Potential of soil fungi to produce penicillin V acylase and its optimization conditions

Fifty fungal isolates representing 31 species, related to 8 genera, were isolated from soil and screened for their abilities to produce endoand exocellular penicillin V acylase enzymes. For endocellular enzyme, eleven isolates (represent 22% of total isolates) exhibited high enzyme activity and 9 isolates (18%) had moderate ability. However, twenty isolates (40%) were low producers and ten isolates (20%) had no ability to produce the enzyme. For exocellular enzyme, four isolates only (representing 8% of total isolates) exhibited high enzyme activity, and fifteen isolates (30%) were found to be moderate. However, twenty two isolates (44%) were low producers and nine isolates (18%) had no activity. The most active fungal isolates were Aspergillus terreus and Penicillium chrysogenum, producing 325 and 280 U/ml, respectively. Maximum activity of penicillin V acylase produced by A. terreus and P. chrysogenum were obtained after 7 and 6 days of incubation, respectively at 35°C and initial pH 6. Dextrin or glucose was the best carbon source for penicillin V acylase produced by A. terreus and P. chrysogenum, respectively. However, peptone was the best nitrogen source. Inoculation of cultures by 3 discs of fungi and incubation of cultures at 180 rpm shaking condition improved the enzyme production.


INTRODUCTION
Penicillin acylase is one of the most important enzymes applied in the pharmaceutical industry for large scale production of 6-aminopenicillanic acid.This enzyme is the starting material for the manufacture of penicillin derivatives.β-lactam antibiotics, like penicillins and cephalosporins represent one of the major world's biotechnology markets, with annual sales of 15 billion Dollars and about 65% of the total antibiotic market (Chou et al., 2000;Parmar et al., 2000;Elander, 2003;Chandel et al., 2008).
Due to the worldwide demand for semisynthetic penicillins, production of 6-aminopenicillanic acid has been increased up to 7000 tons (Javadpour et al., 2002).
Recently, penicillin G acylase DNA of Shigella boydii was isolated, amplified and sequenced.The sequencing gene composed a polypeptide of 846 amino acid residues, and the highest homology was about 96% (Hassan et al., 2009).Penicillin V acylase is produced mainly by moulds and actinomycetes.They are mainly intracellular enzymes and also could produce from genus Fusarium (Lowe et al., 1986;Sudhakaran and Shewale, 1993) and actinomycetes Streptomyces lavendulae (Torres-Bacete et al., 2000) as extracellular acylases.
The Fusarium sp.SKF 235 produced penicillin V acylase intracellularly and extracellulary.The maximum yield of penicillin V acylase was 430 IU/g dry cell wet.This maximum activity was achieved at pH 6.5.Penicillin V acylase from Fusarium moniliforme and Fusarium avenaceum showed maximum activity at pH values of 8.5 and 7.5, respectively (Vanderhaeghe et al., 1968;Vandamme and Voets, 1972).The optimum pH values for the penicillin V acylase from Fusarium sp.SKF 235 is therefore lower than that from other Fusarium isolates.This low optimum pH value (6.5) is advantageous for the commercial production of 6-aminopenicillanic acid from penicillin V since the stability of penicillin V in solution is maximum at pH 5.7.On the other hand, pharmacological studies of some plant extracts showed various antibacterial and antifungal activities such as Peganum harmala (Asgarpanah and Ramezanloo, 2012), Viburnum grandiflorum (Alam et al., 2012) and Allium cepa (Nasri et al., 2012).This research was carried out to investigate the incidence of penicillin V acylase enzyme in different genera and species of soil fungi as well as the optimum conditions for the production of this enzyme by the most active fungal isolates.

Cultivation and culture conditions
The medium was distributed in Erlenmeyer flasks (250 ml), each flask contained 100 ml of the medium.The flasks were sterilized at 121°C for 20 min.Each flask was inoculated with 10 mm mycelial disc cut out from 5 days fungal colony grown on glucose-Czapek's agar medium.The inoculated flasks were incubated under shaking condition (180 rpm) at 30°C for 7 days.At the end of the incubation period, the content of each flask was filtrated.

Determination the activity of penicillin V acylase
Three grams of wet biomass was weighed and suspended in a 6 ml mixture of 0.1 M phosphate buffer (pH = 7), 20% sucrose and 1 mM EDTA (ethylene diamine tetraacetic acid).0.5 g of glass beads were added and the microtubes were placed in a bead mill at 4°C for 25 min, followed by centrifugation at 13,000 rpm for 5 min in case of intracellular enzyme but in case of extracellular enzyme, the supernatant was used directly after filtration and centrifugation of samples.6-aminopenicillanic acid was determined by colorimetric method as described by Balasingham et al. (1972).The mixture of 0.25 ml of 0.1 M phosphate buffer (pH = 7) containing 2.5 mg of penicillin V, 0.25 ml of cell suspension (intracellular enzyme) or supernatant (extracellular enzyme) was incubated at 37°C for 30 min, 3 ml of solution made by mixing 2 ml of glacial acetic acid with 1 ml of 0.05 M NaOH was added to the reaction mixture and centrifuged.0.5 ml of 0.5% P-dimethylaminobenzaldehye in methanol was added to the supernatant, and the ultra violet (UV)absorbance was measured at 415 nm.One unit of enzyme activity was defined as the quantity of the enzyme which produced 1 µmol of 6-aminopenicillanic acid per 1 min.

Effect of incubation periods on penicillin V acylase production by Aspergillus terreus and Penicillium chrysogenum
A. terreus and P. chrysogenum were found to be the most active penicillin V acylase (endoenzyme) producers.So these fungi were used for further studies.The medium previously described by Bashir et al. (2008) was employed.

Cultivation and culture conditions
The influence of different incubation periods (2, 4, 6, 7, 8, 10 and 12 days) on the production of penicillin V acylase and fungal dry weight were measured.The flasks were sterilized at 121°C for 20 min, inoculated with 10 mm disc cut out from 5 days fungal colony grown on glucose-Czapek's agar medium.Two replicates were used for each treatment.Inoculated flasks were incubated under shaking condition (180 rpm) at 30°C.At the end of different incubation periods, fungal mycelium was separated from the growth medium by suction filtration through pre-weighed Whatman #1 filter paper placed in a buchner funnel.The filter paper with mycelium was dried in an oven at 85°C for 24 h.The mycelial dry weight was recorded and also the activity of the enzyme was determined according to the method described by Balasingham et al. (1972).

Aspergillus terreus and Penicillium chrysogenum
The influence of various temperature values (15, 20, 25, 30, 35 and 40°C) on the activity of penicillin V acylase and fungal dry weight were tested by incubating the tested fungi at different temperatures in liquid synthetic medium for 7 days.After the incubation period, fungal dry weight and penicillin V acylase activity in two replicates were measured according to method described by Balasingham et al. (1972).

Effect of pH on penicillin V acylase production by Aspergillus terreus and Penicillium chrysogenum
The influence of different pH values (2, 4, 6, 7, 8, 10 and 12) on penicillin V acylase production as well as fungal dry weight of tested fungi was measured by incubating A. terreus and P. chrysogenum at 30°C in liquid synthetic medium for 7 days.After the incubation period, fungal dry weight and penicillin V acylase activity in two replicates were measured according to the method described by Balasingham et al. (1972).

Aspergillus terreus and Penicillium chrysogenum
A. terreus and P. chrysogenum were grown in Erlenmeyer flasks (250 ml) containing 100 ml liquid medium.Nine carbon sources (glucose, fructose, maltose, lactose, sucrose, cellulose, starch, dextrin and glycerol) were added individually to the basal medium by 20 g/L.Flasks were sterilized at 121°C for 20 min, inoculated with 10 mm disc cut out from 5 days fungal colony grown on glucose-Czapek's agar medium.The inoculated flasks were incubated under shaking condition (180 rpm) at 30°C for 7 days.At the end of the incubation period, the dry weight of mycelium and the activity of penicillin V acylase enzyme were determined according to method described by Balasingham et al. (1972).

Aspergillus terreus and Penicillium chrysogenum
A. terreus and P. chrysogenum were grown in Erlenmeyer flasks (250 ml) containing 100 ml liquid medium.Nine nitrogen sources (Peptone, NH4Cl, (NH4)2SO4, NH4NO3, (NH4)2HPO4, NaNO3, KNO3, Ca(NO3)2.4H2Oand CH3COONH4) were added individually to the basal medium by 5 g/L.The flasks were sterilized at 121°C for 20 min, inoculated with 10 mm disc cut out from 5 days fungal colony grown on glucose-Czapek's agar medium.Two replicates were used for each nitrogen source.Inoculated flasks were incubated under shaking condition (180 rpm) at 30°C for 7 days.At the end of the incubation period, the mycelial dry weight was recorded and also the activity of the enzyme was determined according to method described by Balasingham et al. (1972).

Aspergillus terreus and Penicillium chrysogenum
The influence of inoculum size on the production of penicillin V acylase was tested by inoculating of culture medium with 1 disc, 2 discs and 3 discs (10 mm in diameter) cut out from 5 days colony of the tested fungi grown on glucose-Czapek's agar medium.The flasks were incubated under shaking condition (180 rpm) at 30°C for 7 days.After the incubation period, penicillin V acylase activity was measured in two replicates according to method described by Balasingham et al. (1972).Fungal dry weight was also determined.

Aspergillus terreus and Penicillium chrysogenum
The production of penicillin V acylase enzyme by tested fungi was estimated by using different random speed of rotary shaker (160, 180 and 200 rpm).The two fungi were grown in Erlenmeyer flasks (250 ml) containing 100 ml liquid medium.The flasks were divided into 3 groups: The first group placed on rotary shaker at 160 rpm, the second placed on rotary shaker at 180 rpm and the third group placed on rotary shaker at 200 rpm for 7 days at 30°C.After the incubation period, the activity of the enzyme was determined according to method described by Balasingham et al. (1972).Fungal dry weight was also determined.

Aspergillus terreus and Penicillium chrysogenum
This experiment occurred by using 2 volumes of liquid synthetic medium (50 and 100 ml) in 250 ml Erlenmeyer flasks.The flasks were inoculated with tested fungi and incubated under shaking condition (180 rpm) at 30°C for 7 days.After the incubation period, penicillin V acylase was measured in two replicates according to the method described by Balasingham et al. (1972).Fungal dry weight was also determined.

Screening of fungi for their abilities to produce penicillin V acylase enzyme
Screening of fifty fungal isolates for production of endocellular pencillin V acylase showed that eleven isolates represented 22% of total isolates which showed high enzyme activity.Nine isolates contributed 18% of total isolates found to be moderate activity.Twenty isolates represented 40% of total isolates which were low producers of penicllin V acylase enzyme.Ten isolates represented 20% of total isolates which had no enzyme activity (Table 1).
For exocellular penicillin V acylase, four isolates (represent 8% of total isolates) only showed high enzyme activity and fifteen isolates (30%) were moderate.However, twenty two isolates (44%) were low producers of the enzyme.Nine isolates (18%) had no activity.A. terreus and P. chrysogenum were the most active producer isolates of fungi for penicillin V acylase.These fungi produced 325 and 280 U/ml, respectively (Table 1).Bashir et al. (2008) screened 12 different strains of fungi for their abilities to produce penicillin G acylase enzyme under submerged condition.They showed that A. niger had the highest enzyme activity.

Effect of incubation periods on growth and penicillin V acylase by Aspergillus terreus and Penicillium chrysogenum
A. terreus and P. chrysogenum exhibited maximum penicillin V acylase production at 7 and 6 days of incubation, respectively.These fungi had enzyme activity of 9062.15 and 7826.3U/ml, respectively.At 8 days of incubation, the two tested fungi produced relatively considerable amounts of penicillin V acylase enzyme, however decreasing of incubation period to 2 or 4 days had negative effect on the enzyme production by the two tested fungi.Increasing of incubation period to 10 or 12 days decreased the production of penicillin V acylase enzyme by A. terreus and P. chrysogenum.The results recorded for the effect of incubation time on mycelial growth of A. terreus and P. chrysogenum was nearly similar to those reported for penicillin V acylase enzyme (Table 2).In this respect, maximum production of penicillin G acylase of A. niger was recorded between 20 to 24 h of incubation under shaking conditions (Bashir et al., 2008).

Effect of temperatures on fungal growth penicillin V acylase production by A. terreus and P. chrysogenum
Temperature greatly influenced the production of penicillin V acylase by A. terreus and P. chrysogenum.Maximum production of the enzyme was recorded at 35°C by the two tested fungi producing 9062.15and 7785.36U/ml, respectively.Decreasing of incubation temperature below 35°C decreased the enzyme production.Decreasing of the incubation temperature to 15°C prevented the secretion of the enzyme by the two fungi.Also increasing of incubation temperature to 40°C decreased the production of penicillin V acylase.The results obtained for the effect of incubation temperature on mycelial growth of A. terreus and P. chrysogenum was variable with different incubation temperatures, with maximum growth at 25 and 35°C, respectively (Table 3).
The optimum temperature for maximal penicillin G acylase enzyme of A. fumigatus and Mucor gryseoclanum was at 40°C (Jose et al., 2003).Abedi et al. (2004) showed that the optimum temperature for penicillin G acylase synthesis activity was at 25°C.Souza et al. (2005) reported that penicillin G acylase produced by Bacillus megaterium showed maximum hydrolysis rate at 37°C.Zuza et al. (2007) found that the optimal temperature of Escherichia coli penicillin acylase activity were 27.5 and 31.5°C for free and immobilized enzyme, respectively.

Effect of pH values on growth and penicillin V acylase production by A. terreus and P. chrysogenum
The pH value was greatly affected the production of penicillin V acylase by A. terreus and P. chrysogenum.Maximum production of the enzyme was recorded at pH 6 by the two fungi, producing 9062.15and 7002.49U/ml, respectively.Decreasing of pH value to pH 4 exhibited an inhibitive effect on penicillin V acylase production.Also, increasing of pH value to 7, 8 or 10 decreased the enzyme production by the two tested fungi.No enzyme production was recorded at pH 2 and 12 by the two fungal species.Data recorded for the effect of pH value on mycelial growth of A. terreus and P. chrysogenum was nearly similar to those reported for penicillin V acylase enzyme (Table 4).Javadpour et al. (2002) reported that the optimal pH of penicillin acylase produced by E. coli was determined to be 8.The optimum pH rang for dialyzed preparations of penicillin G acylase produced by A. fumigatus and M. gryseoclanum was 7 to 8 and 7.5 to 8.5, respectively (Jose et al., 2003).Abedi et al. 2004 showed that the optimum pH for penicillin G acylase synthesis activity was 6. Souza et al. (2005) reported that penicillin G acylase produced by Bacillus megaterium showed maximum hydrolysis rate at pH 8. Zuza et al. (2007) reported that the optimal pH of E. coli penicillin acylase activity was 8.5 for both free and immobilized enzyme.

Effect of different carbon sources on growth and Penicillin V acylase production by A. terreus and P. chrysogenum
Carbon source is one of the most essential components in the microbial medium.The effect of different carbon sources on growth and penicillin V acylase production by A. terreus and P. chrysogenum was variable.Glucose and dextrin were the most favorable carbon sources for penicillin V acylase production by P. chrysogenum and A. terreus, respectively.Maximum production of the enzyme was achieved by incorporation of glucose and dextrin in the culture medium for enzyme production, producing 8897.51and 7702.89U/ml for A. terreus and P. chrysogenum, respectively.Addition of other carbon sources in the fungal cultures decreased the enzyme production by the two fungal species.Mycelial growth of A. terreus and P. chrysogenum was not greatly affected by the addition of different carbon sources to the culture medium for fungi (Table 5).Among 7 carbon sources tested for penicillin G acylase, 0.4% lactose was the best for mycelial growth and enzyme production of A. niger (Bashir et al., 2008).Other carbon sources such as glucose, sorbitol glycerol, sucrose, PAA (Phenyl Acetic Acid) and tryptone increased the activity of Penicillin G acylase by E. coli and Bacillus megaterium (Chandel et al., 2008).

Effect of different nitrogen sources on growth and penicillin V acylase production by A. terreus and P. chrysogenum
Nitrogen source is one of the most essential components in the microbial medium because the nitrogen element  penicillin G acylase activity by B. megaterium (Chandel et al., 2008).

Effect of shaker speed on growth and penicillin V acylase production by A. terreus and P. chrysogenum
Speed of shaker had an effect on growth and penicillin V acylase production by A. terreus and P. chrysogenum.A. terreus and P. chrysogenum showed maximum penicillin V acylase production (9479.39 and 7723.36U/ml, respectively) at 180 rpm.Mycelial growth of A. terreus and P. chrysogenum was not greatly affected by incubation shaking of fungal cultures (Table 8).Recently, Saleem et al. (2012) reported that the endocellular Damino acid oxidase enzyme produced by Fusarium heterosporum and Nectria haematococca showed maximum activity in cultures incubated at 160 rpm shaking condition.

Effect of medium volume on growth and penicillin V acylase production by A. terreus and P. chrysogenum
The effect of medium volume on growth and penicillin V acylase production by A. terreus and P. chrysogenum was studied by using different volumes of cultivation medium.A. terreus and P. chrysogenum produced higher amounts of penicillin V acylase (9981.11and 8672.31U/ml, respectively) in flask containing 100 ml of medium.Decreasing of the amount of medium to 50 ml decreased the enzyme production by nearly half amount (4077.79 and 4736.95U/ml, respectively).Mycelial growth of A. terreus and P. chrysogenum was not greatly affected by the amount of medium (Table 9).Saleem et al. (2012) found that D-amino acid oxidase enzyme and mycelial growth of F. heterosporum and Nectria haematococca were higher in flasks containing 100 ml of medium than those containing 50 ml of medium by nearly half amount.

Conclusion
This article would contribute to finding new sources of penicillin acylases by fungi and increase the production of these enzymes by optimizing cultural conditions which will lead to the production of some antibiotics on a large scale and development of some antibiotic manufacture.

Table 1 .
Screening of fungi for their abilities to produce endo-and exoenzyme penicillin V acylase.

Table 2 .
Effect of incubation periods on growth and penicillin V acylase production by Aspergillus terreus and Penicillium chrysogenum.

Table 3 .
Effect of temperatures on growth and penicillin V acylase production by Aspergillus terreus and Penicillium chrysogenum.

Table 4 .
Effect of pH values on growth and penicillin V acylase production by Aspergillus terreus and Penicillium chrysogenum.

Table 7 .
Effect of inoculum size on growth and penicillin V acylase production by Aspergillus terreus and Penicillium chrysogenum.

Table
Effect of medium volume on growth and penicillin V acylase production by Aspergillus terreus and Penicillium chrysogenum.

Table 9 .
Effect of shaker speed on growth and penicillin V acylase production by Aspergillus terreus and Penicillium chrysogenum.