Kinetic study on the production and degradation of leupeptin in streptomyces exfoliatus smf13

ELSEVIER

Journal of Biotechnology 42 (1995) 35-44

Kinetic study on the production and degradation of leupeptin in Streptomyces exfoliatus SMF13 In Seop Kim, Kye Joon Lee

*

Department of Microbiology, College of Natural Science, Research Center for Molecular Microbiology, Seoul National University, Seoul 151-742, South Korea

Received 8 February 1994; revised 3 February 1995; accepted 6 April 1995

Abstract Medium

composition

and cultivation

conditions

were constructed

for the optimum

production

of leupeptin

by

exfolkztusSMF13. The production of leupeptin was related to mycelial growth, being optimum in the cultivation with glucose-excess, phosphate-limited, and casamino acids medium. However, leupeptin-inactivating enzyme (LIE) was produced in the cultivation with glucose-limited, phosphate-excess, and Na-caseinate medium where mycelium degradation was accompanied. LIE was one of the most important factors in optimizing the leupeptin productivity. Kinetic parameters calculated from batch and chemostat cultivations revealed that qlpt was closely related to qs and CL,but qLIE was increased after p declined to near zero, and followed by k,. Optimum production process of leupeptin was determined with phosphate-limited continuous cultivation, which did not permit LIE production. The maximum productivity (0.24 g 1-l h-‘) and production yield (1.64 g leupeptin per g glucose) of phosphate-limited chemostat cultivation were 2.4- and 4-times larger than those of batch cultivation, respectively. This is the first cultivation kinetic analysis for leupeptin production and its inactivation by LIE in relation to mycelium differentiation. Streptomyces

Keywora?

Streptomyces

exfoliates;

Leupeptin

production;

1. Introduction A number of protease found from Streptomyces

inhibitors They

spp.

have have

been been

Abbreviations: LIE: leupeptin-inactivating enzyme; CTP: chymotrypsin-like protease; TLP: trypsin-like protease; MTP: metallo-protease; CL:specific growth rate (h-l); k,: specific mycelium degradation rate (h-l); qs: specific glucose uptake rate (g g-’ h-l); q,,t: specific leupeptin production rate (g g-’ h-‘); q LIE specific leupeptin-inactivating enzyme production rate (U g-’ h-‘) Corresponding author. l

Leupeptin

inactivating

enzyme

interesting due to the strong potential for therapeutic uses as well as their physiological roles in the producing microorganisms (Aoyagi, 1989; Umezawa, 1988). Leupeptin and its analogues were the protease inhibitors isolated from various strains of Streptomyces spp. These compounds inhibit serine proteases and thiol proteases, and may participate in a wide variety of cellular functions (Chi et al., 1989; Sonaka et al., 1984; Kim and Lee, 1995). The structures of the inhibitors are R-LeuL,eu-Arginal (leupeptin), R-Val-Val-Arginal, and

0168-1656/95/$09.50 0 1995 Elsevier Science B.V. AI1 rights reserved SSDI 0168-1656(95)00061-5

36

I.S. fim, KJ. Lee /Journal

R-Ile-lie-Arginal, where R is propionyl or acetyl (Aoyagi et al., 1969b; Kawamura et al., 1969; Kondo et al., 1969; Maeda et al., 1971). Leupeptin is nonribosomally synthesized by a three enzyme system consisting of leucine acyltransferase, leupeptin acid synthetase, and leupeptin reductase. The first step of the synthesis is the acylation of leucine by the leucine acyltransferase (Suzukake et al., 19801. Leupeptin acid synthetase is a multifunctional enzyme by which leupeptin acid (Acetyl-Leu-Leu-Arg) is synthesized in the medium containing acetyl-leucine, leucine, arginine, and ATP (Suzukake et al., 1979). Finally, leupeptin acid is reduced to leupeptin by the leupeptin acid reductase which requires ATP and NADPH (Suzukake et al., 1981). This mechanism allowed some substitution of amino acid components by structurally related ones. Some evidence of possible involvement of a plasmid in leupeptin biosynthesis and its possible transfer by conjugation have been also proposed (Umezawa et al., 1978). However, little is known about production of leupeptin and kinetic analysis of the process, although appreciable advances in the biochemical characterization of leupeptin have been made. We isolated a strain of Streptomyces exfoiiatus producing leupeptin and leupeptin analogues (Kim et al., 1992,1993X S. exfoliatus SMF13 produced extracellular proteases, such as, chymotrypsin-like protease (CTP), trypsin-like protease (TLP), metallo-protease (MTP), and leupeptin-inactivating enzyme (LIE). The physiological roles of CTP, TLP, leupeptin, and LIE were thought to be as follows: CTP participates primarily in utilization of proteinaceous nitrogen source; TLP functions as an essential enzyme involved in the metabolism of mycelium protein; leupeptin inhibits the activity of TLP; LIE inactivates leupeptin. The cascade of regulatory actions of the compounds may provide selective advantages in adverse cultivation conditions (Kim and Lee, 1995). In this report, the optimum cultivation conditions for the production of leupeptin have been evaluated in conjunction with LIE production and morphological differentiation which are affected by the medium composition. Kinetic pa-

of Biotechnology 42 (1995) 35-44

rameters of mycelial growth and leupeptin production were calculated and an optimum leupeptin production process has been constructed. This information will be useful in optimization of the production of other protease inhibitors and it provides some insight into the role of the protease inhibitors in the differentiation of Streptomyces spp.

2. Materials and methods 2.1. Microorganism and cultivation conditions The microorganism

used in this study was

Streptomyces exfoliatus SMF13 isolated from soil

(Kim et al., 1992,1993). Stock cultivation medium consisted of (w/v): 1% glucose, 0.2% casamino acids, 0.1% yeast extract, 0.1% beef extract, and 1.8% agar for solid cultivation. Seed cultivation medium contained (w/v): 3.0% glucose, 1.8% soytone, 0.3% peptone, and 0.4% CaCO,. Basal components of main cultivation medium were followed as (w/v): 0.22% KH,PO,, 0.03% MgSO,. 7H,O, 0.03% NaCl, 0.001% FeSO, * 7H,O, 0.001% CuSO, * 5H,O, 0.001% CaCl, * 2H,O, and 0.0003% MnCl, * 4H,O. Carbon and nitrogen sources were selected on the basis of leupeptin productivity as described in the text. The concentration of phosphate (KH,PO,) was varied from 0 to 0.22% for the optimum production of leupeptin or LIE. Each of the salts were separately sterilized by membrane filtration (0.2 pm; Millipore). The strain was transferred to slopes of stock cultivation medium each month, and stored at 4°C. Spores from the stock cultivations were suspended in saline solution and filtered through cotton (Hopwood et al., 1985). The spore suspension was kept at -20°C. The spore suspension was inoculated (lo6 ml-‘) to 50 ml of seed cultivation medium contained in a 500-ml baffled flask, and cultured at 28°C for 36 h in a rotary shaking incubator (200 i-pm). The seed cultivation was used to inoculate (5% v/v) 3 1 of the main cultivation medium contained in a jar reactor (5 1; Korea Fermentor Co.) for batch cultivations. Continuous cultivations were carried out using a

IS. Kim, RJ. Lee /Journal

2-1 glass reactor (Korea Fermentor, RF-2L) with a working volume of 1 1. The medium was fed into the glass vessel by a peristaltic pump (Eyela, MV-MSC 1). A steady-state condition was taken to show no significant change in any of the measured parameters over a period of at least three residence times. The cultivation temperature was maintained at 28°C and the pH was controlled at 7.0 k 0.1 using a two-way pH controller by the addition of 1 N HCl and 1 N NaOH. Agitation and aeration were 200 rpm and 1 wm, respectively. 2.2. Analytical methods Biomass was estimated as dried cell weight that was determined as follows: Mycelia were washed twice with physiological saline solution and once with distilled water, then collected by vacuum filtration (Whatman filter paper GF/C), and dried at 80°C for 24 h. The concentration of glucose was measured with dinitrosalicylic acid (Miller, 1959). The phosphate concentration was measured by the method of Pierpoint (1957). Cell-free supernatant obtained from centrifugation (10000 X g for 10 min) was used for the analysis of leupeptin and LIE. Leupeptin concentration was determined as follows: Inhibition activity of leupeptin was measured using 80 pg of papain as the target protease. The amount of leupeptin was calculated from the standard inhibition curve using leupeptin purchased from Sigma Co. (Kim and Lee, 1995). The activity of LIE was determined as follows: 1.0 ml of mycelium-free supernatant was preincubated with 50 pg of leupeptin at 4°C and pH 7.5 (Tris-HCl buffer, 0.1 M) for 10 min in order to compensate for the possible interaction between leupeptin and TLP existing in the supematant. The preincubated reaction mixture was incubated at 35°C for 10 min, then heated for 5 min at 80°C for complete inactivation of any protease and LIE in the reaction mixture. The remaining activity of leupeptin was assayed (A>. In parallel, the preincubated reaction mixture was heated at 80°C for 5 min for complete inactivation of LIE, then incubated at 35°C for 10 min. The remaining activity of leupeptin was assayed (B). The differ-

of Biotechnology 42 (1995) 35-44

37

ence between A and B was defined as the leupeptin-inactivating activity. 1 unit of LIE was defined as the amount of enzyme needed for the inactivation of 10 fig of leupeptin per min (Kim and Lee, 1995). 2.3. Analysis of cultivation kinetic parameters The growth kinetic parameters, specific growth rate (IL), specific mycelium degradation rate (k,), specific glucose uptake rate (qS), specific leupeptin production rate (qlpt), and specific leupeptin-inactivating enzyme production rate (qLIE) in batch cultivations and chemostat cultivations were analyzed as follows (Pirt, 1975): b = ln(x,/xl)/(t, - tl) and k, = -ln(x,/q)/(t, tl), where x and t are dry cell weight (g 1-l) and time (h), respectively; qS = (ds/dt)/x, where ds/dt are changes in concentration of glucose during infinitesimal time (g 1-l h-l); qlpt = (d,,,/dt)/x, where d,,,/dt is the increase in the concentration of leupeptin during infinitesimal time (g 1-l hh’); qLIE= (d,,/dt)/x, where dr,,/dt is the increase in the activity of LIE during infinitesimal time (U 1-l h-l). Curve fitting was carried out by the least-squares method programmed with FORTRAN. Batch kinetic parameters were calculated by the package with data estimated at 30-min intervals in the fitted curves. Kinetic parameters in chemostat cultivations were calculated as follows: p = D, qS = D(s, -s> x-1, where s, is the concentration of glucose in the fresh medium (g l-l), S is the steadystate concentration of glucose (g l-l), and X is the steady-state concentration of biomass (g l-l>, respectively; qlpt = D p/Z, where jI is the steadystate concentration of leupeptin (g 1-l); YX,, = X/(s, - S) and Y,pt,x =F/Z, where YX,, is growth yield (g gg’) and Y,pt,x is leupeptin production yield (g gg’), respectively. 2.4. Chemicals, reagents, and reproducibility Leupeptin and synthetic substrate were purchased from Sigma Chemical Co. All other chemicals were of reagent grade. Experiments were carried out in triplicate and the mean values are given.

38

IS. Kim, KJ. Lee /Journal

of Biotechnology 42 (1995) 35-44

‘I’llllC

(h)

Tlrnr

i h)

Fig. 1. Effect of nitrogen sources on the growth (A) and production of leupeptin (B) and leupeptin-inactivating enzyme Na-caseinate (O), 1% casitone (01, and 1% casamino acids (0) were used as the nitrogen sources in the chemically medium containing 1% glucose.

(C). 1% defined

ues at the begining of the stationary phase. Moreover, leupeptin produced in the cultivation with casamino acids was maintained at maximum value and LIE was not produced until the end of the batch cultivation. However, leupeptin produced in the cultivation with casitone or Na-caseinate was degradaded rapidly at the decline phase from which a significant amount of LIE was produced. Hence casamino acids was selected as the choice of nitrogen source for the production of leupeptin using S. exfoliates SMF13.

3. Results 3.1. Effect of nitrogen source on growth and production of leupeptin and LIE Mycelial growth and production of leupeptin and LIE in batch cultivation were varied with organic-nitrogen source, viz. Na-caseinate, casitone and casamino acids, as a sole nitrogen source (Fig. lA-C). Mycelial growth in cultivation with Na-caseinate was slightly lower and turned to decline phase without any distinct stationary phase, whereas the decline of biomass did not happen in the cultivation with casamino acids. The leupeptin productions were closely related to mycelial growth and reached their maximum val-

3.2. Effect of glucose concentration on growth and leupeptin production

Various carbohydrates were evaluated as the substrate (carbon and energy source) for the pro-

B

Tlrn~ (11) Fig. 2. Effect of glucose concentration on the growth and 2.5% ( q ) glucose were used in the chemically source.

Time

(11)

Time

(h)

(A), glucose utilization (B), and leupeptin production (00.5% CO), 1.0% CO), defined medium containing 1.0% casamino acids as the optimized nitrogen

I.S. Kim, KJ. Lee/Journal

of Biotechnology 42 (1995) 35-44

A

duction of leupeptin, from which glucose was found to support mycelial growth and also the production of leupeptin. The effect of initial concentration of glucose on mycelial growth and leupeptin production was evaluated in batch cultivations using casamino acids as the nitrogen source (Fig. 2A-C). Mycelial growth and the production of leupeptin were enhanced by increasing glucose concentration. In order to demonstrate more clearly the effects of glucose on the production of leupeptin and LIE in S. exfoliutus SMF13, the 100 mM of glucose was pulse-fed at 35 h to the cultivations using Na-caseinate as a sole nitrogen source. The production of leupeptin was induced by the addition of glucose (Fig. 3A). However, the production of LIE was repressed completely by the pulse-addition of glucose (Fig. 3B). From the results, it was very clear that glucose concentration was extremely critical for the production of

Time

(h)

C

0

20

40 Tune

60 (h)

80

0

20

40 Time

60

80

(h)

Fig. 4. Effect of phosphate concentration on glucose utilization (A), phosphate utilization (B), growth (0, and leupeptin production (D). Initial PO:- concentration was 0.002% cm), 0.014% CO), and 0.047% (0).

B o-O

leupeptin by S. exfoliates SMF13 and that the production of leupeptin was inversely related to the production of LIE. 3.3. Effect of phospha te concentration and aeration on growth and leupeptin production

0

20

40

60

80

‘rime (h) Fig. 3. Effect of addition of glucose on the production of leupeptin (A) and leupeptin-inactivating enzyme (B) in batch cultivations of S. afoliati SMF13. Culture were grown in a chemically defined medium containing 0.5% glucose and Nacaseinate as the sole nitrogen source. 100 mM of glucose (0) was added to the cultures at early stationary phase (1). Control cultivations were added with saline (0).

The effect of inorganic phosphate on growth and leupeptin produetion was evaluated (Fig. 4). The consumption of glucose was clearly regulated by the initial concentration of phosphate (Fig. 4A). Phosphate-limited cultivation was evident when the initial concentration of PO:- was 0.02 g 1-l (Fig. 4B), where mycelial growth was limited (Fig. 4C). However, the production of leupeptin was remarkably increased by the limitation of phosphate from the early part of the batch cultivation (Fig. 4D). From the results, it was concluded that phosphate is a major negative factor for the over-production of leupeptin.

40

1.X Kim, &I. Lee /Journal

of Biotechnology

42 (1995) 35-44

A

Fig, 5. Effect of aeration

on

growth (A), glucose utilization (B) and leupeptin production

(0.

0.1 wm (Ok

0.5 wm (ok and 1.0 -

(0).

The effect of aeration on growth and leupeptin production was evaluated with optimized nutrient condition. As shown in Fig. 5A-C, both mycelial growth and leupeptin production were enhanced with increasing aeration rate. 3.4. Kinetics of growth, substrate uptake and production of leupeptin in batch cultivations Leupeptin production was low in the cultivation with glucose-limited, phosphate-excess, and Na-caseinate medium. In this cultivation condi-

tion, physiological differentiation (viz. production of leupeptin and LIE) and morphological differentiation (viz. lysis of mycelium) were evident. Mycelial growth turned to stationary phase and immediately to death phase when glucose was completely utilized, where LIE started to be produced. Leupeptin produced during mycelial growth was inactivated with the rise of LIE activity (Fig. 6A). Leupeptin production was optimum in the cultivation with glucose-excess, phosphate-limited, and casamino acids medium (Fig. 6B). Leupeptin

T1m.e(h) Fig. 6. Comparison of batch cultivations with glucose-limited, phosphate-excess, and Na-caseinate medium (A) and glucose-excess, phosphate-limited, and casamino acids medium (B). 0.5% glucose, 0.047% PO:-, and 1% Na-caseinate (A) and 2.5% glucose, 0.002% PO:-, and 1% casamino acids (B). Mycelium growth (a), residual glucose CO), leupeptin (m ), and LIE (0 ).

IS. Kim, RJ. Lee /Journal

production was very closely associated with mycelial growth, but ceased when the growth was turned to stationary phase. Leupeptin was maintained throughout stationary phase where the residual glucose concentration was high and LIE was not produced. Also, lysis of mycelium did not occur. The changes in specific growth rate (CL), specific mycelium degradation rate (k,), specific glucose uptake rate (qJ, specific leupeptin production rate (qlpt), and specific LIE production rate (qLIE) calculated from both cultivations are compared in Fig. 7A and B. It was clear that II, qs and q,,t in both cultivations were changed with similar patterns. In the cultivation with glucoselimited, phosphate-excess and Na-caseinate medium, qlpt very rapidly declined with the increase in the qLIE and the changes in qLIE were followed by an increase in k, (Fig. 7A). Higher values of qlpt and qS were obtained from the cultivation with glucose-excess, phosphate-limited and casamino acids medium, although p was much lower (Fig. 7B). Biomass growth yield (YX,,) and leupeptin production yield (Y,pt,,> to the glucose consump-

of Biotechnology 42 (1995) 35-44

Table 1 Kinetic parameters for S. exfoliatus SMF13 batch cultivations with glucose-limited, phosphate-excess, and Na-caseinate medium (A), and glucose-excess, phophosphate-limited, and casamino acids medium (B) Kinetic parameters

A

B

Y, ,S (g biomass per g glucose) Y,pt,s (g leupeptin per g glucose) YIDt,X(g leupeptin per g biomass)

1.000 0.196 0.196

0.315 0.467 1.482

tion are compared in Table 1. A very high value of 5,s was obtained in the cultivation of glucose-limited, phosphate-excess, and Na-caseinate medium. The yield was about 3-times higher than that obtained in the cultivation of glucose-excess, phosphate-limited, and casamino acids medium. The data indicated that Na-caseinate was used not only as a nitrogen source but also as carbon and energy source when glucose was limiting. Also, the declination of mycelium after the staionary phase might have resulted from the endogenous metabolism in the cultivation condition of carbon and energy limitation. However, it is still very interesting that a remarkably high

B

A

c

41

_I0.4

0.3 5 b 7 w s 2

0.2

01

00 0

20

40 60 Time (h)

80

0

20

40 60 Time (h)

80

Fig. 7. Changes of kinetic parameters such as specific growth rate (P, . . . . . ), specific mycelium degradation rate (k,, --1, specific glucose uptake rate (qs, -_), specific leupeptin production rate (q$,, -. . -), and specific LIE production rate (qLIE, -. -1 during the batch cultures shown in Fig. 6.

of Biotechnology 42 (1995) 35-44

I.S. Kim, XJ. Lee/Journal

42

20

~ 0 12 r

'

0 09

.c

/,, id009 ?;'

.c

00

0 16

=

o0000

0025

0050

n-

0

J

0 00

0

0 075 0 100

00

t

1

.-L--A

0 000 0025

Dllutlon rate (h-l)

0

Dllutlon rate (h-l)

Fig. 8. Phosphate-limited chemostat cultivation for the production of leupeptin. (A) Residual phosphate (0 ), and leupeptin production ( n 1. (B) Specific glucose uptake rate ( A ) and specific Productivity of leupeptin (0) and biomass (*I. (D) Y,nt,,, (0) and Y,pt,x ( w).

between

batch

cultivation

glucose (01, growth (01, residual leupeptin production rate ( A ). (C)

3.5. Kinetics of growth, substrate uptake and production of leupeptin in phosphate-limited chemostat

value of leupeptin production yield was obtained in the glucose-excess, phosphate-limited, and casamino acids medium. Leupeptin production yield (YPt,X 1 to the biomass production in the optimum cultivation for leupeptin was 7.5-times higher than that obtained from the cultivation with glucose-limited, phosphate-excess, and Nacaseinate medium. Therefore, it was concluded that leupeptin production was enhanced with high concentration of glucose and low concentration of phosphate. And leupeptin produced with mycelial growth was inactivated by LIE when carbon and nitrogen sources were limiting to mycelial growth.

Table 2 Comparison of optimized results leupeptin in S. exfoliates SMF13

00

0 050 0 075 0 100

In order to optimize the leupeptin production and to evaluate the production kinetic parameters, phosphate-limited chemostat cultivations with glucose-excess and casamino acids medium were carried out. Steady-state residual concentration of phosphate was undetectable up to dilution rate of 0.075 h-’ where the steady-state concentration of mycelium was relatively constant value of 2.1 g l- ‘. Wash out of biomass occurred at the dilution rate of 0.1 hh’. Leupeptin production

and phosphated-limited

continuous

cultivation

Kinetic parameters

Batch culture

Continuous

Maximum concentration of leupeptin (g I-‘) Leupeptin production yield, Y, t,s (g gg ‘) Productivity of leupeptin (g l-ph-r)

4.30 0.47 0.10

3.20 1.64 0.24

for the production

culture

of

1.X Kim, K.J. Lee /Journal

of Biotechnology 42 (1995) 35-44

increased with increase in dilution rate and maximum value of leupeptin was obtained at 0.075 h- ’ (Fig. 8A). The specific leupeptin production rate was increased linearly with the increase of dilution rate up to 0.075 h-‘, although the specific glucose uptake rate was increased steadily to the critical dilution rate (Fig. 8B). Maximum productivity of biomass and leupeptin was evident at 0.075 hh’ (Fig. 80. The leupeptin production yield (Y,pt,s and Yipt,x ) increased with the increase of dilution rate (Fig. 8D). Therefore it was concluded that the optimum dilution rate for leupeptin production in the phosphate-limited chemostat cultivation was 0.075 hh’. 4. Discussion Medium composition was very critical for the production of leupeptin, glucose and casamino acids being selected as the choice of carbon and nitrogen source, respectively. Leupeptin production was related to mycelial growth and that was enhanced by higher concentration of glucose and higher aeration but limitation of phosphate. Leupeptin production was remarkably stimulated at the phosphate-limited cultivation condition, although mycelial growth was severely retarded. The data indicated that the biosynthesis of leupeptin was negatively regulated by phosphate and that the rates of mycelial growth and glucose consumption would not be directly related to the production of leupeptin. Production of trypsin inhibitor from Cephalosporium sp. KM388 was also related to cell growth and glucose concentration was a critical factor for maintaining the activity of inhibitor at the stationary phase. The production rate and the maximum yield of the inhibitor were increased when the temperature and pH were controlled to produce high growth. However, the glucose effect on maintaining the activity of inhibitor at the stationary phase was not elucidated (Tsuchiya and Kimura, 1977). Production of proteinaceous protease inhibitor from Streptomyces fradiae was also related to the cell growth and regulated by aeration rate (Chung et al., 1990; Lee et al., 1990).

43

It was interesting to notice that leupeptin accumulated during mycelial growth was maintained throughout stationary phase and LIE was not produced in the glucose-excess condition. However, leupeptin was inactivated by LIE which was produced in the condition of glucose exhaustion and declination of mycelium. In a previous report, it was found that production and inactivation of leupeptin and the production of LIE in batch cultivation were closely related to the mycelium morphological differentiation. Production of leupeptin in surface cultivation was associated with substrate mycelium growth and it was rapidly inactivated before aerial mycelium formation. The addition of leupeptin retarded aerial mycelium formation in surface cultivation and reduced mycelium degradation rate in the submerged cultivation of S. exfoliatus SMF13 Kim and Lee, 1995). Therefore, understanding the relationships between the biosynthetic regulation of leupeptin and mycelium differentiation is a key factor for the construction of optimum production of leupeptin. From the analysis of cultivation kinetic parameters of batch cultivations, it was concluded that qlpt was extended longer with the higher value of qs in the glucose-excess and phosphate-limited cultivation condition and that the qLIE was closely related to k, in the glucose-limited condition. A cultivation system for the optimum production of leupeptin was constructed with phosphate-limited batch and chemostat cultivations, from which a very high productivity of leupeptin was obtained with high yield and high concentration of leupeptin (Table 2). The maximum concentration of leupeptin produced by S. exfoliatus SMF13 in batch cultivation was 4-times higher than the previous report where the maximum concentration of leupeptin produced by Streptomyces roseus was 1 g 1-l (Aoyagi et al., 1969a). The productivity and leupeptin production yield (Y,pt,s) of phosphate-limited chemostat cultivation were 2.4and 4-times larger than those of batch cultivation, respectively. Therefore it was concluded that the phosphate-limited chemostat cultivation was a very useful system for the production of leupeptin in S. exfoliates SMF13. This is the first cultivation kinetic analysis for leupeptin production and its

44

IS. Kim, K.J. Lee /Journal

inactivation by LIE in relation differentiation.

with mycelium

Acknowledgements

This work was supported by a research grant from the Research Center for Molecular Microbiology (RCMM) sponsored by the Korea Science and Engineering Foundation (KOSEF).

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