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 of nucleophile 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).

1. Kinetically controlled synthesis of β-lactam antibiotics
catalyzed by penicillin acylase from Achromobacter sp.
Michal Grulich1, Stanislav Bečka1, Václav Štěpánek1, Rajasekar W. Vyasarayani 2, Anupama Datla2, Trupti Ashar2, Pavel Kyslík1
1 Laboratory of enzyme technology, Institute of Microbiology, v.v.i., Academy of Sciences of the Czech Republic, Prague, Czech Republic
2 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 of
nucleophile 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 preparation
of 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.coli
BL21(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 1.0
mM CaCl2
The 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 6.5
The NPGA was significantly more efficient at ampicillin and amoxicillin syntheses when compared to PGA
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)
Media and culture conditions:
The strain E.coli BL21(pKX1P1) was grown in LB medium (shaken flask cultures) or in stirred bioreactor
in 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. The
homogenate was subjected to thermal treatment as described Datla et al.2 . The NPGA was precipitated
with ammonium sulphate, dialyzed and applied onto ion exchange Fractogel COO- column using a linear
gradient 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 sodium
phosphate buffer (pH 7.0) containing 150 mM NaCl was used as a mobile phase. The enzyme solution
was 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 constant
Kcat 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 of
purified 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 ampicillin
and cephalexin, pH 6.5 and 40% MetOH for PEN G and Daoc G.
Syntheses of antibiotics at high concentrations of substrates 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 of
the reaction vessel and washing the catalyst. Conversions were performed at 27.5°C, frequency of
stirring was 400 rpm and pH was maintained at 6.28 with ammonia water (12.5%). The initial conversion
rates 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 into
polyacrylamide gel matrix: the catalysts Fermase NA®-150 (NPGA). At the nucleophile concentration
above 100 mM, NPGA was more efficient than PGA in both the amoxicillin and the ampicillin
syntheses.
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 product
synthesis - 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.
Degreeofconversion(%),
Hydrolyticactivityofcatalyst
(U/gww)
0
1
2
3
4
5
6
7
8
9
10
Initialconversionrate(%/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
Conversion
of
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 weightFermentation
time
Medium (added
CaCl2)
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 the
maximum was shifted to 65°C and the optimum of pH stab ility falls in the range from 4.0 to 6.5. The
half-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