Optimization of bioconversion conditions for vitamin D3 to 25-hydroxyvitamin D using Pseudonocardia autotrophica CGMCC5098

Article abstract of DOI:10.1080/10242422.2016.1268130

The optimal culture conditions for bioconversion of vitamin D₃ to calcifediol (25(OH)D₃) were investigated by varying carbon and nitrogen sources, metal salt concentrations, initial pH, temperature, solvents, surfactants, and agitation speed. In the process of this microbial hydroxylation, the timing of the addition of vitamin D₃, which is dissolved in ethanol, is of critical importance. Besides, the concentration of ethanol in zymotic fluid is the key factor to get high conversion ratio of vitamin D₃. In particular, the optimal culture conditions were 1.5% glucose, 1.5% soybean cake meal, 0.5% yeast extract, 0.5% corn steep liquor, 0.3% CaCO₃, 0.1% NaCl, 0.2% KH₂PO₄, pH 7.2 at 27 °C and the timing of the addition of vitamin D₃ dissolved in 5% (v/v) ethanol was 48 h followed by the inoculation of seed culture broth. Under the optimized conditions, the conversion of vitamin D₃ (1 g/L) by Pseudonocardia autotrophica CGMCC5098 in 50 L fermenter resulted in about 61.31% bioconversion ratio (639 mg/L) of 25(OH)D₃ on the 5th day.

Full text of DOI:10.1080/10242422.2016.1268130

Biocatalysis and Biotransformation
ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: http://www.tandfonline.com/loi/ibab20
Optimization of bioconversion conditions
for vitamin D to 25-hydroxyvitamin D using
3
Pseudonocardia autotrophica CGMCC5098
Jinqi Luo, Fang Jiang, Weizhen Fang & Qun Lu
To cite this article: Jinqi Luo, Fang Jiang, Weizhen Fang & Qun Lu (2016): Optimization
of bioconversion conditions for vitamin D to 25-hydroxyvitamin D using
3
Pseudonocardia autotrophica CGMCC5098, Biocatalysis and Biotransformation, DOI:
10.1080/10242422.2016.1268130
To link to this article: http://dx.doi.org/10.1080/10242422.2016.1268130
Published online: 26 Dec 2016.
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Date: 26 December 2016, At: 05:30

Biocatalysis and Biotransformation, 2016; Early Online: 1–8
RESEARCH ARTICLE
Optimization of bioconversion conditions for vitamin D to
3
25-hydroxyvitamin D using Pseudonocardia autotrophica
CGMCC5098
JINQI LUO, FANG JIANG, WEIZHEN FANG & QUN LU
School of Life Science and Engineering, Southwest Jiaotong University, Chengdu, China
Abstract
3 3
The optimal culture conditions for bioconversion of vitamin D to calcifediol (25(OH)D ) were investigated by varying
carbon and nitrogen sources, metal salt concentrations, initial pH, temperature, solvents, surfactants, and agitation speed. In
the process of this microbial hydroxylation, the timing of the addition of vitamin D , which is dissolved in ethanol, is of
3
critical importance. Besides, the concentration of ethanol in zymotic fluid is the key factor to get high conversion ratio of
vitamin D . In particular, the optimal culture conditions were 1.5% glucose, 1.5% soybean cake meal, 0.5% yeast extract,
3
ꢀ
0
.5% corn steep liquor, 0.3% CaCO , 0.1% NaCl, 0.2% KH PO , pH 7.2 at 27 C and the timing of the addition of vitamin
3
2
4
D
3
dissolved in 5% (v/v) ethanol was 48 h followed by the inoculation of seed culture broth. Under the optimized conditions,
(1 g/L) by Pseudonocardia autotrophica CGMCC5098 in 50 L fermenter resulted in about
1.31% bioconversion ratio (639 mg/L) of 25(OH)D on the 5th day.
the conversion of vitamin D
3
6
3
Keywords: Bioconversion, calcifediol, Pseudonocardia autotrophica, fermentation
Introduction
approaches (hydroxylation then epoxidation/reduc-
tion) and subsequently to 25(OH)D3 via photo-
chemical transformations, the desired product is
separated from the photo-products by crystallization.
Worth mentioning, the precursor compound
is produced by a fermentation process using
Saccharomyces cerevisiae. However, the isomer 25-
Vitamin D3 is biologically inactive and must be
converted to its physiologically active forms
(
bone and intestine of mammals. The active vitamin
25(OH)D3 and 1a,25(OH) D ) to function in
2
3
D are closely associated with calcium absorption,
3
calcium deposition, and phosphate homeostasis in
mammals. Besides, they could control multiple
cellular responses, such as cell differentiation and
proliferation (DeLuca and Schnoes 1983; Ikekawa
OH-previtamin D is also formed in the above path,
3
thus extra purification process is needed (Nakagawa
et al. 1992; Feldman et al. 2011). To the best of our
knowledge, there are few detailed description about
this method.
1987; Seino et al. 1987; Fujii et al. 2009). 25(OH)D3
is the first metabolite of vitamin D3 normally
hydroxylated in the liver and then converted to
Note that some reactions are difficult to achieve or
can’t achieve through chemical synthesis, while it
could be catalyzed by microbial transformation easily
and efficiently. More importantly, microorganisms
or enzymes are mostly positional selective and chiral,
which is important for designing and obtaining target
chiral chemicals. Therefore, to mass-produce such
fine chemicals, the microbial transformation could
be useful than chemical synthesis. In fact, the present
1
a,25(OH) D by P450s in the kidney (DeLuca and
2
3
Schnoes 1983); 25(OH)D3 is more potent than
vitamin D and has been approved as feed additives
3
by European Food Safety Authority (EFSA 2009;
Burild et al. 2016). Up to date, 25(OH)D is mainly
3
manufactured by
a
semi-synthetic method.
Specifically, the precursor 5,7,24-cholestatrienol is
converted to 25-OH-pro-vitamin D3 via chemical
Correspondence: Weizhen Fang; Qun Lu, Department of Chemistry and Chemical Engineering, Southwest Jiaotong University, Chengdu 610031, China.
E-mail: fangweizhen@home.swjtu.edu.cnluqun1125@126.com
(Received 25 May 2016; revised 30 October 2016; accepted 30 November 2016)
ISSN 1024-2422 print/ISSN 1029-2446 online ß 2016 Informa UK Limited, trading as Taylor & Francis Group
DOI: 10.1080/10242422.2016.1268130

2
J. Luo et al.
process of microbiological producing 25(OH)D is
3
spore suspension was transferred into 500 mL
Erlenmeyer flask containing 100 mL of Glucose
Peotone Yeast extract (GPY) medium composed of
1.5% glucose, 1.5% peptone, 0.75% yeast extract,
0.5% corn steep liquor, 0.5% NaCl, 0.3%
(NH ) SO , 0.2% KH PO , 0.1% MgSO , 0.1%
more convenient and efficient than that of chemical
synthesis (Imoto et al. 2011). Some actinomycete,
such as P autotrophica and Amycolata autotrophica,
have the capacity to convert vitamin D to the active
3
forms. Vitamin D direct microbial transformation to
3
4 2
4
2
4
4
2
5(OH)D3 have been reported by several groups
Sasaki et al. 1992; Takeda et al. 1994; Donghu et al.
004; Kang et al. 2006). For instance, a strain
A autotrophica was successfully used for the trans-
CaCO , pH 7.2. The P. autotrophica CGMCC5098
was cultivated by rotary shaking (200 rpm) at 27 C
3
ꢀ
(
2
for 48 h. 10 mL of this culture was transferred into
100 mL of the same medium in 500 mL flask and
cultivated for 48 h. Then the conversion of vitamin
D3 was carried out by the addition of 50 mg of
formation of 25(OH)D with about 26.4% (55 mg/mL)
yield (Takeda et al. 1994). Subsequently, Kang (2006,
2016) studied the optimal culture conditions for the
3
vitamin D in various volume solvent with surfac-
3
bioconversion of vitamin D3 for industrial-scale
production and 25(OH)D3 reached 356.2 mg/L.
Since the reported conversion yields were very low,
some groups try to make improvements by using
recombinant strains (Rahmaniyan et al. 2005;
Hayashi et al 2008; Sallam et al. 2010; Yasutake
et al. 2010; Sakaki et al. 2011; Imoto et al. 2011).
Unfortunately, to date several issues still exist, such
as low efficiency, mutant stability, etc (Yasutake and
Tamura 2011). Therefore, it is highly desirable to
develop a practical process with good efficiency.
This paper focuses on the bioconversion of vitamin
D to 25(OH)D , we herein report a simpler and
tants and cyclodextrin. The bioconversion was
carried out on a rotary shaker for another three
days. 2 mL samples of culture broth were taken at
appropriate times for analysis.
Bioconversion in 7 L and 50 L fermenter jars
Scale-up conversion experiments of vitamin D were
3
done in 7 L and 50 L fermenter jars. P. autotrophica
CGMCC5098 was cultured in five flasks containing
ꢀ
1
00 mL of the GPY medium at 27 C for 48 h. All of
this flask seed broth was transferred into a 7 L jar
fermentor containing 5 L GPY medium supple-
mented by 0.02% soybean oil as antifoam. After
3
3
more efficient procedure by using P. autotrophica
CGMCC5098 via a single-step process. Not only the
culture parameters (supply rate and ways of precur-
sor, solvents, additive agents, pH, temperature, aer-
ation concentration, agitation speed, etc), but also the
influences of induction solvent concentration have
been examined, cell wall inhibitors and surfactants
have been evaluated. It is a pleasant surprise that the
ꢀ
cultivating at 27 C for 48 h, vitamin D was added at
3
various concentrations in ethanol with Tween 80 and
cyclodextrin. The conversion culture was carried out
for another three days with the agitation speed of
3
00 rpm, an aeration concentration of 1 vvm.
A 50 L tank fermenter was used to analysis the
optimal conditions of vitamin D conversion. 10 mL
3
7/
of the spores’ suspension in 10 mL concentration
yield of 25(OH)D achieve 61.31% (639 mg/L).
3
was inoculated into 100 mL fermentation GPY
ꢀ
medium in a 500 mL Erlenmeyer flask at 27 C on
Materials and methods
a rotary shaker (250 rpm). After two days’ cultiva-
tion, all the three flasks seed broth was transferred
into a 7 L jar fermentor containing 3 L GPY medium
with 0.02% soybean oil as antifoam. The cultivation
was conducted with 1 vvm aeration and 350 rpm
Bacterial strain
P. autotrophica CGMCC5098 was isolated from soil
samples collected in China.
ꢀ
agitation at 27 C for 48 h. All of this 3 L seed broth
was transferred into a 50 L jar fermentor containing
Media and bioconversion methods
3
0.01% XX-09 (antifoam; Chenguang Chemical Co.
0 L GPY medium with 0.02% soybean oil and
P. autotrophica CGMCC5098 was cultured on agar
medium (1.5% glucose, 1.5% soybean cake meal,
ꢀ
Ltd, Chengdu). The culture conditions were 27 C
temperature, 200 rpm agitation with 3-blade turbine
0.5% yeast extract, 0.5% NaCl, 0.2% CaCO , 2%
agar, pH 7.2 adjusted by 10% NaOH) at 27 C for
3
ꢀ
and 60 L/min aeration. After 48 h, 30 g of vitamin D
3
ꢀ
seven days and then stored at 4 C for later use.
in 150 mL of ethanol with 60 g partially methylated
CD (PMCD) and 1 g Tween 80 in 150 mL of water
were added into the tank. The culture was continued
for a further 72 h with the agitation speed raised to
300 from 200 rpm and the aeration rate to 100 from
60 L/min.
During the cultivation, the cells could produce large
number of white spores. For inoculum development,
spores from heavily sporulated working stock were
used. Then these spores were suspended in sterile
7
water to a final concentration of 10 per mL. 10 mL

Optimal culture conditions for bioconversion of vitamin D to calcifediol
3
3
Analytical methods
found 401.0 (Figure 2); UVꢀmax(EtOH) ¼ 265nm,
1
UVꢀmin(EtOH) ¼ 228nm; NMR H ((CD ) SO,
3 2
A total of 2 mL of the culture broth was extracted
with methanol-dichloromethane (1/1, v/v) by the
modified Bligh & Dyer method (Lund and DeLuca
4
00 MHz): ꢁ: 6.16 (d, J ¼ 11.3 Hz, 1H), 5.95 (d,
J ¼ 11.3 Hz, 1H), 5.01 (m, 1H), 4.70 (m, 1H), 3.64
(m, 1H), 2.80 (dd, J ¼ 2.8, 12.4 Hz, 1H), 2.51–2.50
(m, 1H), 2.45 (dd, J ¼ 2.7, 13.3 Hz, 1H), 2.34–2.30
(m, 1H), 2.05–1.93 (m, 4H), 1.85–1.82 (m, 2H),
1966) for HPLC analysis. The HPLC conditions
were as follows: column, Waters SiO ; column size,
2
Ø3.9 ꢁ 300mm; mobile phase, n-hexane: 2-propanol
1
.62–1.60 (m, 2H), 1.43–1.20 (m, 13H), 1.05 (s,
1
(
perature, 25 C; detection, absorbance at 265 nm.
85/15, v/v); flow rate, 1.0 mL/min; column tem-
ꢀ
3
6H), 0.90 (d, J ¼ 6.3 Hz, 3H), 0.48 (s, 3H).
C
((CD ) SO, 100 MHz): ꢁ: 145.4, 140.6, 136.4,
3 2
25(OH)D and vitamin D could be distinguished by
this HPLC system according to the differences in
3
3
1
21.0, 117.0, 111.8, 68.7, 67.9, 56.0, 55.6, 45.9,
their retention times (Figure 1). 25(OH)D MS
3
45.2, 44.1, 36.2, 35.6, 35.4, 32.2, 30.6, 29.4, 29.2,
28.3, 27.2, 23.1, 21.8, 20.3, 18.6, 11.8.
+
(
ESI): m/z calcd. for C H O (M + H) 401.3,
2
7
45
2
Figure 1. HPLC of standard (25(OH)D
3
) and ethanol extracted culture broth sample. Column: Waters SiO
ꢀ
2
; column size: Ø3.9 ꢁ 300mm;
mobile phase: n-hexane: 2-propanol (85/15, v/v); flow rate: 1.0 mL/min; column temperature: 25 C; detection: absorbance at 265 nm.

4
J. Luo et al.
Figure 2. MS of ethanol extracted culture broth sample.
ꢀ
Figure 3. Effects of solvents on hydroxylation of vitamin D
3
. The culture conditions were as follows: 27 C, 7 days, 250 rpm, pH 7.0.
Results and discussion
to vitamin D3 solution in ethanol. As shown in
Figure 4, Tween 80 significantly increased
2
5(OH)D3 productivity. The increase of the
5(OH)D3 bioconversion rate by the addition of
Effects of substrate solvents on hydroxylation of
vitamin D3
2
Tween 80 was possibly because it would disperse
vitamin D3 better in the cultivation medium and
have less toxicity to the microorganism and enzyme.
The bioconversion of vitamin D to 25(OH)D was
Vitamin D substrate dissolved in different solvent
3
(
methanol, ethanol, DMF, DMSO, acetone and 2-
propanol), which was added to the medium with a
concentration of 0.5 g/L and the flasks were shaken
for another three days. The solvents had varying
effects on the productivity as can be seen in Figure 3.
Among them, DMF was found to be the most
3
3
enhanced about 2-fold above the control in the GPY
medium when Tween 80 was added. It is clear that
the proper regulation of Tween 80 concentration is
important during the bioconversion of vitamin D3.
The optimum concentration of Tween 80 was
effective in increasing 25(OH)D . The conversion of
3
ethanol is slightly lower than that of DMF. As it is
easy to dissolve vitamin D substrate, so ethanol was
chosen to use for the further experiments.
0
.02% (Figure 5).
3
Effects of cyclodextrin (CD) on the hydroxylation
of vitamin D3
Effects of surfactants on hydroxylation of vitamin D3
In order to further improve the solubility of vitamin
D in culture medium, we added various surfactants
To improve the solubility of steroid in the fermen-
tation broth, CD is often added to the conversion
3

Optimal culture conditions for bioconversion of vitamin D to calcifediol
5
3
3 3 3
Figure 4. Effects of various surfactants on bioconversion of vitamin D to 25(OH)D . Vitamin D solution, which contains 50 mg vitamin
D
3
in 0.5 mL of ethanol with various kinds of surfactants were added to 100 mL GPY medium in a 500 mL Erlenmeyer flask after being
ꢀ
diluted with 2 mL water. The culture conditions were as follows: 27 C, 7 days, 250 rpm, pH 7.0.
Figure 5. Effects of Tween 80 addition on bioconversion of vitamin D
3 3 3
to 25(OH)D . Vitamin D solution which contains 50 mg vitamin
D
3
in 0.5 mL of ethanol with various concentration of Tween 80 were added to 100 mL GPY medium in a 500 mL Erlenmeyer flask after
ꢀ
being diluted with 2 mL water. The culture conditions were as follows: 27 C, 7 days, 250 rpm, pH 7.0.
culture along with steroid. Application of CD to
microbial conversion of vitamin D3 has been
reported (Feldman et al. 2011) and was found that
CD had the ability to enhance the hydroxylation of
vitamin D . Vitamin D is dissolved by tapping it in
Effects of concentration on the hydroxylation of
vitamin D
3
During the vitamin D bioconversion, we found that
3
the productivity and bioconversion of 25(OH)D is
3
3
3
associated with ethanol concentration and addition
time. In order to determine the effects of ethanol
the cyclic structure of CD. All kinds of CD had been
tested, except for the PMCD (Partially Methylated
b-CD) reported (Feldman et al. 2011), we found
that HP-b-CD (hydroxypropyl-b-CD) also can
greatly increase the productivity and conversion
concentration on vitamin D bioconversion, we used
3
various amounts of ethanol to dissolve vitamin D3.
The maximum productivity was achieved with 5%
(v/v) ethanol to dissolve vitamin D . Figure 7 shows
3
rates of 25(OH)D (Figure 6). Optimal concentra-
3
the importance of the concentration of ethanol in
tion of PMCD and Tween 80 provide the maximal
conversion yield of 25(OH)D . The optimum con-
centration of PMCD was 0.3%. Productivity of
culture. The bioconversion yields for 25(OH)D3
3
increased in proportion to the increase in ethanol
concentration. Optimal ethanol concentration pro-
25(OH)D was clearly improved by the addition of
PMCD.
3
vides the proper solubility of vitamin D and the
3

6
J. Luo et al.
Figure 6. Effects of CD concentration on the transformation from vitamin D
vitamin D and 20 mg Tween 80 in 0.5 mL of ethanol with various concentration of CD were added to 100 mL GPY medium in a 500 mL
Erlenmeyer flask after being diluted with 2 mL water. The culture conditions were as follows: 27 C, 7 days, 250 rpm, pH 7.0.
3 3 3
to 25(OH)D . Vitamin D solution which contains 50 mg
3
ꢀ
Figure 7. Effects of ethanol concentration on the transformation from vitamin D
3 3 3
to 25(OH)D . Vitamin D solution which contains 50 mg
vitamin D and 20 mg Tween 80 in varied ethanol with CD (0.3%) were added to 100 mL GPY medium in a 500 mL Erlenmeyer flask. The
3
ꢀ
culture conditions were as follows: 27 C, 7 days, 250 rpm, pH 7.0.
optimal level of P450 hydroxylase. It is clear that the
proper regulation of ethanol concentration is import-
ant during the bioconversion of vitamin D3 to
the production of 25(OH)D . Based on the above
3
results, 50 L tank fermentation was conducted to
confirm the enhancement of the hydroxylation of
25(OH)D3.
vitamin D (Figure 8). P. autotrophica CGMCC5098
3
was cultured in five flasks containing 100 mL of GPY
medium (1.5% glucose, 1.5% tryptone, 0.75% yeast
extract, 0.75% corn steep liquor, 0.4% soybean
meal, 0.5% NaCl, 0.3% (NH3) 2SO4, 0.2%
Scale-up of production of 25(OH)D in 50 L
fermenter jar
3
In order to better evaluate the generality of our
bioconversion system, we also tested production of
KH
2
PO
4
, 0.1% MgSO
4
, 0.1% CaCO
3
, pH 7.2) at
ꢀ
27 C for 48 h. Then, all of these flasks seed broth
was transferred into a 50 L jar fermentor containing
30 L GPY medium with 0.02% soybean oil and
0.01% XX-09 (antifoam; Chenguang Chemical Co.
25(OH)D in 50 L fermenter jar. The fermentation
3
conditions such as agitation speed, dissolved oxygen
concentration, pH, etc. should also be considered for

Optimal culture conditions for bioconversion of vitamin D to calcifediol
7
3
Figure 8. Bioconversion of vitamin D
ꢀ
3
to 25(OH)D
3
in a tank fermentor. The culture conditions were as follows: 30 L of PYG medium in a
(1 g/L) were added after 2 d cultivation.
5
0 L tank, 27 C, pH 7.0. Agitation is 300 rpm. Aeration is 100 L/min. vitamin D
3
Ltd, Chengdu) as antifoam. The cultivation was
conducted with 0.2 L/h aeration and 250 rpm agita-
25(OH)D in a 500 L fermentation tank with over
3
60% bioconversion ratio also succeeded.
ꢀ
tion at 27 C. After two days, 30 g of vitamin D in
3
1500 mL of ethanol with 60 g PMCD (partially
methylated CD) and 6 g Tween 80 in 1500 mL of
water were added into the tank. The culture was
continued for a further four days with the agitation
speed raised to 300 from 250 rpm and the aeration
rate to 100 from 60 L/min. Maximum 25(OH)D3
productivity and bioconversion rate reached a value
of 0.639 g/L and 61.31% respectively on the 5th day.
The results obtained in this study demonstrate that
combined use of PMCD and proper ethanol
increases the conversion yield of 25(OH)D3.
Acknowledgements
Science & Technology Department of Sichuan
Province supported this study.
Declaration of interest
The authors declare that they have no conflict of
interest.
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