Biotechnol Lett (2015) 37:1895–1904 DOI 10.1007/s10529-015-1862-9
ORIGINAL RESEARCH PAPER
Bioconversion of vitamin D3 to calcifediol by using resting cells of Pseudonocardia sp. Dae-Jung Kang . Jong-Hyuk Im . Jae-Hoon Kang . Kyoung Heon Kim
Received: 31 March 2015 / Accepted: 12 May 2015 / Published online: 21 May 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Objectives Resting cells of Pseudonocardia sp. KCTC 1029BP were used for the bioconversion of vitamin D3 to calcifediol which is widely used to treat osteomalacia and is industrially produced by chemical synthesis. Results To obtain the maximum bioconversion yield of calcifediol by the microbial conversion of vitamin D3, a two-step optimization process was used, including the Plackett–Burman and the central composite designs. Six variables, namely agitation speed, aeration rate, resting cell concentration, vitamin D3 concentration, temperature, and pH, were monitored. Of these, aeration rate, resting cell concentration, and temperature were selected as key variables for calcifediol production and were optimized using the central composite design. Optimal bioconversion conditions obtained were as follows: aeration rate of 0.2 vvm, resting cell concentration of 4.7 % w/v, and temperature of 33 °C. Conclusion Using the optimal conditions, 356 mg calcifediol l-1 was obtained with a bioconversion
D.-J. Kang K. H. Kim (&) Department of Biotechnology, Graduate School, Korea University, Seoul 136-713, Republic of Korea e-mail: [email protected] D.-J. Kang J.-H. Im J.-H. Kang Research Laboratories, Ildong Pharmaceutical, Hwaseong 445-170, Republic of Korea
yield of 59.4 % in a 75 l fermentor. These are the highest values reported to date. Keywords Bioconversion Calcifediol Central composite design Plackett–Burman design Pseudonocardia sp. Vitamin D3
Introduction Vitamin D3 (cholecalciferol) is converted to 25-hydroxycholecalciferol (calcifediol) (Fig. 1a) by 25-hydroxylase in the liver (DeLuca and Schnoes 1983; Madhok and DeLuca 1979). Calcifediol is the active form of vitamin D3, which is used for treating osteomalacia and other diseases (Brooks et al. 1978; Clements et al. 1987; Seino et al. 1987). It promotes Ca2? absorption in humans and animals (Eisman et al. 1977). Calcifediol is therefore commercially used as an important poultry feed supplement (Bar et al. 1987; Soares et al. 1995; Vieth 1999). Traditionally, organic synthesis and bioconversion are used for the production of calcifediol (Kametani and Furuyama 1987; Sasaki et al. 1992). Organic synthesis is highly complicated and expensive since a hydroxyl group has to be introduced in a stereo- and regio-specific manner at either C1 or C25 of the carbon chain of the substrate, vitamin D3 (Andrews et al. 1986; Hatakeyama et al. 1991; Kametani and Furuyama 1987; Lythgoe 1980; Vanmaele et al. 1985). The bioconversion method was developed to circumvent the obstacles of the organic
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Fig. 1 a Chemical structures of vitamin D3 (cholecalciferol) and calcifediol. b High performance liquid chromatogram of vitamin D3, calcifediol, and calcitriol from the bioconversion of vitamin D3 by Pseudonocardia sp. KCTC 1029BP for 5 days
synthesis (Sasaki et al. 1991). This process, utilizing microorganisms, is stereo- and regio-specific (Sasaki et al. 1991, 1992). Additionally, the microbial hydroxylation method is more cost-effective than chemical synthesis (Lau et al. 2004; Wang et al. 2001). Various microorganisms such as the actinomycete Pseudonocardia autotrophica (formerly Amycolata autotrophica) (Sasaki et al. 1991) and other microorganisms and their mutants (Imoto et al. 2011; Kang et al. 2006; Yasutake et al. 2013) have been used for the conversion of vitamin D3 to calcifediol. These microbial conversions of vitamin D3 to calcifediol showed two main problems with low titers and by-product generation in culture media due to the simultaneous cell growth and conversion of vitamin D3 to calcifediol. Calcifediol was obtained at only 8.3 mg l-1 (Sasaki et al. 1992) and 28 mg l-1 (Kang et al. 2006) which are too low for industrial applications. In addition, since whole culture media were used in these studies, issues such as by-product generation and side-reactions were unavoidable (Imoto et al. 2011; Kang et al. 2006; Sasaki et al. 1992). In particular, the presence of by-products necessitate additional purification steps, which contribute to increased production costs.
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To avoid these problems using growing cells, resting cells have been used to convert vitamin D3 to calcitriol which is another derivative of vitamin D3 (Kang et al. 2015). This conversion is affected by several reaction conditions including pH, temperature, and precursors used (Flores et al. 1997; Kulprecha et al. 1985; Uzura et al. 2001). Therefore, optimization of bioconversion conditions is essential for effective production of calcifediol. Traditionally, fermentation optimization is performed using the ‘‘one-factor-at-atime’’ approach, which frequently fails to address interactions between parameters (Xiong et al. 2008). Therefore, statistical methods considering mutual interactions among the parameters are recommended (Anderson and Whitcomb 2005; Mandenius and Brundin 2008; Raissi and Farsani 2009). In this study, to maximize calcifediol production vitamin D3, the bioconversion process using resting cells of Pseudonocardia sp., which can convert vitamin D3 to calcifediol and calcitriol (Fig. 1b), was optimized. The bioconversion parameters optimized included the supply rate of the precursor, pH, temperature, aeration, and agitation, and the system was scaled up from a flask to a 7.5 l jar fermentor and then to a 75 l fermentor. The results provide important clues for the industrial applications of the microbial conversion of vitamin D3 to calcifediol.
Materials and methods Bacterial strain and growth Pseudonocardia sp. ID9302 was isolated from soil collected from Yongin, Korea. The activity of converting vitamin D3 to calcifediol was enhanced by a series of UV irradiation-induced mutations. The mutant strain, deposited at the Korean Collection for Type Cultures (KCTC) (accession no. 1029BP), namely, Pseudonocardia sp. KCTC 1029BP was used in this study. It was initially grown on glucose/soytone (GS) agar medium [1.5 % (w/v) glucose, 1.5 % (w/v) soytone, 0.5 % (w/v) NaCl, 0.2 % (w/v) CaCO3, and 2 % (w/v) agar] at 28 °C. A loopful of cells was transferred into 50 ml sterile GS broth in a 250 ml Erlenmeyer flask, and grown at 28 °C with shaking at 180 rpm for 3 days. This culture was then inoculated into 50 ml fresh GS broth in a 250 ml Erlenmeyer flask with baffles, and grown as before for 3 days. This
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culture was inoculated into 50 ml glucose/soytone yeast extract/fish meal (GSYF) medium, composed of 1 % (w/v) glucose, 0.5 % soytone, 0.5 % yeast extract, 1 % fish meal, 0.01 % K2HPO4, 0.01 % NaF, 0.2 % CaCO3, and 0.2 % NaCl at pH 7, in a 250 ml Erlenmeyer flask. The culture was incubated at 28 °C on a rotary shaker at 180 rpm for 5 days. Preparation of resting cells for bioconversion of vitamin D3 to calcifediol After incubating KCTC 1029BP in GSYF medium, cells were harvested by centrifugation (11,0009g for 10 min) and washed twice with bioconversion medium which was composed of 15 mM Trizma base, 25 mM sodium succinate, 2 mM MgSO4, 0.03 % (w/ v) MnCl2, and 0.1 % (w/v) b-cyclodextrin (Kang et al. 2015). The washed, resting cells were resuspended into 50 ml bioconversion medium in a 250 ml Erlenmeyer flask with baffles. Microbial bioconversion using the resting cells was initiated by adding 100 ll 30 % (w/v) vitamin D3 dissolved in ethanol and incubated at 28 °C with shaking at 180 rpm for 5 days. Optimization of the bioconversion of vitamin D3 to calcifediol in a 7.5 l fermentor For the optimization of bioconversion parameters, all experiments were carried out in a 7.5 l fermentor jar (New Brunswick Scientific, Edison, NJ, USA). To prepare the seed culture, cells were inoculated into 140 ml GS medium in a 500 ml Erlenmeyer flask. This seed culture was inoculated into a 7.5 l fermentor containing 3.5 l GSYF medium. To prepare resting cells for bioconversion of vitamin D3 to calcifediol, batch cultivation of Pseudonocardia sp. KCTC 1029BP was performed for 5 days in the 7.5 l fermentor with 3.5 l working volume. The cultivation was at 28 °C, aeration was 1 vvm, and agitation was 500 rpm. Agitation was provided by two 6-blade, disk turbine impellers. To obtain resting cells, cells were then centrifuged and washed as previously mentioned and resuspended into the 7.5 l fermentor containing 3.5 l bioconversion medium. Bioconversion was initiated by adding 7 ml 30 % (w/v) vitamin D3 into the 3.5 l bioconversion medium, and the initial concentration of vitamin D3 in the bioconversion mixture was
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600 mg l-1. The bioconversion conditions included an agitation at 500 rpm and aeration at 1 vvm at 28°C for 5 days. Scale-up of the bioconversion of vitamin D3 to calcifediol in a 75 l fermentor To validate the optimal bioconversion parameters obtained in the 7.5 l fermentor, the bioconversion of vitamin D3 to calcifediol was performed in a 75 l fermentor (Bioengineering, Wald, Switzerland). The seed culture was cultivated in 1.8 l GS medium in a 2 l fermentor (Marubishi, Tokyo, Japan), and was inoculated into 45 l GSYF medium in a 75 l fermentor. Cells were incubated for 5 days in the 75 l fermentor with a 45 l working volume to prepare resting cells. Cultivation was at 28 °C with aeration at 1 vvm and stirring at 500 rpm. Agitation was provided by two 6-blade disk turbine impellers. The diameters of the impeller and the 75 l fermentor vessel were 120 and 350 mm, respectively. Cells from the 75 l fermentor were harvested and washed with bioconversion medium. For the bioconversion of vitamin D3 to calcifediol, 3 kg washed cells (total wet weight) was resuspended into the 75 l fermentor containing 45 l bioconversion medium. Bioconversion was initiated by adding 90 ml 30 % (w/v) vitamin D3, and the resulting initial concentration of vitamin D3 in the fermentor reaction mixture was 600 mg l-1. The bioconversion conditions included agitation 500 rpm and aeration at 1 vvm at 28 °C for 5 days. Analytical methods To analyze vitamin D3 and calcifediol in the postbioconversion mixture, a modified Bligh–Dyer method (Lund and DeLuca 1966) was used to extract the mixture. Briefly, 6 ml methanol/dichloromethane (1:1, v/v) was added to 3 ml post-bioconversion mixture and combined in a vortex mixer. To facilitate phase separation, the mixture was centrifuged at 11,0009g for 10 min at room temperature. The bottom organic phase, was analyzed by HPLC equipped with a photodiode array and an octadecylsilyl column (4.6 9 250 mm) being eluted with 80 % methanol (v/v) at 1 ml/min. Concentrations of vitamin D3 and calcifediol were determined using calibration curves prepared using specific standards (Sigma).
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Plackett–Burman design
y ¼ b0 þ b1 x1 þ b2 x2 þ b3 x3 þ b11 x11 þ b22 x22 þ b33 x33 þ b12 x1 x2 þ b13 x1 x3 þ b23 x2 x3
The Plackett–Burman design was used in this study to optimize the bioconversion of vitamin D3 to calcifediol. This method identifies the critical nutrient and process variables required for maximizing calcifediol production by screening n variables in n ? 1 experiments (Plackett and Burman 1946). The variables chosen for this study were temperature, pH, agitation speed, aeration rate, resting cell concentration, and vitamin D3 concentration. Six assigned variables at 2 levels, -1 for the low level and ?1 for the high level (Table 1), generated 12 experiments as per the Plackett–Burman design (Table 2). This design is based on the first-order model as given in Eq. 1:
where y is the response (calcifediol concentration); b0 is the intercept; b1, b2, and b3 are the linear coefficients; b11, b22, and b33 are quadratic coefficients; b12, b13, and b23 are interaction coefficients; and x1, x2, and x3 are the coded independent variables. The Plackett–Burman design and the central composite design were developed and the results were analyzed using MINITAB 16 software (Minitab, PA, USA).
y ¼ b0 þ R b i x i ;
Results and discussion
ð1Þ
where y is the response (calcifediol concentration), b0 is the model intercept, bi are variable estimates, and xi are independent variables. All experiments were performed in triplicate. The variables with a confidence level higher than 90 % were considered to significantly influence the bioconversion of vitamin D3 to calcifediol. Central composite design After critical factors were identified by the Plackett– Burman design, the central composite design was used to obtain a quadratic model. The effect of temperature, aeration rate, and resting cell concentration on the bioconversion of vitamin D3 to calcifediol was studied at 5 different levels: -a, -1, 0, ?1, and ?a (see Table 3). The statistical design of the factorial, center, and axial points are provided in Table 4. The actual levels of variables for the central composite design were selected taking the parameters of the initial bioconversion condition as the center points. The linear quadratic model with 3 factors is expressed as:
ð2Þ
Screening key variables for calcifediol production using the Plackett–Burman design In bioconversions using enzymes or resting cells, the agitation speed, aeration rate, cell concentration, substrate concentration, pH, and temperature are the major operating conditions affecting the final product yields and concentrations (de Carvalho 2011). The starting values of six variables were determined based on our preliminary results (data not shown). In the 12 bioconversion runs of vitamin D3 to calcifediol using the Plackett–Burman design, the final concentration of calcifediol in bioconversion media ranged from 2.2 to 78.6 mg l-1 (Table 2). To find the significant variables affecting calcifediol concentration, the 12 runs of Plackett–Burman design were statistically analyzed using MINITAB 16.0 software (Table 3). Among the six variables, the aeration rate, resting cell concentration, and temperature showed positive signs of effect on calcifediol concentration. All other factors showed negative signs. [When the sign of effect of a tested variable is positive, the calcifediol concentration
Table 1 Actual values of bioconversion variables Variables
Agitation speed (rpm)
Aeration rate (vvm)
Resting cell conc. based on wet wt (%)
Vitamin D3 (%, w/v)
Temperature (°C)
pH
Low level (-1)
200
0.2
1
0.02
20
4
High level (?1)
600
2
5
0.20
40
8
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Table 2 Plackett–Burman design for 12 runs of calcifediol production by bioconversion Run
Agitation speed (rpm)
Aeration rate (vvm)
Resting cell conc. (%)
Vitamin D3 (%, w/v)
Temperature (°C)
pH
Calcifediol (mg l-1)
1
?1
?1
?1
-1
?1
?1
78.6
2
-1
-1
-1
-1
-1
-1
2.2
3
?1
?1
-1
?1
?1
-1
49.9
4
-1
?1
?1
-1
?1
-1
71.8
5
?1
?1
-1
?1
-1
-1
38.1
6
?1
-1
-1
-1
?1
?1
10.6
7
-1
?1
?1
?1
-1
?1
70.2
8
?1
-1
?1
?1
-1
?1
47.5
9
-1
?1
-1
-1
-1
?1
38.7
10
-1
-1
?1
?1
?1
-1
52.0
11
-1
-1
-1
?1
?1
?1
30.2
12
?1
-1
?1
-1
-1
-1
35.9
Table 3 Statistical analysis of Plackett–Burman design for the bioconversion of vitamin D3 to calcifediol Variable
Effect
Coefficient
43.8083
1.337
32.77
0.000
100
Agitation speed
-0.7700
-0.3850
1.337
-0.29
0.785
21.5
Aeration rate
28.1633
14.0817
1.337
10.53
0.000a
100a
a
100a
Constant
Resting cell conc. Vitamin D3 conc. Temperature pH a
Standard error of coefficient
t value
p value
31.0367
15.5183
1.337
11.61
0.000
Confidence level (%)
8.3667
4.1833
1.337
3.13
0.026
97.4
10.0867
5.0433
1.337
3.77
0.013a
99.9a
4.3000
2.1500
1.337
1.61
0.169
83.1
Indicates significance at the corresponding confidence level
Table 4 The 3 selected variables and their values in the central composite design Factor
Variable
A
Aeration rate (vvm)
B C
Resting cell conc. (%) Temperature (°C)
-a (-1.682) Actual value 0.2 1.3 21.6
increases as the variable increases, and when negative, their relation is the opposite (Gangadharan et al. 2008).] As shown in Table 3, the coefficient of determination, R2, was 0.982, which implies that the 98.2 % variation in calcifediol concentration was attributed to independent
Low (-1)
0.5 2 25
Mean (0)
1 3 30
High (?1)
1.5 4 35
?a (?1.682)
1.8 4.7 38.4
variables. The coefficient of adjusted determination R2 (adj) was calculated to be 0.963, indicating a good agreement between the experimental and predicted values of calcifediol concentration. On the basis of the confidence levels, the aeration rate (i.e. confidence level 100 %), resting cell concentration (i.e. confidence level
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100 %), and temperature (i.e. confidence level 99.9 %) were found to be the significant variables that affected calcifediol concentration (Table 3). These three key variables were selected for further optimization using the central composite design (Table 4).
Optimization of three key variables for calcifediol production using the central composite design In the central composite design using the three selected variables: aeration rate, resting cell concentration, and temperature (Table 4), the final calcifediol concentrations from the bioconversion of vitamin D3 to calcifediol in the 20 runs ranged from 23.9 to 268.8 mg l-1 (Table 5). Among the estimated regression coefficients for the central composite design, p values were used to determine the significance of each coefficient, which indicates interactions between variables of the model (Table 6). Smaller p values indicate higher significance of the corresponding coefficient (Sreekumar and Krishnan 2010). Among the variables analyzed, the individual terms: aeration
rate (p value = 0.000), resting cell concentration (p value = 0.000), and temperature (p value = 0.016) as well as the interaction terms: resting cell concentration 9 resting cell concentration (p value = 0.025), temperature 9 temperature (p value = 0.000), and aeration rate 9 resting cell concentration (p value = 0.001), were significant to the calcifediol concentration during bioconversion. The correlation coefficient (R2 = 97.6 %) and the adjusted coefficient (R2 [adj] = 95.45 %) were also high, which indicates the significance of the following regression model (Yee and Blanch 1993): y ¼ 147:571 57:128x1 þ 40:553x2 þ 11:043x3 3:521x1 x1 9:806x2 x2 20:881x3 x3 24:306x1 x2 9:471x1 x3 1:001x2 x3 ; ð3Þ where y is the predicted response of calcifediol concentration from the bioconversion, and x1, x2, and x3 are the coded values of aeration rate, resting cell concentration, and temperature, respectively. From the analysis of variance of the regression model, an f value of 45.26 and p value of 0.000 were
Table 5 Central composite design of factors in ceded value for optimization of calcifediol production Run
Standard order
Type
Aeration rate (vvm)
Resting cell conc. (%, w/v)
Temperature (°C)
Calcifediol (mg l-1) Actual
Predicted
1
14
Axial
0
0
?1.682
87.4
107.1
2
15
Center
0
0
0
154.7
147.6
3
19
Center
0
0
0
135.9
147.6
4
12
Axial
0
?1.682
0
187.9
188.0
5
8
Factorial
?1
?1
?1
6
18
Center
0
0
7
16
8
6
75.8
73.1
0
146.6
147.6
150.9
147.6
51.0
42.6
226.7
233.7
Center
0
0
0
Factorial
?1
-1
?1
9
9
Axial
-1.682
0
0
10
4
Factorial
?1
?1
-1
63.9
71.9
11
7
Factorial
-1
?1
?1
268.8
254.9
12
3
Factorial
-1
?1
-1
207.8
215.8
13
5
Factorial
-1
-1
?1
135.6
127.2
14
2
Factorial
?1
-1
-1
23.9
37.4
15 16
17 10
Center Axial
0 ?1.682
0 0
0 0
136.8 47.9
147.6 41.5
17
20
18
1
19 20
Center
0
0
0
160.8
147.6
Factorial
-1
-1
-1
81.8
84.1
11
Axial
0
-1.682
0
51.2
51.6
13
Axial
0
0
-1.682
89.1
69.9
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Table 6 Estimated regression coefficients of the central composite design for the bioconversion of vitamin D3 to calcifediol Term Constant
Coefficient
Standard error of coefficient
t value
p value
147.571
5.762
25.610
Aeration rate
x1
-57.128
3.823
-14.943
0.000a
Resting cell concentration Temperature
x2 x3
40.553 11.043
3.823 3.823
10.607 2.888
0.000a 0.016a
Aeration rate 9 Aeration rate
x1x1
-3.521
3.722
-0.946
0.366
Resting cell concentration 9 Resting cell concentration
x2x2
-9.806
3.722
-2.635
0.025a
Temperature 9 Temperature
x3x3
-20.881
3.722
-5.610
0.000a
Aeration rate 9 Resting cell concentration
x1x2
-24.306
4.995
-4.866
0.001a
Aeration rate 9 Temperature
x1x3
-9.471
4.995
-1.896
0.087
Resting cell concentration 9 Temperature
x2x3
-1.001
4.995
-0.200
0.845
a
0.000
Indicates significance at the corresponding confidence level
Table 7 Analysis of variance of the regression model for the bioconversion of vitamin D3 to calcifediol Source
Regression Linear
Degrees of freedom 9
Sum of square
Adjusted sum of square
81,308.7
81,308.7
Mean of square
f value
9034.3
45.26
p value
0.000
3
68,694.8
68,694.8
22,898.3
114.71
0.000
Aeration rate
1
44,570.3
44,570.3
44,570.3
223.28
0.000
Resting cell concentration
1
22,459.1
22,459.1
22,459.1
112.51
0.000
Temperature
1
1665.4
1665.4
1665.4
8.34
0.016
3
7161.8
7161.8
2387.3
11.96
0.001
Square Aeration rate 9 Aeration rate
1
8.2
178.7
178.7
0.90
0.366
Resting cell concentration 9 Resting cell concentration
1
870.2
1385.7
1385.7
6.94
0.025
Temperature 9 Temperature
1
6283.4
6283.4
6283.4
31.48
0.000
Interaction Aeration rate 9 Resting cell concentration Aeration rate 9 Temperature Resting cell concentration 9 Temperature
3
5452.0
5452.0
1817.3
9.10
0.003
1 1
4726.4 717.6
4726.4 717.6
4726.4 717.6
23.68 3.60
0.001 0.087
0.04
0.845
3.08
0.121
1
8.0
8.0
8.0
10
1996.2
1996.2
199.6
Lack-of-fit
5
1506.5
1506.5
301.3
Pure error
5
489.6
489.6
97.9
19
83,304.8
Residual error
Total
obtained (Table 7). It is thus evident that the linear (p value = 0.000), square (p value = 0.001), and interaction terms (p value = 0.003) of variables significantly affected the final concentration of calcifediol (Table 7). To investigate the interaction between the variables and to determine the optimal value of each variable to achieve the maximum concentration of calcifediol by bioconversion, three-dimensional response surfaces
were plotted on the basis of the regression model (Fig. 2). The effects of varying the aeration rate and one of the other variables are shown in Fig. 2a and b, where calcifediol production increases as the aeration rate decreases and the resting cell concentration increases (Fig. 2a). The interaction of aeration rate and temperature revealed that calcifediol production increased below 0.5 vvm of aeration rate and at 30–35 °C (Fig. 2b). The aeration rate was the most
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1902
a
400 Non-optimized conditions Optimal conditions
Calcifediol (mg/L)
significant variable for calcifediol production. In our preliminary experiment, calcifediol was not produced under anaerobic conditions (data not shown). However, the regression model indicates that calcifediol production decreases by approx. 30 % when aeration increased from 0.2 to 0.3 vvm. This is unexpected because the bioconversion requires O2. With regard to the resting cell concentration, up to 4.7 % (w/v) of a resting cell concentration and at 30–35 °C, the highest level of calcifediol production is anticipated (Fig. 2c).
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300
200
100
0 0
1
2
3
4
5
6
Time (day)
b
700 Non-optimized conditions Optimal conditions
Vitamin D3 (mg/L)
600 500 400 300 200 100 0 0
1
2
3
4
5
6
Time (day)
Bioconversion yield of calcifediol from vitmain D3 (%, w/w)
c
80 Non-optimized conditions Optimal conditions
70 60 50 40 30 20 10 0 0
1
2
3
4
5
6
Time (day)
Fig. 2 Response surface plots of interactions between factors during calcifediol production. a Aeration rate and resting cell concentration. b Aeration rate and temperature. c Resting cell concentration and temperature
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Fig. 3 Pilot-scale bioconversion of vitamin D3 to calcifediol conducted in a 75 l fermentor. a Production of calcifediol. b Consumption of vitamin D3. c Bioconversion yield of calcifediol from vitamin D3. Non-optimized conditions: aeration rate of 1.0 vvm, resting cell concentration of 4.0 % (w/v), and temperature of 28 °C. Optimal conditions: aeration rate of 0.2 vvm, resting cell concentration of 4.7 %, w/v, and temperature of 33 °C
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The response optimizer in MINITAB 16.0 was used to find the optimal values of variables for the maximum calcifediol concentration from the bioconversion. The optimal value of the variables in the actual unit was predicted for aeration at 0.2 vvm, a resting cell concentration of 4.7 % (w/v), and temperature of 33 °C with the predicted maximum calcifediol concentration of 350.6 mg l-1. Under these optimal bioconversion conditions, the experimentally obtained calcifediol in the 7.5 l fermentor was 353.2 mg l-1.
resting cells of randomly mutated Pseudonocardia sp., calcifediol reached 356.2 mg l-1 and the bioconversion of 59.4 % (w/w) in a 75 l fermentor. To the best of our knowledge, these are the highest values reported for the microbial conversion of vitamin D3 to calcifediol. The aeration rate, resting cell concentration and temperature were the key variables for calcifediol production. These results serve as valuable information for the commercial-scale production of calcifediol from vitamin D3.
Bioconversion of vitamin D3 to calcifediol in a pilot-scale fermentor
Acknowledgments KHK acknowledges the Grant support by the Ministry of Trade, Industry & Energy (10048684) and the facility support at the Korea University Food Safety Hall for the Institute of Biomedical Science and Food Safety.
To validate the optimal bioconversion conditions on a pilot scale, the bioconversion of vitamin D3 to calcifediol was performed using a 75 l fermentor with a 45 l working volume. Bioconversions were performed under two different conditions: (i) non-optimized conditions including aeration at 1 vvm, resting cells at 4 % (w/v), and at 28 °C; (ii) using the optimal conditions obtained from Plackett–Burman design and the central composite design, namely aeration at 0.2 vvm, resting cells at 4.7 %, w/v, and at 33 °C (Fig. 3). The final concentration of calcifediol after 5 days of bioconversion under optimal conditions (356.2 mg l-1) was 3.2 times higher than that obtained under non-optimized conditions (112.9 mg l-1). When the non-optimized conditions were applied, the bioconversion yield was only 18.8 % (w/w), which increased to 59.4 % (w/w) when optimal conditions were applied. Therefore, optimization of operating conditions significantly contributed to the enhancement in the production of calcifediol. In addition, calcifediol production is generally performed at 28 °C for simultaneous cell growth and bioconversion of vitamin D3 (Imoto et al. 2011; Kang et al. 2006, 2015; Sasaki et al. 1991, 1992; Takeda et al. 1994; Yasutake et al. 2013). In our study, using the approach of separating cell growth from the bioconversion process, using resting cells at a higher temperature (33 °C) was optimal for the increased bioconversion of vitamin D3 to calcifediol.
Conclusion By optimizing the operating conditions for the bioconversion of vitamin D3 to calcifediol using
References Anderson MJ, Whitcomb PJ (2005) RSM simplified: optimizing processes using response surface methods for design of experiments. Productivity Press, Boca Raton Andrews DR, Barton DH, Hesse RH, Pechet MM (1986) Synthesis of 25-hydroxy-and 1 alpha., 25-dihydroxy vitamin D3 from vitamin D2 (calciferol). J Org Chem 51:4819–4828 Bar A, Rosenberg J, Perlman R, Hurwitz S (1987) Field rickets in turkeys: relationship to vitamin D. Poult Sci 66:68–72 Brooks MH, Bell NH, Love L, Stern PH, Orfei E, Queener SF, Hamstra AJ, DeLuca HF (1978) Vitamin-D-dependent rickets type II: resistance of target organs to 1, 25-dihydroxyvitamin D. New Engl J Med 298:996–999 Clements M, Johnson L, Fraser D (1987) A new mechanism for induced vitamin D deficiency in calcium deprivation. Nature 32:62–66 de Carvalho CC (2011) Enzymatic and whole cell catalysis: finding new strategies for old processes. Biotechnol Adv 29:75–83 DeLuca HF, Schnoes HK (1983) Vitamin D: recent advances. Annu Rev Biochem 52:411–439 Eisman J, Shepard R, DeLuca H (1977) Determination of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in human plasma using high-pressure liquid chromatography. Anal Biochem 80:298–305 Flores ER, Pe´rez F, de la Torre M (1997) Scale-up of Bacillus thuringiensis fermentation based on oxygen transfer. J Ferment Bioeng 83:561–564 Gangadharan D, Sivaramakrishnan S, Nampoothiri KM, Sukumaran RK, Pandey A (2008) Response surface methodology for the optimization of a amylase production by Bacillus amyloliquefaciens. Biores Technol 99:4597–4602 Hatakeyama S, Sugawara K, Numata H, Takano S (1991) A novel convergent synthesis of (?)-1a, 25-dihydroxyvitamin D3 using a chromium (II)-mediated coupling reaction. J Org Chem 56:461–463 Imoto N, Nishioka T, Tamura T (2011) Permeabilization induced by lipid II-targeting lantibiotic nisin and its effect on the bioconversion of vitamin D3 to 25-hydroxyvitamin D3
123
1904 by Rhodococcus erythropolis. Biochem Biophys Res Commun 405:393–398 Kametani T, Furuyama H (1987) Synthesis of vitamin D3 and related compounds. Med Res Rev 7:147–171 Kang D, Lee H, Park J, Bang J, Hong S, Kim T (2006) Optimization of culture conditions for the bioconversion of vitamin D3 to 1a, 25-dihydroxyvitamin D3 using Pseudonocardia autotrophica ID 9302. Biotechnol Bioprocess Eng 11:408–413 Kang D, Im J, Kang J, Kim K (2015) Whole cell bioconversion of vitamin D3 to calcitriol using Pseudonocardia sp. KCTC 1029BP. Bioproc Biosyst Eng. doi:10.1007/s00449-0151368-9 Kulprecha S, Ueda T, Nihira T, Yoshida T, Taguchi H (1985) Optimum conditions for ursodeoxycholic acid production from lithocholic acid by Fusarium equiseti M41. Appl Environ Microbiol 49:338–344 Lau J, Tran C, Licari P, Galazzo J (2004) Development of a high cell-density fed-batch bioprocess for the heterologous production of 6-deoxyerythronolide B in Escherichia coli. J Biotechnol 110:95–103 Lund J, DeLuca HF (1966) Biologically active metabolite of vitamin D3 from bone, liver, and blood serum. J Lipid Res 7:739–744 Lythgoe B (1980) Simonsen Lecture. Synthetic approaches to vitamin D and its relatives. Chem Soc Rev 9:449–475 Madhok T, DeLuca H (1979) Characteristics of the rat liver microsomal enzyme system converting cholecalciferol into 25-hydroxycholecalciferol. Evidence for the participation of cytochrome p-450. Biochem J 184:491–499 Mandenius C, Brundin A (2008) Bioprocess optimization using design of experiments methodology. Biotechnol Prog 24:1191–1203 Plackett R, Burman J (1946) The design of optimum multifactorial experiments. Biometrika 33:305–325 Raissi S, Farsani R (2009) Statistical process optimization through multi-response surface methodology. World Acad Sci Eng Technol 51:267–271 Sasaki J, Mikami A, Mizoue K, Omura S (1991) Transformation of 25- and 1a-hydroxyvitamin D3 to 1a, 25-dihydroxyvitamin D3 by using Streptomyces sp. strains. Appl Environ Microbiol 57:2841–2846 Sasaki J, Miyazaki A, Saito M, Adachi T, Mizoue K, Hanada K, Omura S (1992) Transformation of vitamin D3 to 1a,
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Biotechnol Lett (2015) 37:1895–1904 25-dihydroxyvitamin D3 via 25-hydroxyvitamin D3 using Amycolata sp. strains. Appl Microbiol Biotechnol 38:152–157 Seino Y, Tanaka H, Yamaoka K, Yabuuchi H (1987) Circulating 1a, 25-dihydroxyvitamin D levels after a single dose of 1a, 25-dihydroxyvitamin D3 or 1a-hydroxyvitamin D3 in normal men. Bone Miner 2:479–485 Soares J, Kerr J, Gray R (1995) 25-hydroxycholecalciferol in poultry nutrition. Poult Sci 74:1919–1934 Sreekumar G, Krishnan S (2010) Enhanced biomass production study on probiotic Bacillus subtilis SK09 by medium optimization using response surface methodology. Afr J Biotechnol 9:8078–8084 Takeda K, Asou T, Matsuda A, Kimura K, Okamura K, Okamoto R, Sasaki J, Adachi T, Omura S (1994) Application of cyclodextrin to microbial transformation of vitamin D3 to 25-hydroxyvitamin D3 and 1a, 25-dihydroxyvitamin D3. J Ferment Bioeng 78:380–382 Uzura A, Katsuragi T, Tani Y (2001) Conversion of various aromatic compounds by resting cells of Fusarium moniliforme strain MS31. J Biosci Bioeng 92:381–384 Vanmaele L, De Clercq P, Vandewalle M (1985) An efficient synthesis of 1a, 25-dihydroxy vitamin D3. Tetrahedron 41:141–144 Vieth R (1999) Vitamin D supplementation, 25-hydroxyvitamin D concentrations, and safety. Am J Clin Nutr 69:842–856 Wang Z, Zhuge J, Fang H, Prior B (2001) Glycerol production by microbial fermentation: a review. Biotechnol Adv 19:201–223 Xiong Z, Tu X, Tu G (2008) Optimization of medium composition for actinomycin X2 production by Streptomyces spp. JAU4234 using response surface methodology. J Ind Microbiol Biotechnol 35:729–734 Yasutake Y, Nishioka T, Imoto N, Tamura T (2013) A single mutation at the ferredoxin binding site of p450 Vdh enables efficient biocatalytic production of 25 hydroxyvitamin D3. ChemBioChem 14:2284–2291 Yee L, Blanch H (1993) Defined media optimization for growth of recombinant Escherichia coli X90. Biotechnol Bioeng 41:221–230
ORIGINAL RESEARCH PAPER
Bioconversion of vitamin D3 to calcifediol by using resting cells of Pseudonocardia sp. Dae-Jung Kang . Jong-Hyuk Im . Jae-Hoon Kang . Kyoung Heon Kim
Received: 31 March 2015 / Accepted: 12 May 2015 / Published online: 21 May 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Objectives Resting cells of Pseudonocardia sp. KCTC 1029BP were used for the bioconversion of vitamin D3 to calcifediol which is widely used to treat osteomalacia and is industrially produced by chemical synthesis. Results To obtain the maximum bioconversion yield of calcifediol by the microbial conversion of vitamin D3, a two-step optimization process was used, including the Plackett–Burman and the central composite designs. Six variables, namely agitation speed, aeration rate, resting cell concentration, vitamin D3 concentration, temperature, and pH, were monitored. Of these, aeration rate, resting cell concentration, and temperature were selected as key variables for calcifediol production and were optimized using the central composite design. Optimal bioconversion conditions obtained were as follows: aeration rate of 0.2 vvm, resting cell concentration of 4.7 % w/v, and temperature of 33 °C. Conclusion Using the optimal conditions, 356 mg calcifediol l-1 was obtained with a bioconversion
D.-J. Kang K. H. Kim (&) Department of Biotechnology, Graduate School, Korea University, Seoul 136-713, Republic of Korea e-mail: [email protected] D.-J. Kang J.-H. Im J.-H. Kang Research Laboratories, Ildong Pharmaceutical, Hwaseong 445-170, Republic of Korea
yield of 59.4 % in a 75 l fermentor. These are the highest values reported to date. Keywords Bioconversion Calcifediol Central composite design Plackett–Burman design Pseudonocardia sp. Vitamin D3
Introduction Vitamin D3 (cholecalciferol) is converted to 25-hydroxycholecalciferol (calcifediol) (Fig. 1a) by 25-hydroxylase in the liver (DeLuca and Schnoes 1983; Madhok and DeLuca 1979). Calcifediol is the active form of vitamin D3, which is used for treating osteomalacia and other diseases (Brooks et al. 1978; Clements et al. 1987; Seino et al. 1987). It promotes Ca2? absorption in humans and animals (Eisman et al. 1977). Calcifediol is therefore commercially used as an important poultry feed supplement (Bar et al. 1987; Soares et al. 1995; Vieth 1999). Traditionally, organic synthesis and bioconversion are used for the production of calcifediol (Kametani and Furuyama 1987; Sasaki et al. 1992). Organic synthesis is highly complicated and expensive since a hydroxyl group has to be introduced in a stereo- and regio-specific manner at either C1 or C25 of the carbon chain of the substrate, vitamin D3 (Andrews et al. 1986; Hatakeyama et al. 1991; Kametani and Furuyama 1987; Lythgoe 1980; Vanmaele et al. 1985). The bioconversion method was developed to circumvent the obstacles of the organic
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Fig. 1 a Chemical structures of vitamin D3 (cholecalciferol) and calcifediol. b High performance liquid chromatogram of vitamin D3, calcifediol, and calcitriol from the bioconversion of vitamin D3 by Pseudonocardia sp. KCTC 1029BP for 5 days
synthesis (Sasaki et al. 1991). This process, utilizing microorganisms, is stereo- and regio-specific (Sasaki et al. 1991, 1992). Additionally, the microbial hydroxylation method is more cost-effective than chemical synthesis (Lau et al. 2004; Wang et al. 2001). Various microorganisms such as the actinomycete Pseudonocardia autotrophica (formerly Amycolata autotrophica) (Sasaki et al. 1991) and other microorganisms and their mutants (Imoto et al. 2011; Kang et al. 2006; Yasutake et al. 2013) have been used for the conversion of vitamin D3 to calcifediol. These microbial conversions of vitamin D3 to calcifediol showed two main problems with low titers and by-product generation in culture media due to the simultaneous cell growth and conversion of vitamin D3 to calcifediol. Calcifediol was obtained at only 8.3 mg l-1 (Sasaki et al. 1992) and 28 mg l-1 (Kang et al. 2006) which are too low for industrial applications. In addition, since whole culture media were used in these studies, issues such as by-product generation and side-reactions were unavoidable (Imoto et al. 2011; Kang et al. 2006; Sasaki et al. 1992). In particular, the presence of by-products necessitate additional purification steps, which contribute to increased production costs.
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To avoid these problems using growing cells, resting cells have been used to convert vitamin D3 to calcitriol which is another derivative of vitamin D3 (Kang et al. 2015). This conversion is affected by several reaction conditions including pH, temperature, and precursors used (Flores et al. 1997; Kulprecha et al. 1985; Uzura et al. 2001). Therefore, optimization of bioconversion conditions is essential for effective production of calcifediol. Traditionally, fermentation optimization is performed using the ‘‘one-factor-at-atime’’ approach, which frequently fails to address interactions between parameters (Xiong et al. 2008). Therefore, statistical methods considering mutual interactions among the parameters are recommended (Anderson and Whitcomb 2005; Mandenius and Brundin 2008; Raissi and Farsani 2009). In this study, to maximize calcifediol production vitamin D3, the bioconversion process using resting cells of Pseudonocardia sp., which can convert vitamin D3 to calcifediol and calcitriol (Fig. 1b), was optimized. The bioconversion parameters optimized included the supply rate of the precursor, pH, temperature, aeration, and agitation, and the system was scaled up from a flask to a 7.5 l jar fermentor and then to a 75 l fermentor. The results provide important clues for the industrial applications of the microbial conversion of vitamin D3 to calcifediol.
Materials and methods Bacterial strain and growth Pseudonocardia sp. ID9302 was isolated from soil collected from Yongin, Korea. The activity of converting vitamin D3 to calcifediol was enhanced by a series of UV irradiation-induced mutations. The mutant strain, deposited at the Korean Collection for Type Cultures (KCTC) (accession no. 1029BP), namely, Pseudonocardia sp. KCTC 1029BP was used in this study. It was initially grown on glucose/soytone (GS) agar medium [1.5 % (w/v) glucose, 1.5 % (w/v) soytone, 0.5 % (w/v) NaCl, 0.2 % (w/v) CaCO3, and 2 % (w/v) agar] at 28 °C. A loopful of cells was transferred into 50 ml sterile GS broth in a 250 ml Erlenmeyer flask, and grown at 28 °C with shaking at 180 rpm for 3 days. This culture was then inoculated into 50 ml fresh GS broth in a 250 ml Erlenmeyer flask with baffles, and grown as before for 3 days. This
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culture was inoculated into 50 ml glucose/soytone yeast extract/fish meal (GSYF) medium, composed of 1 % (w/v) glucose, 0.5 % soytone, 0.5 % yeast extract, 1 % fish meal, 0.01 % K2HPO4, 0.01 % NaF, 0.2 % CaCO3, and 0.2 % NaCl at pH 7, in a 250 ml Erlenmeyer flask. The culture was incubated at 28 °C on a rotary shaker at 180 rpm for 5 days. Preparation of resting cells for bioconversion of vitamin D3 to calcifediol After incubating KCTC 1029BP in GSYF medium, cells were harvested by centrifugation (11,0009g for 10 min) and washed twice with bioconversion medium which was composed of 15 mM Trizma base, 25 mM sodium succinate, 2 mM MgSO4, 0.03 % (w/ v) MnCl2, and 0.1 % (w/v) b-cyclodextrin (Kang et al. 2015). The washed, resting cells were resuspended into 50 ml bioconversion medium in a 250 ml Erlenmeyer flask with baffles. Microbial bioconversion using the resting cells was initiated by adding 100 ll 30 % (w/v) vitamin D3 dissolved in ethanol and incubated at 28 °C with shaking at 180 rpm for 5 days. Optimization of the bioconversion of vitamin D3 to calcifediol in a 7.5 l fermentor For the optimization of bioconversion parameters, all experiments were carried out in a 7.5 l fermentor jar (New Brunswick Scientific, Edison, NJ, USA). To prepare the seed culture, cells were inoculated into 140 ml GS medium in a 500 ml Erlenmeyer flask. This seed culture was inoculated into a 7.5 l fermentor containing 3.5 l GSYF medium. To prepare resting cells for bioconversion of vitamin D3 to calcifediol, batch cultivation of Pseudonocardia sp. KCTC 1029BP was performed for 5 days in the 7.5 l fermentor with 3.5 l working volume. The cultivation was at 28 °C, aeration was 1 vvm, and agitation was 500 rpm. Agitation was provided by two 6-blade, disk turbine impellers. To obtain resting cells, cells were then centrifuged and washed as previously mentioned and resuspended into the 7.5 l fermentor containing 3.5 l bioconversion medium. Bioconversion was initiated by adding 7 ml 30 % (w/v) vitamin D3 into the 3.5 l bioconversion medium, and the initial concentration of vitamin D3 in the bioconversion mixture was
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600 mg l-1. The bioconversion conditions included an agitation at 500 rpm and aeration at 1 vvm at 28°C for 5 days. Scale-up of the bioconversion of vitamin D3 to calcifediol in a 75 l fermentor To validate the optimal bioconversion parameters obtained in the 7.5 l fermentor, the bioconversion of vitamin D3 to calcifediol was performed in a 75 l fermentor (Bioengineering, Wald, Switzerland). The seed culture was cultivated in 1.8 l GS medium in a 2 l fermentor (Marubishi, Tokyo, Japan), and was inoculated into 45 l GSYF medium in a 75 l fermentor. Cells were incubated for 5 days in the 75 l fermentor with a 45 l working volume to prepare resting cells. Cultivation was at 28 °C with aeration at 1 vvm and stirring at 500 rpm. Agitation was provided by two 6-blade disk turbine impellers. The diameters of the impeller and the 75 l fermentor vessel were 120 and 350 mm, respectively. Cells from the 75 l fermentor were harvested and washed with bioconversion medium. For the bioconversion of vitamin D3 to calcifediol, 3 kg washed cells (total wet weight) was resuspended into the 75 l fermentor containing 45 l bioconversion medium. Bioconversion was initiated by adding 90 ml 30 % (w/v) vitamin D3, and the resulting initial concentration of vitamin D3 in the fermentor reaction mixture was 600 mg l-1. The bioconversion conditions included agitation 500 rpm and aeration at 1 vvm at 28 °C for 5 days. Analytical methods To analyze vitamin D3 and calcifediol in the postbioconversion mixture, a modified Bligh–Dyer method (Lund and DeLuca 1966) was used to extract the mixture. Briefly, 6 ml methanol/dichloromethane (1:1, v/v) was added to 3 ml post-bioconversion mixture and combined in a vortex mixer. To facilitate phase separation, the mixture was centrifuged at 11,0009g for 10 min at room temperature. The bottom organic phase, was analyzed by HPLC equipped with a photodiode array and an octadecylsilyl column (4.6 9 250 mm) being eluted with 80 % methanol (v/v) at 1 ml/min. Concentrations of vitamin D3 and calcifediol were determined using calibration curves prepared using specific standards (Sigma).
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Plackett–Burman design
y ¼ b0 þ b1 x1 þ b2 x2 þ b3 x3 þ b11 x11 þ b22 x22 þ b33 x33 þ b12 x1 x2 þ b13 x1 x3 þ b23 x2 x3
The Plackett–Burman design was used in this study to optimize the bioconversion of vitamin D3 to calcifediol. This method identifies the critical nutrient and process variables required for maximizing calcifediol production by screening n variables in n ? 1 experiments (Plackett and Burman 1946). The variables chosen for this study were temperature, pH, agitation speed, aeration rate, resting cell concentration, and vitamin D3 concentration. Six assigned variables at 2 levels, -1 for the low level and ?1 for the high level (Table 1), generated 12 experiments as per the Plackett–Burman design (Table 2). This design is based on the first-order model as given in Eq. 1:
where y is the response (calcifediol concentration); b0 is the intercept; b1, b2, and b3 are the linear coefficients; b11, b22, and b33 are quadratic coefficients; b12, b13, and b23 are interaction coefficients; and x1, x2, and x3 are the coded independent variables. The Plackett–Burman design and the central composite design were developed and the results were analyzed using MINITAB 16 software (Minitab, PA, USA).
y ¼ b0 þ R b i x i ;
Results and discussion
ð1Þ
where y is the response (calcifediol concentration), b0 is the model intercept, bi are variable estimates, and xi are independent variables. All experiments were performed in triplicate. The variables with a confidence level higher than 90 % were considered to significantly influence the bioconversion of vitamin D3 to calcifediol. Central composite design After critical factors were identified by the Plackett– Burman design, the central composite design was used to obtain a quadratic model. The effect of temperature, aeration rate, and resting cell concentration on the bioconversion of vitamin D3 to calcifediol was studied at 5 different levels: -a, -1, 0, ?1, and ?a (see Table 3). The statistical design of the factorial, center, and axial points are provided in Table 4. The actual levels of variables for the central composite design were selected taking the parameters of the initial bioconversion condition as the center points. The linear quadratic model with 3 factors is expressed as:
ð2Þ
Screening key variables for calcifediol production using the Plackett–Burman design In bioconversions using enzymes or resting cells, the agitation speed, aeration rate, cell concentration, substrate concentration, pH, and temperature are the major operating conditions affecting the final product yields and concentrations (de Carvalho 2011). The starting values of six variables were determined based on our preliminary results (data not shown). In the 12 bioconversion runs of vitamin D3 to calcifediol using the Plackett–Burman design, the final concentration of calcifediol in bioconversion media ranged from 2.2 to 78.6 mg l-1 (Table 2). To find the significant variables affecting calcifediol concentration, the 12 runs of Plackett–Burman design were statistically analyzed using MINITAB 16.0 software (Table 3). Among the six variables, the aeration rate, resting cell concentration, and temperature showed positive signs of effect on calcifediol concentration. All other factors showed negative signs. [When the sign of effect of a tested variable is positive, the calcifediol concentration
Table 1 Actual values of bioconversion variables Variables
Agitation speed (rpm)
Aeration rate (vvm)
Resting cell conc. based on wet wt (%)
Vitamin D3 (%, w/v)
Temperature (°C)
pH
Low level (-1)
200
0.2
1
0.02
20
4
High level (?1)
600
2
5
0.20
40
8
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Table 2 Plackett–Burman design for 12 runs of calcifediol production by bioconversion Run
Agitation speed (rpm)
Aeration rate (vvm)
Resting cell conc. (%)
Vitamin D3 (%, w/v)
Temperature (°C)
pH
Calcifediol (mg l-1)
1
?1
?1
?1
-1
?1
?1
78.6
2
-1
-1
-1
-1
-1
-1
2.2
3
?1
?1
-1
?1
?1
-1
49.9
4
-1
?1
?1
-1
?1
-1
71.8
5
?1
?1
-1
?1
-1
-1
38.1
6
?1
-1
-1
-1
?1
?1
10.6
7
-1
?1
?1
?1
-1
?1
70.2
8
?1
-1
?1
?1
-1
?1
47.5
9
-1
?1
-1
-1
-1
?1
38.7
10
-1
-1
?1
?1
?1
-1
52.0
11
-1
-1
-1
?1
?1
?1
30.2
12
?1
-1
?1
-1
-1
-1
35.9
Table 3 Statistical analysis of Plackett–Burman design for the bioconversion of vitamin D3 to calcifediol Variable
Effect
Coefficient
43.8083
1.337
32.77
0.000
100
Agitation speed
-0.7700
-0.3850
1.337
-0.29
0.785
21.5
Aeration rate
28.1633
14.0817
1.337
10.53
0.000a
100a
a
100a
Constant
Resting cell conc. Vitamin D3 conc. Temperature pH a
Standard error of coefficient
t value
p value
31.0367
15.5183
1.337
11.61
0.000
Confidence level (%)
8.3667
4.1833
1.337
3.13
0.026
97.4
10.0867
5.0433
1.337
3.77
0.013a
99.9a
4.3000
2.1500
1.337
1.61
0.169
83.1
Indicates significance at the corresponding confidence level
Table 4 The 3 selected variables and their values in the central composite design Factor
Variable
A
Aeration rate (vvm)
B C
Resting cell conc. (%) Temperature (°C)
-a (-1.682) Actual value 0.2 1.3 21.6
increases as the variable increases, and when negative, their relation is the opposite (Gangadharan et al. 2008).] As shown in Table 3, the coefficient of determination, R2, was 0.982, which implies that the 98.2 % variation in calcifediol concentration was attributed to independent
Low (-1)
0.5 2 25
Mean (0)
1 3 30
High (?1)
1.5 4 35
?a (?1.682)
1.8 4.7 38.4
variables. The coefficient of adjusted determination R2 (adj) was calculated to be 0.963, indicating a good agreement between the experimental and predicted values of calcifediol concentration. On the basis of the confidence levels, the aeration rate (i.e. confidence level 100 %), resting cell concentration (i.e. confidence level
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100 %), and temperature (i.e. confidence level 99.9 %) were found to be the significant variables that affected calcifediol concentration (Table 3). These three key variables were selected for further optimization using the central composite design (Table 4).
Optimization of three key variables for calcifediol production using the central composite design In the central composite design using the three selected variables: aeration rate, resting cell concentration, and temperature (Table 4), the final calcifediol concentrations from the bioconversion of vitamin D3 to calcifediol in the 20 runs ranged from 23.9 to 268.8 mg l-1 (Table 5). Among the estimated regression coefficients for the central composite design, p values were used to determine the significance of each coefficient, which indicates interactions between variables of the model (Table 6). Smaller p values indicate higher significance of the corresponding coefficient (Sreekumar and Krishnan 2010). Among the variables analyzed, the individual terms: aeration
rate (p value = 0.000), resting cell concentration (p value = 0.000), and temperature (p value = 0.016) as well as the interaction terms: resting cell concentration 9 resting cell concentration (p value = 0.025), temperature 9 temperature (p value = 0.000), and aeration rate 9 resting cell concentration (p value = 0.001), were significant to the calcifediol concentration during bioconversion. The correlation coefficient (R2 = 97.6 %) and the adjusted coefficient (R2 [adj] = 95.45 %) were also high, which indicates the significance of the following regression model (Yee and Blanch 1993): y ¼ 147:571 57:128x1 þ 40:553x2 þ 11:043x3 3:521x1 x1 9:806x2 x2 20:881x3 x3 24:306x1 x2 9:471x1 x3 1:001x2 x3 ; ð3Þ where y is the predicted response of calcifediol concentration from the bioconversion, and x1, x2, and x3 are the coded values of aeration rate, resting cell concentration, and temperature, respectively. From the analysis of variance of the regression model, an f value of 45.26 and p value of 0.000 were
Table 5 Central composite design of factors in ceded value for optimization of calcifediol production Run
Standard order
Type
Aeration rate (vvm)
Resting cell conc. (%, w/v)
Temperature (°C)
Calcifediol (mg l-1) Actual
Predicted
1
14
Axial
0
0
?1.682
87.4
107.1
2
15
Center
0
0
0
154.7
147.6
3
19
Center
0
0
0
135.9
147.6
4
12
Axial
0
?1.682
0
187.9
188.0
5
8
Factorial
?1
?1
?1
6
18
Center
0
0
7
16
8
6
75.8
73.1
0
146.6
147.6
150.9
147.6
51.0
42.6
226.7
233.7
Center
0
0
0
Factorial
?1
-1
?1
9
9
Axial
-1.682
0
0
10
4
Factorial
?1
?1
-1
63.9
71.9
11
7
Factorial
-1
?1
?1
268.8
254.9
12
3
Factorial
-1
?1
-1
207.8
215.8
13
5
Factorial
-1
-1
?1
135.6
127.2
14
2
Factorial
?1
-1
-1
23.9
37.4
15 16
17 10
Center Axial
0 ?1.682
0 0
0 0
136.8 47.9
147.6 41.5
17
20
18
1
19 20
Center
0
0
0
160.8
147.6
Factorial
-1
-1
-1
81.8
84.1
11
Axial
0
-1.682
0
51.2
51.6
13
Axial
0
0
-1.682
89.1
69.9
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Biotechnol Lett (2015) 37:1895–1904
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Table 6 Estimated regression coefficients of the central composite design for the bioconversion of vitamin D3 to calcifediol Term Constant
Coefficient
Standard error of coefficient
t value
p value
147.571
5.762
25.610
Aeration rate
x1
-57.128
3.823
-14.943
0.000a
Resting cell concentration Temperature
x2 x3
40.553 11.043
3.823 3.823
10.607 2.888
0.000a 0.016a
Aeration rate 9 Aeration rate
x1x1
-3.521
3.722
-0.946
0.366
Resting cell concentration 9 Resting cell concentration
x2x2
-9.806
3.722
-2.635
0.025a
Temperature 9 Temperature
x3x3
-20.881
3.722
-5.610
0.000a
Aeration rate 9 Resting cell concentration
x1x2
-24.306
4.995
-4.866
0.001a
Aeration rate 9 Temperature
x1x3
-9.471
4.995
-1.896
0.087
Resting cell concentration 9 Temperature
x2x3
-1.001
4.995
-0.200
0.845
a
0.000
Indicates significance at the corresponding confidence level
Table 7 Analysis of variance of the regression model for the bioconversion of vitamin D3 to calcifediol Source
Regression Linear
Degrees of freedom 9
Sum of square
Adjusted sum of square
81,308.7
81,308.7
Mean of square
f value
9034.3
45.26
p value
0.000
3
68,694.8
68,694.8
22,898.3
114.71
0.000
Aeration rate
1
44,570.3
44,570.3
44,570.3
223.28
0.000
Resting cell concentration
1
22,459.1
22,459.1
22,459.1
112.51
0.000
Temperature
1
1665.4
1665.4
1665.4
8.34
0.016
3
7161.8
7161.8
2387.3
11.96
0.001
Square Aeration rate 9 Aeration rate
1
8.2
178.7
178.7
0.90
0.366
Resting cell concentration 9 Resting cell concentration
1
870.2
1385.7
1385.7
6.94
0.025
Temperature 9 Temperature
1
6283.4
6283.4
6283.4
31.48
0.000
Interaction Aeration rate 9 Resting cell concentration Aeration rate 9 Temperature Resting cell concentration 9 Temperature
3
5452.0
5452.0
1817.3
9.10
0.003
1 1
4726.4 717.6
4726.4 717.6
4726.4 717.6
23.68 3.60
0.001 0.087
0.04
0.845
3.08
0.121
1
8.0
8.0
8.0
10
1996.2
1996.2
199.6
Lack-of-fit
5
1506.5
1506.5
301.3
Pure error
5
489.6
489.6
97.9
19
83,304.8
Residual error
Total
obtained (Table 7). It is thus evident that the linear (p value = 0.000), square (p value = 0.001), and interaction terms (p value = 0.003) of variables significantly affected the final concentration of calcifediol (Table 7). To investigate the interaction between the variables and to determine the optimal value of each variable to achieve the maximum concentration of calcifediol by bioconversion, three-dimensional response surfaces
were plotted on the basis of the regression model (Fig. 2). The effects of varying the aeration rate and one of the other variables are shown in Fig. 2a and b, where calcifediol production increases as the aeration rate decreases and the resting cell concentration increases (Fig. 2a). The interaction of aeration rate and temperature revealed that calcifediol production increased below 0.5 vvm of aeration rate and at 30–35 °C (Fig. 2b). The aeration rate was the most
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1902
a
400 Non-optimized conditions Optimal conditions
Calcifediol (mg/L)
significant variable for calcifediol production. In our preliminary experiment, calcifediol was not produced under anaerobic conditions (data not shown). However, the regression model indicates that calcifediol production decreases by approx. 30 % when aeration increased from 0.2 to 0.3 vvm. This is unexpected because the bioconversion requires O2. With regard to the resting cell concentration, up to 4.7 % (w/v) of a resting cell concentration and at 30–35 °C, the highest level of calcifediol production is anticipated (Fig. 2c).
Biotechnol Lett (2015) 37:1895–1904
300
200
100
0 0
1
2
3
4
5
6
Time (day)
b
700 Non-optimized conditions Optimal conditions
Vitamin D3 (mg/L)
600 500 400 300 200 100 0 0
1
2
3
4
5
6
Time (day)
Bioconversion yield of calcifediol from vitmain D3 (%, w/w)
c
80 Non-optimized conditions Optimal conditions
70 60 50 40 30 20 10 0 0
1
2
3
4
5
6
Time (day)
Fig. 2 Response surface plots of interactions between factors during calcifediol production. a Aeration rate and resting cell concentration. b Aeration rate and temperature. c Resting cell concentration and temperature
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Fig. 3 Pilot-scale bioconversion of vitamin D3 to calcifediol conducted in a 75 l fermentor. a Production of calcifediol. b Consumption of vitamin D3. c Bioconversion yield of calcifediol from vitamin D3. Non-optimized conditions: aeration rate of 1.0 vvm, resting cell concentration of 4.0 % (w/v), and temperature of 28 °C. Optimal conditions: aeration rate of 0.2 vvm, resting cell concentration of 4.7 %, w/v, and temperature of 33 °C
Biotechnol Lett (2015) 37:1895–1904
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The response optimizer in MINITAB 16.0 was used to find the optimal values of variables for the maximum calcifediol concentration from the bioconversion. The optimal value of the variables in the actual unit was predicted for aeration at 0.2 vvm, a resting cell concentration of 4.7 % (w/v), and temperature of 33 °C with the predicted maximum calcifediol concentration of 350.6 mg l-1. Under these optimal bioconversion conditions, the experimentally obtained calcifediol in the 7.5 l fermentor was 353.2 mg l-1.
resting cells of randomly mutated Pseudonocardia sp., calcifediol reached 356.2 mg l-1 and the bioconversion of 59.4 % (w/w) in a 75 l fermentor. To the best of our knowledge, these are the highest values reported for the microbial conversion of vitamin D3 to calcifediol. The aeration rate, resting cell concentration and temperature were the key variables for calcifediol production. These results serve as valuable information for the commercial-scale production of calcifediol from vitamin D3.
Bioconversion of vitamin D3 to calcifediol in a pilot-scale fermentor
Acknowledgments KHK acknowledges the Grant support by the Ministry of Trade, Industry & Energy (10048684) and the facility support at the Korea University Food Safety Hall for the Institute of Biomedical Science and Food Safety.
To validate the optimal bioconversion conditions on a pilot scale, the bioconversion of vitamin D3 to calcifediol was performed using a 75 l fermentor with a 45 l working volume. Bioconversions were performed under two different conditions: (i) non-optimized conditions including aeration at 1 vvm, resting cells at 4 % (w/v), and at 28 °C; (ii) using the optimal conditions obtained from Plackett–Burman design and the central composite design, namely aeration at 0.2 vvm, resting cells at 4.7 %, w/v, and at 33 °C (Fig. 3). The final concentration of calcifediol after 5 days of bioconversion under optimal conditions (356.2 mg l-1) was 3.2 times higher than that obtained under non-optimized conditions (112.9 mg l-1). When the non-optimized conditions were applied, the bioconversion yield was only 18.8 % (w/w), which increased to 59.4 % (w/w) when optimal conditions were applied. Therefore, optimization of operating conditions significantly contributed to the enhancement in the production of calcifediol. In addition, calcifediol production is generally performed at 28 °C for simultaneous cell growth and bioconversion of vitamin D3 (Imoto et al. 2011; Kang et al. 2006, 2015; Sasaki et al. 1991, 1992; Takeda et al. 1994; Yasutake et al. 2013). In our study, using the approach of separating cell growth from the bioconversion process, using resting cells at a higher temperature (33 °C) was optimal for the increased bioconversion of vitamin D3 to calcifediol.
Conclusion By optimizing the operating conditions for the bioconversion of vitamin D3 to calcifediol using
References Anderson MJ, Whitcomb PJ (2005) RSM simplified: optimizing processes using response surface methods for design of experiments. Productivity Press, Boca Raton Andrews DR, Barton DH, Hesse RH, Pechet MM (1986) Synthesis of 25-hydroxy-and 1 alpha., 25-dihydroxy vitamin D3 from vitamin D2 (calciferol). J Org Chem 51:4819–4828 Bar A, Rosenberg J, Perlman R, Hurwitz S (1987) Field rickets in turkeys: relationship to vitamin D. Poult Sci 66:68–72 Brooks MH, Bell NH, Love L, Stern PH, Orfei E, Queener SF, Hamstra AJ, DeLuca HF (1978) Vitamin-D-dependent rickets type II: resistance of target organs to 1, 25-dihydroxyvitamin D. New Engl J Med 298:996–999 Clements M, Johnson L, Fraser D (1987) A new mechanism for induced vitamin D deficiency in calcium deprivation. Nature 32:62–66 de Carvalho CC (2011) Enzymatic and whole cell catalysis: finding new strategies for old processes. Biotechnol Adv 29:75–83 DeLuca HF, Schnoes HK (1983) Vitamin D: recent advances. Annu Rev Biochem 52:411–439 Eisman J, Shepard R, DeLuca H (1977) Determination of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in human plasma using high-pressure liquid chromatography. Anal Biochem 80:298–305 Flores ER, Pe´rez F, de la Torre M (1997) Scale-up of Bacillus thuringiensis fermentation based on oxygen transfer. J Ferment Bioeng 83:561–564 Gangadharan D, Sivaramakrishnan S, Nampoothiri KM, Sukumaran RK, Pandey A (2008) Response surface methodology for the optimization of a amylase production by Bacillus amyloliquefaciens. Biores Technol 99:4597–4602 Hatakeyama S, Sugawara K, Numata H, Takano S (1991) A novel convergent synthesis of (?)-1a, 25-dihydroxyvitamin D3 using a chromium (II)-mediated coupling reaction. J Org Chem 56:461–463 Imoto N, Nishioka T, Tamura T (2011) Permeabilization induced by lipid II-targeting lantibiotic nisin and its effect on the bioconversion of vitamin D3 to 25-hydroxyvitamin D3
123
1904 by Rhodococcus erythropolis. Biochem Biophys Res Commun 405:393–398 Kametani T, Furuyama H (1987) Synthesis of vitamin D3 and related compounds. Med Res Rev 7:147–171 Kang D, Lee H, Park J, Bang J, Hong S, Kim T (2006) Optimization of culture conditions for the bioconversion of vitamin D3 to 1a, 25-dihydroxyvitamin D3 using Pseudonocardia autotrophica ID 9302. Biotechnol Bioprocess Eng 11:408–413 Kang D, Im J, Kang J, Kim K (2015) Whole cell bioconversion of vitamin D3 to calcitriol using Pseudonocardia sp. KCTC 1029BP. Bioproc Biosyst Eng. doi:10.1007/s00449-0151368-9 Kulprecha S, Ueda T, Nihira T, Yoshida T, Taguchi H (1985) Optimum conditions for ursodeoxycholic acid production from lithocholic acid by Fusarium equiseti M41. Appl Environ Microbiol 49:338–344 Lau J, Tran C, Licari P, Galazzo J (2004) Development of a high cell-density fed-batch bioprocess for the heterologous production of 6-deoxyerythronolide B in Escherichia coli. J Biotechnol 110:95–103 Lund J, DeLuca HF (1966) Biologically active metabolite of vitamin D3 from bone, liver, and blood serum. J Lipid Res 7:739–744 Lythgoe B (1980) Simonsen Lecture. Synthetic approaches to vitamin D and its relatives. Chem Soc Rev 9:449–475 Madhok T, DeLuca H (1979) Characteristics of the rat liver microsomal enzyme system converting cholecalciferol into 25-hydroxycholecalciferol. Evidence for the participation of cytochrome p-450. Biochem J 184:491–499 Mandenius C, Brundin A (2008) Bioprocess optimization using design of experiments methodology. Biotechnol Prog 24:1191–1203 Plackett R, Burman J (1946) The design of optimum multifactorial experiments. Biometrika 33:305–325 Raissi S, Farsani R (2009) Statistical process optimization through multi-response surface methodology. World Acad Sci Eng Technol 51:267–271 Sasaki J, Mikami A, Mizoue K, Omura S (1991) Transformation of 25- and 1a-hydroxyvitamin D3 to 1a, 25-dihydroxyvitamin D3 by using Streptomyces sp. strains. Appl Environ Microbiol 57:2841–2846 Sasaki J, Miyazaki A, Saito M, Adachi T, Mizoue K, Hanada K, Omura S (1992) Transformation of vitamin D3 to 1a,
123
Biotechnol Lett (2015) 37:1895–1904 25-dihydroxyvitamin D3 via 25-hydroxyvitamin D3 using Amycolata sp. strains. Appl Microbiol Biotechnol 38:152–157 Seino Y, Tanaka H, Yamaoka K, Yabuuchi H (1987) Circulating 1a, 25-dihydroxyvitamin D levels after a single dose of 1a, 25-dihydroxyvitamin D3 or 1a-hydroxyvitamin D3 in normal men. Bone Miner 2:479–485 Soares J, Kerr J, Gray R (1995) 25-hydroxycholecalciferol in poultry nutrition. Poult Sci 74:1919–1934 Sreekumar G, Krishnan S (2010) Enhanced biomass production study on probiotic Bacillus subtilis SK09 by medium optimization using response surface methodology. Afr J Biotechnol 9:8078–8084 Takeda K, Asou T, Matsuda A, Kimura K, Okamura K, Okamoto R, Sasaki J, Adachi T, Omura S (1994) Application of cyclodextrin to microbial transformation of vitamin D3 to 25-hydroxyvitamin D3 and 1a, 25-dihydroxyvitamin D3. J Ferment Bioeng 78:380–382 Uzura A, Katsuragi T, Tani Y (2001) Conversion of various aromatic compounds by resting cells of Fusarium moniliforme strain MS31. J Biosci Bioeng 92:381–384 Vanmaele L, De Clercq P, Vandewalle M (1985) An efficient synthesis of 1a, 25-dihydroxy vitamin D3. Tetrahedron 41:141–144 Vieth R (1999) Vitamin D supplementation, 25-hydroxyvitamin D concentrations, and safety. Am J Clin Nutr 69:842–856 Wang Z, Zhuge J, Fang H, Prior B (2001) Glycerol production by microbial fermentation: a review. Biotechnol Adv 19:201–223 Xiong Z, Tu X, Tu G (2008) Optimization of medium composition for actinomycin X2 production by Streptomyces spp. JAU4234 using response surface methodology. J Ind Microbiol Biotechnol 35:729–734 Yasutake Y, Nishioka T, Imoto N, Tamura T (2013) A single mutation at the ferredoxin binding site of p450 Vdh enables efficient biocatalytic production of 25 hydroxyvitamin D3. ChemBioChem 14:2284–2291 Yee L, Blanch H (1993) Defined media optimization for growth of recombinant Escherichia coli X90. Biotechnol Bioeng 41:221–230
