Optimization of biotransformation from phytosterol to androstenedione by a mutant Mycobacterium neoaurum ZJUVN-08

Optimization of biotransformation from phytosterol to androstenedione by a mutant Mycobacterium neoaurum ZJUVN-08 1 first-author 2 1 1 1 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:gqhe@zju.edu.cn †E-mail:gqhe@zju.edu.cn gqhe@zju.edu.cn Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310058, China Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310058, China Department of Food Science and Technology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China Department of Food Science and Technology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China Biotransformation of phytosterol (PS) by a newly isolated mutant Mycobacterium neoaurum ZJUVN-08 to produce androstenedione has been investigated in this paper. The parameters of the biotransformation process were optimized using fractional factorial design and response surface methodology. Androstenedione was the sole product in the fermentation broth catalyzed by the mutant M. neoaurum ZJUVN-08 strain. Results showed that molar ratio of hydroxypropyl-β-cyclodextrin (HP-β-CD) to PS and substrate concentrations were the two most significant factors affecting androstenedione production. By analyzing the statistical model of three-dimensional surface plot, the optimal process conditions were observed at 0.1 g/L inducer, pH 7.0, molar ratio of HP-β-CD to PS 1.92:1, 8.98 g/L PS, and at 120 h of incubation time. Under these conditions, the maximum androstenedione yield was 5.96 g/L and nearly the same with the non-optimized (5.99 g/L), while the maximum PS conversion rate was 94.69% which increased by 10.66% compared with the non-optimized (84.03%). The predicted optimum conditions from the mathematical model were in agreement with the verification experimental results. It is considered that response surface methodology was a powerful and efficient method to optimize the parameters of PS biotransformation process. Phytosterol Mycobacterium neoaurum ZJUVN-08 Androstenedione Hydroxypropyl-β-cyclodextrin Introduction 1 As two important pharmaceutical steroid precursors, androst-4-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD), which belong to 17-ketosteroid family can be further used to produce a wide range of pharmaceutical steroid derivatives (Fernandes et al., 2003 2003 ). Being an alternative to chemical methods, bioconversion of steroids has been studied extensively (Malaviya and Gomes, 2008 2008 ). Selective side chain degradation of sterols to 17-ketosteroids is one of the most widely used biotransformation reactions of steroids. It is well-known that phytosterols (PSs) are suitable raw materials for microbial degradation to 17-ketosteroids because of low cost and easy availability (Fernandes and Cabral, 2007 2007 ). In recent years, microbial selective cleavages of PS to AD and ADD have been reported by many researchers (Huang et al., 2006 2006 ; Pérez et al., 2006 2006 ; Sripalakit et al., 2006 2006 ; Wang et al., 2006 2006 ). All things considered, the major obstacle of microbial transformation of PS is the low aqueous solubility (1 μmol/L) of substrate (Malaviya and Gomes, 2008 2008 ). Consequently, many efforts have been devoted to overcoming the low solubility of sterols and improving product yield. These include the application of various water-miscible organic solvents (Simon et al., 1998 1998 ), water-immiscible organic solvents (Cruz et al., 2001 2001 ), silicone oil (Kutney et al., 2000 2000 ; Stefanov et al., 2006 2006 ), polypropylene glycol (Kutney et al., 2000 2000 ; Stefanov et al., 2006 2006 ), cloud point system (Wang et al., 2004 2004 ; 2006 2006 ), and microemulsions (Stefan et al., 2002 2002 ). However, organic solvents present many drawbacks such as toxicity to microbial cells and environmental hazards (Schimid et al., 1998 1998 ; Kim et al., 2007 2007 ). Thus, the main focus of steroid biotransformation is to increase the solubility of hydrophobic substrates as well as retain the activity and stability of microbial cells. Cyclodextrins (CDs) are cyclic oligosaccharides with 6–8 glucopyranose units bonded by α-1,4-linkage and have a structure that conveys a hydrophobic central cavity in the molecule (Nalluri et al., 2005 2005 ). Based on this molecular structure, CDs have been widely used to form host-guest complexes with hydrophobic compounds in their cavities. By inclusion with CDs, the hydrophobic compounds are encapsulated in this CD cavity. The hydrophilic outer surface of the inclusion complex shows advantageous solubility and bioavailability, favoring the solubility of hydrophobic compounds in aqueous media (Szejtli, 1998 1998 ). Thus, the aqueous solubility of hydrophobic compounds is subsequently improved (Loftsson and Brewster, 1996 1996 ). As a result, CDs have been extensively used in the microbial transformation of steroids (Hesselink et al., 1989 1989 ; Roglič et al., 2005 2005 ; 2007 2007 ). Hydroxypropyl-β-cyclodextrin (HP-β-CD) is one kind of chemically modified β-cyclodextrin derivate, which has higher water solubility and complexing ability than CD, and has been widely added in medium to improve steroid biotransformation (Manosroi et al., 2008 2008 ; Ma et al., 2009 2009 ; Zhang et al., 2009 2009 ). This work used response surface methodology (RSM) to optimize the biotransformation process of PSs. RSM is an efficient mathematical and statistical technique applied to investigate the relationship between several independent variables and the generated responses (Senanayake and Shahidi, 2002 2002 ). The application of RSM contributes to the rapid screening of experimental factors and factors interactions, thus obtaining the optimal process parameters, which significantly reduces the cost of experimentation (Ghosh and Hallenbeck, 2010 2010 ). Moreover, this method has been extensively used to optimize the operational conditions of bioconversion process (Goswami et al., 2009 2009 ; Liu et al., 2010 2010 ). In recent years, several studies have reported that Mycobacterium neoaurum strain is able to degrade PS and yield AD (Rodina et al., 2008 2008 ; Wei et al., 2010 2010 ). In the preliminary study, an M . neoaurum strain (designated as M. neoaurum ZJUVN), which could degrade PS, was successfully isolated from soil. The strain was capable of converting PS to AD and ADD. Further, this strain was bred using low-energy nitrogen ion (N + ) implantation method for accumulation of AD as the only product. A genetically stable mutant, which could substantially accumulate AD as the sole product, M. neoaurum ZJUVN-08 was screened and used in this work. The pathway of PS side chain degradation to AD is shown in Fig. 1 1 . In the present study, the influence of inducer concentration, substrate concentration, molar ratio of HP-β-CD to substrate, pH, and incubation time on AD yield and PS conversion was studied using RSM. The optimized PS bioconversion conditions by M. neoaurum ZJUVN-08 were obtained. Materials and methods 1 Materials 2 PS (95%) was obtained from Zhejiang DAVI Biochemistry Co., Ltd. (China). AD and ADD were obtained from Sigma (USA). HP-β-CD was obtained from Jiangsu Kunshan Chemical Industries Co., Ltd. (China). Methanol was of high performance liquid chromatography (HPCC) grade. All other chemicals and solvents in this work were of analytical grade. Microorganisms 2 The soil samples were obtained from Taian suburb, Shandong, China. The strain M. neoaurum ZJUVN was isolated using gradient dilution of soil samples. The genotypical and morphological identifications were performed by 16S ribosomal RNA (rRNA) sequence analysis and biochemical tests (Dogra and Qazi, 2001 2001 ), respectively. The nucleotide sequence of this strain was submitted to GenBank and obtained the accession number JN935007. M. neoaurum ZJUVN-08 (CGMCC No. 5447) was evolved from M. neoaurum ZJUVN using a low-energy N + implantation method. The low-energy N + implantation was conducted with an ion beam bioengineering instrument at the Institute of Bioengineering, Zhejiang University of Technology, China. The energy of N + used in this work was 10 keV. The implantation doses ranged from 20×2.6×10 13 to 180×2.6×10 13 ions/cm 2 , and were finally chosen at 100×2.6×10 13 ions/cm 2 . The mutation of the strain was carried out as described by Liu et al. ( 2007 2007 ). After ion implantation treatment, samples were diluted by physiological sodium chloride solution, and then placed onto seed medium plates containing 1.0 g/L PS. Visible colonies were picked and further used to test PS degradation capabilities by thin layer chromatography (TLC) and HPLC. The selected mutant M. neoaurum ZJUVN-08 exhibited enhanced ability to produce AD and could accumulate AD as the sole product. Culture medium 2 The stock culture was maintained on a medium which contained (g/L): yeast extract, 5.0; glycerol, 10.0; MgSO 4 ·7H 2 O, 0.5; K 2 HPO 4 , 0.5; NH 4 Cl, 1.0; agar power, 20.0 (pH 7.0). The seed medium contained (g/L): glucose, 5.0; glycerol, 20.0; NH 4 NO 3 , 2.0; MgSO 4 ·7H 2 O, 0.5; citric acid, 2.0; K 2 HPO 4 , 0.5; ferric ammonium citrate, 0.05 (pH 7.0). Fermentation medium contained (g/L): glucose, 10.0; K 2 HPO 4 , 0.5; citrate acid, 2.0; MgSO 4 ·7H 2 O, 0.5; (NH 4 ) 2 HPO 4 , 3.5; ferric ammonium citrate, 0.05 (pH 7.0). Biotransformation process 2 PS biotransformation by growing-cell culture was performed in two consecutive steps, namely, activation culture in seed medium and biotransformation culture in fermentation medium. The activation culture was grown in 250-ml shake flasks containing 50 ml seed medium at 30 °C, 200 r/min, and 48 h. PS was served as the inducer of bioconversion process, and was added into the seed medium with 0.1% Tween 80 after 24 h of incubation. After activation culture, 5 ml of seed culture liquid was extracted and transferred to 50 ml fermentation medium, then incubated in 250-ml shake flasks at 30 °C, 200 r/min, and 96–144 h. PS was added to the fermentation medium as an aqueous HP-β-CD solution, and sonicated for 15 min until complete homogenization before sterilization. As the optimization procedure was carried out, inducer concentration, substrate concentration, molar ratio of HP-β-CD/PS, pH value, and incubation time were varied to the designed conditions. Substrate controls and culture controls were grown under the same conditions. All experiments were carried out in triplicate. Separation, purification, and structural elucidation of biotransformation products 2 At the end of fermentation period, 10 ml culture broth was taken from the flasks and extracted twice by vigorous shaking for 2 h with the same volume of ethyl acetate. Then, 5 μl samples were applied onto one TLC plate which was spread by silica gel GF254 (0.25 mm). The solvent system used for TLC was petroleum ether:ethyl acetate (6:4, v/v) and the compounds were visualized by iodine staining. Steroid products were observed as orange spots. The products were isolated by preparative TLC (silica gel GF254, 1.0 mm). The spots obtained were scraped off and dissolved in methanol. After centrifugation, the products were further purified by preparative HPLC (Waters Sunfire C 18 column, 5 μm particles, and 250 mm×10 mm). The mobile phase was composed of methanol and water (80:20, v/v), flow rate was 5 ml/min, and detection wavelength was 254 nm. The compounds obtained were identified by mass spectrum (MS), infrared spectrum (IR), and nuclear magnetic resonance (NMR) analyses. MS was performed using Waters Q-TOF micro MS (USA). IR spectra were obtained on Nicolet 6700 FT-TR spectrometer (USA). 1 H and 13 C NMR spectra were carried out on a Bruker AVIII500 MHz NMR spectrometer (Sweden) at 500 and 125 MHz, respectively. CDCl 3 was used as solvent. Quantification of biotransformation product 2 Quantification of products was carried out using HPLC on Waters Symmetry C 18 column (5 μm particles, 250 mm×4.6 mm). The mobile phase was composed of methanol and water (80:20, v/v), the flow rate was 1 ml/min and column temperature at 30 °C. The sample volumes injected were 10 μl and the detection wavelength was 254 nm. Conversion rate of PS to AD was estimated using the following equation: $${\text{CR}} = \frac{{({m_{{\text{AD}}}}/{\text{M}}{{\text{W}}_{{\text{AD}}}})}}{{({m_{\text{p}}}/{\text{M}}{{\text{W}}_{\text{p}}})}} \times 100\%$$ , where m ad and m p are the weights of AD and PS, respectively; MW AD and MW p are the molecular weights of AD and PS, respectively (Sripalakit et al., 2006 2006 ). Experimental design 2 In order to optimize the conditions of biotransformation for AD yield and PS conversion, a set of statistically designed experiments were conducted. First, fractional factorial design (FFD) was used to identify the significant factors affecting AD yield and PS conversion rate by M. neoaurum ZJUVN-08. Then central composite design (CCD) was utilized to optimize the most important factors for the maximum AD yield and PS conversion rate. A 2 5−1 FFD which included 16 sets of trials with four replicates at center point was carried out in duplicate to screen the critical parameters influencing AD yield and PS conversion rate (Chen et al., 2002 2002 ). The coded values of variables were determined by the following equation: $${X_i} = ({x_i} - {x_0})/\Delta {x_i}$$ , where X i stands for the coded value, x i stands for the actual value, x 0 stands for the real value at the center point, and Δ x i stands for the step change value (Chen et al., 2008 2008 ). The coded and actual values of variables are given in Table 1 1 . The AD yield and PS conversion rate are considered as responses Y AD and Y Conversion , respectively. After the analysis of FFD, a first-order model was gained. A five-level CCD was performed to fit the empirical quadratic polynomial model (Myers and Montgomery, 2002 2002 ). The quadratic model equation was as follows: $$y = {b_0} + \sum {{b_i}} {x_i}\sum {{b_{ii}}x_i^2} + \sum {{b_{ij}}{x_i}{x_j}}$$ , where y is predicted response, b 0 is offset term, b i is linear offset, b ii is squared offset, b ij is interaction coefficient, x i and x j stand for independent variables (Chen et al., 2002 2002 ). x 1 : inducer concentration; x 2 : pH; x 3 : molar ratio of HP-β-CD/PS; x 4 : substrate concentration; x 5 : incubation time; X : coded value X x 1 (g/L) x 2 x 3 x 4 (g/L) x 5 (h) −1 0.05 6.5 1:1 5 96 0 0.10 7.0 2:1 10 120 1 0.15 7.5 3:1 15 144 The regression and graphical analysis of data were performed using Design Expert software (Version 6.0, Stat-Ease Inc., Minneapolis, USA). Model verification and time-course experiment 2 Biotransformation process was performed by a procedure similar to normal transformation experiments using the optimized conditions under a 5-L fermenter (BIOF-2000, Shanghai Gaoji Bioengineering, China) containing 3 L fermentation medium. The pH was monitored by a pH probe (F-635-B120-DH, China) and maintained at 7.0 by addition of 5 mol/L NaOH. The fermenter was run at 30 °C, 350 r/min, and aeration rate of 1 vvm (air volume/culture volume/min). After the seed was cultured for two days, PS was added into the transformation medium to start the conversion process. At a regular time interval, samples were taken to evaluate the yield of AD and PS conversion rate as described earlier, performed in triplicates. Results 1 Bioconversion products of PS using M. neoaurum ZJUVN-08 2 As can be seen from Fig. 2 2 , in bioconversion medium inoculated with parent M. neoaurum ZJUVN strain, PS was transformed into two products, the rate of flow ( R f ) value were 0.31 and 0.48, respectively, which were in agreement with those of the authentic ADD and AD. There was only one product in the bioconversion medium inoculated with mutant M. neoaurum ZJUVN-08 strain. The R f value (0.48) and HPLC peak occurred at the retention time of 5.2 min, which were identical with those of the authentic AD. It is proposed that the compound obtained after bioconversion was AD. Lane 1: standard phytosterols; Lane 2: standard AD; Lane 3: standard ADD; Lane 4: bioconversion medium inoculated M . neoaurum ZJUVN strain; Lane 5: bioconversion medium inoculated mutant M . neoaurum ZJUVN-08 strain Structure characterization of purified product 2 The purified compound was white powder and was further identified using NMR, MS, and IR analyses. MS data of the purified compound exhibited a molecular ion [M+H] + at m / z 287, which indicated that the molecular mass was 286 and is in line with that of authentic AD. IR V max (cm −1 ): 2 920, 2 850, 1 736, 1 661, and 1 615. IR spectra peaks at 1 736 cm −1 supported the structure of 17-C=O, 1 661 cm −1 (3-C=O), 1 617 cm −1 (4-CH=C), 2 920 cm −1 (-CH), and 2 850 cm −1 (-CH-CH 3 ), which are in full agreement with the structure of AD. The data obtained from 1 H NMR (500 MHz, CDCl 3 ) were as follows ( δ ): 0.924, 1.223, and 5.747. Chemical shifts at δ 5.747 suggested the presence of 4-ene (de Brabandere et al., 1997 1997 ). Chemical shifts at δ 0.924, and δ 1.223 demonstrated the C 18 and C 19 methyl groups, respectively. 13 C NMR (125 MHz, CDCl 3 ) spectra data from C 1 to C 19 were as follows ( δ ): 35.70, 33.89, 199.31, 124.14, 170.32, 32.56, 30.75, 35.15, 53.82, 38.64, 20.31, 31.28, 47.49, 50.84, 21.74, 35.84, 220.12, 13.70, and 17.37. The chemical shifts of C-17 at δ 220.12 verified the structure of 17-C=O and were identical with the data of AD reported by the recent studies (Lin et al., 2009 2009 ; Hegazy et al., 2012 2012 ). From the above results, we concluded that the purified product was AD. FFD analysis for process variables affecting AD yield and PS conversion 2 A screening design was performed to estimate the effects of five factors, namely inducer concentration ( X 1 ), pH ( X 2 ), molar ratio of HP-β-CD/PS ( X 3 ), substrate concentration ( X 4 ), and incubation time ( X 5 ). The design and results of FFD are indicated in Table 2 2 . Effects of the five variables were analyzed by multiple regression analysis method and are illustrated in Table 3 3 . The results indicated that the molar ratio of HP-β-CD/PS ( X 3 ) and substrate concentration ( X 4 ) were highly significant factors ( P < 0.0001). However, inducer concentration ( X 1 ), pH ( X 2 ), and incubation time ( X 5 ) were insignificant factors, which exhibited a probability level below 90%. As shown in Table 3 3 , coefficient estimates of molar ratio of HP-β-CD/PS and substrate concentration were both negative, which indicated that the decreases of molar ratio of HP-β-CD/PS and substrate concentration would lead to the increases of AD yield and PS conversion rate. Consequently, molar ratio of HP-β-CD/PS and the substrate concentration were chosen to be investigated in the further optimization experiment. Run X 1 X 2 X 3 X 4 X 5 Y AD (g/L) Y Conversion (%) 1 1 1 1 1 1 1.01 9.51 2 −1 −1 −1 1 −1 2.11 19.93 3 −1 −1 −1 −1 1 2.67 75.83 4 −1 1 1 −1 1 2.11 59.88 5 1 −1 1 −1 1 2.10 59.83 6 0 0 0 0 0 6.00 85.26 7 −1 −1 1 −1 −1 2.06 58.47 8 1 −1 −1 1 1 2.38 22.56 9 −1 1 1 1 −1 0.96 9.07 10 −1 1 −1 1 1 1.81 17.09 11 −1 1 −1 −1 −1 2.63 74.75 12 1 −1 −1 −1 −1 2.61 74.18 13 −1 −1 1 1 1 0.95 8.99 14 1 1 −1 1 −1 2.07 19.58 15 1 −1 1 1 −1 0.90 8.55 16 0 0 0 0 0 5.95 84.49 17 0 0 0 0 0 6.06 86.04 18 0 0 0 0 0 5.99 85.12 19 1 1 1 −1 −1 2.08 59.14 20 1 1 −1 −1 1 2.68 76.05 Significance at 95% confidence level df : degree of freedom Response Variable Coefficient estimate df F -value Probe > F 5 AD yield X 1 0.034 1 5.39 0.116 X 2 −0.028 1 3.81 0.157 X 3 −0.420 1 1 431.12 < 0.0001 * * X 4 −0.420 1 1 419.71 < 0.0001 * * X 5 0.018 1 2.54 0.209 5 PS conversion X 1 0.340 1 4.48 0.125 X 2 −0.200 1 1.65 0.289 X 3 −6.660 1 1 748.92 < 0.0001 * * X 4 −26.430 1 27 554.93 < 0.0001 * * X 5 0.380 1 5.58 0.106 The analysis of variance (ANOVA) of FFD was carried out and the results are shown in Table 4 4 . P values for AD yield and PS conversion rate were 0.0005 and 0.0001, respectively. The higher values of determination coefficient ( R 2 =0.9990, 0.9999 for AD yield and PS conversion rate, respectively) further confirmed the effectiveness of the models. The variance between mean value of center points and factorial points in Table 2 2 was evaluated by t -test, which suggested that the difference was significant ( P < 0.01). Hence, a conclusion could be drawn that the optimum point was in the range of our design and the steepest decent experiment could be omitted. df : degree of freedom Response Source df Mean square F -value Probe > F 4 AD yield Model 15 0.42 207.05 0.0005 Curvature 1 52.61 26 172.13 <0.0001 Pure error 3 0.0020 Cor total 19 4 PS conversion Model 15 795.32 1 961.04 <0.0001 Curvature 1 6 305.33 15 547.29 <0.0001 Pure error 3 0.41 Cor total 19 Optimization of the significant factors by CCD and RSM 2 CCD and RSM were employed to optimize the significant factors for AD yield and PS conversion. Based on the above results, the value of inducer concentration ( X 1 ), pH ( X 2 ) and incubation time ( X 5 ) were fixed at the center-point level. The final optimization experiment of biotransformation conditions was performed using a two-variable-five-level CCD with five repetitions at the center points, thus the total number of experiments needed was n =4+4+5=13. The central composite design and its results are summarized in Tables 5 5 and 6 6 . The experimental data of CCD were fitted to a second-order polynomial model obtained by multiple regression analysis. ANOVA was used to test the adequacy of the model and the results are presented in Table 7 7 . The F -value (33.34 and 71.23) and Probe > F -value ( P < 0.0001 and P < 0.0001) of the models for AD yield and PS conversion rate implied that the models were very significant. The high values of determination coefficients ( R 2 ) were found to be 0.9597, 0.9807 for AD yield and PS conversion rate, respectively, which indicates the experimental data and the predicted values are of good consistency. The quadratic polynomial equations in terms of coded factors were given below: $${Y_{{\text{AD}}}} = {\text{6}}.00 - 0.{\text{52}}{X_{\text{3}}} - 0.{\text{33}}{X_{\text{4}}} - {\text{1}}.{\text{57}}X_3^2 - 0.{\text{75}}X_4^2 - 0.0{\text{89}}{X_{\text{3}}}{X_{\text{4}}}$$ , $${Y_{{\text{Conversion}}}} = {\text{85}}.{\text{23}} - {\text{7}}.{\text{36}}{X_{\text{3}}} - {\text{17}}.{\text{29}}{X_{\text{4}}} - {\text{22}}.{\text{2}}0X_3^2 - {\text{7}}.{\text{24}}X_4^2 + 0.{\text{39}}{X_{\text{3}}}{X_{\text{4}}}$$ . x 3 : molar ratio of HP-β-CD/PS; x 4 : substrate concentration; * X 3 =( x 3 −2)/0.5; X 4 =( x 4 −10)/2 X x 3 x 4 (g/L) −1.414 1.29 7.17 −1 1.5 8 0 2 10 1 2.5 12 1.414 2.71 12.83 Run X 3 X 4 Y AD (g/L) Y Conversion (%) 1 0 0 6.03 85.69 2 0 0 5.93 84.26 3 −1 −1 4.43 78.47 4 0 0 6.01 85.38 5 1 −1 3.44 61.02 6 0 −1.414 4.97 98.34 7 0 0 5.97 84.76 8 −1.414 0 3.78 53.64 9 0 1.414 4.59 50.37 10 1.414 0 2.50 35.56 11 0 0 6.06 86.05 12 −1 1 3.57 42.21 13 1 1 2.22 26.33 Response Source Sum of squares Mean square F -value Probe > F 9 AD yield Model 22.16 4.43 33.34 <0.0001 X 3 2.13 2.13 16.01 0.0052 X 4 0.85 0.85 6.42 0.0390 X 3 2 17.06 17.06 128.29 <0.0001 X 4 2 3.89 3.89 29.26 0.0010 X 3 X 4 0.032 0.032 0.24 0.6388 Residual 0.93 0.13 Lack of fit 0.92 0.31 119.73 0.0002 Pure error 0.010 0.00256 9 PS conversion Model 6 387.45 1 277.49 71.23 <0.0001 X 3 433.64 433.64 24.18 0.0017 X 4 2 390.19 2 390.19 133.27 <0.0001 X 3 2 3 429.69 3 429.69 191.23 <0.0001 X 4 2 364.29 364.29 20.31 0.0028 X 3 X 4 0.62 0.62 0.034 0.8582 Residual 125.54 17.93 Lack of fit 123.47 41.16 79.60 0.0005 Pure error 2.07 0.52 The significance of linear terms and quadratic terms for X 3 and X 4 is summarized in Table 7 7 , and the results implied that the molar ratio of HP-β-CD/PS and substrate concentration had a highly significant effect ( P < 0.01) on the yield of AD as well as the PS conversion. The interaction effect of X 3 and X 4 was not significant ( P > 0.05). As can be seen from the response surface plot (Fig. 3 3 ), a maximum value in this surface plot was observed. With the coded level of −0.16 ( X 3 ) and −0.56 ( X 4 ), AD yield and PS conversion rate reached the maximum, which further verifies that the maximum point existed in the fitted surface. The predicted maximum responses from the regression models for AD yield and PS conversion were 5.98 g/L and 93.26%, respectively. The residuals analysis of CCD data, presented in Fig. 4 4 , further validates that the models simulated the actual process of PS biotransformation under the present system. X 3 is the molar ratio of HP-β-CD/PS, X 4 is the substrate concentration In order to confirm the validity of the models, experiments were carried out with the optimized parameters. The obtained values of AD yield and PS bioconversion rate were (5.93±0.075) g/L and ((94.74±1.19)%), respectively. This further indicates the validity of optimization design. Biotransformation process examination under the optimum conditions in the 5-L fermenter 2 As the influencing factors had been optimized, biotransformation in a 5-L fermenter was examined and compared between the optimized and the non-optimized conditions. Fig. 5 5 shows the changes of AD yield and PS conversion rate versus time. After 24 h biotransformation, AD was accumulated and maximized at 120 h for both the optimized and non-optimized conditions. With the prolongation of biotransformation time, the concentration of final product in fermentation broth was decreased. This could be caused by the product degradation of mycobacteria cells (Malaviya and Gomes, 2008 2008 ). Under the optimized conditions, with lower substrate concentration (8.89 g/L) and lower molar ratio of HP-β-CD/PS (1.92:1), the maximum yield of AD was 5.96 g/L and was nearly the same with the non-optimized (5.99 g/L), while maximum PS conversion rate was 94.69% which increased by 10.66% compared with the non-optimized (84.03%). When the concentration of substrate and molar ratio of HP-β-CD/PS were decreased from 10 to 8.89 g/L and 2:1 to 1.92:1, the costs of PS and HP-β-CD are also decreased by 11% and 15%, respectively. This can result in significant cost savings when the biotransformation is implemented to large-scale production. Discussion 1 HP-β-CD has been demonstrated to improve steroid biotransformation in aqueous media such as dehydrogenation of cortisone acetate (Ma et al., 2009 2009 ) and the production of androstadienedione from progesterone (Manosroi et al., 2008 2008 ). The results obtained in this paper indicated that the addition of HP-β-CD in transformation media was in favor of increasing substrate solubility. The yield of AD and PS conversion rate varied significantly according to the molar ratio of HP-β-CD/PS. As the present data were evaluated, the best molar ratio of HP-β-CD/PS was 1.92:1. This might be due to the formation of 1.92:1 HP-β-CD and PS complex. As shown in Table 2 2 , when the molar ratio of HP-β-CD/PS were at 1:1 and 3:1, both the PS conversion rate and AD yield were very low. The reason might be that when low concentration of HP-β-CD was used, efficient inclusion complex cannot be formed between the PS molecule and HP-β-CD; and at high concentration of HP-β-CD, too much HP-β-CD could hinder the effective contact of enzyme and substrate. Several researchers have reported that when a higher concentration of substrate was used, HP-β-CD may serve as a reservoir of available substrate (Loftsson and Brewster, 1996 1996 ; Zhang et al., 2009 2009 ). Therefore, the presence of HP-β-CD makes it possible to maintain higher concentrations of PS in biotransformation media. Moreover, mycobacteria cells were proven to have good tolerance to HP-β-CD due to the unique composition of their cell wall (Shen et al., 2011 2011 ), which facilitates the application of HP-β-CD in PS bioconversion. The yield of the final product and the bioconversion rate are two critical elements to measure the economy of biotransformation procedure (Wang et al., 2004 2004 ). It can be seen from Table 2 2 that as low substrate concentration was used, the PS conversion rate increased but the yield of product decreased. This made it more difficult for the downstream product separation, thus increasing the production cost from an economical viewpoint. With increased substrate concentration, the yield of AD was enhanced but the PS conversion rate decreased due to the low solubility of high concentration of substrate and could not effectively contact the microorganisms. Therefore, a threshold concentration of substrate must exist. Under this concentration, the highest product yield and conversion rate are both achieved. In the present work, the optimal substrate concentration was 8.89 g/L at 1.92:1 molar ratio of HP-β-CD/PS. Many biotransformation approaches have been performed for improving of steroid bioconversion. As reported earlier, in organic media, the highest conversion rate from sitosterol to AD using immobilized Mycobacterium sp. NRRL B-3805 cells on Celite 545 was 89% when the initial concentration of substrate was 5.28 g/L (Cabral et al., 1997 1997 ). In an organic-aqueous two phase media (dioctyl phthalate (DOP)-buffer system), also using Mycobacterium sp. NRRL B-3805 cells to catalyze the process, full conversion of 12 mmol/L sitosterol could be achieved at 24 h when biomass was 70 g/L (Staebler et al., 2004 2004 ). In poly propylene glycol (PPG)/complex fermentation media, the conversion rate of PS to AD by Mycobacterium MB 3683 was found to be 90% when the initial concentration of substrate was 5 g/L at 168 h of incubation time (Kutney et al., 2000 2000 ). Bioconversion of cholesterol to produce AD and ADD in cloud point system was studied using growing Mycobacterium sp. NRRL B-3683 cells, the obtained conversion rate was 93% at 168 h when the initial concentration of substrate was 14.5 g/L (Wang et al., 2004 2004 ). Again, using resting mycobacterial cells, microbial conversion of PS to ADD in an optimized cloud point system was achieved. The yield of AD reached up to 12 g/L and incubation time was shortened to 96 h when the initial substrate concentration increased to 25 g/L (Wang et al., 2006 2006 ). In a liquid polymer bioconversion system, silicone B oil was proven to be the best substrate carrier, and the conversion rate of sitosterol to AD at 120 h was 83%. It was also found that when the oil phase increased, the efficiency of conversion process decreased accordingly (Carvalho et al., 2009 2009 ). In this paper, the highest yield of AD (5.96 g/L) and PS conversion rate (94.69%) were obtained at 120 h of incubation time when the initial concentration of substrate was 8.98 g/L. Compared with biotransformation processes catalyzed by industrial mycobacteria strains in different transformation systems, the conversion of PS catalyzed by mutant M. neoaurum ZJUVN-08 in HP-β-CD solution showed a certain advantage over the processes in organic media, PPG/complex fermentation media and liquid polymer of silicone B oil systems, which indicated it was a useful bioconversion process and further confirmed the improvement of the biotransformation process when HP-β-CD was added. Moreover, AD was the sole product in the fermentation broth, which made the mutant M. neoaurum ZJUVN-08 a useful strain for large-scale production of AD from PS. Conclusions 1 In the present work, bioconversion of PS by M. neoaurum ZJUVN-08 was thoroughly investigated. AD was the sole product in the final fermentation broth catalyzed by the new mutant strain. FFD was conducted to screen the significant factors influencing the biotransformation process. Then CCD and RSM were employed for further optimization process. The optimized conditions were observed at inducer concentration of 0.01 g/L, pH 7.0, molar ratio of HP-β-CD/PS 1.92:1, substrate concentration of 8.89 g/L, and incubation time at 120 h. Under the optimum condition, more than 94% of PS was degraded and was obtained the maximum yield of AD 5.96 g/L, which is a cost-efficient process compared to the non-optimized condition. It can be seen that RSM is an effective method to optimize the PS bioconversion conditions. For in-depth study, further studies should be undertaken on the mode of adding HP-β-CD in fermentation medium. References JMS Cabral , MR Aires-Barros , H Pinheiro , DMF Prazeres . Biotransformation in organic media by enzymes and whole cells . J Biotechnol , 1997 . 59 ( 1-2 ): 133 - 143 . 9487721 DOI: 10.1016/s0168-1656(97)00176-4 http://doi.org/10.1016/s0168-1656(97)00176-4 . Biotransformation in organic media by enzymes and whole cells F Carvalho , MPC Marques , CCCR de Carvalho , JMS Cabral , P Fernandes . Sitosterol bioconversion with resting cells in liquid polymer based systems . Biores Technol , 2009 . 100 ( 17 ): 4050 - 4053 . DOI: 10.1016/j.biortech.2009.03.044 http://doi.org/10.1016/j.biortech.2009.03.044 . Sitosterol bioconversion with resting cells in liquid polymer based systems QH Chen , GQ He , AMA Mokhtar . Optimization of medium composition for the production of elastase by Bacillus sp. EL31410 with response surface methodology . Enzyme Microb Technol , 2002 . 30 ( 5 ): 667 - 672 . DOI: 10.1016/s0141-0229(02)00028-5 http://doi.org/10.1016/s0141-0229(02)00028-5 . Optimization of medium composition for the production of elastase by Bacillus sp. EL31410 with response surface methodology QH Chen , ML Fu , J Liu , HF Zhang , GQ He , H Ruan . Optimization of ultrasonic-assisted extraction (UAE) of betulin from white irch bark using response surface methodology . Ultrason Sonochem , 2008 . 16 ( 5 ): 599 - 604 . 19110462 DOI: 10.1016/j.ultsonch.2008.11.009 http://doi.org/10.1016/j.ultsonch.2008.11.009 . Optimization of ultrasonic-assisted extraction (UAE) of betulin from white irch bark using response surface methodology A Cruz , P Fernandes , JMS Cabral , HM Pinheiro . Whole-cell bioconversion of β-sitosterol in aqueous-oganic two-phase systems . J Mol Catal B: Enzym , 2001 . 11 ( 4-6 ): 579 - 585 . DOI: 10.1016/s1381-1177μ00)00047-3 http://doi.org/10.1016/s1381-1177μ00)00047-3 . Whole-cell bioconversion of β-sitosterol in aqueous-oganic two-phase systems VI de Brabandere , LM Thienpont , D Stockl , AP de Leenheer . 13 C-NMR and mass spectral data of steroids with a 17,17-dialkyl-l8-nor-l3(14)-ene substructure . J Lipid Res , 1997 . 38 ( 4 ): 780 - 789 . 9144092 . 13C-NMR and mass spectral data of steroids with a 17,17-dialkyl-l8-nor-l3(14)-ene substructure N Dogra , GN Qazi . Steroid biotransformation by different strains of Micrococcus sp . Folia Microiol , 2001 . 46 ( 1 ): 17 - 20 . DOI: 10.1007/BF02825877 http://doi.org/10.1007/BF02825877 . Steroid biotransformation by different strains of Micrococcus sp P Fernandes , JMS Cabral . Phytosterols: applications and recovery methods . Biores Technol , 2007 . 98 ( 12 ): 2335 - 2350 . DOI: 10.1016/j.biortech.2006.10.006 http://doi.org/10.1016/j.biortech.2006.10.006 . Phytosterols: applications and recovery methods P Fernandes , A Cruz , B Angelova , HM Pinheiro , JMS Cabral . Microbial conversion of steroid compounds: recent developments . Enzyme Microb Technol , 2003 . 32 ( 6 ): 688 - 705 . DOI: 10.1016/s0141-0229(03)00029-2 http://doi.org/10.1016/s0141-0229(03)00029-2 . Microbial conversion of steroid compounds: recent developments D Ghosh , PC Hallenbeck . Response surface methodology for process parameter optimization of hydrogen yield by the metabolically engineered strain Escherichia coli DJT135 . Bioresour Technol , 2010 . 101 ( 6 ): 1820 - 1825 . 19897361 DOI: 10.1016/j/biortech.2009.10.020 http://doi.org/10.1016/j/biortech.2009.10.020 . Response surface methodology for process parameter optimization of hydrogen yield by the metabolically engineered strain Escherichia coli DJT135 D Goswami , R Sen , JK Basu , S De . Maximization of bioconversion of castor oil into ricinoleic acid by response surface methodology . Bioresour Technol , 2009 . 100 ( 18 ): 4067 - 4073 . 19419859 DOI: 10.1016/j/biortech.2008.11.040 http://doi.org/10.1016/j/biortech.2008.11.040 . Maximization of bioconversion of castor oil into ricinoleic acid by response surface methodology MEF Hegazy , AM Gamal-Eldeen , AM Ei-Halawany , AEHH Mohamed , PW Paré . Steroidal metabolites transformed by Marchantia polymorpha cultures block breast cancer estrogen biosynthesis . Cell Biochem Biophys , 2012 . 63 ( 1 ): 85 - 96 . 22350385 DOI: 10.1007/s12013-012-9343-4 http://doi.org/10.1007/s12013-012-9343-4 . Steroidal metabolites transformed by Marchantia polymorpha cultures block breast cancer estrogen biosynthesis PGM Hesselink , SV Vliet , HD Vries , B Witholt . Optimization of steroid side chain cleavage by Mycobacterium sp. in the presence of cyclodextrins . Enzyme Microb Technol , 1989 . 11 ( 7 ): 398 - 404 . DOI: 10.1016/0141-0229(89)90133-6 http://doi.org/10.1016/0141-0229(89)90133-6 . Optimization of steroid side chain cleavage by Mycobacterium sp. in the presence of cyclodextrins CL Huang , YR Chen , WH Liu . Production of androstenones from phytosterol by mutants of Mycobacterium sp . Enzyme Microb Technol , 2006 . 39 ( 2 ): 296 - 300 . DOI: 10.1016/j.enzmictec.2005.10.017 http://doi.org/10.1016/j.enzmictec.2005.10.017 . Production of androstenones from phytosterol by mutants of Mycobacterium sp PY Kim , DJ Pollard , JM Woodley . Substrate supply for effective biocatalysis . Biotechnol Prog , 2007 . 23 ( 1 ): 74 - 82 . 17269674 DOI: 10.1021/bp060314b http://doi.org/10.1021/bp060314b . Substrate supply for effective biocatalysis JP Kutney , RK Milanova , CD Vassilev , 等 . Process for the Microbial Conversion of Phytosterol to Androstenedione and Androstadienedione . US Patent , 6071714 . 2000 . Process for the Microbial Conversion of Phytosterol to Androstenedione and Androstadienedione YL Lin , X Song , J Fu , JQ Lin , YB Qu . Microbial transformation of phytosterol in corn flour and soybean flour to 4-androstene-3,17-dione by Fusarium moniliforme sheld . Bioresour Technol , 2009 . 100 ( 5 ): 1864 - 1867 . 19006663 DOI: 10.1016/j/biortech.2008.09.040 http://doi.org/10.1016/j/biortech.2008.09.040 . Microbial transformation of phytosterol in corn flour and soybean flour to 4-androstene-3,17-dione by Fusarium moniliforme sheld J Liu , ML Fu , QH Chen . Biotransformation optimization of betulin into betulinic acid production catalysed by cultured Armillaria luteo-virens sacc ZJUQH 100-6 cells . J Appl Microbiol , 2010 . 110 ( 1 ): 90 - 97 . 20860772 DOI: 10.1111/j.1365-2672.2010.04857.x http://doi.org/10.1111/j.1365-2672.2010.04857.x . Biotransformation optimization of betulin into betulinic acid production catalysed by cultured Armillaria luteo-virens sacc ZJUQH 100-6 cells ZQ Liu , JF Zhang , YG Zheng , YC Shen . Improvement of astaxanthin production by a newly isolated Phaffia rhodozyma mutant with low-energy ion beam implantation . J Appl Microbiol , 2007 . 104 ( 3 ): 861 - 872 . 17953683 DOI: 10.1111/j.1365-2672.2007.03603.x http://doi.org/10.1111/j.1365-2672.2007.03603.x . Improvement of astaxanthin production by a newly isolated Phaffia rhodozyma mutant with low-energy ion beam implantation T Loftsson , ME Brewster . Pharmaceutical applications of cyclodextrins. 1. Drug solubilization and stabilization . J Pharm Sci-US , 1996 . 85 ( 10 ): 1017 - 1025 . DOI: 10.1021/js950534b http://doi.org/10.1021/js950534b . Pharmaceutical applications of cyclodextrins. 1. Drug solubilization and stabilization YH Ma , M Wang , Z Fan , YB Shen , LT Zhang . The influence of host-guest inclusion complex formation on the biotransformation of cortisone acetate Δ 1 -dehydrogenation . J Steroid Biochem Mol Biol , 2009 . 117 ( 4 ): 146 - 151 . 19744560 DOI: 10.1016/j.jsbmb.2009.08.007 http://doi.org/10.1016/j.jsbmb.2009.08.007 . The influence of host-guest inclusion complex formation on the biotransformation of cortisone acetate Δ1-dehydrogenation A Malaviya , J Gomes . Androstenedione production by biotransformation of phytosterols . Biores Technol , 2008 . 99 ( 15 ): 6725 - 6737 . DOI: 10.1016/j.biortech.2008.01.039 http://doi.org/10.1016/j.biortech.2008.01.039 . Androstenedione production by biotransformation of phytosterols A Manosroi , S Saowakhon , J Manosroi . Enhancement of androstadienedione production from progesterone by biotransformation using the hydroxypropyl-β-cyclodextrin complexation technique . J Steroid Biochem , 2008 . 108 ( 1-2 ): 132 - 136 . DOI: 10.1016/j.jsbmb.2007.05.032 http://doi.org/10.1016/j.jsbmb.2007.05.032 . Enhancement of androstadienedione production from progesterone by biotransformation using the hydroxypropyl-β-cyclodextrin complexation technique RH Myers , DC Montgomery . Response Surface Methodology: Process and Product Optimization Using Designed Experiment , 2002 . : New York Wiley-Interscience Publication , 219 - 264 . . Response Surface Methodology: Process and Product Optimization Using Designed Experiment BN Nalluri , KPR Chowdary , KVR Murthy , V Satyanarayana , AR Hayman , G Becket . Inclusion complexation and dissolution properties of nimesulide . J Incl Phenom Macrocycl Chem , 2005 . 53 ( 1-2 ): 103 - 110 . DOI: 10.1007/s10847-005-1676-9 http://doi.org/10.1007/s10847-005-1676-9 . Inclusion complexation and dissolution properties of nimesulide C Pérez , A Falero , HL Duc , Y Balcinde , BR Hung . A very efficient bioconversion of soybean phytosterols mixtures to androstanes by mycobacteria . J Ind Microbiol Biotechnol , 2006 . 148 ( 8 ): 719 - 723 . DOI: 10.1007/s10295-006-0148-6 http://doi.org/10.1007/s10295-006-0148-6 . A very efficient bioconversion of soybean phytosterols mixtures to androstanes by mycobacteria NV Rodina , MA Molchanova , NE Voishvillo , VA Andryushina , TS Stytsenko . Conversion of phytosterols into androstenedione by Mycobacterium neoaurum . Appl Biochem Microbiol , 2008 . 44 ( 1 ): 48 - 54 . DOI: 10.1134/S0003683808010080 http://doi.org/10.1134/S0003683808010080 . Conversion of phytosterols into androstenedione by Mycobacterium neoaurum U Roglič , P Žnidaršič-Plazl , L Plazl . The influence of β-cyclodextrin on the kinetics of progesterone transformation by Rhizopus nigricans . Biocatal Biotransfor , 2005 . 23 ( 5 ): 299 - 305 . DOI: 10.1080/10242420500175929 http://doi.org/10.1080/10242420500175929 . The influence of β-cyclodextrin on the kinetics of progesterone transformation by Rhizopus nigricans U Roglič , L Plazl , P Žnidaršič-Plazl . Batch and continuous transformation of progesterone by Rhizopus nigricans pellets in the presence of β-cyclodextrin . Biocatal Biotransfor , 2007 . 25 ( 1 ): 16 - 23 . DOI: 10.1080/10242420601060954 http://doi.org/10.1080/10242420601060954 . Batch and continuous transformation of progesterone by Rhizopus nigricans pellets in the presence of β-cyclodextrin A Schimid , A Kollmer , RG Mathys , B Witholt . Developments toward large-scale bacterial bioprocesses in the presence of bulk amounts of organic solvents . Extremophiles , 1998 . 2 ( 3 ): 249 - 256 . 9783172 DOI: 10.1007/s007920050067 http://doi.org/10.1007/s007920050067 . Developments toward large-scale bacterial bioprocesses in the presence of bulk amounts of organic solvents SPJN Senanayake , F Shahidi . Lipase-catalyzed incorporation of docosahexaenoic acid (DHA) into borage oil: optimization using response surface methodology . Food Chem , 2002 . 77 ( 1 ): 115 - 123 . DOI: 10.1016/s0308-8146(01)00311-9 http://doi.org/10.1016/s0308-8146(01)00311-9 . Lipase-catalyzed incorporation of docosahexaenoic acid (DHA) into borage oil: optimization using response surface methodology YB Shen , M Wang , LT Zhang , YH Ma , B Ma , Y Zheng , H Liu , JM Luo . Effects of hydroxypropyl-β-cyclodextrin on cell growth, activity, and integrity of steroid-transformation Arthrobacter simplex and Mycobacterium sp . Appl Microbiol Biotechnol , 2011 . 90 ( 6 ): 1995 - 2003 . 21468712 DOI: 10.1007/s00253-011-3214-6 http://doi.org/10.1007/s00253-011-3214-6 . Effects of hydroxypropyl-β-cyclodextrin on cell growth, activity, and integrity of steroid-transformation Arthrobacter simplex and Mycobacterium sp LM Simon , K László , A Vértesi , K Bagi , B Szajáni . Stability of hydrolytic enzymes in water-organic solvent systems . J Mol Catal B: Enzym , 1998 . 4 ( 1-2 ): 41 - 45 . DOI: 10.1016/s1381-1177(97)00019-2 http://doi.org/10.1016/s1381-1177(97)00019-2 . Stability of hydrolytic enzymes in water-organic solvent systems P Sripalakit , U Wichai , A Saraphanchotiwitthaya . Biotransformation of various natural sterols to androstenones by Mycobacterium sp. and some steroid-converting microbial strains . J Mol Catal B: Enzym , 2006 . 41 ( 1-2 ): 49 - 54 . DOI: 10.1016/j.molcatb.2006.04.007 http://doi.org/10.1016/j.molcatb.2006.04.007 . Biotransformation of various natural sterols to androstenones by Mycobacterium sp. and some steroid-converting microbial strains A Staebler , A Cruz , W van der Goot , HM Pinheiro , JMS Cabral , P Fernandes . Optimization of androstenedione production in an organic-aqueous two-liquid phase system . J Mol Catal B: Enzym , 2004 . 29 ( 1-6 ): 19 - 23 . DOI: 10.1016/j.molcatb.2004.01.013 http://doi.org/10.1016/j.molcatb.2004.01.013 . Optimization of androstenedione production in an organic-aqueous two-liquid phase system A Stefan , G Palazzo , A Ceglie , E Panzavolta , A Hochkoeppler . Water-in-oil microemulsions to sustain long-term viability of microbial cells in organic solvents . Biotechnol Bioeng , 2002 . 81 ( 3 ): 323 - 328 . DOI: 10.1002/bit.10476 http://doi.org/10.1002/bit.10476 . Water-in-oil microemulsions to sustain long-term viability of microbial cells in organic solvents S Stefanov , D Yankov , V Beschkov . Biotransformation of phytosterols to androstenedione in two phase water-oil systems . Chem Biochem Eng Q , 2006 . 20 ( 4 ): 421 - 427 . . Biotransformation of phytosterols to androstenedione in two phase water-oil systems J Szejtli . Introduction and general overview of cyclodextrin chemistry . Chem Rev , 1998 . 98 ( 5 ): 1743 - 1754 . 11848947 DOI: 10.1021/cr970022c http://doi.org/10.1021/cr970022c . Introduction and general overview of cyclodextrin chemistry ZL Wang , FS Zhao , XQ Hao , DJ Chen , DT Li . Microbial transformation of hydrophobic compound in cloud point system . J Mol Catal B: Enzym , 2004 . 27 ( 4-6 ): 147 - 153 . DOI: 10.1016/j.molcatb.2003.11.002 http://doi.org/10.1016/j.molcatb.2003.11.002 . Microbial transformation of hydrophobic compound in cloud point system ZL Wang , FS Zhao , DJ Chen , DT Li . Biotransformation of phytosterol to produce androsta-diene-dione by resting cells of Mycobacterium in cloud point system . Process Biochem , 2006 . 41 ( 3 ): 557 - 561 . DOI: 10.1016/j.procbio.2005.09.014 http://doi.org/10.1016/j.procbio.2005.09.014 . Biotransformation of phytosterol to produce androsta-diene-dione by resting cells of Mycobacterium in cloud point system W Wei , SY Fan , FQ Wang , DZ Wei . A new steroid-transformation strain of Mycobacterium neoaurum and cloning of 3-ketosteroid 9α-hydroxylase in NwIB-01 . Appl Biochem Biotechnol , 2010 . 162 ( 5 ): 1446 - 1456 . 20204712 DOI: 10.1007/s12010-010-8919-y http://doi.org/10.1007/s12010-010-8919-y . A new steroid-transformation strain of Mycobacterium neoaurum and cloning of 3-ketosteroid 9α-hydroxylase in NwIB-01 LT Zhang , M Wang , YB Shen , YH Ma , JM Luo . Improvement of steroid biotransformation with hydroxypropyl-β-cyclodextrin induced complexation . Appl Biochem Biotechnol , 2009 . 159 ( 3 ): 642 - 654 . 19189059 DOI: 10.1007/s12010-008-8499-2 http://doi.org/10.1007/s12010-008-8499-2 . Improvement of steroid biotransformation with hydroxypropyl-β-cyclodextrin induced complexation 10.1631/jzus.B1200067 Project (No. 31130042) support by the National Natural Science Foundation of China 2012-03-06 2013-01-03 2012-07-29 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2013 2013 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 02 14