Appl Microbiol Biotechnol DOI 10.1007/s00253-013-5476-7

BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS

Improved activity and pH stability of E. coli ATCC 11105 penicillin acylase by error-prone PCR Huseyin Balci & Merve Tuzlakoglu Ozturk & Tjaard Pijning & Saliha Issever Ozturk & Fusun Gumusel

Received: 30 September 2013 / Revised: 13 December 2013 / Accepted: 15 December 2013 # Springer-Verlag Berlin Heidelberg 2014

Abstract Penicillin G acylase is the key enzyme used in the industrial production of β-lactam antibiotics. This enzyme hydrolyzes penicillin G and related β-lactam antibiotics releasing 6-aminopenicillanic acid, which is an intermediate in the production of semisynthetic penicillins. To improve the enzymatic activity of Escherichia coli penicillin acylase, sequential rounds of error-prone polymerase chain reaction were applied to the E. coli pac gene. After the second round of evolution, the best mutant M2234 with enhanced activity was selected and analyzed. DNA sequence analyses of M2234 revealed that one amino acid residue (K297I), located far from the center of the catalytic pocket, was changed. This mutant (M2234) has a specific activity 4.0 times higher than the parent enzyme and also displayed higher stability at pH 10. Keywords Directed evolution . Error-prone PCR . E. coli penicillin acylase . 3D homology model . Catalytic activity

Introduction Penicillin G acylase (PA; penicillin amidase or penicillin amidohydrolase, EC 3.5.1.11) is a member of a large enzyme group known as β-lactam acylases which are industrially used Electronic supplementary material The online version of this article (doi:10.1007/s00253-013-5476-7) contains supplementary material, which is available to authorized users. H. Balci (*) : M. T. Ozturk : S. I. Ozturk : F. Gumusel Department of Molecular Biology and Genetics, Gebze Institute of Technology (GIT), 41400 Kocaeli, Turkey e-mail: [email protected] T. Pijning Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands

for the synthesis of semisynthetic β-lactam antibiotics (Valle et al. 1991). Penicillin acylases hydrolyze penicillin G to 6aminopenicillanic acid (6-APA) and phenylacetic acid (Calleri et al. 2004). Although their major industrial application is the production of 6-APA, which is the key intermediate for the synthesis of semisynthetic penicillins, via the hydrolysis reaction of the acyl side chain of penicillin G and related β-lactam antibiotics, and production of valuable antibiotics, in which the enzyme catalyzes the condensation of the appropriate Damino acid derivative with a β-lactam nucleus, they can be used in several other biotechnological applications, such as peptide synthesis and racemic resolution (Arroyo et al. 2003; Giordano et al. 2006). The in vivo role of PA has not yet been elucidated, although it has been suggested that PA could be involved in the metabolism of aromatic compounds to generate a carbon source. Penicillin acylases are classified into three groups based on substrate specificity: PAs, penicillin V acylases, and ampicillin acylases (Parmar et al. 2000; Arroyo et al. 2003; Calleri et al. 2004). Mature Escherichia coli PA is a heterodimer with a 23-kDa α subunit and a 65-kDa β subunit; the two monomer chains consist of 209 and 557 amino acid residues, respectively (Bruns et al. 1985). Crystallographic studies have indicated that the two chains of the enzyme are closely intertwined and form a pyramidal structure that contains a deep cone-shaped depression at the bottom of which is the active site. PA is a member of the N-terminal nucleophilic hydrolase superfamily, a class of enzymes which share a common fold around the active site and contain a catalytic serine, cysteine, or threonine at the N-terminal position (Brannigan et al. 1995). The mature E. coli PA (86 kDa) is located in the periplasmic space and is processed from a single precursor, which has a 26-amino-acid signal transport sequence (residues 1–26), a 209-amino-acid α subunit (residues 27–235), a 54-amino-acid spacer peptide (residues 236–289), and a 557-amino-acid β subunit (residues 290–846) (Schumacher et al. 1986; Sizmann

Appl Microbiol Biotechnol

et al. 1990; Duggleby et al. 1995; McVey et al. 2001). Cleavage of the spacer peptide is autocatalytic (Kasche et al. 1999) or endoproteolytic (Bruns et al. 1985; Schumacher et al. 1986) and exposes the active site of the mature enzyme. Naturally occurring enzymes often lack features necessary for commercial applications and, therefore, efforts are continuously being undertaken towards enhancing enzyme stability and functionality. Protein engineering techniques have been used to improve enzyme properties such as activity, substrate specificity, thermostability, and activity in organic solvents. Directed enzyme evolution has emerged as a powerful alternative to rational approaches for engineering biocatalysts (Chen 2001; Jaeger et al. 2001; Wang et al. 2006). Enzymes with the desired property or set of properties are identified by selection or screening, and mutants may serve as starting points for additional rounds of mutagenesis to accumulate beneficial mutations. One of the most popular directed evolution methods to improve enzyme properties is error-prone polymerase chain reaction (PCR), which is based on the increasing frequency of mismatched incorporation of nucleotides into newly synthesized PCR products (Leung et al. 1989; Cadwell and Joyce 1992, 1994; Lin-Goerke et al. 1997; Xu et al. 1999). Very few studies aimed at improving penicillin acylase properties with the directed evolution approach have been reported. In these reported studies, DNA family shuffling method was used to evolve mutants with increased β-lactam antibiotic synthetic activity (Zheng et al. 2003; Jager et al. 2007). To our knowledge, there was no report in which the error-prone PCR method was used to improve the properties of penicillin acylase. In the present study, we applied the error-prone PCR method, coupled with a high-throughput screening assay, to improve the activity of penicillin acylase from E. coli ATCC 11105. The mutant PA (M2234) with the highest activity was selected and sequenced. Then, the purified enzyme was characterized biochemically and the results were compared with that of the parent enzyme.

Materials and methods Chemicals Penicillin G and 6-APA were a kind gift from Mustafa Nevzat Pharmaceuticals (Istanbul, Turkey). All other chemicals, reagents, and media supplements were purchased from SigmaAldrich (St. Louis, MO, USA), Merck (Darmstadt, Germany), and Becton Dickinson and Company (Franklin Lakes, NJ, USA). Restriction endonucleases, T4 DNA ligase, Taq DNA polymerase, deoxynucleotide triphosphate (dNTP) mix, ampicillin, lysozyme, and isopropyl β-D-1-thiogalactopyranoside (IPTG) were obtained from Roche (Mannheim, Germany). Pfu DNA polymerase was obtained from Stratagene (La Jolla,

CA, USA) and used as recommended by the manufacturer. Oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, IA, USA). E. coli ATCC 11105 chromosomal DNA was isolated by using a Wizard DNA Purification Kit (Promega, Madison, WI, USA). DNA fragments were isolated from agarose gel with a Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA). Protein markers were purchased from Thermo Scientific Pierce (Rockford, IL, USA) and used as recommended by the manufacturer. Strains, plasmids, and media E. coli JM109 [(recA1, endA1, gyrA96, thi, hsdR17, supE44, relA1, Δ(lac-pro AB)/F′ (traD36, proAB+, lacIq, lacZΔM 15)] was used as a host strain for DNA manipulation and gene expression. Plasmid pUC19 (Yanisch-Perron et al. 1985) was used as vector for cloning, expression, and DNA sequencing. E. coli ATCC 11105 chromosomal DNA was used as the source of the penicillin acylase gene. E. coli JM109 and E. coli ATCC 11105 were grown at 37 °C in Luria–Bertani (LB) medium [1 % (w/v) nutrient broth, 0.5 % (w/v) yeast extract, and 0.8 % (w/v) NaCl]. For expression studies, E. coli transformants were grown at 26 °C in casein medium [0.4 % (w/v) casein, 0.8 % (w/v) yeast extract, 0.42 % (w/v) K2HPO4, and 0.3 % (w/v) KH2PO4] supplemented with ampicillin (100 μg/ml). If necessary, IPTG (0.1 mM) was added to the medium for inducing synthesis of PA. Cloning of the pac gene from E. coli ATCC 11105 All DNA manipulations were conducted based on the standard methods as described by Sambrook et al. (1989) and Ausubel et al. (2001). As previously described by Gümüşel et al. (2001), the ~3.5-kb DNA fragment from E. coli ATCC 11105 encoding the pac gene was amplified by PCR using Taq DNA polymerase. PCR amplification was performed using the forward primer 5′-TAC GTA AGC TTC GTT GCT AGT ATC AAT TCG-3′ and the reverse primer 5′-TAC GTG AAT TCC GGC GAA GTC TCC GTT G-3′. PCR assay was performed in a reaction mixture containing 1× PCR buffer, 80 ng each primer, 200 ng DNA, 0.2 mM dNTP, 2 mM MgCl2, and 0.9 U Taq DNA polymerase in a final volume of 25 μl. The temperature profile was initiated with a hot start at 95 °C for 5 min, followed by 30 cycles of 94 °C for 1 min, 64 °C for 1 min, and 72 °C for 3.5 min. The reaction was terminated after an additional 10 min at 72 °C. The amplified fragment was digested with EcoRI/HindIII and ligated with the pUC19 vector, which has been digested with the same enzymes also. This construct was used to transform the E. coli JM109 cells and then they were grown on LB agar plates containing ampicillin. Plasmids were extracted from

Appl Microbiol Biotechnol

transformants, digested with EcoRI/HindIII, and then the desired fragments were determined on 1 % agarose gel. Error-prone PCR and library construction of pac gene To generate a library of pac variants, mutations were introduced using error-prone PCR. PCR conditions for the generation of mutagenic pac gene were performed according to Cadwell and Joyce (1994), with some modifications. In addition to a control PCR, a range of mutagenic conditions were chosen for the first round of mutation. The control reaction contained 1.5 mM MgCl2 and 0.2 mM of each nucleotide. The 100-μl mutagenic reaction mixture contained 1× PCR buffer, 0.2 mM dATP, 0.2 mM dGTP, 1 mM dCTP, 1 mM dTTP, 7 mM MgCl2, 20 fmol template, 30 pmol of each primer, 0.05–0.5 mM MnCl2, and 5 U Taq DNA polymerase. One mutant with slightly improved activity was identified after initial screening and testing and then subjected to a second round of mutagenesis, using only 0.25 mM MnCl2 and 2 fmol template DNA. The pUC19 plasmid containing the ~3.5-kb wild-type pac gene was used as the template for the first round, while one mutant plasmid was used as the template for the sequential round of random mutagenesis. Primers FG13-For (5′-GTT TTC CCA GTC ACG ACG TTG TAA AAC GAC GGC C-3′) and FG15-Rev (5′-CAC ACA GGA AAC AGC TAT GAC CAT GAT TAC GCC AAG C-3′), both of which anneal to the pUC19 vector, were used for the PCRs. After preheating at 94 °C for 3 min, the cycling conditions were conducted as 30 cycles of the following program: 94 °C for 30 s, 64 °C for 30 s, 72 °C for 3.5 min, and the final extension step at 72 °C for 10 min. After gel extraction of PCR fragments, the products were used as a template for cloning PCR as described by An et al. (2010) and Tuzlakoglu-Ozturk et al. (2013). The 50-μl cloning reaction mixture composed of 250 ng vector pUC19 (linearized with EcoRI/HindIII), template DNA (1:5 molar ratio), 0.2 mM dNTPs, 1× PCR buffer, and 2.5 U Taq/Pfu polymerase (1:1). The PCR cycling conditions were used as described previously, except the primer annealing and extension steps. At which steps, annealing was performed at 55 °C for 30 s and synthesis was performed at 72 °C for 7 min. Then, the PCR product was used to transform E. coli JM109 competent cells. Screening for variants with improved PA activity A three-step screening protocol was used for the selection of the active transformants (Fig. 1). In the first step of screening, transformant colonies were selected with the 6-nitro3-(phenylacetamido)benzoic acid (NIPAB) filter paper assay (Zhang et al. 1986). Sterile filter paper disks (Whatman no. 1, 8.5 cm diameter) were saturated with 2 mg/ml NIPAB

solution in 20 mM potassium phosphate buffer, pH 7.7, and dried before used. Single colonies of transformed E. coli were grown for 24 h at 26 °C onto casein agar plates supplemented with ampicillin (100 μg/ml) and 0.1 mM IPTG. Replica plates were also prepared. The assay was conducted by gently placing the filter paper onto the agar surface of the master plate. The filter paper saturated with fluid was removed from agar and incubated at 37 °C for 15 min in a sterile Petri dish. The colonies on the filter paper were then scored (as judged by eye); yellow color from the hydrolyzed product p-nitrophenol indicated the presence of penicillin acylase, while the natural color showed its absence. Then, the PA-positive colonies were selected for the second screening step. In the second step, PA-positive colonies were transferred from the replica plates to a well of a 96-well microtiter plate containing 200 μl casein medium supplemented with ampicillin (100 μg/ml) and 0.1 mM IPTG. After 24 h of incubation at 26 °C under horizontal shaking (150 rpm) on Certomat® BS-T incubator–shaker (Sartorius, Göttingen, Germany), PA activity was assayed quantitatively using NIPAB as substrate. Ten microliters of the cell culture was transferred to a new plate and 100 μl NIPAB dissolved in 50 mM potassium phosphate buffer, pH 8.0 (250 μM final concentration), was added. After incubation at 40 °C for 30 min, the resulting reactions were monitored at 405 nm using a Fluostar Omega Microplate Reader (BMG Labtech, Ortenberg, Germany). The activity of each well was normalized based on the corresponding culture density at 600 nm and compared with the parent strain; variants exhibiting a higher normalized activity were selected. For the third selection, positive clones that showed higher activity in the second screening step were cultivated in casein medium supplemented with ampicillin (100 μg/ml) at 26 °C for 24 h. The cells were induced with 0.1 mM IPTG when OD600 reached 0.6–0.8. PA activity was determined using penicillin G as a substrate based on the method described by Balasingham et al. (1972). The colonies with higher PA activity were selected and DNA sequences of their pac gene were determined. Production and purification of PA Wild-type and mutant strains were grown to 0.6–0.8 OD600 in casein medium at 26 °C and then induced with IPTG to a final concentration of 0.1 mM. The culture was allowed to incubate for 24 h at 100 rpm. The cells were collected by centrifugation at 8,000×g for 20 min at 4 °C. They were resuspended in 50 mM potassium phosphate buffer, pH 8.0. The cell suspension was sonicated using a Sonifier® S-250D digital sonifier (Branson, Danbury, CT, USA) and then centrifuged at 10,000×g for 30 min at 4 °C, and the supernatant was treated with 0.7 % (w/v) streptomycin sulfate for further clarification. After stirring in cold for 30 min, the lysate was centrifuged for

Appl Microbiol Biotechnol

Fig. 1 Schematic presentation for the screening and selection procedure of potential PA clones with enhanced activity. IPTG isopropyl β-D-1thiogalactopyranoside (0.1 mM); ampicillin (100 μg/ml); NIPAB, 6-nitro-3-(phenylacetamido)benzoic acid

Appl Microbiol Biotechnol

30 min at 8,000×g. The supernatant was mixed slowly with ammonium sulfate at 4 °C to a final concentration of 40 % and then 60 % saturation. The final precipitate was dissolved in 10 mM potassium phosphate buffer, pH 7.0, and dialyzed overnight against the same buffer. The sample was centrifuged and the supernatant was loaded onto a DEAE-Sepharose column (9.5×1.8 cm), which was previously equilibrated with 10 mM potassium phosphate buffer, pH 7.0. The fractions containing PA activity were pooled and checked for purity using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli 1970). Protein concentration was determined using the Bradford assay with bovine serum albumin as a standard (Bradford 1976).

30 % concentrations; the mixed preparations were preincubated at 50 and 55 °C for 30 min. The activities observed in the absence of any stabilizing agent were taken as 100 %. Substrate specificity The substrate specificity of the purified enzymes was studied in the presence of various substrates (penicillin G, ampicillin, penicillin V, amoxicillin, cephalexin, cloxacillin, dicloxacillin, and cephalosporin C; 15 mM final concentration) dissolved in 50 mM potassium phosphate buffer, pH 8.0, under optimal assay conditions. PA enzyme assay

Characterization of wild-type and mutant enzymes

The effects of various metal ions, chelating agents, and organic solvents were tested by incubating the enzyme solution with the respective chemicals (Supplementary Material Figs. S1, S2, and S4) for 30 min at 25 °C. The remaining activities were expressed as a percentage of the activity of the untreated control taken as 100 %.

Enzyme activity for expression studies was determined as follows: Cells from 10 ml of culture were collected by centrifugation at 4,000×g for 20 min at 4 °C and then suspended in 1 ml of 50 mM potassium phosphate buffer, pH 8.0. The cell suspension was sonicated using a Sonifier® S-250D digital sonifier (Branson, Danbury, CT, USA) and then centrifuged at 10,000×g for 30 min at 4 °C. PA activity in a clear supernatant was determined using penicillin G as a substrate based on the method described by Balasingham et al. (1972). Fifty microliters of the enzyme (clear supernatant) was mixed with 450 μl of substrate solution containing 15 mM penicillin G in 50 mM potassium phosphate buffer, pH 8.0, and the reaction mixture was incubated for 1 h at 40 °C with gentle shaking. The reaction was stopped by the addition of 3 ml sodium acetate buffer, pH 2.5, and then 500 μl of 0.5 % (w/v) pdimethylaminobenzaldehyde in methanol was added to this mixture. Activity was determined by measuring the absorbance at 415 nm. One unit of PA activity is defined as the amount of enzyme producing 1 μmol of 6-APA from penicillin G per minute under specified conditions. Enzyme activity assays for characterization studies were performed with the purified wild-type and mutant enzymes having a specific activity of 1.34 and 5.51 U/mg, respectively. Twenty microliters of the enzyme was added to 480 μl of 15 mM penicillin G in 50 mM potassium phosphate buffer, pH 8.0 (in the determination of optimal pH, different pH buffers were used at 50 mM concentrations), and the reaction mixture was incubated for 3 min at optimal temperature (in the determination of optimal reaction temperature, different temperatures were used in the range of 25–70 °C) with gentle shaking. Activity was determined by measuring the absorbance at 415 nm.

Effects of stabilizing agents on PA thermostability

Molecular modeling studies

To determine the effects of stabilizing agents on PA thermostability, glycerol, ethylene glycol (EG), and dimethyl sulfoxide (DMSO) were added to the enzyme solutions at 10 and

A molecular model for the M2234 mutant of PA was generated using the one-to-one threading protocol implemented in Phyre (Kelley and Sternberg 2009), based on the structure of

All measurements in this section were conducted in triplicate, and the mean value is presented with the standard deviation. Effects of temperature and pH on PA activity and stability For determination of optimal reaction temperature, enzyme activity was measured at different temperatures in the range of 25–70 °C. To determine thermal stability, the purified enzymes were preincubated for 30 min at 25–55 °C and the residual activity was analyzed under standard assay conditions. A temperature stability profile of the remaining activities was drawn by taking the residual activity of the untreated sample as a control (100 % activity). The optimal pH of the enzymes was determined by measuring relative activity using different pH buffers at 50 mM concentrations (sodium acetate–acetic acid buffers for pH 3– 6; potassium phosphate buffer for pH 6–8; Tris–HCl buffer for pH 8.5–9; and glycine–NaOH buffer for pH 9.5–10). The pH stabilities were also ascertained by incubating the enzymes at related pH values at 4 °C for 48 h; the remaining activities were calculated based on the PA activities of non-incubated enzymes. Effects of some additives on enzyme activity

Appl Microbiol Biotechnol

the E. coli PA published by Hewitt et al. (2000) (PDB ID: 1E3A). In addition to the K297I mutation, the template and the model have three other amino acids differences, resulting in a sequence identity of nearly 100 %.

Results Cloning and expression of the E. coli pac gene A ~3.5-kbp DNA fragment containing the coding region for PA and a short upstream noncoding region carrying part of the regulatory sequences was PCR amplified from the E. coli ATCC11105 chromosomal DNA and cloned into the pUC19 plasmid. The ligation mixture was transformed into E. coli JM109. Ten colonies were selected randomly and their plasmids were extracted by using the boiling miniprep method as described by Ausubel et al. (2001). After the digestion of plasmids with EcoRI and HindIII, insert DNA fragments of ~3.5 kb were observed in three colonies on agarose gel electrophoresis. For these three colonies, expression studies were conducted for 24 h at 26 °C in casein medium. The cells were induced with 0.1 mM IPTG when OD600 reached 0.6–0.8. All three transformants were found positive for PA production, and PA activities of the three clones were 0.21, 0.22, and 0.24 U/ml, respectively. The strain, which has 0.24 U/ml PA activity, was labeled as pUC19-pacwt11 and used for further studies. Error-prone PCR and screening of mutant library The ~3.5-kb fragment from pUC19-pacwt11 carrying the pac gene was used as a template for the construction of mutants by error-prone PCR. The mutant library was transformed into E. coli JM109 and subjected to the NIPAB filter paper assay for the first screening step. Of the 2,000 colonies screened on filter papers in the first round of error-prone PCR, 100 potential variants showing yellow color formation were selected. Then, all of these selected 100 clones were subjected to the second screening step. At this stage of screening, 10 clones showing a higher PA activity than the parent strain were selected for a third screening step, and of these 10 clones, Table 1 DNA and amino acid substitutions in M2234 PA

one variant showing the highest activity was used as a template for generating the second generation error-prone PCR library. In the second generation error-prone PCR library, 2,500 colonies were screened by the NIPAB filter paper assay, and 150 clones showed yellow color formation. All 150 variants were subjected to the 96-well microplate assay. From these, the 15 clones showing a higher activity than the wild-type strain were selected for a third screening step. One clone (M2234) in particular was found to be four times more active than the wild-type PA. DNA sequencing of the randomly mutated pac gene showed seven base changes (A738T, A890T, T1026A, G1029T, A1119C, G2226A, and T2418A), resulting in the replacement of one amino acid (K297I) located in the N-terminal region of the β subunit (Table 1).

Purification and characterization of the randomly mutated penicillin acylase The wild-type and randomly mutated PAs were purified using a DEAE-Sepharose column as described in the “Materials and methods” section. The results of the purification are summarized in Table 2. While the wild-type PA was purified 33-fold with 1.34 U/mg specific activity and an overall yield of 44 %, the mutant PA was purified 43-fold with 5.51 U/mg specific activity and overall yield of 50 %. As expected, SDS-PAGE analysis (Fig. 2) showed that both enzymes consisted of two dissimilar subunits (α and β) with approximate molecular masses of 24 and 63 kDa, respectively. After the biochemical characterization studies, the optimum reaction temperature was found to be 60 °C for both the parent and mutant PAs (Fig. 3a). The residual activity profiles for both enzymes after 30 min of incubation at 25– 55 °C were similar (Fig. 3b). Both enzymes were stable up to 45 °C for 30 min, but the activity was lost steadily at higher temperatures and at 55 °C, decreasing fast to near-zero values. The optimum activity for both enzymes was observed at pH 8.0 and gradually decreased to 35 % at pH 4.0 and to 85– 90 % at pH 10 (Fig. 4a). The mutant PA was more stable at pH 10 than the wild-type PA after incubating the enzymes for 48 h at 4 °C (Fig. 4b). Wild-type enzyme lost approximately

Base

Base substitution

Position in codon

Amino acid

Amino acid substitution

738 890 1,026 1,029 1,119 2,226 2,418

A→T A→T T→A G→T A→C G→A T→A

3 2 3 3 3 3 3

246 297 342 343 373 742 806

Silent Lys → Ile Silent Silent Silent Silent Silent

Appl Microbiol Biotechnol Table 2 Purification table for wild-type and mutant PAs

Crude extract 40 % (NH4)2SO4 precipitation 60 % (NH4)2SO4 precipitation DEAE-Sepharose FF

Total activity (U)

Total protein (mg)

Specific activity (U/mg)

Purification fold

Yield (%)

WT

M2234

WT

M2234

WT

M2234

WT

M2234

WT

M2234

36.62 31.61 23.96 16.35

60.46 55.25 44.51 30.68

890.27 628.04 379.56 12.23

470.92 309.96 193.12 5.57

0.04 0.05 0.06 1.34

0.13 0.18 0.23 5.51

1.00 1.25 1.50 33.50

1.00 1.39 1.80 42.90

100.00 86.32 65.43 44.65

100.00 91.38 73.62 50.74

30–35 % of its activity at pH 10, whereas the mutant PA was clearly more stable, retaining 94 % of its activity at pH 10. The effects of various metal ions and ammonium salts at 10 mM concentration on enzyme activity were also assessed. In general, metal ions (10 mM) did not affect enzyme activity significantly (Supplementary Material Fig. S1); the residual activity profiles of both enzymes were similar against metal ions, and the remaining activities were in the range of 61– 99 %. CuCl2 had the most prominent effect on the enzyme activity, as it decreased the activity of both the parent and mutant enzymes to approximately 40 % of their initial activities. FeCl2 also inhibited the activity of both the parent and mutant enzymes, with remaining activities of 78 and 87 %,

Fig. 2 SDS-PAGE profile for the purified PAs. Lane 1 molecular weight marker, lane 2 purified wild-type PA (pUC19-pacwt11), lane 3 purified mutant PA (M2234)

respectively. No considerable effect was also seen upon treatment of both the parent and mutant PAs with ammonium salts. Phenylmethylsulfonyl fluoride (PMSF) (1 or 10 mM) strongly inhibited the activity of both the parent and mutant enzymes, while the presence of ethylenediaminetetraacetic

Fig. 3 Effect of temperature on activity (a) and stability (b) of the wildtype and mutant PAs. To determine thermal stability, the purified enzymes were preincubated for 30 min at the indicated temperatures and the remaining activity was determined under standard assay conditions. Squares wild-type PA (pUC19-pacwt11), triangles mutant PA (M2234). Error bars represent the standard deviations

Appl Microbiol Biotechnol

activity at 50 °C. However, at 55 °C, the effect was more significant, especially for the parent PA. For most of the tested organic solvents, the inhibiting effect on the activity of the parent and mutant enzymes at 10 % concentration was small. The inhibitory effect was stronger at higher concentration (30 %) and was more pronounced for isoamyl alcohol and acetonitrile at this concentration. The mutant enzyme showed a tolerance to ethanol and DMSO at 10 % concentration, and generally, the mutant enzyme showed more tolerance to all tested organic solvents at 10 or 30 % concentration than the parent enzyme (Supplementary Material Fig. S4). The substrate specificity of both the parent and mutant enzymes were determined using different substrates (Supplementary Material Fig. S5). Penicillin G was the best substrate for both enzymes. The mutant enzyme showed a slightly higher activity toward cephalexin, amoxicillin, ampicillin, and penicillin V than the parent enzyme. In contrast, both enzymes did not show any activity towards cloxacillin, dicloxacillin, and cephalosporin C. Molecular modeling studies

Fig. 4 Effect of pH on the activity (a) and stability (b) of the wild-type and mutant PAs. To determine pH stability, the purified enzymes were preincubated at 4 °C for 48 h at the indicated pH values and the remaining activity was determined under standard assay conditions. Squares wildtype PA (pUC19-pacwt11), triangles mutant PA (M2234). Error bars represent the standard deviations

acid (EDTA) (1 or 10 mM) slightly inhibited the activity of both enzymes. β-Mercaptoethanol (1 or 10 mM) had a similar effect on the activity of the parent PA, but it had almost no effect on the mutant PA activity (Supplementary Material Fig. S2). The effects of various stabilizing agents on thermal stability of both enzymes at 50 or 55 °C were also assessed by adding 10 or 30 % of glycerol, EG, or DMSO to the reaction mixture (Supplementary Material Fig. S3). EG and DMSO at 10 or 30 % concentration had no effect on thermal stabilities of both the parent and mutant enzymes at 50 or 55 °C. Glycerol at 10 or 30 % concentrations slightly stabilized both enzymes Fig. 5 Stereo figure of the model for E. coli PA mutant M2234. Light orange chain A, orange chain B. The first β-strand of chain B is shown in blue, with the side chains of the catalytic serine (S290) and the mutation K297I represented as sticks

The model for the PA mutant M2234 obtained from Phyre is shown in Fig. 5. The K297I mutation is located in a surface loop following the β-strand of which the catalytic serine S290 is the first residue. The distance between S290 and K297I is approximately 24 Å (Cα–Cα); the hydrophobic side chain of isoleucine points into the solvent.

Discussion We are interested in the production of higher activity PA using directed evolution to explore its use in novel biotechnological applications. Directed evolution is one of the most powerful tools presently available to improve the characteristics of enzymes (Jaeger et al. 2001; Wang et al. 2006). The success of the directed evolution approach depends on the efficient formation of a random mutant library and a highly quantitative and high-throughput screening system. In this paper, we applied a three-step screening protocol to screen expected

Appl Microbiol Biotechnol

mutants. M2234 was selected from the error-prone PCR library by screening for PA activity. For rational enzyme design, amino acid residues located close to the active center and/or the substrate-binding pocket are often targeted because it is more plausible that they affect the catalytic reaction by affecting the active site architecture. Evidently, it is more difficult to demonstrate functions of amino acids located far from the site where the reaction takes place. In our case, in mutant M2234, a single amino acid mutation (K297I) occurred in the N-terminal region of the β subunit. Given the long distance (~24 Å) to the active site previously identified in PA (Duggleby et al. 1995), it is difficult to speculate on its effects on enzymatic activity. Possibly, the K297I mutation may enhance catalytic activity by indirect long-distance effects or by changes occurring elsewhere as a consequence of the mutation. Similarly, for other enzymes, it has been reported that mutations far from the active site can improve the catalytic properties (Oue et al. 1999; Jung et al. 2003; Xu et al. 2003; Fan et al. 2007; Stephens et al. 2007; Zuo et al. 2007). On the other hand, PA is transcribed as a single-chain precursor that undergoes posttranslational proteolytic processing to generate the mature, active form of the enzyme (Sizmann et al. 1990; Choi et al. 1992; Kasche et al. 1999). Determination of the N-terminal amino acid sequence of the β subunit has revealed that the amino-terminal region of the large subunit generally consists of hydrophobic amino acids (Schumacher et al. 1986) and is important in the endoprotease cleavage site recognition. On the other hand, Lee et al. (2000) have reported that S290 and K299 were critical residues for the autocatalytic processing of the PA precursor and K299 was responsible for the pH-dependent activation of the autocatalytic processing events in the periplasm. In our case, introduction of the K297I mutation increases the hydrophobicity of this region and may contribute to the higher affinity for endoprotease. Penicillin acylases are involved mainly in the industrial production of semisynthetic penicillins and cephalosporins, which remain the most widely used group of antibiotics (Arroyo et al. 2003; Giordano et al. 2006). In addition, penicillin acylases are useful as biocatalysts in many potentially valuable reactions such as protection of amino and hydroxyl groups in peptide synthesis, as well as in the resolution of racemic mixtures of chiral compounds. Biotechnological processes for the production of semisynthetic penicillins and cephalosporins can be performed by either thermodynamically or kinetically controlled synthesis (Kasche 1986); the usefulness of penicillin acylases in such biotransformations depends on the effects of organic solvents and a broad range of temperature and pH stability. The addition of cosolvents to the reaction medium has recently been reported for the improvement of penicillin acylase-catalyzed hydrolysis of penicillins for 6-APA production (Arroyo et al. 1999). The use of organic

solvents or water–cosolvent mixtures has increased the performance of enzymatic β-lactam synthesis (Illanes and Fajardo 2001; Wei and Yang 2003). Thermodynamic equilibrium could be shifted towards synthesis using hydrophobic solvents in the reaction medium (Rajendhran and Gunasekaran 2004; Samanta 2012). Given that PA loses activity in organic solvents or aqueous–organic mixtures, the stabilization of PA in organic solvents requires attention to accelerate synthetic reaction. Our results indicate that, for many of the organic solvents tested, mutant M2234 has a higher catalytic activity than the parent enzyme, promoting its use as a possible biocatalyst in industrial applications. Thermal unfolding of penicillin acylases has been linked to their conformational mobility in water. The mobility can be reduced by reducing the amount of free water available. This may be achieved by adding stabilizing agents such as polyol compounds, neutral salts, and sugars (Arroyo et al. 2000; Parmar et al. 2000). The protective effect of glycerol and EG seems to be correlated with the number of hydroxyl groups (Azevedo et al. 1999). EG, with two hydroxyl groups, had no stabilizing effect, whereas glycerol, with three hydroxyl groups, increased the enzyme stability. Furthermore, Arroyo et al. (2000) reported that there was an improvement of the stabilization related to the polarity of glycol: the lower the logP value, the higher stabilization factor value. In our experiment, EG did not affect enzyme stability, and we can consider it as an inert solvent for PA, whereas glycerol is a protective agent. Glycerol has the lower logP value than that of EG, so stabilization was even higher than that observed in the presence of EG. There was an improvement of stability proportional to glycerol concentration for both enzymes at 55 °C. None of the metal ions, except CuCl2 and FeCl2, affected the activity of the parent and mutant PAs significantly. The minimal effect of the metal-chelating agent EDTA on the activity of the parent enzyme and M2234 suggests that no metals are required for the catalytic mechanism (Ignatova et al. 2005). The strong inhibition by the serine-specific reagent PMSF indicates the role of serine as an active site residue. With respect to substrate specificity, M2234 PA exhibited maximum activity with penicillin G followed by cephalexin, amoxicillin, and ampicillin, respectively, similar to the parent enzyme. Our results suggest that M2234 PA efficiently hydrolyzes cephalexin and amoxicillin. However, Forney et al. (1989) have reported that the activity of the E. coli PA with cephalexin was 100-fold lower than that with penicillin G. Even though we screened for increased activity as the desired property, M2234 surprisingly displayed an increase in pH stability as well. Although both the parent and mutant PAs showed their optimum activity at pH 8.0, the mutant PA was considerably more stable at pH 10, retaining 94 % of its initial activity after 48 h at 4 °C. Increased stability at alkaline pH should be a valuable attribute for the utilization of penicillin acylase in bioreactors employed to convert penicillins

Appl Microbiol Biotechnol

into 6-APA, a precursor of semisynthetic penicillins. In these systems, base is added for pH control, which results in local alkaline conditions that promote enzyme inactivation. Hydrolysis and synthesis reactions are also pH-dependent (Del Rio et al. 1995). The formation of the acyl–enzyme complex demands that the terminal amine of Serβ1 be deprotonated. The pK of free α-amine group ranges between 6.8 and 7.9; this is the reason we used pH values >8 to hydrolyze penicillin G (Giordano et al. 2006). Therefore, the mutant PA with enhanced stability at alkaline pH would be advantageous for the industrial production of 6-APA. We have used successive rounds of error-prone PCR combined with ligase-independent cloning to generate variants of E. coli PA that exhibited increased activity and pH stability. Based on our current knowledge, our study is the first application of error-prone PCR to generate E. coli PA mutants. After two rounds of error-prone PCR, a mutant PA having four-fold increased specific activity, as well as improved pH stability at pH 10 was obtained. This increase in catalytic activity is due to a single mutation outside the active site, revealing that there is still opportunity for further evolution of the enzyme towards higher activity or for other performance improvements by changing the error-prone PCR conditions and by increasing the number of rounds. The activating effect of the mutation was subject to preliminary analysis. Further examination of the effect of the single mutation on the activity in M2234 will be conducted with additional studies. Acknowledgments During this work, Prof. Dr. Fusun Gumusel passed away. So, we remember her with respect. The authors would like to thank Prof. Dr. Tamer Yagci for the general help and Mustafa Nevzat Pharmaceuticals for providing penicillin G and 6-APA.

References An Y, Wu W, Lv A (2010) A convenient and robust method for construction of combinatorial and random mutant libraries. Biochim 92: 1081–1084. doi:10.1016/j.biochi.2010.03.016 Arroyo M, Torres R, de le Mata I, Castillon MP, Acebal C (1999) Interaction of penicillin V acylase with organic solvents. Catalytic activity modulation on the hydrolysis of penicillin V. Enzyme Microb Technol 25:378–383. doi:10.1016/S0141-0229(99)00057-5 Arroyo M, Torres-Guzman R, de le Mata I, Castillon MP, Acebal C (2000) Activation and stabilization of penicillin V acylase from Streptomyces lavendulae in the presence of glycerol and glycols. Biotechnol Prog 16:368–371. doi:10.1021/bp000023h Arroyo M, de la Mata I, Acebal C, Castillon MP (2003) Biotechnological applications of penicillin acylases: state-of-the-art. Appl Microbiol Biotechnol 60:507–514. doi:10.1007/s00253-002-1113-6 Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (2001) Current protocols in molecular biology. Wiley, New York Azevedo AM, Fonseca LP, Prazeres DMF (1999) Stability and stabilisation of penicillin acylase. J Chem Technol Biotechnol 74:1110–1116. doi: 10.1002/(SICI)1097-4660(199911)74:113.0. CO;2-B

Balasingham K, Warburton D, Dunnill P, Lilly MD (1972) The isolation and kinetics penicillin amidases from Escherichia coli. Biochim Biophys Acta 276:250–256. doi:10.1016/0005-2744(72)90027-7 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. doi:10.1016/0003-2697(76) 90527-3 Brannigan JA, Dodson G, Duggleby HJ, Moody PC, Smith JL, Tomchick DR, Murzin AG (1995) A protein catalytic framework with an Nterminal nucleophile is capable of self-activation. Nature 378:416– 419. doi:10.1038/378416a0 Bruns W, Hoppe J, Tsai H, Brüning HJ, Maywald F, Collins J, Mayer H (1985) Structure of the penicillin acylase gene from Escherichia coli: a periplasmic enzyme that undergoes multiple proteolytic processing. J Mol Appl Genet 3(1):36–44 Cadwell RC, Joyce GF (1992) Randomization of genes by PCR mutagenesis. Genome Res 2:28–33. doi:10.1101/gr.2.1.28 Cadwell RC, Joyce GF (1994) Mutagenic PCR. Genome Res 3:S136– S140 Calleri E, Temporini C, Massolini G, Caccialanza G (2004) Penicillin G acylase-based stationary phases: analytical applications. J Pharm Biomed Anal 35:243–258. doi:10.1016/ S0731-7085(03)00587-9 Chen R (2001) Enzyme engineering: rational redesign versus directed evolution. Trends Biotechnol 19(1):13–14. doi:10.1016/S01677799(00)01522-5 Choi KS, Kim JA, Kang HS (1992) Effects of site-directed mutations on processing and activities of penicillin G acylase from Escherichia coli ATCC 11105. J Bacteriol 174(19):6270–6276 Del Rio G, Rodriguez ME, Munguia ME, Lopez-Mungui A, Soberon X (1995) Mutant Escherichia coli penicillin acylase with enhanced stability at alkaline pH. Biotechnol Bioeng 48(2):141–148. doi:10. 1002/bit.260480208 Duggleby HJ, Tolley SP, Hill CP, Dodson EJ, Dodson G, Moody PCE (1995) Penicillin acylase has a single-amino-acid catalytic centre. Nature 373:264–268. doi:10.1038/373264a0 Fan Y, Fang W, Xiao Y, Yang X, Zhang Y, Bidochka MJ, Pei Y (2007) Directed evolution for increased chitinase activity. Appl Microbiol Biotechnol 76:135–139. doi:10.1007/s00253-007-0996-7 Forney LJ, Wong DC, Ferber DM (1989) Selection of amidases with novel substrate specificities from penicillin amidase of Escherichia coli. Appl Environ Microbiol 55:2550–2555 Giordano RC, Ribeiro MPA, Giordano RLC (2006) Kinetics of β-lactam antibiotics synthesis by penicillin G acylase (PGA) from the viewpoint of the industrial enzymatic reactor optimization. Biotechnol Adv 24:27–41. doi:10.1016/j.biotechadv.2005.05.003 Gümüşel F, İşsever-Öztürk S, Kiremit-Korkut N, Gelegen Ç, Bermek E (2001) Cloning and expression of the penicillin acylase gene (pac) from E. coli ATCC 11105. Enzyme Microb Technol 29:499–505. doi:10.1016/S0141-0229(01)00410-0 Hewitt L, Kasche V, Lummer K, Lewis RJ, Murshudov GN, Verma CS, Dodson GG, Wilson KS (2000) Structure of a slow processing precursor penicillin acylase from Escherichia coli reveals the linker peptide blocking the active-site cleft. J Mol Biol 302:887–898. doi: 10.1006/jmbi.2000.4105 Ignatova Z, Wischnewski F, Notbohm H, Kasche V (2005) Pro-sequence and Ca2+-binding: implications for folding and maturation of Ntnhydrolase penicillin amidase from E. coli. J Mol Biol 348:999–1014. doi:10.1016/j.jmb.2005.03.005 Illanes A, Fajardo A (2001) Kinetically controlled synthesis of ampicillin with immobilized penicillin acylase in the presence of organic cosolvents. J Mol Catal B: Enzym 11:587–595. doi:10.1016/ S1381-1177(00)00048-5 Jaeger KE, Eggert T, Eipper A, Reetz MT (2001) Directed evolution and the creation of enantioselective biocatalysts. Appl Microbiol Biotechnol 55:519–530. doi:10.1007/s002530100643

Appl Microbiol Biotechnol Jager SAW, Jekel PA, Janssen DB (2007) Hybrid penicillin acylases with improved properties for synthesis of β-lactam antibiotics. Enzyme Microb Technol 40:1335–1344. doi:10.1016/j.enzmictec.2006.10.014 Jung HC, Ko S, Ju SJ, Kim EJ, Kim MK, Pan JG (2003) Bacterial cell surface display of lipase and its randomly mutated library facilitates highthroughput screening of mutants showing higher specific activities. J Mol Catal B: Enzym 26:177–184. doi:10.1016/j.molcatb.2003.05.007 Kasche V (1986) Mechanism and yields in enzyme catalysed equilibrium and kinetically controlled synthesis of β-lactam antibiotics, peptides and other condensation products. Enzyme Microb Technol 8:4–16. doi:10.1016/0141-0229(86)90003-7 Kasche V, Lummer K, Nurk A, Piotraschke E, Rieks A, Stoeva S, Voelter W (1999) Intramolecular autoproteolysis initiates the maturation of penicillin amidase from Escherichia coli. Biochim Biophys Acta 1433:76–86. doi:10.1016/S0167-4838(99)00155-7 Kelley LA, Sternberg MJE (2009) Protein structure prediction on the web: a case study using the Phyre server. Nat Protoc 4:363–371. doi: 10.1038/nprot.2009.2 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. doi:10.1038/ 227680a0 Lee H, Park OK, Kang HS (2000) Identification of a new active site for autocatalytic processing of penicillin acylase precursor in Escherichia coli ATCC 11105. Biochem Biophys Res Commun 272:199–204. doi:10.1006/bbrc.2000.2729 Leung DW, Chen E, Goeddel DV (1989) A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique 1(1):11–15 Lin-Goerke JL, Robbins DJ, Burczak JD (1997) PCR-based random mutagenesis using manganese and reduced dNTP concentration. Biotechniques 23(3):409–412 McVey CE, Walsh MA, Dodson GG, Wilson KS, Brannigan JA (2001) Crystal structures of penicillin acylase enzyme–substrate complexes: structural insights into the catalytic mechanism. J Mol Biol 313:139–150. doi:10.1006/jmbi.2001.5043 Oue S, Okamoto A, Yano T, Kagamiyama H (1999) Redesigning the substrate specificity of an enzyme by cumulative effects of the mutations of non-active site residues. J Biol Chem 274:2344– 2349. doi:10.1074/jbc.274.4.2344 Parmar A, Kumar H, Marwaha SS, Kennedy JF (2000) Advances in enzymatic transformation of penicillins to 6-aminopenicillanic acid (6-APA). Biotechnol Adv 18:289–301. doi:10.1016/S0734-9750(00)00039-2 Rajendhran J, Gunasekaran P (2004) Recent Biotechnological interventions for developing improved penicillin G acylases. J Biosci Bioeng 97(1):1–13. doi:10.1016/S1389-1723(04)70157-7 Samanta TB (2012) Enzymatic synthesis of β-lactams: constraints and control. Indian J Biotechnol 11:7–15 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, New York

Schumacher G, Sizmann D, Haug H, Buckel P, Böck A (1986) Penicillin acylase from E. coli: unique gene–protein relation. Nucleic Acids Res 14(14):5713–5727. doi:10.1093/nar/14.14. 5713 Sizmann D, Keilmann C, Böck A (1990) Primary structure requirements for the maturation in vivo of penicillin acylase from Escherichia coli ATCC 11105. Eur J Biochem 192:143–151. doi:10.1111/j.14321033.1990.tb19207.x Stephens DE, Rumbold K, Permaul K, Prior BA, Singh S (2007) Directed evolution of the thermostable xylanase from Thermomyces lanuginosus. J Biotechnol 127(3):348–354. doi:10.1016/j.jbiotec. 2006.06.015 Tuzlakoglu-Ozturk M, Akbulut N, Issever-Ozturk S, Gumusel F (2013) Ligase-independent cloning of amylase gene from a local Bacillus subtilis isolate and biochemical characterization of the purified enzyme. Appl Biochem Biotechnol 171(2):263–278. doi:10.1007/ s12010-013-0331-y Valle F, Balbas P, Merino E, Bolivar F (1991) The role of penicillin amidase in nature and in industry. Trends Biochem Sci 16:36–40. doi:10.1016/0968-0004(91)90014-M Wang TW, Zhu H, Ma XY, Zhang T, Ma YS, Wei DZ (2006) Mutant library construction in directed molecular evolution. Mol Biotechnol 34:55–68. doi:10.1385/MB:34:1:55 Wei DZ, Yang L (2003) Effects of ethylene glycol on the synthesis of ampicillin using immobilized penicillin G acylase. J Chem Technol Biotechnol 78:431–436. doi:10.1002/jctb.749 Xu H, Petersen EI, Petersen SB, El-Gewely MR (1999) Random mutagenesis libraries: optimization and simplification by PCR. Biotechniques 27(6):1102–1108 Xu HF, Zhang XE, Zhang ZP, Zhang YM, Cass AEG (2003) Directed evolution of E. coli alkaline phosphatase towards higher catalytic activity. Biocatal Biotransformation 21(1):41–47. doi:10.1080/ 1024242031000087493 Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119. doi:10.1016/ 0378-1119(85)90120-9 Zhang Q, Zhang L, Han H, Zhang Y (1986) A method for screening penicillin G acylase-producing bacteria by means of 2-nitro-5phenylacetaminobenzoic acid test paper. Anal Biochem 156:413– 416. doi:10.1016/0003-2697(86)90274-5 Zheng Z, Ai-Hui Z, Jing-Ru W, Mao-Lin C, Ren-Bao L, Sheng Y, ZhongYi Y (2003) Improving the specific synthetic activity of a penicillin G acylase using DNA family shuffling. Acta Biochim Biophys Sin 35(6):573–579 Zuo ZY, Zheng ZL, Liu ZG, Yi QM, Zou GL (2007) Cloning, DNA shuffling and expression of serine hydroxymethyltransferase gene from Escherichia coli strain AB90054. Enzyme Microb Technol 40: 569–577. doi:10.1016/j.enzmictec.2006.05.018