Production by Clonostachys compactiuscula of a lovastatin esterase that converts lovastatin to monacolin J

Enzyme and Microbial Technology 39 (2006) 1051–1059

Production by Clonostachys compactiuscula of a lovastatin esterase that converts lovastatin to monacolin J Lin-Cheng Chen a , Yiu-Kay Lai a , Suh-Chin Wu a , Chih-Chien Lin a , Jia-Hsin Guo b,∗ a

b

Department of Life Science and Institute of Biotechnology, National Tsing Hua University, Hsinshu 30013, Taiwan, ROC Department of Food Science, National Pingtung University of Science and Technology, No. 1, Shuehfu Road, Neipu, Pingtung 91201, Taiwan, ROC Received 16 November 2005; received in revised form 2 February 2006; accepted 7 February 2006

Abstract The conversion of monacolin K (lovastatin) to monacolin J, a core structure in the synthesis of other statins, was achieved using the fungus Clonostachys compactiuscula and optimized with response surface methodology. To study the proposed second-order polynomial model, a central composite experimental design with multiple linear regression was used to estimate the model coefficients of five selected factors believed to influence the conversion process. The experimental results indicated that the optimal conditions for growth of C. compactiuscula mycelium were as follows: 2.0 g glucose/L, initial pH of the medium 8.5, and incubation of the mycelium for 4 days. These conditions yielded an optimal concentration of the substrate lovastatin of 1 mg/mL, and a conversion time of 15 h. A lovastatin esterase was isolated and purified from the mycelium of C. compactiuscula by ammonium sulfate precipitation, size-exclusion chromatography, and ion-exchange chromatography. Following SDS-PAGE, the purified enzyme appeared as a single band with an apparent molecular mass of 28 kDa. The converted product, monacolin J, was isolated and purified, and its structure was analyzed by NMR. © 2006 Elsevier Inc. All rights reserved. Keywords: Clonostachys compactiuscula; Hypercholesterolemia; Lovastatin; Monacolin esterase; Response surface methodology

1. Introduction The endogenous synthesis of cholesterol is carried out by the mevalonate pathway, in which the rate-limiting reaction, the conversion of (S)-HMG-CoA to (R)-mevalonate, is catalyzed by 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase (EC. 1.1.1.34). The series of drugs referred to as statins are often prescribed to control hypercholesterolemia. All statins function similarly, i.e., by binding to the active site of HMGCoA reductase, thereby inhibiting cholesterol biosynthesis and causing a marked reduction of serum cholesterol levels [1,2]. The statin lovastatin [3–6], known as monacolin K, is administered as the pharmacologically active lactone form, which has the chemical structure 1 ,2 ,6 ,7 ,8a -hexahydro-3,5-dihydroxy2 ,6 -dimethyl-8 -2 -methyl-1 -oxobutoxy)-1-naphtalene heptanoic acid-5-lactone [7]. Schimmel and Borneman [8] reported that an enzyme produced by the fungus Clonostachys compactiuscula selectively hydrolyzes the 2-methylbutyryloxy side-

∗

Corresponding author. Tel.: +886 8 770 3202x7449; fax: +886 8 7740378. E-mail address: [email protected] (J.-H. Guo).

0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2006.02.018

chain of lovastatin to generate monacolin J and methyl butyrate. This enzyme was named lovastatin esterase, and its occurrence in nature was also described by Komagata et al. [9], who purified the enzyme from dried mycelia of the fungus Emericella unguis. Monacolin J [8] has also been shown to inhibit HMG-CoA reductase [8–10] and thus may also be useful as a cholesterollowering agent [10]. The compound is also potentially valuable as an intermediate in the synthesis of other, semi-synthetic HMG-CoA reductase inhibitors [8,11–13]. For example, monacolin J could serve as a core molecule that could be variously modified to generate different, and possibly novel, HMG-CoA reductase inhibitors [8,11,14]. Thus, the enzymatic hydrolysis of lovastatin, and the subsequent production of monacolin J may have a large number of important therapeutic applications. In a preliminary study, four strains of mold capable of hydrolyzing lovastatin to form monacolin J were examined. Of these, C. compactiuscula strain BCRC 33575, had the highest hydrolytic activity and was therefore chosen for further study. The growth conditions that yielded a cell mass with a high level of lovastatin hydroxylation activity were determined. Enzyme activity was measured by assaying monacolin J production in vegetative-phase mycelia. To further maximize the enzyme

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activity of the mycelia, a second-order polynomial model of response surface methodology (RSM) was applied to determine the optimal conditions for conversion of the substrate, lovastatin ammonium salt, to monacolin J. The lovastatin esterase produced under these conditions was purified, and the properties and structure of the product, monacolin J, obtained in lactone form, were analyzed by HPLC and NMR. Our results will be useful in establishing a comprehensive conversion system for monacolin J production and may provide a foundation for further development of better anti-hypercholesterolemia drugs. 2. Materials and methods 2.1. Chemicals The anti-hypercholesterolemia drug lovastatin was purchased from Yung Shin Pharmaceutical Industrial (Taichung, Taiwan, R.O.C.). Glucose and corn steep liquor were purchased from Sigma–Aldrich (St. Louis, MO); yeast extract, malt extract, beef extract, peptone, methanol, Tris base, Tris hydrochloride, and NaCl were from Difco Laboratories (Detroit, MI); and agar powder was obtained from Amresco (Solon, OH). Chemical-grade cyclohexane, n-hexane, and phosphoric acid were purchased from J.T. Baker (Phillipsburg, NJ). Ethyl acetate and acetonitrile (HPLC grade) were from Mallinckrodt (Hazelwood, MO); methanol, sodium hydroxide, hydrogen chloride and other chemicals (chemical grade) were from Sigma–Aldrich. Isopropyl alcohol was obtained from Nihon Shiyaku Industries (Tokyo, Japan), ammonium sulfate from Merck (Whitehouse Station, NJ), and Celluclast from TRUMP Chemical (Taipei, Taiwan, R.O.C.).

2.2. Maintenance of C. compactiuscula and fermentation conditions Clonostachys compactiuscula strain BCRC 33575 was purchased from the Bioresources Collection and Research Center (Food Industry Research and Development Institute, Taiwan, R.O.C.). The fungus was maintained on yeast extract-malt extract (YME) agar, containing 4 g yeast extract/L, 10 g malt extract/L, 4 g glucose/L, and 20 g agar (pH 7.0)/L. Freshly inoculated cultures were incubated at 28 ◦ C for 5 days, after which stock cultures were kept at 4 ◦ C and transferred to fresh medium monthly. C. compactiuscula strain BCRC 33575 was grown in liquid medium by inoculating one loop of stock culture into a 500-mL Erlenmeyer flask containing 50 mL of yeast extract–beef extract (YBE) growth medium (containing 10 g glucose/L, 2 g peptone/L, 1 g beef extract/L, 1 g yeast extract/L, and 3 g corn steep liquor/L, pH 7.2) and incubating the culture at 28 ◦ C on a rotary shaker at 220 rpm. Lovastatin esterase activity was induced by the addition of lovastatin ammonium salt (LAS) to each flask to a final concentration of 0.5 mg/mL. The culture was then allowed to incubate for another day before it was harvested. Growth curves of the culture were obtained by harvesting four culturecontaining flasks daily, beginning at day one. The wet and dry weights of the growing mycelium of each flask were measured. The wet weight of the mycelium was determined by vacuum-filtering the culture through a paper filter and weighing the mycelium, which remained on the filter. The mycelium was then dried in an oven at 60 ◦ C for one day and the dry weight was measured.

2.3. Conversion of lovastatin to LAS Since the lactone form of lovastatin is water-insoluble, the water-soluble sodium form was prepared by dissolving 50 g lovastatin (lactone form) in 900 mL cyclohexane containing 9.92 g NaOH, 49.6 mL water, and 240 mL isopropyl alcohol. The mixture was stirred at room temperature for about 12 h, after which the organic solvents were removed by vacuum concentration. Water was then added up to 1.5 L and the mixture stirred at room temperature for another 12 h. The pH of the solution was adjusted to 4.0 by the addition of 4 M H3 PO4 , 1.5 L of a mixture of n-hexane and ethyl acetate (1:2; v/v) was added, and the sample was stirred again for about 12 h. After centrifugation at 12,000 rpm for 30 min, the upper organic level was collected and purged with ammonia gas (Toyo Gas,

Taichung, Taiwan). The resulting product, LAS, was then crystallized as a white precipitate, filtered, and vacuum-dried for experimental use.

2.4. Statistical procedure Response surface methodology (RSM) was used to optimize the enzymatic conversion of lovastatin to monacolin J. Five experimental factors capable of influencing the enzymatic conversion ratios of lovastatin to monacolin J were selected for analysis: initial glucose concentration of the medium, initial pH of the medium, culture incubation time, LAS converting time, and added LAS concentration. For the first three experimental factors, conditions were chosen so as to avoid inhibition of either microbial growth or the bioconversion process. The LAS concentration and the conversion times were chosen based on practical considerations. The different experimental levels used in this study are listed in Table 1. For each factor, five levels are given, consistent with a second-order model. For this study, the model comprised 16 experiments organized in a 25–1 fractional factorial design. Second-order coefficients were assessed by 10 “star” points (one factor being at level −2 or +2 and all the others maintained at the zero level) and 10 replications of the central point (all factors at the zero level). A total of 36 experiments were necessary for the estimation of the model.

2.5. The effect of various concentrations of LAS on its enzymatic conversion to monacolin J by C. compactiuscula mycelial extract It was previously reported that lovastatin esterase activity may be hindered by a high concentration of lovastatin [8]. In order to determine the lovastatin concentration yielding the highest amount of lovastatin esterase activity in C. compactiuscula mycelial extract, various concentrations of LAS were tested. C. compactiuscula strain BCRC 33575 mycelium (4-days post-inoculation) was harvested by vacuum filtration, washed three times with 50 mM Tris buffer (pH 8.5), frozen in liquid nitrogen, and ground using a mortar and pestle. The ground mycelium was then suspended in 50 mM Tris buffer (pH 8.5), centrifuged at 8000 rpm, and the supernatant was collected. Enzymatic conversion tests were conducted as follows: various concentrations of LAS (final concentration range: 0.1–10 g/L) were prepared in 60 ␮L of 50 mM Tris buffer (pH 8.5) containing 6 ␮L methanol. After the addition of 50 ␮L of crude enzyme supernatant, the mixture was incubated at 28 ◦ C for 60 min. The conversion of LAS to monacolin J was quantified by isocratic reverse-phase chromatography. The HPLC set-up consisted of a Pecosphere octyldecyl silane column with a column length of 3.3 cm and particle size of 3 mm (Perkin-Elmer, Norwalk, CT). The mobile phase of aqueous phosphoric acid (1 g/L) and acetonitrile at a ratio of 50:50 (v/v) was kept at a constant flow rate of 1 mL/min. The concentrations of LAS and monacolin J were determined spectrophotometrically at 238 nm.

2.6. Preparation of mycelial extract for enzyme purification C. compactiuscula strain CCRC 33575 mycelium was harvested from a 4day-old flask fermentation by centrifugation at 8000 rpm for 15 min at 4 ◦ C. The Table 1 Boundaries of the experimental domain and spacing of levels, expressed in coded and natural unitsa Code

Glucose (%, w/v)

pH

Incubation time (days)

Conversion time (h)

LAS concentration (mg/mL)

−2 −1 0 1 2

0.50 0.75 1.00 1.25 1.50

5.2 6.2 7.2 8.2 9.2

3.0 3.5 4.0 4.5 5.0

4 8 12 16 20

1 2


Xb

0.25

1.0

0.5

4

1

a

4 5

For a description of the coded and natural values, please refer to the text.
X is the increment of the experimental factor natural values, corresponding to one unit of the coded variable. b

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mycelium was washed three times with 50 mM Tris buffer (pH 8.5), homogenized over liquid nitrogen in a pre-chilled mortar, and ground with a ceramic pestle. The frozen homogenate was suspended in 50 mM Tris buffer (pH 8.5), after which 0.1 mL Celluclast and Triton X-100 to a final concentration of 25 g/L were added. The suspension was incubated at 28 ◦ C for 30 min, homogenized for 10 min over an ice bath using a Polytron at 20,000 rpm, and then centrifuged at 6000 rpm for 30 min at 4 ◦ C to remove cell debris. The supernatant, containing crude lovastatin esterase, was collected and filtered through a 0.22-␮m pore-size filter. A series of ammonium sulfate gradients (range 10–70%, with 10% increments in saturation) was conducted at 0 ◦ C for salt-out precipitation of various proteins in the mycelium homogenate. Each cycle of ammonium sulfate precipitation was followed by centrifugation at 20,000 rpm for 15 min at 4 ◦ C using a Beckman L8-70M ultracentrifuge with a SW41 rotor. The pellets obtained after each precipitation were suspended in 50 mM Tris buffer (pH 8.5) and assayed for lovastatin esterase activity. The pellet found to contain lovastatin esterase activity was saved for enzyme purification.

residual amount of monacolin K, was collected after centrifugation of the mixture at 12,000 rpm for 30 min. Since monacolin J is slightly more hydrophilic than monacolin K, phosphate buffer (pH 5.0) was added and the mixture was stirred for about 12 h at room temperature to extract monacolin J. Again, the mixture was centrifuged at 12,000 rpm for 30 min. The lower level (aqueous phase), which contained most of the monacolin J, was collected and applied to a C-18 column. Phosphoric acid (1 g/L) and acetonitrile, at a ratio of 50/50 (v/v), was used to elute monacolin J. About 120 mL of monacolin J solution was collected and then freeze-dried. The freeze-dried monacolin J was dissolved in 200 ␮L methanol and then further purified by preparative thin layer chromatography (TLC) using 60 F254 precoated silica-gel plates (E. Merck, Germany) and a mobile phase of methanol:ethyl acetate at a ratio of 2:8 (v/v). Spots on the TLC gel that contained pure monacolin J were scraped off and was extracted with methanol. The supernatant of the extraction was subjected to filtration through a 0.22-␮m membrane and then freeze-dried for analysis by NMR spectroscopy.

2.7. Purification of lovastatin esterase

2.11. NMR spectra

The crude lovastatin esterase fraction was applied onto a Superdex 75 pg size-exclusion column (Pharmacia, Piscataway, NJ.) and washed with 150 mM sodium chloride in 20 mM Tris buffer (pH 8.5) using fast protein liquid chromatography (FPLC). The FPLC, column, and fraction collector were kept at 4 ◦ C. Fractions were collected at a flow rate of 1 mL/min and assayed for lovastatin esterase activity. Fractions containing enzyme activity were combined, concentrated to 3 mL in an Amicon protein concentrator (Centrplus YM-20, 20-kDa cutoff), and then subjected to MonoQ anion-exchange column chromatography (Pharmacia) by FPLC. Lovastatin esterase was eluted from the column with a linear gradient of 0–800 mM sodium chloride in 20 mM Tris buffer (pH 8.5) at a flow rate of 1 mL/min.

Purified monacolin J or lovastatin was dissolved in 1 mL D-methanol and subjected to NMR spectroscopy. 1 H NMR spectra were recorded on a Varian Inova 600 spectrometer located at the Instrument Center of National Chung Hsing University, Taichung, Taiwan, R.O.C.

2.8. Lovastatin esterase assay Lovastatin esterase activity was measured in a reaction containing 50 mM Tris buffer (pH 8.5), 100 mg methanol/mL, 5 mg LAS/mL, 50 ␮L of purified enzyme, and water to a final volume of 110 ␮L. The mixture was incubated for 1 h at 28 ◦ C. Enzyme activity of C. compactiuscula mycelium or in the culture broth was determined as described above, with a known amount of mycelium or a known volume of whole broth substituted for the purified enzyme. Any other variation to these standard conditions is described in the Results. The conversion of LAS to monacolin J was quantified by isocratic reverse-phase HPLC using a Pecosphere octyldecyl silane column with a 3.3-cm column length and 3mm particle size (Perkin-Elmer). The mobile phase of aqueous phosphoric acid (1 g/L) and acetonitrile, at a ratio of 50/50 (v/v), was kept constant at a flow rate of 1 mL/min. The concentrations of LAS and monacolin J were measured spectrophotometrically at 238 nm.

3. Results and discussion 3.1. Maintenance and fermentation of C. compactiuscula BCRC 33575 C. compactiuscula strain BCRC 33575 was routinely cultured on YME agar. The mycelium initially showed a light yellow color. After 5 days of incubation, the surface of the culture was fully covered with powder-like spores with a snow-white color. For fermentation experiments, one loop of the culture was inoculated into YBE growth medium, and the culture was incubated at 28 ◦ C on a rotary shaker at 220 rpm until light-yellow pellets floating in the medium were obtained. According to the literature [8], lovastatin esterase activity in C. compactiuscula is enhanced by adding LAS as an inducer. Similarly, the addition of lovastatin or lovastatin-related compounds, such as simvastatin, to the culture medium of C. com-

2.9. Gel electrophoresis The purity and apparent molecular mass of lovastatin esterase were determined by SDS-PAGE. The purified enzyme preparation was separated in a 12.5% acrylamide (w/v) separating gel and 4.75% acrylamide (w/v) stacking gel in SDS electrophoresis buffer on a Bio-Rad Mini-PROTEIN II electrophoresis cell. Protein standards purchased from Sigma were used to determine the molecular mass. The gel was subjected to silver staining after electrophoresis as described by Nesterenko et al. [15].

2.10. Production and isolation of monacolin J Conversion of LAS to monacolin J was carried out in a reaction containing 50 mM Tris buffer (pH 8.5), purified lovastatin esterase, 20 mL methanol, and 2 g LAS in a total volume of 200 mL. The reaction was incubated at 28 ◦ C for 24 h. At the end of the reaction, monacolin J and residual LAS were converted to their lactone forms by adding 4 M H3 PO4 to bring the pH below 5.0. Subsequently, 150 mL of n-hexane:ethyl acetate at a ratio of 1:2 (v/v) was added to the final reaction and the mixture was stirred for about 12 h at room temperature. The upper level, which contained mostly the lactone form of monacolin J but also a

Fig. 1. Growth curve of Clonostachys compactiuscula strain BCRC 33575. Diamonds (
or ♦) and squares ( or ) indicate wet and dry weights of mycelia, respectively. The solid lines (and the solid symbols) and the dashed lines (and the empty symbols) indicate, respectively, in the absence or presence of LAS. The data points and the vertical bars show the means and SDs of four duplicates, respectively.

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Fig. 2. Factorial analyses of lovastatin esterase production. The weight of each factor influencing lovastatin esterase production was ranked after factorial analyses.

pactiuscula was required to induce enzyme expression [8]. To determine the optimal time point for adding LAS, a growth curve of C. compactiuscula in culture was obtained by measuring the wet and the dry weights of the mycelia everyday during the incubation period. As shown in Fig. 1, cell growth entered exponential phase after 24 h of incubation. The dry weight of the mycelial mass was maximum after 4 days of incubation and began to decrease at day 5 of incubation. Thus, LAS was added to the culture at day 3 of incubation, i.e., one day before the culture reached the end of the exponential phase of growth. However, we found that, when LAS was added the mycelia stopped growing, and there was no additional induction of lovastatin esterase activity, in contrast to a previous report [8]. Our results suggest that the production of lovastatin esterase by C. compactiuscula correlates with the amount of mycelial mass. In subsequent experiments, maximal growth of mycelia and high amounts of lovastatin esterase were obtained by culturing C. compactiuscula for about 4 days without the addition of LAS, unless otherwise stated.

Table 2 Central composite design consisting of 36 experiments for the study of five experimental factors Run no.

Glucose (%, w/v)

pH

Incubation time (days)

Hydrolytic time (h)

LAS concentration (%, w/v)

Production of monacolin J (␮M)

Conversion ratio (%, w/w)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

−1 1 −1 1 −1 1 −1 1 −1 1 −1 1 −1 1 −1 1

−1 −1 1 1 −1 −1 1 1 −1 −1 1 1 −1 −1 1 1

−1 −1 −1 −1 1 1 1 1 −1 −1 −1 −1 1 1 1 1

−1 −1 −1 −1 −1 −1 −1 −1 1 1 1 1 1 1 1 1

−1 1 1 −1 1 −1 −1 1 1 −1 −1 1 −1 1 1 −1

171.61 71.80 391.34 223.68 344.40 243.80 826.08 46.08 940.88 1439.14 230.78 59.57 1521.19 701.82 982.31 453.28

7.25 1.52 8.27 9.45 7.28 10.30 34.91 0.97 19.88 60.81 9.75 1.26 64.28 14.83 20.75 19.15

17 18 19 20 21 22 23 24 25 26

−2 2 0 0 0 0 0 0 0 0

0 0 −2 2 0 0 0 0 0 0

0 0 0 0 −2 2 0 0 0 0

0 0 0 0 0 0 −2 2 0 0

0 0 0 0 0 0 0 0 −2 2

2844.35 44.97 36.85 3324.85 35.58 523.11 814.25 39.84 22.41 882.89

80.13 1.27 1.04 93.66 1.00 14.74 22.94 1.12 1.89 14.92

27 28 29 30 31 32 33 34 35 36

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

1639.15 51.92 35.74 130.58 475.77 42.05 160.96 323.10 333.75 2667.61

46.18 1.46 1.01 3.68 13.40 1.18 4.53 9.10 9.40 75.15

Conversion ratio (%, w/w) = (converted monacolin J/initial LAS) × 100%.

L.-C. Chen et al. / Enzyme and Microbial Technology 39 (2006) 1051–1059

3.2. Optimization of lovastatin esterase production and enzymatic conversion using RSM The conversion of lovastatin to monacolin J was demonstrated by the addition of LAS to cultures of C. compactiuscula at the time of harvest. For RSM analysis, the boundaries of the experimental domain and the spacing of levels for glucose, pH, incubation time, conversion time, and LAS concentration were established (Table 1). The actual values of the five experimental

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factors were assigned code numbers (−2, −1, 0, 1, and 2) for purposes of the study. The amount of monacolin J conversion resulting from each experiment was determined in order to evaluate the catalytic capability of lovastatin esterase. In addition, the concentrations of residual LAS and of monacolin J produced were measured by HPLC analysis. The ratio of monacolin J to lovastatin for each of the 36 experiments is presented in Table 2. The goodness of fit of the experimental design was checked with different weighted and ranked factors (Fig. 2), with pH and

Fig. 3. Surface response of lovastatin esterase production. (A) Glucose concentration in the medium vs. media pH; (B) glucose concentration in the medium vs. culture incubation time; (C) culture time vs. medium pH; (D) hydrolytic time of the mycelia vs. medium pH; (E) growing time of the mycelia vs. hydrolytic time of the mycelia; (F) growing time of the mycelia vs. LAS concentration.

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Table 3 Critical values for monacolin J yield Factors

Monacolin J yield (%; based on LAS)

Glucose concentration (%, w/v) pH Incubation time (days) Hydrolysis time (h) LAS concentration (mg/mL) a

Observed minimum

Critical valuesa

Observed maximum

0.25 5.20 3.00 4.00 0.50

0.59 7.48 4.08 15.18 1.00

0.75 9.20 5.00 20.00 2.50

Solution: saddle point.

glucose concentration of the medium being the most important. The fitted surface plots (Fig. 3(A)) indicated that high pH (≥8.5) and a glucose concentration of the medium <0.59% resulted in a good conversion yield. The fitted surface plots (Fig. 3(B)) also showed that high pH was more effective, but only in association with a long culture time (≥4.0 days), in improving the conversion yield. Similarly, as shown in Fig. 3(C), a low glucose concentration together with a long culture time (≥4.0 days) result in a better yield. Taken together, the results indicate that high pH and a low glucose concentration in the medium are preferable. Moreover, higher conversion yields are obtained with longer culture times of C. compactiuscula. Indeed, in this study, all three factors were also the three most important individual factors (significant effectors), as shown in Fig. 2. These three factors also influenced growth of the mycelia of C. compactiuscula, implying that the production of lovastatin esterase by this fungus is strongly related to the production of mycelial mass. The interactions between other factors were also studied. The fitted surface plots showed that a low glucose concentration of the medium enhances the production of lovastatin esterase, whereas the factor of reaction time of LAS hydrolysis by mycelia had no significant effect (data not show). However, when this same factor was associated with a medium pH ≥ 8.5, the reaction time of LAS hydrolysis by the mycelia was shorter (<16 h) (Fig. 3(D)). Thus, it seems that pH not only influences mycelial growth, but it may also influence enzyme activity directly. The optimal culture time of mycelia for production of lovastatin esterase is 4.08 days, and the optimal reaction time of LAS hydrolysis by the mycelia is 15.18 h (Fig. 3(E)).

Fig. 4. Specific enzyme activity of the mycelial lysate harvested after various growing times.

Since high concentrations of the substrate lovastatin may inhibit lovastatin esterase activity [8], the interactions between this factor and others were studied. In mycelial growth medium, a lower glucose concentration (≤0.59%) and a higher pH (≥8.5) could influence the optimal concentration of lovastatin and thus of lovastatin esterase activity (data not show). Thus, under the above-defined conditions for glucose and pH, the optimal substrate concentration of lovastatin and the optimal culture time of mycelia before adding LAS to induce lovastatin esterase activity were found to be 1 mg/mL and 4.08 days, respectively (Fig. 3(F)). Furthermore, under such conditions, when the inter-

Fig. 5. The effect of LAS concentration on enzymatic conversion by the mycelial lysate. Empty squares () indicate the conversion ratios of monacolin J from lovastatin (see right Y axis); solid diamonds (
) indicate monacolin J production (left Y axis).

Table 4 Enzyme purification Purification step

V

A

PC

TA

SA

Y

PF

1 2 3 4

360.0 17.5 4.0 1.8

12.93 126.74 187.16 375.22

10.29 57.14 1.13 0.32

4658.25 2217.95 748.64 675.40

1.25 2.22 165.63 1172.56

100.00 47.61 16.07 14.50

1.00 1.77 132.50 938.05

Purification step: (1) Cell disruption—supernatant collected after polytron and centrifugation; (2) Ammonium sulfate (60%) fractionation (after desalting); (3) Size-exclusion chromatography (Superdex 75 pg); (4) Ion-exchange chromatography (MonoQ). Designations: V, volume (mL); A, activity (U/mL; one U is defined as the amount of lovastatin esterase required to hydrolyze 1 ␮mol of LAS per min); PC, protein concentration (mg/mL); TA, total activity (U; TA = A × V); SA, specific activity (U/mg; SA = A/C); Y, yield (%; Y = TAn /TA1 × 100%); PF, purification factor (-fold; PF = SAn /SA1 ). TA1 or TAn represents the total activity of lovastatin esterase obtained within the purification step of 1 or n (n = 1, 2, 3 or 4), respectively. SA1 or SAn represents the specific activity of lovastatin esterase obtained within the purification step of 1 or n (n = 1, 2, 3 or 4), respectively.

L.-C. Chen et al. / Enzyme and Microbial Technology 39 (2006) 1051–1059

Fig. 6. SDS-PAGE. Lanes 1 and 10 are markers; the molecular masses are indicated. Lane 2 is the crude extract obtained from the 60% ammonium sulfate precipitation. Lanes 3–9 are fractions 12–18, respectively, obtained from ion-exchange chromatography using a monoQ column. The box indicates the position of the enzyme migration.

action of substrate (lovastatin) concentration and the reaction time of LAS hydrolysis by the mycelia were cross-investigated, optimal conditions were found to be 1 mg/mL and 15.18 h, respectively.

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The critical (predicted) values of the five selected factors that should result in optimal lovastatin esterase production are listed in Table 3. Under these conditions, a 100% conversion yield of monacolin J may be theoretically obtained. Additional confirmation for these results is described in the following. In order to verify the results of RSM, the factor of mycelial culture time before LAS addition was re-investigated to determine whether the optimal results were reproducible. All other factors were held at the optimal level indicated in Table 3; however, some minor procedures were changed: Crude lovastatin esterase from broken mycelia, instead of directly from mycelia, was used for the conversion of lovastatin to monacolin J. Additionally, the specific activity of lovastatin esterase, instead of the conversion ratio of monacolin J, was used to measure the catalytic capability of lovastatin esterase, since the substrate, LAS, was not varied but was fixed at 1 mg/mL. Fig. 4 indicates that the best specific enzyme activity of lovastatin esterase was obtained at culture day 4, consistent with the previously described results of RSM optimization. The concentration of the substrate LAS, another RSM factor, was also re-investigated to verify the optimization results. LAS substrates in various concentrations (0.1, 0.5, 1, 3, 5, 10, and 20 mg/mL) were subjected to a de-branching reaction using a constant amount of crude lovastatin esterase. The results in Fig. 5

Fig. 7. Purification of monacolin J for NMR analysis. (A) Crude extract (pH 7.82) used in monacolin J production; (B) adjusted to pH 5.0; (C) the organic solvent phase after extraction with n-hexane/ethyl acetate; (D) the phosphate buffer (pH 5.0) phase after extraction; (E) after purification by TLC.

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Fig. 8. Theoretical NMR spectrum. Structures of (A) lovastatin and (B) monacolin J.

show that, under such conditions, monacolin conversion reached the maximum level of 100% when LAS was provided at a lower concentration, i.e., of 0.1, 0.5, or 1 mg/mL. The results agree with the predicted results of the RSM analyses. Interestingly, if the LAS concentration is >1 mg/mL, and more LAS is added, monacolin J production may be further increased, although the conversion ratios decrease. Furthermore, if the concentration of substrate LAS is >5 mg/mL, there is no further increase in the production of monacolin J, but the conversion ratios continue to decline (Fig. 5). 3.3. Purification of lovastatin esterase C. compactiuscula mycelia were homogenized as detailed in Materials and methods. Mycelia lysate was centrifuged at 30,000 × g, and the supernatant was collected and filtered through a 0.2-␮m disk membrane. The filtrate was subjected

to ammonium precipitation. The fraction containing lovastatin esterase was purified by size-exclusion chromatography on an FPLC Superdex 75 pg column and then by anion-exchange chromatography on an FPLC Mono Q FIR 10/10 column. Esterase bound to the anion-exchange resin was eluted with a linear gradient of 0–800 mM sodium chloride in 20 mM Tris buffer (pH 8.5) at a flow rate of 1 mL/min. Using this procedure, lovastatin esterase could be readily purified away from the majority of C. compactiuscula proteins. Table 4 shows the purification parameters for each step of the procedure. Purified lovastatin esterase produced a single band of approximately 28 kDa on SDS-PAGE (Fig. 6). The molecular mass of our enzyme differed from that reported by others [8], but the reasons for the differences remain to be investigated. Lovastatin esterase purified using the above procedure had a specific activity of 1172 U/mg, which was 938fold higher than the activity of the original mycelial lysate. One unit of enzyme activity is defined as the amount of lovastatin

Fig. 9. NMR results. (A) Lovastatin; (B) monacolin J.

L.-C. Chen et al. / Enzyme and Microbial Technology 39 (2006) 1051–1059

esterase that catalyzed the transformation of 1 ␮mol LAS to monacolin J per min. 3.4. Determination of lovastatin and monacolin J structures using NMR spectroscopy LAS was converted into monacolin J by the purified lovastatin esterase. Both monacolin J and residual LAS were transformed to the water-insoluble lactone forms by acidification. Since monacolin J is slightly more hydrophilic than lovastatin, it was extracted with phosphate buffer (pH 5.0) for a few cycles. The partially purified monacolin J was subjected to a C-18 column and then eluted by acidified acetonitrile. Fractions containing monacolin J were combined, concentrated by freeze-drying, and then subjected to preparative TLC separation. The spots were scraped out off and the purified monacolin J was extracted. HPLC was used to determine the purity of monacolin J at each purification step (Fig. 7(A–E)). The structures of the lactone forms of purified monacolin J and lovastatin were determined by NMR spectroscopy. Based on the theoretical NMR spectra generated by a computer program (ChemDraw Ultra 8.0), we detected a 1 H NMR signal at ␦ 0.96 that was inferred to be the methyl singlet of the 2-methylbutyryloxy side-chain of lovastatin (Fig. 8(A)). This signal should be absent from the spectrum of monacolin J (Fig. 8(B)). Indeed, this signal was present in the 1 H NMR spectrum of lovastatin (Fig. 9(A)), but not in that of monacolin J (Fig. 9(B)). This result supports the conclusion that the enzyme produced by C. compactiuscula under the conditions described above is lovastatin esterase. The enzyme generates monacolin J by catalyzing the hydrolysis of the 2-methylbutyryloxy sidechain of lovastatin. In this study, we determined the conditions for optimal production of a hydrolytic enzyme, lovastatin esterase, by C. compactiuscula. The enzyme is capable of specifically cleaving an ester bond in lovastatin to yield monacolin J. Since monacolin J provides a core molecule for a variety of modifications that generate different HMG-CoA reductase inhibitors, the hydrolytic enzyme of C. compactiuscula provides a useful conversion system for monacolin J production. Moreover, we believe that this hydrolytic enzyme can act as the basis for further development of better anti-hypercholesterolemia drugs. Acknowledgments We gratefully acknowledge the excellent technical assistance of Yan-Lin Lai. We also thank Professor Ta-Jung Lu and his

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graduate student, Mr. Cheng-Kun Lin, of the Department of Chemistry, National Chung-Hsing University, Taiwan, R.O.C., for kind assistance with the NMR analyses. This work was supported in part by research grants NSC-90-2313-B-241-004 and NSC-91-2313-B-241-002 from the National Science Council, Taiwan, R.O.C. References [1] Singh NV, Azmi S, Maurya S, Singh UP, Jha RN, Pandey VB. Two plant alkaloids isolated from Corydalis longipes as potential antifungal agents. Folia Microbiol (Praha) 2003;48:605–9. [2] Alberts AW, Chen J, Kuron G, Hunt V, Huff J, Hoffman C, et al. Mevinolin: a highly potent competitive inhibitor of hydroxymethylglutarylcoenzyme A reductase and a cholesterol-lowering agent. Proc Natl Acad Sci USA 1980;77:3957–61. [3] Alberts AW. Discovery, biochemistry and biology of lovastatin. Am J Cardiol 1988;62:10J–5J. [4] Bischoff KM, Rodwell VW. Biosynthesis and characterization of (S)and (R)-3-hydroxy-3-methylglutaryl coenzyme A. Biochem Med Metab Biol 1992;48:149–58. [5] Laws PE, Spark JI, Cowled PA, Fitridge RA. The role of statins in vascular disease. Eur J Vasc Endovasc Surg 2004;27:6–16. [6] Nixon JV. Cholesterol management and the reduction of cardiovascular risk. Prev Cardiol 2004;7:34–9 [Quiz 40-1]. [7] Tobert JA. New developments in lipid-lowering therapy: the role of inhibitors of hydroxymethylglutaryl-coenzyme A reductase. Circulation 1987;76:534–8. [8] Schimmel TW, Borneman CMJ. Purification and characterization of a lovastatin esterase from Clonostachys compactiuscula. Appl Environ Microbiol 1997;63:1307–11. [9] Komagata D, Yamashita H, Endo A. Microbial conversion of compactin (ML-236B) to ML-236A. J Antibiot (Tokyo) 1986;39: 1574–7. [10] Endo A, Komagata D, Shimada H, Monacolin M. a new inhibitor of cholesterol biosynthesis. J Antibiot (Tokyo) 1986;39: 1670–3. [11] Cover W, Dabora RL, Hong A, Reeves C, Steiber RW, Vinci VA. Mutant strains of Aspergillus terreus for producing 7-[1,2,6,7,8,8A(R)hexa-hydro-2(S), 6(R)-dimethyl-8(S)-hydroxy-1(S)-napht hyl]-3(R),5(R)dihydroxyheptanoic acid (triol acid). U.S. Patent 5250435; October 1993. [12] Kimura K, Komagata D, Murakawa S, Endo A. Biosynthesis of monacolins: conversion of monacolin J to monacolin K (mevinolin). J Antibiot (Tokyo) 1990;43:1621–2. [13] Komagata D, Shimada H, Murakawa S, Endo A. Biosynthesis of monacolins: conversion of monacolin L to monacolin J by a monooxygenase of Monascus ruber. J Antibiot (Tokyo) 1989;42:407–12. [14] Sleteinger M, Verhoeven T, Volant R. One step process for C-methylation of 2-methylbutyrates. U.S. Patent 4582915; April 1986. [15] Nesterenko MV, Tilley M, Upton SJ. A simple modification of Blum’s silver stain method allows for 30 minute detection of proteins in polyacrylamide gels. J Biochem Biophys Methods 1994;28: 239–42.