Studies of operational variables in batch mode for genetically engineered Escherichia coli cells containing penicillin acylase

S. Bhattacharya,* V. S. Gupta, A. A. Prabhune, H. SivaRaman, M. Debnath* and P. K. Ranjekar

Biochemical Sciences Division, National Chemical Laboratory, Pune, India and *School o f Biochemical Engineering, Institute o f Technology, BHU, Varanasi, India

A recombinant Escherichia coli was constructed by cloning the penicillin acylase gene from E. coli ATCC 11105. The cloning was carried out using a recombinant plasmid pUSAD2 harboring the pac gene. The recombinant E. coli DH 5 cells were used as a biocatalyst and were studied in a batch reactor for determination of optimum value for some of the process parameters, such as effect of pH, temperature, substrate concentration, kLa and effect of carbon and nitrogen source on penicillin acylase production. These values were then compared with the values obtained with the standard parent strain. Whereas the cloned pac gene was found to produce higher levels of penicillin acylase constitutively, the process parameters remained about the same for both the parent and the recombi- nant.

Keywords: Escherichia coli; penicillin acylase; constitutive, fl-lactamase negative; plasmid pUSAD2; pac gene; DH5 cells; optimization of enzyme production parameters; 6-aminopenicillanic acid (6-APA); lactose broth (LB)

Introduction

Benzylpenicillin acylase (EC 3.5.1.11) catalyzes the hydrolysis of benzylpenicillin to 6-aminopenicillanic acid (6-APA) and phenylacetic acid. The interest in 6- APA production is due to its importance as a starting material for production of semisynthetic penicillins.

Standardization of conditions for hyperproduction of the enzyme has usually been carried out by nutrient- controlled metabolism of the cells and by genotypic changes produced in the cells.~-3 During the last de- cade, gene cloning has been considered to be the method of choice in producing clones with superior properties.

Cloning of the penicillin acylase gene (pac gene) in Escherichia coli has been reported by several workers. A recombinant E. coli HB101 (pPAKS2) is reported to produce penicillin acylase. 4'5 An engineered E. coli strain QE79 bearing a recombinant plasmid containing

Address reprint requests to Dr. Ranjekar at the Biochemical Sciences Division, National Chemical Laboratory, Pune 411 008, India Received 3 September 1992; revised 29 April 1993

the pac gene from E. coli ASI 76 was constructed. 6 The pac gene has also been cloned from E. coli 194, Bacillus megaterium UNI and E. coli ATCC 11105. Using cosmid vector pJC720, cloning of the pac gene in E. coli 5K from E. coli ATCC 11105 led to the isola- tion of a positive clone. After subcloning, the high- yielding constitutive producer strain E. coli 5K/priM12 was obtained. 2'7'8 A systematic analysis of the influence of operation variables on production of penicillin acy- lase recombinant catalyst is still lacking. In the present study, we have constructed a hyperproducer which is/3-1actamase negative and which produces penicillin acylase constitutively from E. coli ATCC 11105.

Materials and methods E. coli ATCC 11105 and Serratia marcescens ATCC 27117 were obtained from the National Collection of Industrial Mi- croorganisms, Pune, and maintained routinely on nutrient agar slants. Plasmid pUSAD1 was derived from pUC18 by digesting it with Dral and religating the 1.975-kb fragment. Plasmid pUSAD2 contained the pac gene derived from E. coli ATCC 11105 in the MCS site of pUSAD1. Restriction enzymes Dral, HindIII, and T4 DNA ligase were purchased from New England BioLabs. All other chemicals used were of the highest purity available locally.

1070 Enzyme Microb. Technol., 1993, vol. 15, December © 1993 Butterworth-Heinemann

Media and culture conditions

Bioassay method. Luria Bertani agar media were used for detecting strains containing the pac gene using a bioassay method of Oostendrop 9 involving Serratia marcescens. A zone of clearance was obtained when penicillin acylase con- verted benzylpenicillin to 6-APA; the zone was due to sensi- tivity of S. marcescens to 6-APA. A zone of clearance was obtained when E. coli ATCC 11105 and recombinant cells were streaked on test plates containing benzylpenicillin but was absent for E. coli DH 5.

Optimization of production parameters. The optimization of the various parameters was performed using LB medium. Synthetic media were used for optimization of carbon and nitrogen sources. The carbon sources used were glucose, xylose, lactose, maltose, fructose and sucrose. The inorganic nitrogen sources used were ammonium dihydrogen ortho- phosphate, ammonium sulfate, sodium nitrate, and urea. The organic sources of nitrogen used were yeast extract, peptone, and glycine.

E. coli containing penicillin acylase: S. Bhattacharya et al.

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Construction o f recombinant plasmid bearing pac gene

All the procedures involving DNA digestion, ligation, and transformation of DNA were carried out as described by Maniatis et al. l° Plasmid pUC18 DNA was digested with Dral in order to remove the/~-lactamase gene. Out of three fragments obtained after Dral digestion, the 1.975-kbp frag- ment was self-ligated and was named pUSAD1.

The genomic DNA of E. coli ATCC 11105 was isolated using the procedure of Marmur H and was digested with HindIII. This digest was ligated with pUSADI/HindIII digest, and the ligated DNA was used to transform E. coli DH5. Transformants were tested for penicillin acylase activity by the bioassay method.

Detection o f the cloned strains containing the pac gene and assay o f penicillin acylase activity

This was primarily done with the bioassay method of zone clearance with S. marcescens. The Southern hybridization experiment using total genomic DNA of E. coli ATCC 11105 as radiolabeled probe (TGD probe) for the presence of the pac gene in pUSAD 2 and using labeled pUSAD2 as a probe (pac probe) for the presence of the gene in E. coli ATCC 11105 and its absence in E. coli DH5 was also carried out to confirm the findings of the plate assay.J° The penicillin acylase activity ofpac was measured by the method of Balasingham et al. 12 and the color of the complex obtained with p-dimethylamino benzaldehyde was measured by the method of Evans.~3

Optimization o f parameters for penicillin acylase production

A New Brunswick jar fermenter (500 ml) and a Labline shaker incubator were used. A Bioengineering AG 1-1 fermentor, compact model, was used for estimation of KLa by the dy- namic method. 14 The cells were grown in LB medium at 28°C and at an agitation speed of 175 rev/min at an air flow rate of 0.6 vvm; the pH of the growth medium was 7.4.

The effect of temperature, pH, nitrogen, and carbon source was tested for optimum production of penicillin acy- lase.~5 Cell concentration was measured spectrophotometri- cally at 600 nm and calculated on a dry weight basis.

Results

Cloning o f the pac gene and determination o f penicillin acylase activity

The presence of the pac gene in the recombinant E. coli DH5 (pUSAD2) cells was confirmed by the bioassay method as well as by Southern hybridization. When the activity of the penicillin acylase in the recombinant E. coli was estimated, it was found to be 2-3 times higher than in the parent strain (Figure 1). The enzyme was produced constitutively and the hybrid was fl-lac- tamase negative. This activity could not be increased further, even by chloramphenicol treatment.

Optimization o f parameters for penicillin acylase production

Penicillin acylase production is found to be affected by the pH of the medium (Figure 2). A pH range of 6.5-9.5 at intervals of 0.5 pH units was used to evaluate optimum pH for production. It was observed that peni- cillin acylase production of the recombinant is optimum at pH 8.0-9.0 (Figure 2).

A temperature range between 20 and 40°C with inter- vals of 5°C was used. The optimum temperature for production of penicillin acylase by the recombinant was found to be between 25 and 30°C (Figure 3).

The effect of sugars on penicillin acylase production was tested, and glucose was found to be the best carbon source, followed by sucrose. The optimum glucose concentrat ion for penicillin acylase production was 7.5 g 1-1 (Figure 4). Penicillin acylase production varies drastically with the type of nitrogen source used. Urea was the best inorganic source, the optimum concentra- tion of urea being 1.0 g 1 -~ (Figure 5). The reason for obtaining increased production of penicillin acylase in the presence of urea is increased cell mass production. Peptone produced the best results among the organic sources; 1.0 g 1-1 was the optimum concentration of

Enzyme Microb. Technol., 1993, vol. 15, December 1071

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peptone; however, higher concentrations of peptone did not make any further difference (Figure 6). The KLa value in a 1-1 BioEngineering AG fermentor in LB broth was 56.4 h -~ for the untransformed E. coli and 58.2 h -~ for the recombinant.

Discussion A genetically engineered strain for the production of penicillin acylase should (1) be/3-1actamase negative, (2) produce penicillin acylase in far greater quantities than the parent, and (3) produce penicillin acylase en- zyme constitutively. By cloning the pac gene into E. coli we have obtained a fl-lactamase negative constitu- tive construct which has 2-3 times the activity ex-

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1072 Enzyme Microb. Technol., 1993, vol. 15, December

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pressed by the standard E. coli ATCC 11105. It is recognized that the standard recombinant DNA tech- nology by pac gene cloning in E. coli does not produce the expected high levels of activity. The reason for this is the posttranslational maturing of the gene product, a large enzymatically inactive precursor which is se- creted into the periplasmic space where the active en- zyme forms and accumulates. The limitation is attrib- uted to the saturation of the secretion mechanism across the cytoplasmic membrane, the number of se- creting channels in E. coli being estimated to be about 20,000.16

Although the pac gene has been cloned and ampli- fied, the performance of E. coli cells harboring the cloned p a c gene has not been tested so far in batch fermentors. In the present work, our main interest was to assess the performance in a batch reactor of E. coli cells harboring the cloned p a c gene in a plasmid. This would be useful for scaleup studies with recombinants. The batch experiments would also be useful to find out approximate continuous mode variables and parame- ters such as dilution rate, which have direct relevance for the industrial use of microorganisms as biocata- lysts. Parameters such as KLa estimated in batch mode most often act as a basis for scaleup calculations. Our studies have clearly indicated that the optimum value of process parameters such as pH, temperature, KLa , carbon source, and nitrogen source for the engineered strain are very similar to the standard parent strain. Further, the recombinant was found to be stable over many trials. This indicates that there is a potential for using recombinants in scaleup operations, provided the production of the enzyme per volume of medium in the reactor is increased. Our efforts in this direction are in progress.

References

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4 Ghosh, T. K. and Tyagi, R. D. Rapid ethanol fermentation of cellulose hydrolysate. I. Batch versus continuous systems. Biotech. Bioeng. 1979, 21, 1387-1390

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12 Balasingham, K., Warkentan, E., Dunnill, P. and Lilly, M. D. The isolation and kinetics of penicillin amidase from Esche- richia coli. Biochim. Biophys. Acta 1972, 276, 250-256

13 Bomstein, J. and Evans, W. G. Automated colorimetric deter- mination of 6-aminopenicillanic acid in fermentation media. Anal. Chem. 1965, 37, 576-578

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