expression tools. A 23 fold increase that the parent strain of genetically engineered E. coli was obtained that was cloned in to a stable vector (pYA292) [44]. Skrob et al. [18] isolated PGA from the bacterial strain Achromobacter sp. CCM4824, consisting of two dissimilar ␣ and ␤ subunits with molecular masses of 27 and 62.4 kDa, respectively. In another attempt, Valesov´a et al. [51] studied the optimization conditions for the host–plasmid interaction in the recombinant E. coli strains overproducing PGA and found that the recombinant plasmid pKA18 bearing the pga gene is segregationally style. The clones with increased synthesis of the enzyme that had an average value of 833 U/g dry mass were isolated with increased ˇ ep´anek et al. [20] sequenced expression of PGA per plasmid. Stˇ and characterized cryptic plasmid pRK2 of the strain E. coli W (ATCC 9637) and classified it as a Col E1-like plasmid. Recombinant plasmids pRS11 (4.91 kbp), pRS 12 (4.91 kbp), pRS 2 (2.996 kbp), and pRS 3 (2.623 kbp) that bear the spectinomycin resistance determinant (SpCRr) were prepared, and these constructs were found to be stable in E. coli cells grown for 63 generations. These studies indicate that a stable recombinant

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microbial strain would provide an easier and cheaper method to convert raw carbohydrate for effective PGA production; therefore, studies utilizing a recombinant microbial strain for PGA fermentation merit further attention. 3. Optimization of fermentation media for PGA production Often, microorganisms can adapt to a variety of fermentation media. However, multiple essential nutrients along with suitable carbon and nitrogen sources are required for any fermentation reaction. A variety of constituents have been optimized for PGA production media with different microorganisms [23–27,52–54]. A comprehensive analysis of fermentation media optimization has been summarized in Table 3, and those optimized specifically for PGA production are indicated. 3.1. Fermentation types for commercial significance PGA production has been broadly studied under various types of fermentation, including batch, fed-batch, and continuous [22,25,44,52–56]. Fed-batch fermentation was found to be the most viable and feasible for PGA production on an industrial scale, and it has been adopted for high biomass production within specific time intervals and conditions [21,27,53]. In a recombinant E. coli, the pga gene was expressed into the periplasmic

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space [54]; therefore, it was found advantageous to generate a substantial amount of biomass during fermentation, which in turn would lead to higher PGA production [21]. In fed-batch fermentation, a lower substrate concentration is recommended to increase the biomass production and, consequently, the PGA production [53]. However, in batch fermentation, microorganisms can survive efficiently at higher substrate concentrations and provide an increased yield of PGA in the later stages of fermentation [25,27]. Due to diminishing productivity and the labor-intensive nature of the process, batch fermentation generally is not preferred for industrial operations [57]. On the other hand, continuous fermentations have not been explored in detail, as they are prone to contamination and other system-specific limitations. Continuous fermentations are usually the choice of industries because they are less labor-intensive and easier to perform than the batch operations [57]. Fermentation media used on an industrial scale must, above all, fulfill economic requirements; therefore, these media are often based on complex, inexpensive carbon and nitrogen sources. The composition of the medium used and other details of the fermentation processes, including production and yields, are normally considered “intellectual property” and generally not disclosed in the public domain. Despite the progress made so far, studies have yet to meet the demand for a simple carbohydrate substrate that does not carry additional costs for substrate clarification and system-specific requirements.

Table 3 Optimization of fermentation media and related parameters for modulated production of penicillin G acylase Organism

Optimized/optimum parameters

PGA activity

References

Mutant strain of E. coli EP1 (pGL-5)

Carbon sources: glucose, sorbitol glycerol and PAA

[21]

B. megaterium ATCC 14945

Carbon and nitrogen sources

B. megaterium ATCC 14945

Inoculum germination phase and inoculum size

Bacillus sp. isolate

2-Level fractional design with seven components

Bacillus sp.

Sucrose, PAA and tryptone

K. citrophila penicillin G acyalse expression in E. coli

TB medium (Tryptone, yeast extract and glycerol), supplemented with IPTG (isopropylthio-beta-d-galactoside) Vitamin solution

Cell concentration-162 g wet weight/L (2.4 times higher compared to that of the original operation), and a specific PGA activity of 37 (IU/g wet weight). Maximum PGA activity-138 IU/L using casein hydrolysate supplemented with 0.6 L of alcalase and cheese whey. Spore concentration-1.5 × 107 spores/mL and germination during 24 and 72 h showed maximum PGA activity. 2-step medium optimization resulted into 2-fold increase in PGA activity. PGA activty was 2 fold higher than the production in basal medium. 2.4 factor increased PGA activity.

B. megaterium ATCC 14945

B. megaterium ATCC 14945

Fed-batch cultivation using free amino acids and cheese whey supplemented media

B. subtillus WB 600 (pMA5)

PGA production under batch and fed-batch culture using starch as carbon source

7-fold improvement in PGA activity. Ca2+ (2.5 mM) increased 2.6 fold specific activity of PGA, Exchange of natural signal peptide increased 1.7 fold activity in place of extracellular lipase Lip A. Maximum PGA activity-220 IU/mL, biomass concentration, 5.5 g/L using, during fed batch cultivation. In batch cultivation, PGA activity – 160 IU/mL with biomass concentration (4.5 g/L). 5-fold increased PGA activity (1960 IU/mL) was found upon feeding of hydrolysed starch and tryptone in media with rate of productivity – 19.6 U/L/h.

[23]

[24]

[25] [26] [19]

[27]

[52]

[53]

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4. Product recovery Beyond purification, recovering the desired product with stable activity and in pure form after fermentation is one of the tedious tasks for fermentation industries. Downstream processing plays a key role in deciding the economic viability of any fermentative product. Regardless the site-specificity (i.e., intracellular and/or extracellular) of an enzyme, product stability adds another step in the recovery process that can add to the overall cost of the desired product in a fermentation reaction [58,59]. The separation of extracellular enzymes from undesired proteins is less tedious than the separation of enzymes generated within the cell organelles. An efficient downstream recovery of PGA from E. coli homogenate has been obtained using Rolquat (quaternary ammonium salt) and adsorption of the enzyme on Amp-Seph (3.8 ␮mole ampicillin cm−3 ) under pseudo-affinity conditions [59]. Cheng et al. [58] extracted 7.5 fold increased PGA from Alcaligenes faecalis when cells were permeabilized with 0.3% (w/v) cetyl-trimethylammonium bromide. The extracted enzyme was found to be stable at 4 ◦ C for up to 30 days. For industrial applications, despite the increasing feasibility of the recovery process, shortcomings such as discontinuity, protein diffusion, and pressure drops in the systems still need to be fixed. Kecilli et al. [60] observed 10.3- and 35.5-fold purification with a recovery of 90% and 89% from Penicillium chrysogenum NRRL 1951 and P. purpurogenum crude extracts respectively, using a monolith column containing methacryloyl antipyrine. In another recent effort, Chen et al. [61] purified and simultaneously immobilized PGA using a bifunctional membrane (epoxy and immobilized copper ion) and reported that 96.3% of the PGA activity could be retained through 26 reactions over more than 2 months. 5. Immobilization and biocatalytic stability of PGA The commercial viability of any enzyme depends on its operational stability and reusability. Enzymes in free form are thermolabile and cannot be reused, owing to their loss during downstream processing and purification of the product [62,63]. Immobilization is the most important technique for stabilizing enzyme activity and enhancing its operational life. Immobilization does not necessarily enhance the enzyme’s stability, but this can be achieved by different modes of the immobilization matrix system [63]. PGA is one of the most common commercially significant examples of enzyme reusability. In the past, a number of immobilization systems have been patented and commercialized for PGA production [62,64]. Whole cells are being entrapped with a certain ratio of polyethylimine and glutaraldehyde and used as a catalyst for antibiotics’ conversion into intermediates. In liquid, PGA has been immobilized on a number of novel carriers, such as ethylene glycol dimethacrylate, Eupergit C, alumina beads, nylon fibers, silica support, zerogel, sepa beads, glycoxyl agarose, and a number of anionic exchangers [64–66]. Hydrophobic interaction chromatography that concentrates and purifies the enzyme in a single-step process was explored by Chen et al. [61], and Wang

et al. [66] by fabricating macroporous weak cation-exchange methacrylate polymers to immobilize PGA. To obtain the desired yield of antibiotic intermediates, attention has been focused toward sturdy, robust supports like sepa beads, Eupergit C [67], and nylon fibers [68] for immobilization. To enhance the thermal stability of enzymes, Bahulekar et al. [69] constructed macroporous beaded polymers of varying pore size coated with polyethyleneimine and further derivatized with glutaraldehyde for use as immobilizers. Using this technique, they demonstrated efficient, highly stable, and specific binding on the supports. They also found that the optimum temperature shifted from 40 to 57 ◦ C and that the Km increased from 31 to 128 ␮mol. The carboxylic groups of PGA and glutaryl acylase were chemically aminated in a controlled way by reaction with ethylene diamine via 1-ethyl-3-dimethyl aminopropyl carbiimide coupling, which showed a noticeable stability [70]. 5.1. Commercial significance of PGA immobilization In industry, the stability of immobilized protein during bioconversion is the most important parameter for obtaining the required yields and economizing the process. A rigid support of sepa beads was able to stand in 90% dioxane without significant loss of PGA activity. The sepa beads were found to be an excellent carrier because of their robustness and the ease with which they separated from the substrate solution [67]. Efficient recovery and reuse of the biocatalyst is a prerequisite for a commercially viable process in terms of obtaining a satisfactory yield of antibiotic intermediates 6-APA and 7-ADCA. Cheng et al. [58] successfully demonstrated the immobilization of permeabilized whole-cell PGA from A. faecalis using pore matrix cross-linking with glutaraldehyde that enhanced PGA activity 7.5-fold. In this bioconversion, the yield of penicillin G to 6APA was 75% in batch reactions up to 15 repeated cycles, and about 65% enzyme activity was retained at the end of the thirtyfirst cycle. Illanes et al. [71] studied cephalexin production at high substrate concentration (750 mM) acyl donor with immobilized and cross-linked aggregates (CIEA) of PGA. To assess the operational stability and global productivity of the biocatalyst, 40.1 and 135.5 g of cephalexin/g of biocatalyst were obtained for PGA-450 and CLEA, respectively. Magnetic hydroxyl particles have been activated with epoxyl chloropropane by suspension polymerization and used for PGA immobilization. The activity of the immobilized enzyme remained constant at ∼94% for up to 80 cycles of the bioconversion process [66]. The use of restrictive conditions during enzyme adsorption onto anionic exchangers allows the adsorption strength and enzyme stability to increase in the presence of organic solvents. This action suggests that the enzyme actively penetrates the polymeric beads, and becomes fully covered with the polymer. After the enzyme is inactivated, it can be desorbed to reuse the support. The possibility of improving the immobilization properties of an enzyme by site-directed mutagenesis of its surface opens a promising new scenario for enzyme engineering. Recently, Montes et al. [72] used site-directed mutagenesis to add eight, new, homogeneously distributed glutamic residues throughout the enzyme’s surface to study reverse immobilization

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using anionic exchangers (DEAE- or polyethyleneimine-coated agarose). Despite the enormous efforts toward biocatalysis with industrial applications, the traditional stoichiometry-based chemistry still needs to be replaced with alternative approaches. Introduction of biocatalysis to existing processes may require extensive modifications that often are not feasible with the lifespan of today’s products. It is therefore of utmost importance to consider the option of biocatalysis from the very beginning of product and process development. An example of the successful application of biocatalysis to produce existing compounds can be found in the manufacturing of ␤-lactam antibiotics. Kallenberg et al. [65] highlighted the benefits of PGA immobilization in the production of semi-synthetic ␤-lactam antibiotics. PGA now plays a central role in ␤-lactam antibiotics chemistry from enzyme isolation to purification, and provides growing insight into enzyme immobilization. 6. Penicillin G acylase and biotransformation of ␤-lactam antibiotics PGA is one of the key pharmaceutical enzymes in the production of polysynthetic ␤-lactam antibiotics. The enzyme catalyzes the hydrolysis of the amidic bonds of penicillin G and cephalosporin G to produce 6-APA and 7-ADCA, key components in the production of ␤-lactam antibiotics. These compounds serve as starting points for the production of semisynthetic penicillins/cephalosporins; PAA is produced as a by-product (Fig. 2). PGA, acting as either a transferase or a hydrolase, catalyzes two undesired side reactions: the hydrolysis of the acyl side-chain precursor (an ester or amide, a parallel reaction) and the hydrolysis of the antibiotic itself (a consecutive reaction) [64]. The biotransformation of penicillin G and cephalosporin G into antibiotic intermediates can be controlled by thermodynamically or kinetically controlled synthesis [64]. It is mainly based on the direct acylation of nucleophiles such as 6-APA or 7ADCA by free acids at low pH [3]. It has been shown that the effective nucleophile reactivity of 6-APA and 7-ADCA in their supersaturated solutions continues to grow proportionally to the nucleophile concentration [33]. The nucleophilic supersaturation controls the rate of enzymatic synthesis and/or hydrolysis into intermediates [33]. This mechanism appears to be effective for the aqueous synthesis of the most widely used ␤-lactam antibiotics, such as amoxicillin, cephalexin, cephadroxil, and cephaclor. In addition, PGA can be used in other useful biotransformations such as peptide synthesis and the resolution of racemic mixtures of chiral compounds [16]. A series of structurally related 2-aryloxy-2-arylacetic acids together with a thioisostere derivative has been synthesized and characterized by GC–MS and 1 H NMR [15]. Cabrera et al. [16] reported that PGA is able to hydrolyze prochiral diethyl and dimethyl phenylmalonate to produce +(−) methyl phenylmalonate, although compared to other substrates, PGA requires a higher activity for this conversion. Shaw et al. [13] reported that using PGA, the conversion yield of cephalothin from 7ADCA and amide derivatives of 2-thienylacetic acid (2-TA)

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