World
Journal
of Microbiology
and Biotechnology,
9, 233-239
Biosynthesis of penicillin V acylase by fusarium sp.: effect of culture conditions V.K. Sudhakaran
and J.G. Shewale*
Penicillin V acylase was produced, both intracellularly and extracellularly, by Fusarium sp. SKF 235 grown in submerged fermentation. When neopeptone was added to the medium, >95% of the penicillin V acylase was extracellular. In the absence of a complex organic nitrogen source, the fungus produced low levels of totally intracellular penicillin V acylase. MgSO, was essential for synthesis of the enzyme, which was induced by phenoxyacetic acid and penicillin V. The maximum yield of penicillin V acylase was 430 IU/g dry cell wt. The optimum pH value and temperature for the penicillin V acylase were 6.5 and 55”C, respectively. Key words: 6Arninopenicillanic
acid, Fusarium sp,, penicillin
6Aminopenicillanic acid (6-APA) is produced by hydrolysis of either penicillin G (Pen G) or penicillin V (Pen V), using penicillin G acylase (PGA) or penicillin V acylase (PVA), respectively (Vandamme 1988; Valle el al. 1991). Both the enzymes hydrolyse the side-chain amide bond present in the penicillin molecules to generate the /3-lactam nucleus, 6-APA, and corresponding side chain acids (Shewale & Sivaraman 1989). It is estimated that 88% of the 6-APA obtained enzymatically results from PGA activity; the rest results from PVA activity (Vandamme 1988). This is mainly because of the development of hyper-producing strains for PGA. Pen V should be preferred over Pen G as bulk raw material for the production of 6-APA, since the stability of Pen V in aqueous solution is greater than that of Pen G (Sieh 1988). Preparations of PVA with optimum activity at pH 5.5 to 6.8 are important in reducing the degradation of 6-APA during the hydrolysis. As it is estimated that annual world production of 6-APA will be 7000 tonnes in the year 2000 (Shewale & Sivaraman 1989), studies on PVA have gained importance. Various bacteria, fungi, yeasts and actinomycetes produce PVA (Sudhakaran & Borkar 1985). After screening several organisms for production of PVA with an acidic optimum pH, Ftts&um sp. SKF 235 was selected for detailed studies since it produced large amounts of PVA (unpublished work). Bacillus sphaericus, Beijerinckia
acylase, penicillin
indica var. penicillinicum, Fusarium avenaceum, Ftcsarium oxysporum, Pleurotus osfreafus {Bovistu plumbea) and Pseudomonas acidovorans are known to produce large amounts of PVA (Vanderhaeghe et al. 1968; Vanderhaeghe 1975; Carlson & Emborg 1981; Lowe et al. 1981; Stoppock & Wagner 1983; Lowe et al. 1986; Ambedkar et al. 1991). In the present paper we describe the effect of culture conditions on PVA production by Fusarium sp. SKF 235.
Materials
@ 1993 Rapid Communications
are with Research and Development, Pune 411 018, India. ‘Corresponding
of Oxford
and Methods
Organism
Fusarium sp. SKF 235 was isolated in our laboratory from root rot on a wilting sorghum plant. The culture was maintained on potatoldextroselagar slopes. Vegetative seed was prepared by growing Fusarium sp. SKF 235 in a seed medium which contained (% w/v): yeast extract, 0.3; malt extract, 0.3; peptone, 0.5; ghrcose, 1.0; at pH 5.8. Sterile distilled water (5.0 ml) was added aseptically to a lo-day-old sporulated slope and the spore suspension thus obtained was used to inoculate 100 ml seed medium in a 500-ml flask. The seed flask was grown for 48 h on a rotary shaker (210 rev/min) at 25°C and 10 ml aliquots of the vegetative seed were inoculated into 500-ml flasks, each with 100 ml of basal medium containing (% w/v): glucose, 2.0; corn steep liquor, 2.0; MgS0,7H,O, 0.2; KH,PO,, 0.15: potassium salt of Pen V, 0.3; at pH 6.0. The fermentation was carried
V.K. Sudhakaran and J.G. Shewale Hindustan Antibiotics Ltd. Pimpri, author.
V acylase.
out on a rotary
shaker
(250 rev/min)
at 25°C
for 120 h.
Determination of Growth Mycelial washed
cells from 10 ml of the whole broth were separated three times with distilled water by centrifugation
and at
Ltd World ]ownal of Micrvbioiogy
and Biotechnology. Vol 9. 1993
233
V. K. Sudhakaran and 1.G. Shewale 8540
g for 10 min and then dried at 80°C was expressed as dry cell wt/l.
x
Growth
to constant
weight.
Enzyme Assays One ml of appropriately diluted whole broth (for whole broth assay) or supematant after centrifugation (for extracellular enzyme assay) was mixed with 1.0 ml of 0.1 M phosphate buffer, pH 6.5, containing 40 mg Pen V. The reaction was stopped by adding 1.0 ml of 2.0 M acetate buffer, pH 3.2, and 0.5 ml of the reaction mixture was centrifuged and then processed for estimation of 6-APA by the p-dimethylaminobenzaldehyde method (Shewale et
al.
1987). One unit
of enzyme activity required for production
enzyme
Results
was defined as the amount of 1 pmol 6-AF’A per min.
of
and Discussion
by Bacillus sphaerincs Beijerinckia indica var. penicillanictlm (Ambedkar et al. 1991), Envinia aroideae (Vandamme & Voets 1972), Pseudomonas (Lowe et al. 1981) and the soil bacterium NRRL 11240 (Diers & Emborg 1979). Pletrrotus osfreattis also produces some PVA intracellularly but some of the enzyme is secreted into the medium (Stoppock et al. 1981). Fusarium strain 7.5-5 also produces PVA both intracellularly and extracellularly, in the proportion 60:40 (Borkar et al. 1966). The barium sp. SKF 235 used in the present study produced PVA intracellularly and extracellularly when grown in basal medium. Attempts were made to induce extracellular PVA and to increase the production of PVA by altering the culture conditions. A two-fold increase in PVA production was achieved and most of the enzyme accumulated in the culture filtrate. The fungus does not produce a B-lactamase. PVA from Fusarium sp. SKF 235 had maximum activity at pH 6.5 and 55°C. The optimum pH and temperature values for PVA isolated from other microorganisms were found to be between 5.6 and 8.5 and 28 to 45”C, PVA
(Carlson
Table Nitrogen (% w/v)
is
produced
& Emborg
1. Effect
intracellularly
1982),
of inorganic
source
nitrogen
Final
source
on the production
pH
None NH&I
broth
Effect of Inorganic Nitrogen Source on the Production of PVA Incorporation of various inorganic nitrogen salts, at equimolar amounts, to basal medium generally increased PVA production, increased the degree of excretion and decreased cell growth; CaNO,, however, supported vegetative growth (Table I). Among the nitrogen sources studied, maximum production of PVA was with NH,H,PO,, which also increased extracellular PVA from 67% to 79% of total PVA. Although excretion was higher (85%) when (NH,),HPO, was used as nitrogen source, the total and specific productions of the enzyme were lower than those on NH,H,PO,. When the concentration of NH,H,PO, in the medium was varied from 0.1% to 0.5% w/v (data not shown), an excretion of 78 to 80% was achieved at 0.15% (w/v) and remained unaltered at higher concentrations. Therefore, NH,H,PO, at 0.15% (w/v) was used in further experiments. Effect of Complex Organic Nitrogen Sources on the Production of PVA The production of PVA by Enuinia aroideae and Beijerinckia indica var. penicillinicum cultures is influenced by peptones and these cultures have a selectivity for a particular type of peptone (Vandamme & Voets 1975; Sudhakaran & Shewale 1990); PVA production is 20% higher when Erwinia aroideae
of penicillin
PVA (IUlml) Whole
respectively. PVA from Fusarium moniliforme, F. avenaceum, Fusarium 7.5-5 and F. oxysporum show maximum activity at pH values of 8.5, 7.5, 7.0 and 7.4, respectively (Borkar et al. 1966; Vanderhaeghe et al. 1968; Vandamme & Voets 1972; Sheng & Ye 1989). The optimum pH value for the PVA 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-APA from Pen V since the stability of Pen V in solution is maximum at pH 5.7.
V acylase Excretion
(PVA).
(%I
Growth (g dry cell wtll)
Specific production (M/g dry cell wt) 240 470
Extracellular
8.9 8.9 8.9
1.2 1.7 1.8
0.8 1.3 1.3
67 76 72
4.9 3.6 4.5
8.9 8.8 7.7
1.6 1.3 1.9
1.1 1.1 1.5
69 85 79
4.0 3.6 3.1
400 400 360 610
NH,acetate (0.17) NaNO, (0.2) KNO, (0.23)
9.0 9.0 9.0
1.4 1.6 1.6
0.9 1.2 1.2
64 75 75
4.0 4.0 4.0
350 400 400
CaNO, (0.38) NH,HCO, (0.18)
8.9 9.0
0.8
0.4 0.8
50 61
5.4 4.5
150 290
(0.126)
W-&SO, (0.15) NH,NO, (0.09) (NH,),HPO, (0.15) NH,H,PO, (0.26)
234
World loumal
of Microbiology
1.3
and Biotechnology, Vol 9, 1993
Fusarium sp. penicillin Table
2. Effect
of complex
organic
Organic N source (2% w/v)
Final
nitrogen
source
pH
*In Basal
Medium
3.2 7.6 a.2
a.0
8.0 7.6 2.7 3.0 2.8 7.1 7.7 2.7 6.8 corn
broth
steep
liquor
0.15%
(w/v)
is grown on tryptone and 10% higher when Bei. in&a var. penicillanicum is grown on Casamino acids, compared with that on other peptones. Similarly, PGA production by hcherichia coli is favoured by Aminobak rather than tryptone (Wojskowicz 1984). The presence of a complex organic nitrogen source was necessary for both production and excretion of PVA by hsarium sp. SKF 235 (Table 2). Without such a nitrogen source, the fungus produced very low amounts of PVA, which was totally intracellular, and acid production predominated during fermentation. Production of PVA was X.3-fold more when the corn steep liquor (CSL) in the basal was replaced with neopeptone and, with medium neopeptone, 80% of the enzyme was excreted into the culture filtrate. Very low levels of PVA, which was mainly cell bound, were produced when corn meal, wheat bran,
Table
3. Effect
of concentration Final
of neopeptone
‘In Basal
4.3 7.6 a.4 a.7 a.8 9.0 a.9 Medium
without
broth
steep
liquor
71 65 74 65 15 5 0 58 71 0 50
(w/v)
70 550 520 420 660 520 390 220 370 310 60 160 150 520 300 150 330
wheat flour or gram flour (chick-pea flour) were used as organic nitrogen sources, although vegetative growth was relatively high. A close relationship was observed in these experiments between final pH value and the amount of PVA produced. When the final pH of the medium dropped below 3.5, the fungus produced less than 1.0 II-I/ml of PVA, cell growth was relatively high, at > 3.5 g dry cell wt/l, and none of the synthesised PVA was secreted into the medium. When the concentration of neopeptone in the medium was varied from 0.5% to 4.0% (w/v) (Table 3), PVA production in the whole broth was highest (about 2.4 III/ml) between 1.0% and 2.0% (w/v) but excretion was maximum (> 95%) between 1.0% and 1.5% (w/v). Higher concentrations of neopeptone favoured cell growth but not enzyme production.
of penicillin
V acylase
(PVA). Growth (g dry cell
Extracellular
0.15%
production (IUlg dry cell wt)
NH,H,PO,.
0.0 1.4 2.3 2.4 1.8 1.1 0.2 and with
SpeciliC
wt/l)
2.7 3.4 4.2 4.2 3.6 4.8 6.1 9.1 6.1 7.3 6.1 5.5 4.5 3.6 7.9 7.3 4.2
80
Excretion w-1
0.2 1.5 2.4 2.5 2.4 1.8 0.6 corn
Growth (g dry cell
0 79 73 72 71
PVA (Ill/ml) Whole
Nil 0.5 1.0 1.5 2.0 3.0 4.0
on the production
pH
(PVA).
Extracellular 0.0 1.5 1.6 1.3 1.7 2.0 1.7 1.3 1.7 1.5 0.1 0.1 0.0 1.1 1.7 0.0 0.7
and with
V acylase
Excretion mo)
0.2 1.9 2.2 1.8 2.4 2.5 2.4 2.0 2.3 2.3 0.4 0.9 0.7 1.9 2.4 1.1 1.4
a.1 a.2 a.5 7.9
without
of penicillin
PVA (NJ/ml) Whole
None Corn steep liquor Yeast extract Casein acid hydrolysate Peptone Neopeptone Tryptone Soyabean meal Soyabean hydrolysate Peanut meal Corn meal Wheat bran Wheat flour Brewer’s yeast powder Pharmamedia Gram flour SVP Scotafarm
on the production
V acylase
0 93 96 96 75 61 33
2.8 3.2 5.5 5.8 6.0 7.5 9.7
wtll)
Specific production (M/g dry cell wt) 70 460 430 430 400 240 60
NH,H,PO,.
Worki Joumai of Microbiology and Biotechnology, Vol 9, 1993
235
V K. Sudhakaran and J G. Shewale Effect of MgSO, and K&PO, concentration on production of PVA MgSO, was essential for PVA biosynthesis but not for the growth of Fusarium sp. SKF 235; in the absence of MgSO, there was no PVA synthesis; in the presence of low concentrations (0.1% to 0.5% w/v) the production of PVA was unaltered. Although PVA production was the same (2.4 B-I/ml) whether or not KH,PO, was present (up to 0.5% w/v), KH,PO, was incorporated in the medium to avoid variation of pH, if any. At this stage, Production Medium I was constituted. This contained (% w/v): glucose, 1.0; neopeptone, 1.0; NH,H,PO,, 0.15; KH,PO,, 0.15; MgS0,.7H,O, 0.2; Pen V (K+ salt), 0.3; pH adjusted to 6.0 prior to autoclaving.
4). The fungus produced low amounts of PVA when sugar alcohols and polysaccharides were used as carbon sources. Vegetative growth was predominant on glycerol, sorbitol, inositol and corn starch. Specific production was found to be higher with sucrose as carbon source than with glucose, so the glucose in Production Medium I was replaced with sucrose in further studies. Citrate enhanced production of PVA by Pleurofus osfreatus and the effect was progressive when the citrate was added to the medium after glucose depletion (Stoppock & Wagner 1983). However, addition of citrate prior to inoculation or at various stages of fermentation did not alter the production of PVA by Fusarium sp. SKF 235 (data not shown).
Effect of Sugars and Other Carbohydrates on PVA Production The synthesis of penicillin G acylases is repressed by glucose, fructose, maltose and glycerol (Shewale & Sivaraman 1989). Although PVA synthesis was not repressed by glucose in Enoinia aroideae (Vandamme & Poets 1975) or Bar. sphaericus (Carlson & Emborg 1981), glucose, galactose and mannose repressed PVA production by Bei. indica var. penicillanicum SKB 3133 (Sudhakaran & Shewale 1990). Different sugars, sugar alcohols and polysaccharides were therefore studied for their effect on growth and PVA production in Fusarium sp. SKF 235. Almost all the sugars studied were utilized for growth; production of PVA was similar in all cases except for lactose and sorbose, which favoured the vegetative growth (Table
Effect of Inducers on PVA Production PVA production by Aspergillus ochraceus, Epidermophyton sp., F. monihforme, Fusarium sp., Rhodotorula glutinis var. glutinis, Trichosporon sp. and Trichophyton sp. is induced by phenoxyacetic acid (POAA) whereas Bat. sphaericus, Bei. indica var. penicillanicum, Erwinia aroideae, Micrococcus ureae, Pleurotus ostreafus and Sfreptomyces lavendulae produce PVA constitutively (Vandamme et al. 1971; Vandamme & Voets 1974, 1975; Schneider and Roehr 1976; Carlson & Emborg 1981; Sudhakaran & Borkar 1985; Shewale & SivaRaman 1989; Sudhakaran & Shewale 1990). Pen V and its synthons, 6-APA and POAA, were incorporated on an equimolar basis into the medium in the present study and their effect on the production of PVA was
Table
4. Effect
of carbon
source Final
Carbon source (1.0% w/v)
on the production
pH
* In Production
236
a.7 a.4 a.5 a.5 a.5 a.5 a.5 8.5 a.5 a.5 a.5 a.3 a.2 a.4 8.4 a.7 a.4 a.4 a.4 a.5 Medium
World]oumal
1 devoid
of Microbiology
V acylase
(PVA).*
PVA (IlJlml) Whole
None Glucose Sucrose Lactose Fructose Galactose Maltose Mannose Ribose Xylose Sorbose Glycerol Sorbitol lnositol Mannitol Cellulose Tapioca starch Corn starch Soluble starch Dextrin
of peniclllln
broth
and with
Growth (g dry cell wtll)
Specific production (IUlg dry cell wt)
3.6 6.6 5.3 7.5 6.0 6.0 6.6 6.0 6.0 6.0 9.7 22.5 19.5 11.2 6.0 6.6 9.7 11.3 7.5 6.6
90 360 480 160 350 390 350 410 360 380 180 60 50 70 260 70 180 140 170 220
Extracellular
0.4 2.4 2.6 1.2 2.2 2.4 2.4 2.5 2.2 2.3 1.8 1.5 1.1 0.8 1.6 0.5 1.8 1.7 1.3 1.5 of glucose
Excretion W) 0.3 2.4 2.5 0.8 1.9 2.4 0.8 2.4 2.2 2.3 1.6 0.9 0.9 0.8 1.3 0.5 1.7 1.5 1.0 1.3
indicated
and Bmtechnology, Vol 9, 1993
carbon
88 99 97 61 87 99 32 97 97 98 89 57 76 97 83 96 99 88 74 90 source.
Fusarium sp. penicillin Table 5. Effect of penicillin V acyiase (PVA).’ Inducer
(% w/v)
V (Pen
Final
V), 6-aminopeniciiianic
pH
l
In Production
7.9 a.7 a.3 a.5 Medium
I in which
and phenoxyacetic
broth
was
0.0 2.6 0.1 1.7 replaced
(POAA)
on the production
Growth (g dry ceil wt/i)
Extracellular
0.0 2.7 0.1 2.1 glucose
acid
Excretion (“/.I
PVA (IUlmi) Whole
None Pen V (0.3) 6-APA (0.17) POAA (0.1)
acid (6-APA)
with
studied (Table 5). In the absence of Pen V, 6-APA and POAA, only vegetative growth was observed; no enzyme was produced. 6-APA was a poor inducer of PVA and the specific production of the enzyme was very low compared with that in cultures with Pen V or POAA. The production of PVA was found to be highest when Pen V was used as inducer. When the concentration of Pen V in the medium was then varied from 0.1% to 1.5% (w/v), concentrations of 0.3% to 0.5% (w/v) were found to be optimum for PVA production (data not shown). Fermentation runs were also conducted in which Pen V was added prior to inoculation, 24 or 48 h after inoculation, or, in low amounts, every 24 h during the fermentation. The best results were obtained when Pen V was incorporated into the medium prior to inoculation. Effect of Surface-active Agents on PVA Production We have observed that production of PVA by Bei. indicu var. penicillanicum UREMS 5 increased by 40% to 46% when soyabean oil or olive oil was added to the medium (Ambedkar et al. 1991). However, addition of various vegetable oils and other surface-active agents, such as soyabean oil, paraffin oil, ground nut oil, white oil (liquid paraffin), palm oil, cottonseed oil, olive oil, sag 471 (silicon emulsion), Tween 80 and oleic acid, at 0.2% (v/v), did not significantly alter PVA production by Fusarium sp. SKF 235 (data not shown).
and Pen V was
MgS0,.7Hz0,
of penicillin
Specific production (NJ/g dry ceil wt)
16.2 6.2 12.0 5.0
98 a3 a0
sucrose
V acylase
replaced
with
the indicated
Pen V (K+ salt), 0.3;
0.2;
430 10 410 inducer.
pH adjusted
to
6.0.
PVA Formation During the Growth Cycle The course of PVA production, growth and pH profile during the growth cycle of Fusarium sp. SKF 235 in Production Medium II are shown in Figure 1. The pH dropped from 6.0 to 5.8 in the first 24 h and thereafter increased slowly to 8.5 at 120 h of fermentation. The initial drop in pH could be due to rapid utilization of carbohydrates. Growth was abundant during the early stages of fermentation, peaking at 13.9 g/l after 72 h and then decreasing rapidly up to 105 h. Production of PVA had a lag of 24 h, beginning once the pH rose above 7.0. Synthesis of PVA did not run parallel to cell growth; most of the PVA was synthesised during the lytic phase. The ratio of extracellular to intracellular PVA was 65 : 35 at 45 h. The accumulation of PVA in the culture filtrate increased during the fermentation and at 120 h almost all (> 95%) of the PVA produced was in the filtrate. Synthesis of penicillin acylases during the lytic phase of Pen&urn and Fusarium growth has been reported by Alfonso et al. (1989). Maximum yield of PVA was 2.4 IU/ml, corresponding to 430 IU/g dry cell wt. The production of PVA by various organisms is summarized in Table 6. Fusarium sp. SKF 235
Efiect of Initial pH on PVA Production During the initial experiments, very low amounts of PVA were produced when the final pH of the medium fell below 5.0. Therefore, the effect of initial pH on the production of PVA was studied. PVA was produced by Fusarium sp. SKF 235 when initial pH was > 4.0 and the optimum value was found to be 6.0. But. sphaericus produces PVA strictly between initial pH values of 7.5 and 8.0 (Carlson & Emborg 1981).
A medium for production by Fusaritrm sp. SKF 235 was optimised following these later results. This medium, Production Medium II, contained (% w/v): sucrose, 1.0; 1.0; NH,H,PO,, 0.15; KH,PO,, 0.15; neopeptone,
Time (h)
Figure 1. Penicillin V acylase (PVA) concentrations in the whole broth (r) and culture filtrate (a), pH (0) and cell growth (0) during the growth cycle of fusarium sp. SKF 235.
World Journal
of
Microbmiogy and Biotechnology. Vol 9, 1993
237
V.K.
Sudhakaran
Table
and ].G.
6. Comparison
Shewale
of penicillin
V acylase
(PVA)
Organism
production PVA (IUlml)
Fusarium Bacillus Bacillus
sp. SKF 235 sphaericus NCTC subtilis”
10338
Beijerinckia indica var. penicillanicum Escherichia co/i HB lOl* Escherichia coli RRI (pOH 50)’ Fusarium avenaceum Fusarfum oxysporum Pleurotus ostreatus NRRL 3824 Pseudomonas acidovorans isolate E
UREMS-5
by various
organlsms.
Specific productlon (ItJIg dry cell wt)
2.4 0.1 NA
430 NA 410
2.2 NA NA NA 0.6 NA
244 110
3200 10
50 120 NA
0.7
Reference
Carlson & Emborg 1981 Olsson et al. 1985 Gattenbeck et a/. 1986 Ambedkar et a/. 1991 Olsson et al. 1985 Olsson & Uhlen 1986 Vanderhaeghe eta/. 1968 Su & Wang 1989 Stoppock & Wagner 1983 Lowe et a/. 1981
‘Recombinant strain. NA-Not available.
produced high levels of PVA and its specific production was higher than that of other, non-recombinant isolates; Escherichia co/i RR I (pOH SO), which produces 3200 IU/g dry cell wt of PVA, is a recombinant strain (Olsson & Uhlen 1986).
Acknowledgements We thank Dr S.R. Naik, General Manager, R & D, for his encouragement during this work and U.V. Gaikwad for his help in isolation of the culture.
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C.,
Cribeiro,
L.
&
Reyes,
F.
1989
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amidohydrolases in fungal autolysis. Microbiology rind Immunology 33, 69-74. Ambedkar, S.S.,Deshpande, B.S.,Sudhakaran, V.K. & Shewale, J.G. 1991 Beijerinckia indica var. penicillunicum penicillin V acylase: enhanced enzyme production by catabolite repression resistant mutant and effect of solvents on enzyme activity. Journal of Industrial
Microbiology
7, 209214.
Borkar, P.S., Vinze, V.L. & Ambedkar, S.S. 1966 Penicillin amidase. I. Factors influencing the induced formation of penicillin amidase in Fusuriwn sp, Hindusfun Anfibiofics Bulletin 9, 1-9. Carlson, F. & Emborg, C. 1981 Bacillus sphaerictls V penicillin acylase. 1. Fermentation. Biotechnology Letters 3, 375-378. Carlson, F. & Emborg, C. 1982 Bacillus sphuericus V penicillin acylase. II. Isolation and characterisation. Io~mal of Chemirai Technology and Biotechnology 32, 808-811. Diers, I.V. & Emborg, C. 1979 Penicillin V acylase. British UK Patent 2,021,119. Gattenbeck, S., Nilsson, B., Olsson, A. & Uhlen, M. 1986 Recombinant DNA molecule, transformed microorganism and process for producing penicillin V amidase. International Patent
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R.P. 1981 Penicillin and the isolation of a
new bacterial penicillin V acylase. Developments in Industrial Microbiology 22, 163-180. Lowe, D.A., Romancik, G. 81 Elander, R.P. 1986 Enzymatic hydrolysis of penicillin V to 6-APA by Fusarium orysponrm. Biotechnology Leffers 8, 15 l-156. Olsson, A., Hagstron, T., Nilsson, B., Uhlen, M. & Gattenbeck, S. 1985 Molecular cloning of Bacilltts sphuerictrs penicillin V acylase gene and its expression in Escherichiu co/i and Bucillus subtilis. Applied and Environmental Microbiology 49, 1081-1089. Olsson, A. & Uhlen, M. 1986 Sequencing and heterologous expression of the gene encoding penicillin V amidase from Bacillus sphuericw. Gene 45, 175-181. Schneider, WJ. & Roehr, M. 1976 Purification and properties of penicillin acylase of Bovistu plumbeu. Biochimicu et Biophysicu Actu
452,177-185. Sheng, G. & Ye, Y. 1989 Some enzymological and kinetic properties of immobilized Flcsurium oxysporurn. Shengwn Gongcheng Xubeuo 5, 129-134. Shewale, J.G., Kumar, K.K. & Ambekar, G.R. 1987 Evaluation of determination of 6-APA by p-dimethylaminobenzaldehyde. Biotechnology Techniqties 1, 69-72. Shewale, J.G. & Sivaraman, H. 1989 Penicillin acylase: enzyme production and its application in the manufacture of 6-APA. Process Biochemistry 24, 146-154. Sieh, D.H. 1988 Potassium penicillin V. In Analytical Profiles of Drug Stibstances, Vol. 17, ed Florey, K. pp. 677-748. New York: Academic Press. Stoppock, E., Schemer, U., Segner, A., Meyer, H. & Wagner, F. 1981 Production of 6-APA from penicillin V and penicillin G by Bovista plttmbeu NRRL 3824 and E. co/i 5K (pHM12). Advances in Biotechnology 3, 547-552. Stoppock, E. & Wagner, F. 1983 Effect of citrate on the synthesis of penicillin V acylase of Pleurotw ostreutus. Biotechnology Letters
5,503-508. Su, Y.C. & Wang, J.S. 1989 The production of penicillin V acylase by Fusarium oxysporum. Taiwan Tang Yeh Yen Chiu So Yen Chiu Hui Pao 123, 29-44. Sudhakaran, V.K. & Borkar, P.S. 1985 Phenoxymethyl penicillin acylase: sources and study-a sum up. Hindusfun Antibiotics Bulletin 27, 44-63. Sudhakaran, V.K. & Shewale, J.G. 1990 Effect of culture conditions on penicillin V acylase production by Beijerinckia indicu var. penicilhnictrm. ]otrrnul of Microbiul Biotechnology 5, 6674.
Fusarium Valle, F., Balbas, P., Merino, E. & Bolivar, F. 1991 The role of penicillin amidases in nature and industry. Trends in Biochemical Sciences 16, 36-40. Vandamme, E.J. 1988 Immobilized biocatalysts and antibiotic production: biochemical, genetical and biotechnical aspects. In Immobilized Enzymes and Cells: Fundamentals and Applicufions, ed Moo-Young, M. pp. 261-286. New York: Marcel Dekker. Vandamme, E.J. & Voets, J.P. 1972 Comparative studies on a fungal and bacterial penicillin V acylase. Mededelingen van de Fuculteit Landbouwwetenschappen Rijksuniversitait Gent 37, 1185-1200. Vandamme, E.J. & Voets, J.P. 1974 Microbial penicillin acylases. Advances in Applied Microbiology 17, 311-361. Vandamme, E.J. & Voets, J.P. 1975 Properties of the purified penicillin V acylase of Erwinin aroideae. Experientia 31, 140-143.
sp. penicillin
V acyluse
Vandamme, E.J., Voets, J.P. & Beyaert, G. 1971 Penicillin V acylase activity of fungal spores. Mededelingen van de Fucukeit LandboMwwetenschappen Rijksuniversiteit Gent 36, 5 77-605. Vanderhaeghe, H. 1975 Penicillin acylase (fungal). Methods in Enzymology 43, 721-728. Vanderhaeghe, H., Claesen, M., Vlietinek, A. & Parmentier, G. 1968 Specificity of penicillin acylase of Fttsaritrm and Penicillium chrysogenum. Applied Microbiology 16, X557-1563. Wojskowicz, J. 1984 Biosynthesis of penicillin amidase by E. coli in organic media. Acta Microbiologia Polonicn 33, 179-183.
(Received
in revised
form
6 October
World ]oumal
of Microbiology
1992; accepted 76 October
1992)
and Biotechnology, Voi 9, 1993
239
Journal
of Microbiology
and Biotechnology,
9, 233-239
Biosynthesis of penicillin V acylase by fusarium sp.: effect of culture conditions V.K. Sudhakaran
and J.G. Shewale*
Penicillin V acylase was produced, both intracellularly and extracellularly, by Fusarium sp. SKF 235 grown in submerged fermentation. When neopeptone was added to the medium, >95% of the penicillin V acylase was extracellular. In the absence of a complex organic nitrogen source, the fungus produced low levels of totally intracellular penicillin V acylase. MgSO, was essential for synthesis of the enzyme, which was induced by phenoxyacetic acid and penicillin V. The maximum yield of penicillin V acylase was 430 IU/g dry cell wt. The optimum pH value and temperature for the penicillin V acylase were 6.5 and 55”C, respectively. Key words: 6Arninopenicillanic
acid, Fusarium sp,, penicillin
6Aminopenicillanic acid (6-APA) is produced by hydrolysis of either penicillin G (Pen G) or penicillin V (Pen V), using penicillin G acylase (PGA) or penicillin V acylase (PVA), respectively (Vandamme 1988; Valle el al. 1991). Both the enzymes hydrolyse the side-chain amide bond present in the penicillin molecules to generate the /3-lactam nucleus, 6-APA, and corresponding side chain acids (Shewale & Sivaraman 1989). It is estimated that 88% of the 6-APA obtained enzymatically results from PGA activity; the rest results from PVA activity (Vandamme 1988). This is mainly because of the development of hyper-producing strains for PGA. Pen V should be preferred over Pen G as bulk raw material for the production of 6-APA, since the stability of Pen V in aqueous solution is greater than that of Pen G (Sieh 1988). Preparations of PVA with optimum activity at pH 5.5 to 6.8 are important in reducing the degradation of 6-APA during the hydrolysis. As it is estimated that annual world production of 6-APA will be 7000 tonnes in the year 2000 (Shewale & Sivaraman 1989), studies on PVA have gained importance. Various bacteria, fungi, yeasts and actinomycetes produce PVA (Sudhakaran & Borkar 1985). After screening several organisms for production of PVA with an acidic optimum pH, Ftts&um sp. SKF 235 was selected for detailed studies since it produced large amounts of PVA (unpublished work). Bacillus sphaericus, Beijerinckia
acylase, penicillin
indica var. penicillinicum, Fusarium avenaceum, Ftcsarium oxysporum, Pleurotus osfreafus {Bovistu plumbea) and Pseudomonas acidovorans are known to produce large amounts of PVA (Vanderhaeghe et al. 1968; Vanderhaeghe 1975; Carlson & Emborg 1981; Lowe et al. 1981; Stoppock & Wagner 1983; Lowe et al. 1986; Ambedkar et al. 1991). In the present paper we describe the effect of culture conditions on PVA production by Fusarium sp. SKF 235.
Materials
@ 1993 Rapid Communications
are with Research and Development, Pune 411 018, India. ‘Corresponding
of Oxford
and Methods
Organism
Fusarium sp. SKF 235 was isolated in our laboratory from root rot on a wilting sorghum plant. The culture was maintained on potatoldextroselagar slopes. Vegetative seed was prepared by growing Fusarium sp. SKF 235 in a seed medium which contained (% w/v): yeast extract, 0.3; malt extract, 0.3; peptone, 0.5; ghrcose, 1.0; at pH 5.8. Sterile distilled water (5.0 ml) was added aseptically to a lo-day-old sporulated slope and the spore suspension thus obtained was used to inoculate 100 ml seed medium in a 500-ml flask. The seed flask was grown for 48 h on a rotary shaker (210 rev/min) at 25°C and 10 ml aliquots of the vegetative seed were inoculated into 500-ml flasks, each with 100 ml of basal medium containing (% w/v): glucose, 2.0; corn steep liquor, 2.0; MgS0,7H,O, 0.2; KH,PO,, 0.15: potassium salt of Pen V, 0.3; at pH 6.0. The fermentation was carried
V.K. Sudhakaran and J.G. Shewale Hindustan Antibiotics Ltd. Pimpri, author.
V acylase.
out on a rotary
shaker
(250 rev/min)
at 25°C
for 120 h.
Determination of Growth Mycelial washed
cells from 10 ml of the whole broth were separated three times with distilled water by centrifugation
and at
Ltd World ]ownal of Micrvbioiogy
and Biotechnology. Vol 9. 1993
233
V. K. Sudhakaran and 1.G. Shewale 8540
g for 10 min and then dried at 80°C was expressed as dry cell wt/l.
x
Growth
to constant
weight.
Enzyme Assays One ml of appropriately diluted whole broth (for whole broth assay) or supematant after centrifugation (for extracellular enzyme assay) was mixed with 1.0 ml of 0.1 M phosphate buffer, pH 6.5, containing 40 mg Pen V. The reaction was stopped by adding 1.0 ml of 2.0 M acetate buffer, pH 3.2, and 0.5 ml of the reaction mixture was centrifuged and then processed for estimation of 6-APA by the p-dimethylaminobenzaldehyde method (Shewale et
al.
1987). One unit
of enzyme activity required for production
enzyme
Results
was defined as the amount of 1 pmol 6-AF’A per min.
of
and Discussion
by Bacillus sphaerincs Beijerinckia indica var. penicillanictlm (Ambedkar et al. 1991), Envinia aroideae (Vandamme & Voets 1972), Pseudomonas (Lowe et al. 1981) and the soil bacterium NRRL 11240 (Diers & Emborg 1979). Pletrrotus osfreattis also produces some PVA intracellularly but some of the enzyme is secreted into the medium (Stoppock et al. 1981). Fusarium strain 7.5-5 also produces PVA both intracellularly and extracellularly, in the proportion 60:40 (Borkar et al. 1966). The barium sp. SKF 235 used in the present study produced PVA intracellularly and extracellularly when grown in basal medium. Attempts were made to induce extracellular PVA and to increase the production of PVA by altering the culture conditions. A two-fold increase in PVA production was achieved and most of the enzyme accumulated in the culture filtrate. The fungus does not produce a B-lactamase. PVA from Fusarium sp. SKF 235 had maximum activity at pH 6.5 and 55°C. The optimum pH and temperature values for PVA isolated from other microorganisms were found to be between 5.6 and 8.5 and 28 to 45”C, PVA
(Carlson
Table Nitrogen (% w/v)
is
produced
& Emborg
1. Effect
intracellularly
1982),
of inorganic
source
nitrogen
Final
source
on the production
pH
None NH&I
broth
Effect of Inorganic Nitrogen Source on the Production of PVA Incorporation of various inorganic nitrogen salts, at equimolar amounts, to basal medium generally increased PVA production, increased the degree of excretion and decreased cell growth; CaNO,, however, supported vegetative growth (Table I). Among the nitrogen sources studied, maximum production of PVA was with NH,H,PO,, which also increased extracellular PVA from 67% to 79% of total PVA. Although excretion was higher (85%) when (NH,),HPO, was used as nitrogen source, the total and specific productions of the enzyme were lower than those on NH,H,PO,. When the concentration of NH,H,PO, in the medium was varied from 0.1% to 0.5% w/v (data not shown), an excretion of 78 to 80% was achieved at 0.15% (w/v) and remained unaltered at higher concentrations. Therefore, NH,H,PO, at 0.15% (w/v) was used in further experiments. Effect of Complex Organic Nitrogen Sources on the Production of PVA The production of PVA by Enuinia aroideae and Beijerinckia indica var. penicillinicum cultures is influenced by peptones and these cultures have a selectivity for a particular type of peptone (Vandamme & Voets 1975; Sudhakaran & Shewale 1990); PVA production is 20% higher when Erwinia aroideae
of penicillin
PVA (IUlml) Whole
respectively. PVA from Fusarium moniliforme, F. avenaceum, Fusarium 7.5-5 and F. oxysporum show maximum activity at pH values of 8.5, 7.5, 7.0 and 7.4, respectively (Borkar et al. 1966; Vanderhaeghe et al. 1968; Vandamme & Voets 1972; Sheng & Ye 1989). The optimum pH value for the PVA 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-APA from Pen V since the stability of Pen V in solution is maximum at pH 5.7.
V acylase Excretion
(PVA).
(%I
Growth (g dry cell wtll)
Specific production (M/g dry cell wt) 240 470
Extracellular
8.9 8.9 8.9
1.2 1.7 1.8
0.8 1.3 1.3
67 76 72
4.9 3.6 4.5
8.9 8.8 7.7
1.6 1.3 1.9
1.1 1.1 1.5
69 85 79
4.0 3.6 3.1
400 400 360 610
NH,acetate (0.17) NaNO, (0.2) KNO, (0.23)
9.0 9.0 9.0
1.4 1.6 1.6
0.9 1.2 1.2
64 75 75
4.0 4.0 4.0
350 400 400
CaNO, (0.38) NH,HCO, (0.18)
8.9 9.0
0.8
0.4 0.8
50 61
5.4 4.5
150 290
(0.126)
W-&SO, (0.15) NH,NO, (0.09) (NH,),HPO, (0.15) NH,H,PO, (0.26)
234
World loumal
of Microbiology
1.3
and Biotechnology, Vol 9, 1993
Fusarium sp. penicillin Table
2. Effect
of complex
organic
Organic N source (2% w/v)
Final
nitrogen
source
pH
*In Basal
Medium
3.2 7.6 a.2
a.0
8.0 7.6 2.7 3.0 2.8 7.1 7.7 2.7 6.8 corn
broth
steep
liquor
0.15%
(w/v)
is grown on tryptone and 10% higher when Bei. in&a var. penicillanicum is grown on Casamino acids, compared with that on other peptones. Similarly, PGA production by hcherichia coli is favoured by Aminobak rather than tryptone (Wojskowicz 1984). The presence of a complex organic nitrogen source was necessary for both production and excretion of PVA by hsarium sp. SKF 235 (Table 2). Without such a nitrogen source, the fungus produced very low amounts of PVA, which was totally intracellular, and acid production predominated during fermentation. Production of PVA was X.3-fold more when the corn steep liquor (CSL) in the basal was replaced with neopeptone and, with medium neopeptone, 80% of the enzyme was excreted into the culture filtrate. Very low levels of PVA, which was mainly cell bound, were produced when corn meal, wheat bran,
Table
3. Effect
of concentration Final
of neopeptone
‘In Basal
4.3 7.6 a.4 a.7 a.8 9.0 a.9 Medium
without
broth
steep
liquor
71 65 74 65 15 5 0 58 71 0 50
(w/v)
70 550 520 420 660 520 390 220 370 310 60 160 150 520 300 150 330
wheat flour or gram flour (chick-pea flour) were used as organic nitrogen sources, although vegetative growth was relatively high. A close relationship was observed in these experiments between final pH value and the amount of PVA produced. When the final pH of the medium dropped below 3.5, the fungus produced less than 1.0 II-I/ml of PVA, cell growth was relatively high, at > 3.5 g dry cell wt/l, and none of the synthesised PVA was secreted into the medium. When the concentration of neopeptone in the medium was varied from 0.5% to 4.0% (w/v) (Table 3), PVA production in the whole broth was highest (about 2.4 III/ml) between 1.0% and 2.0% (w/v) but excretion was maximum (> 95%) between 1.0% and 1.5% (w/v). Higher concentrations of neopeptone favoured cell growth but not enzyme production.
of penicillin
V acylase
(PVA). Growth (g dry cell
Extracellular
0.15%
production (IUlg dry cell wt)
NH,H,PO,.
0.0 1.4 2.3 2.4 1.8 1.1 0.2 and with
SpeciliC
wt/l)
2.7 3.4 4.2 4.2 3.6 4.8 6.1 9.1 6.1 7.3 6.1 5.5 4.5 3.6 7.9 7.3 4.2
80
Excretion w-1
0.2 1.5 2.4 2.5 2.4 1.8 0.6 corn
Growth (g dry cell
0 79 73 72 71
PVA (Ill/ml) Whole
Nil 0.5 1.0 1.5 2.0 3.0 4.0
on the production
pH
(PVA).
Extracellular 0.0 1.5 1.6 1.3 1.7 2.0 1.7 1.3 1.7 1.5 0.1 0.1 0.0 1.1 1.7 0.0 0.7
and with
V acylase
Excretion mo)
0.2 1.9 2.2 1.8 2.4 2.5 2.4 2.0 2.3 2.3 0.4 0.9 0.7 1.9 2.4 1.1 1.4
a.1 a.2 a.5 7.9
without
of penicillin
PVA (NJ/ml) Whole
None Corn steep liquor Yeast extract Casein acid hydrolysate Peptone Neopeptone Tryptone Soyabean meal Soyabean hydrolysate Peanut meal Corn meal Wheat bran Wheat flour Brewer’s yeast powder Pharmamedia Gram flour SVP Scotafarm
on the production
V acylase
0 93 96 96 75 61 33
2.8 3.2 5.5 5.8 6.0 7.5 9.7
wtll)
Specific production (M/g dry cell wt) 70 460 430 430 400 240 60
NH,H,PO,.
Worki Joumai of Microbiology and Biotechnology, Vol 9, 1993
235
V K. Sudhakaran and J G. Shewale Effect of MgSO, and K&PO, concentration on production of PVA MgSO, was essential for PVA biosynthesis but not for the growth of Fusarium sp. SKF 235; in the absence of MgSO, there was no PVA synthesis; in the presence of low concentrations (0.1% to 0.5% w/v) the production of PVA was unaltered. Although PVA production was the same (2.4 B-I/ml) whether or not KH,PO, was present (up to 0.5% w/v), KH,PO, was incorporated in the medium to avoid variation of pH, if any. At this stage, Production Medium I was constituted. This contained (% w/v): glucose, 1.0; neopeptone, 1.0; NH,H,PO,, 0.15; KH,PO,, 0.15; MgS0,.7H,O, 0.2; Pen V (K+ salt), 0.3; pH adjusted to 6.0 prior to autoclaving.
4). The fungus produced low amounts of PVA when sugar alcohols and polysaccharides were used as carbon sources. Vegetative growth was predominant on glycerol, sorbitol, inositol and corn starch. Specific production was found to be higher with sucrose as carbon source than with glucose, so the glucose in Production Medium I was replaced with sucrose in further studies. Citrate enhanced production of PVA by Pleurofus osfreatus and the effect was progressive when the citrate was added to the medium after glucose depletion (Stoppock & Wagner 1983). However, addition of citrate prior to inoculation or at various stages of fermentation did not alter the production of PVA by Fusarium sp. SKF 235 (data not shown).
Effect of Sugars and Other Carbohydrates on PVA Production The synthesis of penicillin G acylases is repressed by glucose, fructose, maltose and glycerol (Shewale & Sivaraman 1989). Although PVA synthesis was not repressed by glucose in Enoinia aroideae (Vandamme & Poets 1975) or Bar. sphaericus (Carlson & Emborg 1981), glucose, galactose and mannose repressed PVA production by Bei. indica var. penicillanicum SKB 3133 (Sudhakaran & Shewale 1990). Different sugars, sugar alcohols and polysaccharides were therefore studied for their effect on growth and PVA production in Fusarium sp. SKF 235. Almost all the sugars studied were utilized for growth; production of PVA was similar in all cases except for lactose and sorbose, which favoured the vegetative growth (Table
Effect of Inducers on PVA Production PVA production by Aspergillus ochraceus, Epidermophyton sp., F. monihforme, Fusarium sp., Rhodotorula glutinis var. glutinis, Trichosporon sp. and Trichophyton sp. is induced by phenoxyacetic acid (POAA) whereas Bat. sphaericus, Bei. indica var. penicillanicum, Erwinia aroideae, Micrococcus ureae, Pleurotus ostreafus and Sfreptomyces lavendulae produce PVA constitutively (Vandamme et al. 1971; Vandamme & Voets 1974, 1975; Schneider and Roehr 1976; Carlson & Emborg 1981; Sudhakaran & Borkar 1985; Shewale & SivaRaman 1989; Sudhakaran & Shewale 1990). Pen V and its synthons, 6-APA and POAA, were incorporated on an equimolar basis into the medium in the present study and their effect on the production of PVA was
Table
4. Effect
of carbon
source Final
Carbon source (1.0% w/v)
on the production
pH
* In Production
236
a.7 a.4 a.5 a.5 a.5 a.5 a.5 8.5 a.5 a.5 a.5 a.3 a.2 a.4 8.4 a.7 a.4 a.4 a.4 a.5 Medium
World]oumal
1 devoid
of Microbiology
V acylase
(PVA).*
PVA (IlJlml) Whole
None Glucose Sucrose Lactose Fructose Galactose Maltose Mannose Ribose Xylose Sorbose Glycerol Sorbitol lnositol Mannitol Cellulose Tapioca starch Corn starch Soluble starch Dextrin
of peniclllln
broth
and with
Growth (g dry cell wtll)
Specific production (IUlg dry cell wt)
3.6 6.6 5.3 7.5 6.0 6.0 6.6 6.0 6.0 6.0 9.7 22.5 19.5 11.2 6.0 6.6 9.7 11.3 7.5 6.6
90 360 480 160 350 390 350 410 360 380 180 60 50 70 260 70 180 140 170 220
Extracellular
0.4 2.4 2.6 1.2 2.2 2.4 2.4 2.5 2.2 2.3 1.8 1.5 1.1 0.8 1.6 0.5 1.8 1.7 1.3 1.5 of glucose
Excretion W) 0.3 2.4 2.5 0.8 1.9 2.4 0.8 2.4 2.2 2.3 1.6 0.9 0.9 0.8 1.3 0.5 1.7 1.5 1.0 1.3
indicated
and Bmtechnology, Vol 9, 1993
carbon
88 99 97 61 87 99 32 97 97 98 89 57 76 97 83 96 99 88 74 90 source.
Fusarium sp. penicillin Table 5. Effect of penicillin V acyiase (PVA).’ Inducer
(% w/v)
V (Pen
Final
V), 6-aminopeniciiianic
pH
l
In Production
7.9 a.7 a.3 a.5 Medium
I in which
and phenoxyacetic
broth
was
0.0 2.6 0.1 1.7 replaced
(POAA)
on the production
Growth (g dry ceil wt/i)
Extracellular
0.0 2.7 0.1 2.1 glucose
acid
Excretion (“/.I
PVA (IUlmi) Whole
None Pen V (0.3) 6-APA (0.17) POAA (0.1)
acid (6-APA)
with
studied (Table 5). In the absence of Pen V, 6-APA and POAA, only vegetative growth was observed; no enzyme was produced. 6-APA was a poor inducer of PVA and the specific production of the enzyme was very low compared with that in cultures with Pen V or POAA. The production of PVA was found to be highest when Pen V was used as inducer. When the concentration of Pen V in the medium was then varied from 0.1% to 1.5% (w/v), concentrations of 0.3% to 0.5% (w/v) were found to be optimum for PVA production (data not shown). Fermentation runs were also conducted in which Pen V was added prior to inoculation, 24 or 48 h after inoculation, or, in low amounts, every 24 h during the fermentation. The best results were obtained when Pen V was incorporated into the medium prior to inoculation. Effect of Surface-active Agents on PVA Production We have observed that production of PVA by Bei. indicu var. penicillanicum UREMS 5 increased by 40% to 46% when soyabean oil or olive oil was added to the medium (Ambedkar et al. 1991). However, addition of various vegetable oils and other surface-active agents, such as soyabean oil, paraffin oil, ground nut oil, white oil (liquid paraffin), palm oil, cottonseed oil, olive oil, sag 471 (silicon emulsion), Tween 80 and oleic acid, at 0.2% (v/v), did not significantly alter PVA production by Fusarium sp. SKF 235 (data not shown).
and Pen V was
MgS0,.7Hz0,
of penicillin
Specific production (NJ/g dry ceil wt)
16.2 6.2 12.0 5.0
98 a3 a0
sucrose
V acylase
replaced
with
the indicated
Pen V (K+ salt), 0.3;
0.2;
430 10 410 inducer.
pH adjusted
to
6.0.
PVA Formation During the Growth Cycle The course of PVA production, growth and pH profile during the growth cycle of Fusarium sp. SKF 235 in Production Medium II are shown in Figure 1. The pH dropped from 6.0 to 5.8 in the first 24 h and thereafter increased slowly to 8.5 at 120 h of fermentation. The initial drop in pH could be due to rapid utilization of carbohydrates. Growth was abundant during the early stages of fermentation, peaking at 13.9 g/l after 72 h and then decreasing rapidly up to 105 h. Production of PVA had a lag of 24 h, beginning once the pH rose above 7.0. Synthesis of PVA did not run parallel to cell growth; most of the PVA was synthesised during the lytic phase. The ratio of extracellular to intracellular PVA was 65 : 35 at 45 h. The accumulation of PVA in the culture filtrate increased during the fermentation and at 120 h almost all (> 95%) of the PVA produced was in the filtrate. Synthesis of penicillin acylases during the lytic phase of Pen&urn and Fusarium growth has been reported by Alfonso et al. (1989). Maximum yield of PVA was 2.4 IU/ml, corresponding to 430 IU/g dry cell wt. The production of PVA by various organisms is summarized in Table 6. Fusarium sp. SKF 235
Efiect of Initial pH on PVA Production During the initial experiments, very low amounts of PVA were produced when the final pH of the medium fell below 5.0. Therefore, the effect of initial pH on the production of PVA was studied. PVA was produced by Fusarium sp. SKF 235 when initial pH was > 4.0 and the optimum value was found to be 6.0. But. sphaericus produces PVA strictly between initial pH values of 7.5 and 8.0 (Carlson & Emborg 1981).
A medium for production by Fusaritrm sp. SKF 235 was optimised following these later results. This medium, Production Medium II, contained (% w/v): sucrose, 1.0; 1.0; NH,H,PO,, 0.15; KH,PO,, 0.15; neopeptone,
Time (h)
Figure 1. Penicillin V acylase (PVA) concentrations in the whole broth (r) and culture filtrate (a), pH (0) and cell growth (0) during the growth cycle of fusarium sp. SKF 235.
World Journal
of
Microbmiogy and Biotechnology. Vol 9, 1993
237
V.K.
Sudhakaran
Table
and ].G.
6. Comparison
Shewale
of penicillin
V acylase
(PVA)
Organism
production PVA (IUlml)
Fusarium Bacillus Bacillus
sp. SKF 235 sphaericus NCTC subtilis”
10338
Beijerinckia indica var. penicillanicum Escherichia co/i HB lOl* Escherichia coli RRI (pOH 50)’ Fusarium avenaceum Fusarfum oxysporum Pleurotus ostreatus NRRL 3824 Pseudomonas acidovorans isolate E
UREMS-5
by various
organlsms.
Specific productlon (ItJIg dry cell wt)
2.4 0.1 NA
430 NA 410
2.2 NA NA NA 0.6 NA
244 110
3200 10
50 120 NA
0.7
Reference
Carlson & Emborg 1981 Olsson et al. 1985 Gattenbeck et a/. 1986 Ambedkar et a/. 1991 Olsson et al. 1985 Olsson & Uhlen 1986 Vanderhaeghe eta/. 1968 Su & Wang 1989 Stoppock & Wagner 1983 Lowe et a/. 1981
‘Recombinant strain. NA-Not available.
produced high levels of PVA and its specific production was higher than that of other, non-recombinant isolates; Escherichia co/i RR I (pOH SO), which produces 3200 IU/g dry cell wt of PVA, is a recombinant strain (Olsson & Uhlen 1986).
Acknowledgements We thank Dr S.R. Naik, General Manager, R & D, for his encouragement during this work and U.V. Gaikwad for his help in isolation of the culture.
References Alfonso,
C.,
Cribeiro,
L.
&
Reyes,
F.
1989
Penicillin
amidohydrolases in fungal autolysis. Microbiology rind Immunology 33, 69-74. Ambedkar, S.S.,Deshpande, B.S.,Sudhakaran, V.K. & Shewale, J.G. 1991 Beijerinckia indica var. penicillunicum penicillin V acylase: enhanced enzyme production by catabolite repression resistant mutant and effect of solvents on enzyme activity. Journal of Industrial
Microbiology
7, 209214.
Borkar, P.S., Vinze, V.L. & Ambedkar, S.S. 1966 Penicillin amidase. I. Factors influencing the induced formation of penicillin amidase in Fusuriwn sp, Hindusfun Anfibiofics Bulletin 9, 1-9. Carlson, F. & Emborg, C. 1981 Bacillus sphaerictls V penicillin acylase. 1. Fermentation. Biotechnology Letters 3, 375-378. Carlson, F. & Emborg, C. 1982 Bacillus sphuericus V penicillin acylase. II. Isolation and characterisation. Io~mal of Chemirai Technology and Biotechnology 32, 808-811. Diers, I.V. & Emborg, C. 1979 Penicillin V acylase. British UK Patent 2,021,119. Gattenbeck, S., Nilsson, B., Olsson, A. & Uhlen, M. 1986 Recombinant DNA molecule, transformed microorganism and process for producing penicillin V amidase. International Patent
86 00 929. Lowe, D.A., acylases-A
Romancik, G. & review of existing
Elander, enzymes
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(Received
in revised
form
6 October
World ]oumal
of Microbiology
1992; accepted 76 October
1992)
and Biotechnology, Voi 9, 1993
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