Strains and expression constructs
The construction of the strains E. coli RE3 (CCM 4228) and RE3(pKA18) have been described earlier [2, 32].
The leader-less pga gene was amplified in two steps by two PCR using the chromosomal DNA of the strain E. coli RE3 as a template. An upstream primer 5'-AGCGGTGAATAAAGCGATTCGTTT derived from a flanking part of 5'end of pga gene and a downstream primer 5'-TTTCGGGCCCAATTATCTCTCTGAAC were used in the first PCR reaction (the ApaI restriction site is highlighted in bold). The specific product from the first PCR reaction was used as a template in the second PCR reaction with pair of primers: upstream primer 5'-ATAATGGAGCAGTCGTCAA-GTGAG (a part of yeast consensus sequence is highlighted in bold) and the same downstream primer as in the first PCR reaction. The specific PCR product was cloned into PmlI/ApaI restriction sites of the vector pPICZ-A (Invitrogen). The constructed plasmid was designated as pPIC-PA and used to transform the host E. coli TOP10. Plasmid bearing the leader-less pga gene from ZeoR transformant was linearized by SacI and transformed by electroporation into the host P. pastoris X-33. All routine techniques for DNA manipulations were performed as described previously .
Media and flasks cultures
E. coli strains were cultured in low-salt Luria-Bertani (LB) medium at 37°C. If required, Zeocin (25 μg ml-1) was supplemented. P. pastoris X-33 was cultured in YP medium (1% yeast extract, 2% peptone) supplemented with 2% glucose (YPD medium) or 2% glycerol (YPGly medium). Solid YPDS medium (1% yeast extract, 2% peptone, 2% glucose, 1 M sorbitol, 2% agar, and 100, 200, 500 or 1000 μg of Zeocin per ml) was used for selection of the yeast transformants.
Screening for ZeoRpositive clones
A screening was carried out on solid YPDS medium supplemented with Zeocin (100 μg ml-1) after incubation for 3 days at 28°C. Subsequent selection of 150 ZeoR isolates was carried out using replica plating on solid YPD medium containing 200, 500 or 1000 μg ml-1 of Zeocin. Thirty clones from the plates with the highest concentration of Zeocin were grown in 10 ml of YPGly medium with biotin (4 × 10-5 %) for 16 h at 28°C. Each culture was centrifuged at room temperature, the cell pellet was resuspended in 20 ml of YP medium to get OD600 of 1.0 and methanol was added to a final concentration of 0.5% to induce the enzyme synthesis. Every 24 hours, an equal dose of methanol was added to maintain the culture growth for 64 hours. Cultures were sampled at given intervals for the enzyme assay.
High-cell density culture in stirred bioreactor
The fed-batch culture was carried out in a stirred bioreactor Biostat MD (B. Braun Biotech International, Melsungen, Germany) with initial working volume of 6 litres at 28°C. The batch culture medium contained per litre: 6 g KOH, 10 g MgSO4.7H2O, 1.3 g KI, 40 ml glycerol and 10 ml of 85% H3PO4. Solution of CaSO4 (1.3 g) and biotin (0.4 mg l-1 of medium) were sterilized separately. PTM1 trace salt stock solution contained per litre: 65 g FeSO4.7H2O, 6 g CuSO4.5H2O, 3 g MnSO4.H2O, 20 g ZnCl2, 0.2 g Na2MoO4.2H2O, 0.8 g KI, 0.2 g H3BO3, 0.5 g CaSO4.2H2O, 5 ml of concentrated H2SO4, and was added to the medium after sterilization (2 ml per litre of the medium). Two feeding solutions were prepared: glycerol (50% water solution) and methanol (99.5% solution). Both solutions were supplemented with trace metals and biotin in amounts stated above. The airflow was kept constant at the maximum of the system (10 litres of air per minute) and the pH of the culture was controlled at 5.0 by NH4OH (25% water solution). A concentration of dissolved oxygen (pO2) was maintained at 30% of the value for air saturation of the medium by cascade regulation of stirring frequency in the course of the initial batch phase. The glycerol as well as methanol feeding was controlled by the value of pO2 that was set up to the value of 30%.
The flask inoculum (10 ml of YPGly medium) was inoculated with a single colony from agar plate with YPD medium and cultured on orbital shaker for 20 hours at 28°C. Two ml of the culture were transferred to 100 ml of YPGly medium, the flask was cultured for 24 hours at 28°C and the whole culture was used to seed the medium in a bioreactor. A standard fed-batch process consisting of four phases was used to express the heterologous pga gene: phase 1 - a batch culture in the medium supplemented with glycerol (about 40 h); phase 2 - feeding of glycerol to reach high concentration of the biomass (OD of about 280); phase 3 - transition of the cell metabolism from glycerol to methanol (90 - 120 min), and phase 4 - induction of the enzyme expression by feeding of methanol.
To maximize the production of hPGAEc, two strategies dealing with the phase 3 were applied. Profile I: a linear increase of methanol concentration in the course of the transition phase up to 0.5 and 1.5% in experiment A and B, respectively, accompanied simultaneously with linear decrease of glycerol feeding rate to zero. Profile II: the culture was starved in the course of the transient phase both for glycerol and methanol; after 120 min, methanol was added in a single dose to reach final concentration of 1.5 and 0.5 % in experiment C and D, respectively.
Assay of hydrolytic activity
The biomass from 1-ml samples of a culture was separated by centrifugation and rinsed with distilled water. The pellet was resuspended in 1 ml of 0.1 M sodium phosphate buffer (pH 8.0), 1.25 ml of glass beads were added (diameter of 0.5 mm, Willy A. Bachofen AG. Basel, Switzerland), and the cells were mechanically disrupted by vortexing for 20 min. The debris of cells was removed by centrifugation and the activity of PGA was measured in the supernatant. The activity of PGA was assayed in 0.05 M sodium phosphate buffer (pH 8.0) supplemented with penicillin G (2% solution) using the method described by . The activity of one unit (U) is defined as the amount of PGA producing 1 μmol of 6-APA min-1 at 37°C. The specific activity is expressed in U per g of cell dry weight (cdw).
Hydrolysis of substrates (penicillin G, penicillin V, ampicillin, amoxicillin, cephalexin, NIPAB and phenylacetamide; concentration of 0.5%) was carried out in 50 mM phosphate buffer, pH 7.5 (hPGAEc) or 8 (PGAEc) at 37°C. The resulting 6-APA and 7-ADCA were monitored by spectrophotometer at 415 nm after coupling with p-dimethylaminobenzaldehyde . Determination of the activity with phenylacetamide was based on the measurement of liberated ammonia . Hydrolysis of NIPAB was measured by spectrophotometer according to . The molar extinction coefficient for 3-amino-6-nitrobenzoic acid (ε405) equalled 9.09 mM-1cm-1. The activity of one unit (U) was defined as the amount of the enzyme that hydrolyzed 1 μmol of NIPAB per min in 50 mM phosphate buffer, pH 7.5 (hPGAEc) or 8 (PGAEc) at 37°C.
Biomass (50 g wet weight) of the strain E. coli RE3(pKA18) from 2 l of fermentation broth obtained as described earlier  was disintegrated and cell-free enzyme extract was prepared by thermal treatment in the presence of polyethyleneimine (45°C, pH 5.0). PGA was precipitated with ammonium sulphate from 50 ml of the enzyme extract at concentration between 40-60% of sulphate saturation. The precipitate was collected by centrifugation (5000 g, 35 min, 4°C), dissolved in 10 mM phosphate buffer (pH 8.0), dialyzed against the same buffer and the enzyme solution was applied to a DEAE-fractogel EMD 6500(S) column. The elution was carried out with the same buffer. The fractions having the activity of PGA were pooled and dialyzed against 1 mM phosphate buffer (pH 7.0). The enzyme solution was applied to a hydroxyapatite column equilibrated with 1 mM phosphate buffer (pH 7.0) and PGA was eluted using a linear gradient 0-100% of 200 mM phosphate buffer. The pooled fractions were concentrated by utrafiltration on Amicon 8010 and washed with 150 mM NaCl in 50 mM phosphate buffer (pH 7.5). The enzyme solution was applied to a Superdex 200 column equilibrated with 50 mM phosphate buffer (pH 7.0) containing 150 mM NaCl. PGA was eluted from the column and the pooled fractions were ultrafiltrated on Amicon 8010, washed as described above and stored at -20°C.
The hPGAEc from P. pastoris X-33(pPIC-PA1) was purified from 11.8 g wet weight of the biomass from the experiment B: the biomass was resuspended in 50 ml of 0.1 M sodium phosphate buffer (pH 8.0) with 0.5 ml of Protease Inhibitor Cocktail (Sigma). 50 ml of glass beads were added to disrupt the cells on Lab-Shaker (A. Kühner AG, Switzerland) at 330 rpm for 1 h. After centrifugation (10000 g, 20 min, 4°C), CYSEP 329 (CYTEC Industries BV, Netherlands) was added to the supernatant up to the final concentration of 7.5%. Precipitated bulk protein was separated by centrifugation and PGA in the supernatant was precipitated with ammonium sulphate (up to 50% of sulphate saturation). The aggregated PGA was collected by centrifugation (5000 g, 35 min, 4°C), dissolved in 10 mM phosphate buffer (pH 8.0), dialyzed against the same buffer and the enzyme solution was applied to a DEAE-fractogel EMD 6500(S) column. The elution was carried out with the same buffer and the fractions having the activity of PGA were pooled and ultrafiltrated on Amicon 8010.
Protein concentrations were determined by means of BCA Protein assay kit (Pierce, Rockford, Ill). Bovine serum albumin was used as a standard.
MALDI-TOF Mass Spectrometry
The subunits of the purified PGA were separated by 12% SDS-PAGE electrophoresis. Coomassie Blue-stained protein bands were cut out and digested "in gel" with a sequencing grade trypsin (Promega). The generated peptides were extracted from the gel and measured by MALDI-TOF MS as described earlier . Positive ion MALDI mass spectra were measured on a Bruker BIFLEX reflectron time-of-flight mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a gridless delayed extraction ion source, and a nitrogen laser (337 nm). Instrument was calibrated externally with a PepMix calibration kit (Bruker Daltonics). Samples of peptides (1 μl) deposited and dried on target were overlaid with 1 μl of diluted matrix α-cyano-4-hydroxy-cinnamic acid (one volume of saturated solution in methanol mixed with two volumes of 50% methanol and 0.3% trifluoroacetic acid (TFA)). All the measured masses are monoisotopic M+H adduct ions.
For determination of the N-terminuses, proteins were labelled at their N-terminuses with 4-sulfophenyl-isothiocyanate reagent (10 mg ml-1 of SPITC in 20 mM NaHCO3) using modified protocol . 10 μl of the reagent were added to the dry protein. Immediately after 30 min incubation at 56°C, the loading sample buffer was added and the samples were treated according to the conventional protocol for SDS-PAGE electrophoresis. The separated proteins were digested with trypsin according to the standard protocol . Before measurement the peptides were desalted as follows: the peptides were deposited on target, dried on air, overlaid with CCA matrix (5 mg ml-1 in 0.1% TFA, 50% acetonitrile), and washed with 5 μl of 0.1% TFA for 5-10 s. The derivatized peptide was sequenced by MALDI TOF-TOF tandem mass spectrometry (Ultraflex III, Bruker Daltonics) to confirm the N-terminal sequence.
Kinetic parameters determination and kinetically controlled syntheses
Kinetic parameters of PGAs for penicillin G were determined by titration of phenylacetic acid with 0.01 M NaOH using an autotitrator (Radiometer, Copenhagen, Denmark) at 37°C. Kinetic parameters for NIPAB were determined by spectrophotometer, as described above. The relationship between the initial rate of the reaction and substrate concentration (1-2000 μM) was determined using the Michaelis-Menten equation and a non-linear regression program (Enzfitter, Elsevier Biosoft). To calculate k
of the purified hPGAEc, molecular weights of 86.4 (the enzyme with α2) and 88.4 (the enzyme with α1) kDa in a ratio 2 to 1 were used. The densities of the subunit bands in SDS-PAGE gel were evaluated using Image analysis by Intelligent Quantifier program (BioImage, Ann Arbor, Mich).
A kinetically controlled enzymatic synthesis of cephalexin, ampicillin or amoxicillin was carried out according to . The reaction was catalyzed by the purified PGAs in 0.05 M potassium phosphate buffer (pH 7.0) at 30°C. The initial concentration of activated acyl donor (D-phenylglycine amide, D-p-hydroxyphenylglycine amide) was 15 mM and the concentration of an appropriate β-lactam nucleophile (7-ADCA or 6-APA) was 25 mM. All reactants were monitored in time by HPLC analysis and the initial rates of formation of the antibiotics and the hydrolysis of acyl donors were determined. The S/H ratio was calculated from the initial rates measured in the experiment.
Effect of pH and temperature on enzyme activity
The temperature optimum for the activity was determined by measuring penicillin G hydrolysis in the range of temperatures 20-60°C. The thermal stability of PGAs was determined by incubation of the purified enzymes for 30 min in the range of temperatures 20-70°C and assaying of the remaining activity at 37°C with penicillin G at pH optima 8.0 and 7.5 for PGAEc and hPGAEc, respectively. The effect of pH on the activity of the enzyme was determined with penicillin G as the substrate at 37°C in Britton-Robinson buffer (40 mM H3PO4, 40 mM acetic acid and 40 mM H3BO3, the required pH was adjusted by 10% NaOH) in the range of pH 4-10. The pH-stability was determined by incubation of the enzyme sample at a given pH (ranging from 3-10, Britton-Robinson buffer) for 60 min at room temperature. Remaining activity was assayed at 37°C with penicillin G at pH optima 8.0 and 7.5 for PGAEc and hPGAEc, respectively.