Kinetically controlled synthesis of β-lactam antibioticscatalyzed by penicillin acylase from Achromobacter sp .

Michal Grulich 1, Stanislav Be čka1, Václav Št ěpánek 1, Rajasekar W. Vyasarayani 2, Anupama Datla 2, Trupti Ashar 2, Pavel Kyslík 1

1 Laboratory of enzyme technology, Institute of Micro biology, v.v.i., Academy of Sciences of the Czech R epublic, Prague, Czech Republic2 Fermenta Biotech Ltd., Thane, Maharashtra, India

Penicillin G acylase from Achromobacter sp. (NPGA) was overproduced by culture of the recombinant E.coli in a stirred bioreactor. Enzyme amount assayed by titration of enzyme catalytic site with inhibitor PMSF corresponded to 0.9 g of NPGA per L of fermentation broth ( 20% of the cell soluble protein). The enzyme was purified, characterized and evaluated in kinetically controlled syntheses of antibiotics ampicillin, amoxicillin, cephalexin and cefadroxil in soluble or immobilized form (Fermase NA®-150). Catalyst synthetic activity calculated from initial rate of product synthesis at 160mM concentration ofnucleophile for ampicillin, amoxicillin, cephalexin were 2.14, 4.14 and 3.86 kg per kg catalyst (dry weight) per hour, respectively. A high operational stability, a half-life of more than 2000 cycles, of the immobilized NPGA was determined for amoxicillin synthesis (230 mM 6-APA, 340 mM HPGMe, 27.5°C, pH 6.25).

References:1Vojtíšk V. and Slezák J.: Penicillinamidohydrolase in Escherichia coli: Folia Microbiol 20: 224-30 (1975)2Datla A., Rajasekar V.W., Kyslik P., Becka S., Krishnakant A.T., Yogesh Z.S., Nikunj K. Process for the preparationof penicillin or cephalosporin antibiotics: Patent 2173892 B1 (2011), Sep.21

To evaluate production of NPGA by the recombinant E.coli, fed-batch cultures of the strain E.coliBL21(pKX1P1) were performed in a stirred bioreactor. The highest production of the enzyme was achieved in the fed-batch cultures containing 0.33 and 1.0 mM CaCl2: specific activity of NPGA reached 1400 U/g cdw (volumetric activity of 33000 U/l) after 22 h of cultivation.

Conclusions:The highest production of the enzyme was achieved in the fed-batch cultures containing 0.33 and 1.0mM CaCl2The NPGA is highly thermostable: maximum activity at 60°C (pH 8.0) or 65°C (at pH 6.0). The optimum of pH stability falls in the range of pH from 4.0 to 6.5The NPGA was significantly more efficient at ampicillin and amoxicillin syntheses when compared to PGAA high operational stability, a half life of more than 2000 cycles, of the immobilized NPGA was determined for amoxicillin synthesis (230 mM 6-APA, 340 mM HPGMe, 27.5°C, pH 6.25)

Media and culture conditions:

The strain E.coli BL21(pKX1P1) was grown in LB medium (shaken flask cultures) or in stirred bioreactorin mineral medium as decribed Vojtíšek et al.1, that was supplemented with casein hydrolyzate (10 g/l) and glycerol (10 g/l) (MCHGly medium) as a carbon source. Inoculum was grown in orbital shaker (200 rpm) for 24 h at 28 °C in medium MCHGlyK that was in oculated with a vial of a glycerol stock culture. The inoculum was diluted 20 times into fresh medium. For fed-batch cultures, E. coli BL21(pKX1P1) was grown in a 10-L stirred bioreactor Biostat MD (B. Braun Biotech International) with an initial working volume of 8.2 L of medium MCHGly. The pH of the medium in the fed-batch phase of the culture was maintained at the value of 6.7 with 25% solution of NH4OH.

Isolation and purification of NPGA:

Frozen biomass suspended in 34 ml of 0.1 M NaCl was desintegrated by Ultrasonic Cell Disruptor. Thehomogenate was subjected to thermal treatment as described Datla et al.2 . The NPGA was precipitatedwith ammonium sulphate, dialyzed and applied onto ion exchange Fractogel COO- column using a lineargradient form 0 to 0.1 M KCl in 0.01 M phosphate buffer (pH 7.0) for 2 hours. After concentrating by ultrafiltration the enzyme solution was applied onto a column with Superdex 200 and 0.01 M sodiumphosphate buffer (pH 7.0) containing 150 mM NaCl was used as a mobile phase. The enzyme solutionwas kept frozen in 0.5 ml aliquotes for further characterization.

Kinetic parameters for substrate hydrolysis:

The parameters of purified enzymes were determined in 0.005 M potassium phosphate buffer (pH 7.5 and 6.5, 25°C). Concentration of the hydrolytic products was monitored by HPLC. The kinetic parameters(Km and Vmax) were calculated using the Hanes-Wolf plot and ANOVA calculator. The catalytic constantKcat for Pen G was calculated from Vmax and molar concentration of enzyme in solution on basis of protein content and NPGA molecular weight of 89 kDa.

Kinetic parameters for ß-lactam antibiotic syntheses

Reaction mixrure containing activated acyldonor and variable concentrations of ß-lactam nucleophile in 0.005 M phosphate buffer (pH 7.0) was temperated to 25°C and the reaction was started by addition ofpurified soluble enzyme. The synthetic reaction was monitored by HPLC using C18 column. Mobile phase consisted of 0.01 M sodium phosphate buffer and methanol and differed for the substrates as follows: pH 3.0 and 10% MetOH for amoxicillin and cephadroxil; pH 5.6 and 30% MetOH for ampicillinand cephalexin, pH 6.5 and 40% MetOH for PEN G and Daoc G.

Syntheses of antibiotics at high concentrations of substra tes using immobilized NPGA:

The reaction was performed in temperated two-coated glass vessel at 25°C, using vertical stirrer and pH-stat filled with 12.5% NH4OH. The nucleophile was dissolved in water by adjusting pH 7.0, then acyl donor was added and required pH 6.3 or lower was adjusted with ammonia. The reaction was started by catalyst addition. The course of reaction was monitored by HPLC assay.

Determination of catalyst operational half-time:

Repetaed cycles of Amoxicillin synthesis were performed in automatic machine using filling/emptying ofthe reaction vessel and washing the catalyst. Conversions were performed at 27.5°C, frequency ofstirring was 400 rpm and pH was maintained at 6.28 with ammonia water (12.5%). The initial conversionrates were calculated in time period of 0-60 min. based on conversion of 6-APA to amoxicillin per min. The final degree of conversion was determined after 130 min.

Tab.1. Fermentation of new penicillin G acylase (NPGA, Achromobacter sp.) from recombinant strain E.coli BL21

(pKX1P1) at different Ca2+ concentrations

a Activity of the enzyme was measured with 2% pncG as the substrate at 37°C. pH 8.0

Methods:

PGA: D-phenylglycine amide; HPGA: D-p-hydroxyphenylglycine amide

17.629.70.842526.471.10.5539Cefadroxil

25.012.72.823612.723.60.8019Cephalexin

9.89.01.541418.3124.30.2227Amoxicillin

15.06.03.572115.049.10.4522Ampicillin

14.61.12192123.01.432434HPGA

20.30.93322917.61.361926PGA

(µmol min-1 mg-1)(mM-1s-1)(mM)(s-1)(µmol min-1 mg-1)(mM-1s-1)(mM)(s-1)

Vmaxkcat/KmKmkcatVmaxkcat/KmKmkcat

Escherichia coli PGAAchromobacter sp. NPGASubstrate

We compared the synthetic performances of both the NPGA and PGA enzymes at high substrate concentrations (140-160 mM nucleophile and 340-350 mM acyl donor methyl ester) (tab. 3). The experiments were done with the enzymes immobilized by entrapment intopolyacrylamide gel matrix: the catalysts Fermase NA®-150 (NPGA). At the nucleophile concentrationabove 100 mM, NPGA was more efficient than PGA in both the amoxicillin and the ampicillinsyntheses.

Tab. 3. Syntheses of Amox, Amp, Cpx by catalyst Fermase NA 150® at high substrate concentrations in water

Temperature of 25°C and 6g ww of the catalyst per 100 ml o f reaction mixture (RM) was used into the reactions (*10g ww of the catalyst per 100 ml of RM and **9 g ww of the catalyst per 100 ml of RM), Amox=amoxicillin, Amp=ampicillin, Cpx=cephalexin, AD=acyldonors: HPGMe(4-hydroxyphenylglycine methyl ester) and PGMe (phenylglycine methyl ester), N= β-lactam nucleophiles: 6-APA (6-aminopenicillanic acid) and 7-ADCA (7-aminodeacetoxycephalosporanic acid), AD/N= molar ratio of acyldonor to nucleophile, (VPs)init. = initial rate of productsynthesis - synthetic activity, (VPh)init. = initial rate of hydrolysis of activated acyldonor – initial rate of production of free aminoacids, (VPs/VPh)init. = S/H ratio

Immobilized NPGA (Fermase NA® -150) was used for synthesis of amoxicillin from 6-APA (230 mM) and D-4-hydroxyphenylglycine methyl ester (340 mM). The conversion degree varied between 90-92% (Fig.2).

0

20

40

60

80

100

120

0 50 100 150 200 250 300

Conversion cycle No.

Deg

ree

of c

onve

rsio

n (%

), H

ydro

lytic

act

ivity

of c

atal

yst

(U/g

ww

)

0

1

2

3

4

5

6

7

8

9

10

Initi

al c

onve

rsio

n ra

te (%

/min

)

Conversion degree (%) Hydrolytic activities (U/g ww) Initial rate of conversion (%/min)

Results:

6.330088.511.2418.54.34208.01.33400300Cpx

6.321091.16.4721.52.90139.01.50360240Cpx**

6.321092.07.0126.43.86185.02.25360160Cpx

5.927094.010.5525.86.59272.21.35540400Amp*

5.930092.06.1756.98.50351.01.50600400Amp

6.320091.12.8560.04.14171.02.19350160Amp

6.324091.34.2517.81.9075.61.50360240Amox*

6.320096.93.1527.02.1485.02.19350160Amox

6.314096.82.9856.04.20167.02.46320130Amox

(min)(%)(x)(µmol ph/min/g

dw)(kgPs/kgcat/h

dw)(µmol ps/min/g

dw)(x)(mM)(mM)(mM)(mM)

pHReact.time

Conversionof

nucleophile

(VPs/VP

h)init.

(VPh)init.(VPs)init.AD/NPGMeHPGMe7ADCA6APAAntibiotic

10001545415.520.03.000

199014003251622.022.01.000

244414003327921.720.00.332

570934816.416.00.000

U/g dw(U/g dw)U/L(g dw/L)(h)(mM)

SA of homogenateSpecific act. of cells a

VACell dry weightFermentationtime

Medium (addedCaCl2)

Fig. 1.

After 308 conversion cycles (Fig.2) the activity of the catalyst activity dropped by 6.6% which corresponds to operational half-life of about 2000 conversion cycles.

NPGA-catalyzed hydrolysis of substrates was studied with the soluble,purified enzyme in phosphate buffer at pH of 7.5 and temperature of 25°C. The list of substrates subjected to hydrolysis is shown in the Table 2.

Fig. 2. Repeated conversions of substrates to Amoxicillin with Fermase® NA 150

At pH of 8.0, the activity maximum of obtained NPGA was achieved at 60°C while at pH of 6.0 themaximum was shifted to 65°C and the optimum of pH stab ility falls in the range from 4.0 to 6.5. Thehalf-life of NPGA activity at pH of 6.0 and 8.0 was 6.9 hours and 24 min, resp.

Tab. 2. Kinetic parameters of NPGA and PGA for acyldonors and antibiotic derivatives, determined in 0.05 M

potassium phosphate, pH7.0 at 30°C