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. 2020 Nov 6;5(45):28972–28976. doi: 10.1021/acsomega.0c02813

Process Development for 6-Aminopenicillanic Acid Production Using Lentikats-Encapsulated Escherichia coli Cells Expressing Penicillin V Acylase

Amol M Sawant ‡,†, Avinash Vellore Sunder ‡, Koteswara Rao Vamkudoth ‡,†,*, Sureshkumar Ramasamy ‡, Archana Pundle ‡,†,*
PMCID: PMC7675567  PMID: 33225127

Abstract

graphic file with name ao0c02813_0006.jpg

Penicillin V acylase (PVA, EC 3.5.1.11) hydrolyzes the side chain of phenoxymethylpenicillin (Pen V) and finds application in the manufacture of the pharmaceutical intermediate 6-aminopenicillanic acid (6-APA). Here, we report the scale-up of cultivation of Escherichia coli whole cells expressing a highly active PVA from Pectobacterium atrosepticum and their encapsulation in polyvinyl alcohol–poly(ethylene glycol) Lentikats hydrogels. A biocatalytic process for the hydrolysis of 2% (w/v) Pen V was set up in a 2 L reactor using the Lentikats-immobilized whole cells, with a customized setup to enable continuous downstream processing of the reaction products. The biocatalytic reaction afforded complete conversion of Pen V for 10 reaction cycles, with an overall 90% conversion up to 50 cycles. The bioprocess was further scaled up to the pilot-scale at 10 L, enabling complete conversion of Pen V to 6-APA for 10 cycles. The 6-APA and phenoxy acetic acid products were recovered from downstream processing with isolated yields of 85–90 and 87–92%, respectively. Immobilization in Lentikats beads improved the stability of the whole cells on storage, maintaining 90–100% activity and similar conversion efficiency after 3 months at 4 °C. The robust PVA biocatalyst can be employed in a continuous process to provide a sustainable route for bulk 6-APA production from Pen V.

1. Introduction

The core β-lactam compound of penicillins, 6-aminopenicillanic acid (6-APA), is an important active pharmaceutical intermediate that can be integrated and repurposed into semisynthetic antibiotics, including amoxicillin, ampicillin, and cephalosporin antibiotics cefadroxil and cephalexin.1 The rising occurrence of antibiotic resistance has increased the need for semisynthetic antibiotics,2,3 and over 75% of the penicillin produced is currently directed toward 6-APA production.4 The enzymatic hydrolysis of penicillins using penicillin acylases offers a milder and more efficient alternative to the chemical process for the synthesis of 6-APA.1,5 Penicillin G acylase (PGA) from Escherichia coli has been extensively developed as an industrial biocatalyst for the deacylation of benzylpenicillin, yielding over 20,000 tons of 6-APA per year.6 Protein engineering of PGAs from E. coli and Alcaligenes faecalis has been explored to improve their catalytic activity and stability,7 while the immobilization of free PGA enzyme or whole cells has been performed to enable its effectiveness as an industrial biocatalyst.8

On the other hand, the hydrolysis of phenoxymethylpenicillin (Pen V) to 6-APA using Pen V acylase (PVA) is relatively less explored,9 although the system boasts of some advantages including the higher activity of PVA and higher chemical stability of Pen V and 6-APA at the lower pH favored for 6-APA extraction, compared to the pH optimum of 7.5–8 for PGA. PVA-producing strains also exhibit better tolerance for phenoxyacetic acid (POA) inhibition.10 However, the application of the PVA–Pen V system in 6-APA production has so far been limited by slightly higher costs of Pen V and low PVA activity. In this context, we have recently reported a highly active PVA enzyme from the Gram-negative Pectobacterium atrosepticum cloned and expressed in E. coli.11

Effective immobilization of recombinant E. coli whole cells or enzymes is a prerequisite for industrial biocatalytic applications, facilitating repeated use of the biocatalyst.12 Immobilization also allows the convenient separation of catalyst from the reaction medium and subsequently the continuous refining and purification of the reaction products.13 While enzymes are generally immobilized by covalent binding to a solid support, whole cells can be encapsulated in natural polymers or synthetic hydrogels including alginate, polyurethane, polyacrylamide, and polyvinyl alcohol.14,15 Lentikats technology16 for encapsulation of cells is based on controlled drying of the polyvinyl alcohol–poly(ethylene glycol) (PEG) hydrogel matrix and chemical stabilization into lens-shaped particles. Lentikats particles offer improved mechanical properties and reduced diffusional limitations14 compared to spherical alginate beads. Several bacterial or yeast whole cells have been encapsulated in Lentikats hydrogels, with applications in biocatalysis, wastewater treatment, and microbial alcohol production.17,18 Optimized processes are also available for the large scale printing of Lentikats beads, enabling rapid immobilization of the required biocatalyst for industrial applications. Cross-linked enzyme aggregates of PGA have been encapsulated in Lentikats and applied in the synthesis of deacetoxycephalosporin G.19 In the case of PVAs, immobilized enzyme preparations Novozyme 217 and Semacylase have been initially employed in the industry for 6-APA production.18,20,21

In the present study, recombinant E. coli whole cells expressing PVA from P. atrosepticum (PaPVA) have encapsulated in Lentikats hydrogels and employed in the conversion of Pen V to 6-APA. The efficiency of conversion and reusability of the immobilized cells were evaluated in a 2 L bioreactor. The biocatalytic reaction was further combined with continuous downstream processing (CDSP) for 6-APA production.

2. Results and Discussion

The PaPVA can efficiently hydrolyze Pen V to 6-APA, exhibiting a higher specific activity (430 U/mg)11 compared to other reported PVAs. Furthermore, the high levels of soluble expression of PaPVA in E. coli up to 250 mg/mL make it ideal for use as an industrial biocatalyst. In this study, recombinant E. coli-PaPVA cells were cultivated using terrific broth in a 2 L bioreactor. A cell biomass yield of 35 g/L with a PVA activity of 42,656 U/L could be achieved following fermentation for 13 h.

In contrast to the variety of immobilization supports explored for PGA,5 reports on the use of immobilized PVA are relatively scarce. Partially purified PVA from Fusarium has been immobilized on cation-exchange resin,22 while the enzyme from Streptomyces lavendulae has been covalently bound to Eupergit C epoxy-acrylic beads.19 We have earlier reported20 the permeabilization of E. coli-PaPVA cells using treatment with CTAB and encapsulation in alginate beads for use in the biotransformation of Pen V. Despite a high initial conversion rate, the mechanical stability of the beads and reusability for continuous cycles required further improvement to make commercial application feasible. To circumvent these limitations, we considered other approaches, including lyophilization and the use of Lentikats as robust synthetic polymer support to encapsulate E. coli-PaPVA cells. Whole cells were chosen for encapsulation as enzyme molecules are generally too small to be retained in Lentikats particles, and attempts at cross-linking PaPVA with glutaraldehyde or covalent binding to epoxy polymers resulted in low recovery (<10%) of enzyme activity.

Initial biotransformation trials with Lentikats-immobilized E. coli-PaPVA cells were performed in 100 mL in a lab-scale reactor to optimize cell loading, Pen V concentration, and aeration conditions. While lyophilized cells were able to facilitate the complete conversion of 2% w/v Pen V in 60 min, similar to free cells, they were limited by the loss of activity on storage or recycling (data not shown). Using Lentikats-immobilized cells (LK-PaPVA, 10% w/w cell loading), about 80% conversion of Pen V to 6-APA was recorded within 30 min. Further extension of the incubation period led to a reduction in reaction rate, but complete conversion could be achieved in 90 min (Figure 1).

Figure 1.

Figure 1

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Time course of the conversion of Pen V to 6-APA production using LK-PaPVA immobilized whole-cell biocatalyst.

The biocatalytic reaction of Pen V hydrolysis with LK-PaPVA was scaled up to 2 L in a continuous stirred tank bioreactor (CSTR) with a cycle time of 90 min. A customized setup (Figure 2) was employed to enable removal and CDSP of 6-APA and byproduct, POA. At the end of every cycle, the reaction mixture was transferred to a separate tank for settling and precipitation of 6-APA, while fresh Pen V substrate and buffer were added to the stirred tank containing LK-PaPVA beads to restart the reaction. As described above, cell biomass (35 g/L) with a PVA activity of 42,656 U/L (21 U/mL) displayed specific activity (430 U/mg) used in transformation studies. A total of 50 reaction cycles were performed for 6-APA production with each batch of LK-PaPVA biocatalyst (80 g beads containing 8 g wet cell biomass). Complete conversion of Pen V to 6-APA was achieved for the first 10 cycles, with conversion efficiency slightly decreasing to 97% for the next 10 cycles. The 6-APA production further reduced to 91.2, 81.8, and 81.7%, respectively, for consecutive batches of 10 cycles (Figure 3). An overall conversion of 90% Pen V to 6-APA could be achieved at the end of 50 cycles. Further biotransformation cycles resulted in decreased conversion to 75–80%. Nevertheless, the immobilization of E. coli-PaPVA cells on Lentikats resulted in a significant enhancement in reusability for several cycles over alginate beads, where the conversion rate was reduced to 80% after only 10 cycles (Figure 4).20

Figure 2.

Figure 2

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Reactor design for biocatalytic reaction system and CDSP of 6-APA.

Figure 3.

Figure 3

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CDSP of 6-APA using LK-PaPVA immobilized E. coli-PaPVA cells.

Figure 4.

Figure 4

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Storage stability and recyclability of LK-PaPVA immobilized whole-cell biocatalyst.

During CDSP, POA was extracted from the reaction mixture in the settling tank using n-butyl acetate as the organic solvent. The water-soluble 6-APA was concentrated in the aqueous phase and precipitated by adjusting the pH to 4.2, followed by isolation and crystallization similar to established industrial procedures. Starting from 20 g/L Pen V as a substrate in the biocatalytic reaction, 10.4 g 6-APA and 7.2 g POA were recovered via downstream processing (DSP). In terms of a gram to gram conversion, this translates to efficiency of ∼90% recovery. In addition to 6-APA, it would also be possible to extract and purify the secondary product POA, which finds importance in the synthesis of 2-phenoxyethanol and the production of certain drugs and agricultural herbicides. CDSP has been applied in combination with immobilized biocatalysts in several industries to improve the processes and reduce the ecological costs.13 The whole process was also further scaled up to a pilot-scale volume of 10 L without any change in the reaction parameters. Complete conversion of Pen V to 6-APA was achieved up to 10 reaction cycles, with an improvement of 6–8% product yield using CDSP over the batch process.23 After successful, DSP both 6-APA and POA displayed 100 and 80% purity, respectively, from the transformed product by high-performance liquid chromatography (HPLC).

It is also desirable that the immobilized biocatalyst retains sufficient activity on storage over time, which allows for immobilization in bulk using cells from the cultivation step and to use them when required. Lentikats beads containing encapsulated E. coli-PaPVA cells could be stored at 4 °C in phosphate buffer, with minimal loss of activity for up to 2 months. The biotransformation of Pen V was performed at 15 days interval for a total period of 60 days, running 50 reaction cycles for each sample. The LK-PaPVA immobilized cells retained over 90% of their initial PVA activity even after 2 months, with a 2–5% gradual decrease in activity noticeable after every 15 days interval. Nevertheless, it was possible to store the LK-PaPVA biocatalyst for up to 6 months with only a 20% reduction in PVA activity. Overall Pen V conversions of 90, 88, 85, and 82% were observed for 50 cycles of the biocatalytic reaction when using cells stored at 4 °C for 15, 30, 45, and 60 days, respectively. Up to 45 days storage, the immobilized cells were able to achieve complete conversion of Pen V to 6-APA in the initial 10–15 biotransformation cycles, while longer storage times reduced the conversion to 90%.

3. Conclusions

In conclusion, the results from the current study describe the development of a functional and robust PVA biocatalyst that can be employed as a commercially viable alternative for 6-APA production. The use of non-toxic immobilization support such as Lentikats and the integration of CDSP could help reduce the ecological footprint and the economic costs associated with the process.

4. Methods

4.1. Cultivation of Recombinant E. coli-PaPVA Cells

PaPVA was cloned and expressed in E. coli with a C-terminal 6X-His-tag as reported earlier.11 Cultivation was performed in Studier’s auto-induction medium ZYM-505224 supplemented with kanamycin (100 μg/mL). The preinoculum grown in LB at 37 °C for 4 h with 200 rpm shaking (OD600 = 1) was transferred (5% v/v) to 2 L terrific broth in a Biostat 2B reactor (Sartorius Biotech, working volume 2 L). The following cultivation for 8 h at 37 °C till the mid-log phase, protein expression, was induced by the addition of lactose to a final concentration of 0.2% and continued for 5 h at 27 °C. The pH was maintained at 7.5 by the controlled addition of 1 M HCl/NaOH, and dissolved oxygen levels were maintained (above 35) with agitation and air sparging. Cells were harvested using centrifugation at 10,000g for 10 min, washed twice using phosphate-buffered saline (PBS, pH 7.2), and directly used for immobilization.

4.2. Whole-Cell Encapsulation on Lentikats

The Lentikats PVOH–PEG hydrogel was liquefied in the water bath by heating at 95 °C and then cooled to room temperature without solidification. Wet cell biomass (35 g) was resuspended in sterile PBS at 37 °C and added to 350 g of Lentikats hydrogel with constant stirring in line with manufacturer’s instructions (Lentikats Biotechnologies, Czech Republic). The gel was cast on polystyrene plates as lens-shaped drops using the LentiPrinter device and then dried in airflow at 37 °C (until ∼70% of the mass of Lentikats liquid had evaporated). These immobilized particles were further reswelled in stabilizer solution (sodium sulfate, 0.1 M) for 30 min and then washed with PBS and stored at 4 °C until use.

4.3. Biotransformation and CDSP

A customized bioreactor was employed for CDSP of 6-APA with a separate reaction tank, control device, and settling tank. Briefly, the biocatalytic reaction was performed on a 2 L scale in a CSTR with agitation at 200 rpm for optimal and homogeneous mixing of Lentikats-immobilized E. coli-PaPVA cells (LK-PaPVA). Buffer (sodium acetate buffer pH 5.0) and substrate (Pen V, 2% w/v) were added via pumps to the reaction mixture in the tank followed by the introduction of LK-PaPVA beads (80 g beads containing 8 g cell biomass). The reaction temperature 37 °C and pH 5.0 were monitored using 1 M HCl or 1 M NaOH supplied via automatically operated pumps. Samples were withdrawn at different time intervals during the catalytic reaction to determine the amount of 6-APA. Following biotransformation, the reaction mixture was transferred to the settling tank using a pump connected to the reaction tank and further processed to recover 6-APA and POA. The biocatalytic reaction was repeated for several cycles by the addition of a fresh Pen V substrate to the reaction tank.

During DSP, the buffer was chilled to 4 °C for 30 min followed by acidification to pH 2.0 with 1 M HCl. The 6-APA in the reaction mixture was extracted thrice with butyl acetate and filtered using a 0.22 μM filter (Millipore, MF). The organic fraction was dried under Na2SO4 and concentrated on a rotary evaporator at 50–60 °C. The purity of the 6-APA and POA was quantified by HPLC.

4.4. Stability and Reusability of the Lentikats-Immobilized System

Immobilized LK-PaPVA cells were evaluated for their stability and reused under long storage conditions. Beads encapsulated with 10% cell loading were stored in sodium acetate buffer at 4 °C, and the biocatalytic reaction was performed at 15 day intervals for a total period of 60 days. Biotransformation was performed with 2% Pen V for 50 consecutive cycles as described above. At the end of each batch, the reaction mixture was drained to the settling tank, and fresh buffer and substrate were added to the immobilized cells in the reaction tank. 6-APA concentration was evaluated after each cycle.

4.5. Analytical Methods

PVA enzyme activity was assayed by monitoring the production of 6-APA through the formation of Schiff’s conjugate with p-dimethyl-aminobenzaldehyde.25 One unit of PVA activity was defined as the amount of enzyme-producing 1 μmol 6-APA in 1 min.

4.5.1. HPLC Analysis of 6-APA and POA

Qualitative determination and separations of 6-APA and POA were analyzed using liquid chromatography, Ultimate 3000 (Thermo Fisher Scientific, USA). The 6-APA (Sigma-Aldrich) and POA (Loba Chemie, Mumbai, India) working standards were prepared for 100 μg/mL. Isocratic chromatographic separation of both 6-APA and POA was carried out using YMC-OSD, C18 column (250 × 4.0 mm). The mobile phase consists of acetonitrile and water (60:40, v/v), and pH 4.0 was maintained with orthophosphoric acid. The injection of 5.0 μL of the sample was at 0.7 ml/min flow rate and scanned under a UV detector at 240 nm.26

Acknowledgments

This work was supported by Fast track translational (FTT) project funding from the Council of Scientific and Industrial Research (CSIR), New Delhi, India, and CSIR-National Chemical Laboratory, Pune, India. The authors thank Scigenics (India), Private Ltd. Chennai, India for the design of bioreactor. Authors also thankful to Sparsh Biotech, Ahmedabad, India for providing penicillin V.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c02813.

  • Scale-up of the process for 2 L bioreactor growth of E. coli-PaPVA cells, CDSP of 6-APA using LK-PaPVA immobilized whole cell biocatalyst in a customized bioreactors, statistical analysis of CDSP of 6-APA, process development and PVA immobilization for production of 6-APA, pilot-scale (10 L) bioprocess and DSP of 6-APA using LK-PaPVA enzyme system, qualitative determination of 6-APA by HPLC, and qualitative determination of POA by HPLC (PDF)

Author Present Address

§ Institute for Organic Chemistry and Biochemistry, Technical University of Darmstadt, 64287 Darmstadt, Germany.

The authors declare no competing financial interest.

Notes

This article does not contain any studies with human or animal subjects performed by any of the authors. All photos displayed in figures or Supporting Information were taken by the authors.

Supplementary Material

ao0c02813_si_001.pdf (35.9MB, pdf)

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Supplementary Materials

ao0c02813_si_001.pdf (35.9MB, pdf)

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