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Enzyme and Microbial Technology 41 (2007) 71–79

The 11�-hydroxylation of 16,17�-epoxyprogesterone and the purificationof the 11�-hydroxylase from Absidia coerulea IBL02

Kun Chen, Wang-Yu Tong ∗, Dong-Zhi Wei ∗, Wei JiangState Key Laboratory of Bioreactor Engineering, Institute of New World Biotechnology, East China University of Science and Technology,

Shanghai 200237, China

Received 22 August 2006; received in revised form 3 December 2006; accepted 4 December 2006

bstract

The 11�-hydroxylation product of 16,17�-epoxyprogesterone (EP), 11�-hydroxy-16,17�-epoxyprogesterone (HEP), is an important interme-iate for many anti-inflammatory drugs. To understand the hydroxylation characteristics of the 11�-hydroxylase from filamentous fungus Absidiaoerulea IBL02, the 11�-hydroxylation process, the purification results and some characteristics of the enzyme (protein P450) from mycelia wererst reported in this paper. When the feeding time of the substrate was set at 27 h and the biotransformation time was for 42 h after fermentationptimization in the whole-cell bioconversion, a satisfied yield of about 4 g/l with above 85% biotransformation rate was achieved. We found the1�-hydroxylase biosynthesis in cell cultivation could be induced by adding analogs of the substrate, and an about three-fold increase in enzymaticmount was achieved by adding progesterone as inducer for about 4 h. Experimental results showed that Ca2+ and Fe2+ could greatly enhance the

nzymatic activity, but Mg2+, Mn2+, Cu2+, and Zn2+ could inhibit it. After the optimization of the purification conditions, the 11�-hydroxylaseprotein P450) with a single band on SDS-PAGE was obtained and estimated to be a MW of about 60 kDa, giving a maximum absorbance at48 nm in reduced carbon monoxide difference spectra.

2006 Elsevier Inc. All rights reserved.

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eywords: 11�-Hydroxylation; 16,17�-Epoxyprogesterone; Absidia coerulea;

. Introduction

Steroid drugs, a large category of compounds with steroiducleus, are mainly used to attenuate the inflammatoryesponse and treat cancers and HIV in clinical therapy1–3]. The 11�-hydroxylation is economically important inhe pharmaceutical industry. The 11�-hydroxylation productFig. 1) of 16,17�-epoxyprogesterone (EP), 11�-hydroxy-6,17�-epoxyprogesterone (HEP), is an important intermediateor many anti-inflammatory drugs like hydrocortisone, pred-isone acetate, dexamethasone and so on.

The steroid hydroxylase in mycelia usually is a monooxy-enase enzyme system [4,5]. These multienzyme complexes

ituate on the microsomal membrane of the endoplasmic retic-lum in cells, and contain a P450 as substrate binding terminalxidase and a flavoprotein for transferring electrons from

∗ Corresponding authors. Tel.: +86 21 64253156; fax: +86 21 64250068.E-mail addresses: tongwy@ecust.edu.cn (W.-Y. Tong),

zhwei@ecust.edu.cn (D.-Z. Wei).

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141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2006.12.002

chrome P450; Purification; Steroid

educed coenzyme to P450, which is called NADPH-P450eductase [6,7].

Biotransformation has being used for the production ofteroid drugs universally [8–10]. Among all sites of steroidolecular structure accessible for microbial hydroxylation, the

1�-, 11�-, 15�- and 16�-hydroxylations are most valuableow in the anti-inflammatory drugs industry [11–15]. The bio-ransformation found in steroid drugs was mainly catalyzedy Rhizopus nigricans [16]. The steroid 11�-hydroxylase, annducible enzyme, was also found in Cochliobolus lunatus12,17] and Curvularia lunata [18,19]. The NADPH-P450eductase from filamentous fungus R. nigricans had alsoeen purified and characterized [20,21]. Many attemptsave been made to purify fungal P450 but few success-ul examples are presented [22,23]. Up to now, no reportsbout the 11�-hydroxylation of 16,17�-epoxyprogesteronend the hydroxylase purification from Absidia coerulea are

ound.

In this paper, the 11�-hydroxylation of 16,17�-poxyprogesterone by the whole cell and its free-cell extract,nd the purification results and some characteristics of the

72 K. Chen et al. / Enzyme and Microbial Technology 41 (2007) 71–79

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Fig. 1. Biotransformation of 16,17�-epoxyprogestero

1�-hydroxylase (especially P450) from filamentous fungus A.oerulea IBL02 are first reported.

. Materials and methods

.1. Materials

16,17�-Epoxyprogesterone (EP) and 11�-hydroxy-16,17�-poxyprogesterone (HEP) were obtained from XianJu Pharmaceuticalompany Ltd. in Zhejiang Province, China. NADPH and NADH wereurchased from Yuanju Bio-Tech Co. Ltd. in Shanghai, China. Progesteroneas purchased from Fluka in Switzerland. Yeast extract was purchased fromxoid Ltd. in England. Phenyl Sepharose Fast Flow and Q Sepharose Fast Flowere purchased from Pharmacia Biotech in Sweden. The methanol for HPLCas purchased from Tedia in USA. All other chemicals were of analyticalrade.

.2. Strain and medium

Strain A. coerulea IBL02 was obtained from Zhejiang University in Zhejiangrovince, China. The agar-slant medium contains: 1% (w/v) glucose, 20% (v/v)otato extract (potato extract preparation: 200 g potato slices were immediatelydded into 1000 ml of tap water and then boiled for about 30 min, filtered throughotton-cloth and finally complemented the filtrate to 1000 ml) and 2% (w/v)gar power. The seed medium contains: 1% (w/v) glucose, 1.2% (v/v) cornteep liquor, 0.25% (w/v) yeast extract and 0.5% (w/v) (NH4)2SO4, pH 6.8.he fermentation medium contains: 1.2% (w/v) glucose, 1.8% (v/v) corn steep

iquor, 0.25% (w/v) yeast extract and 0.5% (w/v) (NH4)2SO4, pH 6.8. Mediumsere sterilized at 121 ◦C and 0.1 MPa for 20 min in an autoclave.

.3. Biotransformation in whole cell

.3.1. Pre-cultivationFirst, A. coerulea IBL02 was grown on an agar-slant at 28 ◦C for 5 days

nd then the spores on the agar-slant were washed down into an autoclavedask with sterile water. The spore solution of 1.5 ml with a concentration of× 107 spores/ml was inoculated into a 250 ml flask with 30 ml seed medium as

nocula for the pre-cultivation and then incubated on a rotatory shaker at 170 rpmt 28 ◦C for 30 h.

.3.2. Medium optimizationThe single factor experiments were performed for medium optimization.

lucose [0%, 0.5%, 0.8%, 1.0%, 1.2%, 1.5% and 2.0% (w/v)], corn steep liquor0%, 0.6%, 1.2%, 1.8%, 2.5% and 4.0% (v/v)], (NH4)2SO4 [0%, 0.25%, 0.5%,.9%, 1.5% and 2.5% (w/v)] and yeast extract [0%, 0.15%, 0.25%, 0.5%, 1.0%nd 1.5% (w/v)] were optimized at different concentrations one by one. One

illilitre of the pre-culture was transferred into a 250 ml flask with 30 ml fer-entation medium and then cultivated for 24 h (the same conditions as in the

re-cultivation). One millilitre of EP solution (135 mg EP/1 ml glycol) was addednto the flask and the biotransformation was carried out for an additional 36 hFig. 1). Finally the fermentation culture was collected for further analysis.

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P). *HEP: 11�-hydroxy-16,17�-epoxyprogesterone.

.3.3. Process parameters optimizationThe single factor experiments were done. The different feeding time of sub-

trate (18 h, 21 h, 24 h, 27 h, 30 h and 36 h) and the different transforming time21 h, 30 h, 36 h, 42 h and 50 h)were optimized one by one after the mediumptimization. One millilitre of the pre-culture was transferred into a 250 ml flaskith 30 ml optimized fermentation medium and cultivated. One millilitre of EP

olution (135 mg EP/1 ml glycol) was added into the flask at different feedingimes (the same conditions as in the pre-cultivation), and the biotransformationas carried out for different transforming times. Finally the fermentation cultureas collected for further analysis.

After fermentation, 30 ml cultures were first divided into two parts by vac-um filtration. The mycelial fraction was dried at 50 ◦C in an oven, and thenulverized by a mortar and extracted with 30 ml chloroform. The flowthroughraction was extracted with a total of 60 ml chloroform twice (30 ml each).ll (90 ml) of the chloroform extracts were pooled together at last. 0.5 ml of

he solution was evaporated by an oven at 100 ◦C for about 20 min and theried-sample was redissolved in 5 ml methanol. The methanol solution of 20 �las diluted to 1 ml with pure ethanol. After centrifugation at 12,000 rpm formin, the supernatant liquor collected was detected by using TLC and HPLC,

espectively.

.4. Biotransformation in cell-free extract

.4.1. Induction of P450One millilitre pre-culture was transferred into 250 ml-flasks with 30 ml of

he fresh optimized medium. After the cells were cultivated for 24 h, 300 �lrogesterone (10 mg progesterone/ml glycol) was added to each flask as inducernd the culture was cultivated for additional 4 h to improve the biosynthesis ofhe P450. The mycelia from non-induced (non progesterone added) and inducedultures were, respectively collected every other 4 h by filtration and disruptedthree parallel flasks). The contents of P450 were measured by reduced carbononoxide difference spectra.

.4.2. Coenzyme screeningOne millilitre pre-culture was transferred into 250 ml flasks with 30 ml

ermentation medium and the cultivation was done for 24 h. An inductioneriod of about12 h was triggered accompanying the addition of 300 �l pro-esterone solution (10 mg progesterone/ml glycol) to each flask as inducer. Theycelia were collected by vacuum filtration and washed with 0.1 M NaOH of

0 ml. 2.5 g of the mycelia were resuspended in 10 ml of suspension buffer0.05 M sodium phosphate buffer, pH 8.0) and disrupted by sonic treatmentthree 6-s intervals, 600 w) in an ice-water bath. The homogenate was cen-rifuged at 10,000 rpm for 10 min at 4 ◦C and the supernatant was collectedthe method for disrupted supernatant preparation is the same unless specifiedtherwise). In order to facilitate the assay of the enzymatic activity, differ-nt coenzymes (1 �M NADPH, 1 �M NADH and 1 �M Na2S2O4) and 50 �lP solution (10 mg EP/ml glycol) as substrate were, respectively put into the

isrupted supernatant of 5 ml (0.05 M sodium phosphate buffer pH 8.0) andhen incubated for about 1.5 h at 30 ◦C, and terminated by addition of 1 mlf chloroform. Product was extracted with 1 ml chloroform twice, dried byacuum and redissolved in 250 �l methanol. Analysis was carried out by anPLC.

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.4.3. Enzymatic stabilityIn order to test the stability of 11�-hydroxylase complexes in cell-free

xtract, 5 ml of the disrupted supernatant [after stored at 30 ◦C for 0 h (as aontrol), 3 h, 8 h and at −20 ◦C for 24 h, respectively] with final concentrationsf 20% glycerol, 0.5 mM PMSF, 0.5 mM DTT after optimization was mixedith 0.5 mg NADPH and 50 �l EP solution (10 mg EP/ml glycol) and then theix was incubated for 1.5 h at 30 ◦C to react. The reaction was terminated by

ddition of 1 ml of chloroform. Product was extracted by using 1 ml chloro-orm twice, dried by vacuum and redissolved in 250 �l methanol. Analysis wasarried out by an HPLC.

.4.4. Effect of temperature and pH on the 11β-hydroxylase activityAfter 50 �l EP solution (10 mg EP/ml glycol) and 0.5 mg NADPH were

ut into the disrupted supernatant of 5 ml (0.05 M sodium phosphate buffer, pH.0), the supernatants were incubated for 1.5 h at different temperatures (20 ◦C,8 ◦C, 30 ◦C, 35 ◦C and 37 ◦C), respectively. These reactions were terminatedy addition of 1 ml of chloroform. Product was extracted by using 1 ml chlo-oform twice, dried by vacuum and redissolved in 250 �l methanol. Analysis

as carried out by an HPLC. Under the optimal temperature, 1.25 ml of 0.2 M

odium phosphate buffers with different pHs (6.5, 7.0, 7.2, 7.4 and 8.0) wereropwise added to 3.75 ml of the disrupted supernatants accompanying withlow stirring in an ice-water bath to adjust the pH, and then 50 �l EP solution10 mg EP/ml glycol) and 0.5 mg NADPH were added into the supernatants and

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able 1he purification procedures of the cytochrome P450a

nit operation Operation processes

EC (1) Resin: 20 ml Q-sepharose Fast FlowColumn: 1.6 cm × 10 cmEquilibriation: 100 ml of 0.05 M sodium phosphate buffer (pLoading sample: 40 ml of the disrupted supernatant, 1.0 ml/mRequilibriation: 40 ml of 0.05 M sodium phosphate buffer (pElution: 25 ml of 0.05 M sodium phosphate buffer (pH 7.0, 0flow rate

IC Resin: 20 ml Phenyl Sepharose Fast FlowColumn: 1.2 cm × 20 cmEquilibriation: 100 ml of 0.05 M sodium phosphate buffer (pof flow rateLoading sample: 60 ml of the flowthrough above with 1 M NElution: 25 ml distilled water, 2.0 ml/min of flow rate

ample treatment (1) The pH of the eluate solution above was first adjusted to pHbuffer and then Triton X-100 was dropwise added into it witha final concentration of 1% Triton X-100. Finally the solutiosodium phosphate buffer (pH 7.65) in an ice-water bath for 3

EC (2) Resin: 8 ml Q-sepharose Fast FlowColumn: 1.6 cm × 10 cmEquilibriation: 40 ml of 0.02 M sodium phosphate buffer (pHLoading sample: about 25 ml of the dialyzed solution, 1.0 mRequilibriation: 20 ml of 0.05 M sodium phosphate buffer (pElution: 10 ml of 0.02 M sodium phosphate buffer (0.5 M Na

ample treatment (2) The 30 ml flowthrough containing the target protein was collsodium phosphate buffer (pH 8.0) in an ice-water bath for 3

EC (3) Resin: 8 ml Q-sepharose Fast FlowColumn: 1.6 cm × 10 cmEquilibriation: 40 ml of 0.02 M sodium phosphate buffer (pHLoading sample: 30 ml of the disrupted supernatant, 1.0 ml/mRequilibriation: 20 ml of 0.02 M sodium phosphate buffer (pElution 1: 10 ml of 0.02 M sodium phosphate buffer (0.3 M NElution 2: 10 ml of 0.02 M sodium phosphate buffer (0.5 M N

EC: ion exchange chromatography; HIC: hydrophobic interaction chromatography. Ised in this experiments for the first time, (2) means the second time, and (3) meansample was treated in this experiments for the first time and (2) means the sample waa All samples in these chromatographic steps were monitored by UV adsorbance a

l Technology 41 (2007) 71–79 73

he supernatants were incubated for 1.5 h, respectively. These reactions wereerminated by addition of 1 ml of chloroform. Products were extracted by usingml chloroform twice, dried by vacuum and redissolved in 250 �l methanol.nalysis was carried out by an HPLC.

.4.5. Effect of metal ions on the11β-hydroxylase activityFifty microlitres of EP solution (10 mg EP/ml ml glycol), 0.5 mg NADPH

nd different metal ions (a final concentration of 1 mM each) were added intohe 5 ml the disrupted supernatants (0.05 M sodium phosphate buffer pH 8.0),espectively, and then the reaction system was held for 1.5 h at 30 ◦C. Theseeactions were terminated by addition of 1 ml of chloroform. Products werextracted by using 1 ml chloroform twice, dried by vacuum and redissolved in50 �l of methanol. Analysis was carried out by an HPLC. A system withoutdding any metal ion was as the control.

.5. Purification of cytochrome P450

Mycelia of 10 g was disrupted by sonic treatment (three 6-s intervals, 600 w)n a tube with 40 ml suspension buffer (0.05 M sodium phosphate buffer pH 7.0,ontaining 10% glycerol). The homogenate was centrifuged at 10,000 rpm for0 min at 4 ◦C and the cytochrome P450 in the supernatant was purified as theescribed procedures in Table 1.

Sample collected

About 60 ml flowthrough

H 7.0, 10% glycerol), 1.0 ml/min of flow ratein of flow rate

H 7.0, 10% glycerol), 1.0 ml/min of flow rate.5 M NaCl, 10% glycerol), 1.0 ml/min of

About 25 ml eluate

H 7.0, 1 M NaCl, 10% glycerol), 1.0 ml/min

aCl, 1.0 ml/min of flow rate

7.65 by using 0.2 M sodium phosphatestirring for 60 min in an ice-water bath until

n was dialyzed against a buffer of 20 mMh

About 25 ml dialyzed solution

About 30 ml flowthrough

7.65), 1.0 ml/min of flow ratel/min of flow rateH 7.0, 10% glycerol), 1.0 ml/min of flow rateCl, pH 7.65), 1.0 ml/min of flow rate

ected and dialyzed against a buffer of 20 mMh

About 30 ml dialyzed solution

About 10 ml eluate

8.0), 1.0 ml/min of flow ratein of flow rate

H 8.0), 1.0 ml/min of flow rateaCl, pH 8.0), 1.0 ml/min of flow rateaCl, pH 8.0), 1.0 ml/min of flow rate

EC (1), IEC (2) and IEC (3): (1) means the ion exchange chromatography wasthe third time. Sample treatment (1) and sample treatment (2): (1) means the

s treated in this experiments for the second time.t 280 nm.

7 crobial Technology 41 (2007) 71–79

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4 K. Chen et al. / Enzyme and Mi

.6. Analytical methods

.6.1. TLCTwenty microlitres of samples of biotransformation were qualitatively

nalyzed by thin-layer chromatography (TLC) on silica gel plates (Huiyouilica Gel Development Co. Ltd. in Yantai, China), using a solvent systemf benzene/ethyl acetate (7:3, v/v) as a mobile phase. Then the plates wereried by hot air, and wetted by 20% H2SO4 using a sprayer to localize thepot. Finally, the spots with different colors were visualized in an oven at00 ◦C.

.6.2. HPLCSamples after biotransformation were quantitatively analyzed by HPLC

Agilent Technologies, 1100 series, USA). The sample of 10 �l was first loadednto a SDB C18 column (4.6 mm × 150 mm, Agilent Technologies, USA) andluted with ethanol/water (60/40, v/v). The flow rate was set at 1 ml/min. Theavelength for UV-detection was set at 245 nm. The temperature of the columnas set at 40 ◦C.

onversion rate (%) = HEP content

original EP content,

urity (%) = HEP content

HEP content + residual EP content

.6.3. SDS-PAGEThe proteins in samples were analyzed, respectively by 8% or 15% sodium

odecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according tochagger et al. [24] and bands were visualized by silver staining. An aliquot of5 �l was loaded for each lane of SDS-PAGE, and a 10-kDa-protein ladder wassed as a molecular mass marker.

.7. Lowry method

The amounts of proteins in samples were measured by the method ofowry [25]. A standard protein concentration curve was constructed by usingovine serum albumin (BSA) and the sample protein concentrations werealculated.

.8. Assay of cytochrome P450

Cytochrome P450 content was assessed by measuring its sodium dithionite-educed CO difference spectrum using UV2300 spectrophotometer [26].

In this work, three replicates were run in every experiment.

. Results

.1. Biotransformation in whole cell

In order to improve the bioconversion rate, medium compo-ents [glucose, corn steep liquor, (NH4)2SO4) and yeast extract],

able 2he optimized results in the single factor experiments by Absidia coerulea IBL02

ptimized elements Optimal level from SFE Conversion rate (%)

lucose 1.2% (w/v) 81.4 ± 2.3orn steep liquor 1.8% (w/v) 86.4 ± 1.7

NH4)2SO4 0.5% (w/v) 81.5 ± 2.4east extraction 0.25% (w/v) 85.7 ± 3.1eeding time of substrate 27 h 84.2 ± 1.5ransforming time 42 h 87.4 ± 3.2

Fst

ig. 2. The optimized results in the single factor experiments by Absidiaoerulea IBL02. Error bar: the variance of three parallel experiments.

eeding time of substrate and transforming time were first opti-ized. The optimized results of single factor experiments (SFE)ere shown in Table 2 and Fig. 2, and the final optimized resultsf TLC and HPLC were shown in both Fig. 3 (TLC) and Fig. 4HPLC). A higher bioconversion rate of 87.4% [0.45% of EPw/v) as substrate] was obtained compared to a common lab-ratory level of the bioconversion rate of 70% [0.25% of EPw/v) as substrate] and ethanol as solvent, indicating that the

ig. 3. The TLC chromatogram of samples. Lane 1, standard of EP; Lane 2,tandard of HEP; Lane 3, the extract of the whole cell biotransformation afterhe optimization, corresponding to the results in the last line in Table 2.

K. Chen et al. / Enzyme and Microbial Technology 41 (2007) 71–79 75

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Fig. 4. HPLC profile of the whole cel

.2. Biotransformation characteristics of the1β-hydroxylase in cell-free extract

.2.1. Induction of P450The induction results of P450 were presented in Fig. 5. We

ould find from the figure that the amount of P450 in the non-nduced cells of A. coerulea IBL02 was very low, only about.025 nmol P450 per mg mycelia. However, after the mycelia ofhe fungus were induced with progesterone for 4 h, the contentsad a distinct increase, about three-fold compared with the resultf no inducer addition.

.2.2. Coenzyme screeningThe 11�-hydroxylase activity in the disrupted super-

atant, after the mycelia were disrupted by sonic treat-ent, could rarely be detected in the absence of coen-

yme addition in our experiments. The effective order ofhe contributors to the enzymatic activity was, respec-ively NADPH (1.7 × 10−4 mg HEP/min/nmol P450), Na2S2O4

0.5 × 10−6 mg/min/nmol), and NADH (n.d.), suggesting thatADPH is a suitable coenzyme for the 11�-hydroxylase activity

rom A. coerulea IBL02.

ig. 5. Time course of cytochrome P450 biosynthesis with and without proges-erone as inducer.

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ransformation after the optimization.

.2.3. Enzymatic stabilityThe 11�-hydroxylase activity in the disrupted supernatant

as not stability also found in our experiments, after the myceliaere disrupted by sonic treatment. In order to test the stability,.5 mg NADPH and 50 �l EP solution (10 mg EP/ml glycol)ere added to 5 ml of the disrupted supernatant [after stored at0 ◦C for 0 h (control), 3 h, 8 h and at −20 ◦C for 24 h, respec-ively] with the final concentrations of 20% glycerol, 0.5 mMMSF and 0.5 mM DTT and then the solution was incubated for.5 h at 30 ◦C. It was found that the relative activity of the enzymen the disrupted supernatant greatly decreased to 25% after storedt 30 ◦C for 3 h, and even close to 0 for 8 h (Table 3). However,nder the optimal conditions of 20% glycerol, 0.5 mM PMSF,.5 mM DTT and storage of −20 ◦C for 24 h the level of thenzyme activity could be significantly reduced and maintainedt 95.3%, indicating that the addition of the suitable additivesnd lower temperature could effectively keep from the loss ofhe enzyme activity.

.2.4. Effect of temperature and pH on the 11β-hydroxylasectivity

The effects of different temperatures and pHs on enzymectivity were investigated and the optimized results for the1�-hydroxylase activity were determined by measuring HEPmounts using an HPLC. As shown in Fig. 6, the optimal bio-ransformation temperature was 30 ◦C.

As shown in Fig. 7, under the optimal temperature of 30 ◦C,he optimal pH for the enzyme activity was determined to be

.0 close to the physiological pH of cells. Higher or lowerhan the pH value of 7.0, the 11�-hydroxylase activity greatlyecreased.

able 3he activity of the 11�-hydroxylase at the different storage conditions

ample Relative activity (%)

ontrol 100t 30 ◦C for 3 h 25.6t 30 ◦C for 8 h n.d.t −20 ◦C for 24 h with the addition of 20%glycerol, 0.5 mM PMSF, 0.5 mM DTT

95.3

.d., not determined. The disrupted supernatant was stored at 30 ◦C for 0 h ashe control.

76 K. Chen et al. / Enzyme and Microbial Technology 41 (2007) 71–79

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Table 4Effect of metal ions on the activity of the 11�-hydroxylases from A. coeruleaIBL02

Metal ions (1 mM) Residual activity (%)

Control 100Mg2+ 52.7Ca2+ 230.5Mn2+ 50.4Cu2+ 11.1Z 2+

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ig. 6. Effect of temperature on the activity of the 11�-hydroxylase from A.oerulea IBL02.

.2.5. Effect of metal ions on the11β-hydroxylase activityThe effect of different metal ions including Mg2+, Ca2+,

n2+, Cu2+, Zn2+ and Fe2+ on the activity of the 11�-ydroxylase were investigated. It was found that Ca2+ and Fe2+

ould greatly enhance the 11�-hydroxylase activity up to aboutwo-folds (230.5% and 193.3%) but Mg2+, Mn2+, Cu2+, andn2+ could inhibited it compared to the control (Table 4). The

nteresting phenomenon was that Ca2+ and Fe2+ could greatly

ig. 7. Effect of pH on the activity of the 11�-hydroxylase from A. coeruleaBL02.

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able 5urification results of P450 from A. coerulea IBL02

tep Volume (ml) Prot

olubilizate 40 102EC (1)-flowthrough pH 7.0 60 72.6IC-elute pH 7.0 25 5.2

EC (2)-elute pH 7.65 20 2EC (3)-elute pH 8.0 10 n.d.

.d., not determined. The disrupted supernatant was stored at 30 ◦C for 0 h as the con

n 26.7e2+ 193.3

system without adding any metal ion was used as the control.

nhance the activity of 11�-hydroxylases (no report found) butther bivalent metal ions including Mg2+, Mn2+, Cu2+, and Zn2+

ecreased the activity of 11�-hydroxylation.

.3. Purification of the 11β-hydroxylase

The procedure for the purification of the 11�-hydroxylaserom the strain was summarized in Table 1 and the results werehown in Table 5 after optimization. The content of P450 wasbout 0.8 nmol/g wet mycelia in the induced cells. First, theicrosomal fraction was primarily purified by both the IEC (1)

nd the following HIC step (the results were shown in Fig. 8).he enzyme activity mainly appeared in the HIC-elute (withclear band near 60 kDa in the gel in Fig. 8) not in HIC-

owthrough (without the band near 60 kDa in the gel in Fig. 8).hen the P450 was solubilized as described above in Table 1.majority of impurity proteins and partial P450 was removed

n the IEC (2)-elute (see Table 1 above) and the results werehown in Fig. 9. The IEC (2)-flowthrough with notable enzymectivity was used in the next step. Finally, the 11�-hydroxylasea P450) and its reductase (a flavoprotein) were separated byEC (3) (see Table 1 above) after sample treatment (2). The450 reductase was obtained by eluting with 0.3 M NaCl. And

he P450 of the 11�-hydroxylase with a single band on theDS-PAGE (Fig. 10) was obtained by eluting with 0.5 M NaCl,iving a maximum absorbance at 448 nm in reduced carbononoxide difference spectra (Fig. 11B) and a MW of about

0 kDa.

.3.1. Spectral properties of P450

Our experiments showed that the P450 alone in reduced

arbon monoxide difference spectra revealed a maximumbsorbance at 448 nm and a smaller at 420 nm, and that in theresence of substrate, the P450 showed absorption spectrums

ein (mg) P450 content (nmol) Yield (%)

8.79 1005.27 58.81.76 19.60.44 4.90.11 1.2

trol.

K. Chen et al. / Enzyme and Microbial Technology 41 (2007) 71–79 77

Fig. 8. SDS-PAGE (8%) analysis of the fungal P450 purified by HIC. ProteinsbLt

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ands were visualized by silver staining. Lane 1, molecular weight standards;ane 2, HIC-flowthrough (without the enzyme activity); Lane 3, HIC-elute (with

he enzyme activity).

ith a maximum at 389, a medium at 368 nm and a minimumt 416 nm, respectively.

. Discussion

A main disadvantage in steroid drugs biotransformation isow yield (about 1–10 g/l) due to their low aqueous solubil-

ty [27]. After the biotransformation condition optimization ofhe 11�-hydroxylation of 16,17�-epoxyprogesterone in wholeells, a good yield of near 4 g/l with a bioconversion rate of7.4% [0.45% of EP (w/v) as substrate and glycol as sol-

ig. 9. SDS-PAGE (8%) analysis of the fungal P450 purified by IEC (2). Proteinsands were visualized by silver staining. Lane 1, molecular weight standards;ane 2, IEC (2)-flowthrough (with the lowest enzyme activity); Lane 3, IEC

2)-elute with 0.15 M NaCl (with the medium enzyme activity); Lane 4, IEC2)-elute with 0.5 M NaCl (with the highest enzyme activity).

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ig. 10. SDS-PAGE (8%) analysis of the fungal P450 purified by IEC (3).roteins bands were visualized by silver staining. Lane 1, molecular weighttandards; Lane 2, IEC (3)-elute with 0.5 M NaCl pH 8.0.

ent] was obtained compared to a common laboratory levelf near 2 g/l with a bioconversion rate of 70% [0.25% of EPw/v) as substrate and ethanol as solvent]. The dissolution ofubstrate is the key for steroid microbial conversion [28,29].he main reason, we think, is that the 11�-hydroxylation of6,17�-epoxyprogesterone occurs intracellularly and hence theelatively high concentration of 16,17�-epoxyprogesterone inhe cultures facilitates the trans-membrane transport of the sub-trate, that is, the intracellular high concentration of the substrateesults in the high yield.

As well known, steroid hydroxylases of P450 usually arenduced enzymes and the levels of the hydroxylases in myceliaan increase several fold upon induction with substrates or suit-ble analogs such as progesterone. Our experimental results for

ig. 11. (A) The P450 absorption spectrum after substrate-binding in reducedarbon monoxide difference spectra (about 200 �g progesterone and 0.08 nmol450); (B) the absorption spectrum of the purified P450 alone in reduced carbononoxide difference spectra.

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he analysis of the P450 support the viewpoint. The amount of450 in the non-induced cells of A. coerulea IBL02 was very

ow, only about 0.025 nmol P450 per mg mycelia. Howeverfter the mycelia of the fungus were induced with proges-erone for 4 h, the contents of the P450 had a distinct increase,bout three-fold compared with the reported level (0.08 nmol450/1 mg mycelia) without the inducer addition [22]. In

he 11�-hydroxylation of EP in the cell-free extract, reactionystems including coenzymes, stability conditions of the 11�-ydroxylase, temperatures, pHs and metal ions were investigatedn order to both improve 11�-hydroxylation level and understandhe 11�-hydroxylase characteristics. It was found that the stor-ge conditions of 20% glycerol, 0.5 mM PMSF, 0.5 mM DTTnd storage temperature of −20 ◦C within 24 h were accept-ble (enzymatic activity of 95.3% saved) and that the optimaliotransformation conditions for the 11�-hydroxylation were asollows: NADPH, 30 ◦C, pH 7.0 and 1.0 mM Ca2+. In the micro-omal P450s, electrons input from pyridine nucleotides to P450ssually follow this mode, NADPH-P450 reductase transferringlectrons from NADPH to P450 [30]. So it is a symbol of micro-omal P450 distinguished from other P450 in cytoplasm thatn the microsomal P450s this hydroxylase activity is supportednly by NADPH. A very valuable phenomenon in our experi-ents was that Ca2+ and Fe2+ could greatly enhance the activity

f the 11�-hydroxylases (no report found) but other bivalentetal ions including Mg2+, Mn2+, Cu2+, and Zn2+ decreased

he activity of the 11�-hydroxylase. The study on the detailolecular mechanism of this phenomenon is significant. Maybe

he bioconversion rate by the whole cell could be increased bydding proper amount of Ca2+ or Fe2+ in medium.

11�-Hydroxylase is composed of two protein fractions, a450 protein as an oxidase and a flavoprotein as a reductase. Theetail purification procedure for the P450 fraction of the 11�-ydroxylase was described in Table 1. The P450 fraction withsingle band on the SDS-PAGE (Fig. 10) was obtained, givingmaximum absorbance at 448 nm in reduced carbon monox-

de difference spectra (Fig. 11B) and a MW of about 60 kDahat corresponds to the reported values of other organism 11�-ydroxylases (P450: about 60 kDa and reductase: about 79 kDa22,25]). Our investigation also indicated that in the presence ofubstrate, the P450 showed three absorption spectrums, a maxi-um at 389 nm, a medium at 368 nm and a minimum at 416 nm,

espectively (Fig. 11A). It was reported that a substrate moleculeas buried in an internal pocket just above the heme distal sur-

ace adjacent to the oxygen binding site [31], and its contractionith oxygen atom could change the spin ferric form. This is

alled as type I substrate binding spectrum, typical for P450,uggesting that the binding of a substrate to P450 near the hemean cause a change in the spin state of the heme from low to high32]. The P450 alone in reduced carbon monoxide differencepectra revealed an absorbance at 420 nm besides a maximumt 448 nm (Fig. 11B), suggesting that the P450 probably lost thectivity, or did not react with CO [33].

In summary, the 11�-hydroxylation of steroids is mediatedy P450 as a part of monooxygenase enzyme system in the fil-mentous fungus A. coerulea IBL02. It is inducible and needsADPH as coenzyme. The optimum reaction pH value and tem-

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l Technology 41 (2007) 71–79

erature are close to pH 7.0 and 30 ◦C, respectively. Ca2+ ande2+ could enhance the enzyme activity. The purified P450 hasmolecular mass of about 60 kDa estimated by SDS-PAGE.

cknowledgements

We thank Mr. Feng-Qing Wang, Xiang-Ping Shen andian Sun for their excellent assistant. This research work wasupported by grant no. F200-C-22 from Returnee Scientificesearch Startup Foundation of the Ministry of Education ofhina, and also by grant no. 044319202 from Science & Tech-ology Committee of Shanghai Municipal Government.

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