Co-immobilised 7α- and 7β-HSDH as recyclable biocatalyst: high-performance production of TUDCA from waste chicken bile

Article abstract of DOI:10.1039/c8ra06798h

Chicken gallbladder has long been considered to be worthless and discarded as waste. The main composition of chicken bile is taurochenodeoxycholic acid (TCDCA), which is the isomeride of tauroursodeoxycholic acid (TUDCA). TUDCA has been effectively used for treatment of many diseases. In this paper, 7α- and 7β-hydroxysteroid dehydrogenases (HSDH) were co-immobilised on modified chitosan microspheres, and used as recyclable biocatalyst for the catalysis of chicken bile. The catalytic reaction reached equilibrium within 4 h compared with 1 h using TCDCA as substrate. After four continuous batch reactions, the conversion of TCDCA was lower than 40% and TUDCA yield was about 15% for the catalysis of chicken bile. TUDCA yield was approximately 62% after equilibrium and the content of TUDCA in reaction product was as high as 33.16%. Furthermore, the experiments showed that activity of enzymes were significantly inhibited by bilirubin, Cu²⁺ and Ca²⁺ in complex substrate. The research described not only widens the utilization of chicken bile, but also provides a clean way for the preparation of TUDCA.

Full text of DOI:10.1039/c8ra06798h

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Co-immobilised 7a- and 7b-HSDH as recyclable
biocatalyst: high-performance production of
TUDCA from waste chicken bile†
Cite this: RSC Adv., 2018, 8, 34192
a
b
Chou Li,c Jinglan Haoa and Wenjing Fenga
*
Qingzhi Ji,
Bochu Wang,
Chicken gallbladder has long been considered to be worthless and discarded as waste. The main composition
of chicken bile is taurochenodeoxycholic acid (TCDCA), which is the isomeride of tauroursodeoxycholic acid
(TUDCA). TUDCA has been effectively used for treatment of many diseases. In this paper, 7a- and 7b-
hydroxysteroid dehydrogenases (HSDH) were co-immobilised on modified chitosan microspheres, and
used as recyclable biocatalyst for the catalysis of chicken bile. The catalytic reaction reached equilibrium
within 4 h compared with 1 h using TCDCA as substrate. After four continuous batch reactions, the
conversion of TCDCA was lower than 40% and TUDCA yield was about 15% for the catalysis of chicken
bile. TUDCA yield was approximately 62% after equilibrium and the content of TUDCA in reaction product
was as high as 33.16%. Furthermore, the experiments showed that activity of enzymes were significantly
inhibited by bilirubin, Cu2+ and Ca2+ in complex substrate. The research described not only widens the
utilization of chicken bile, but also provides a clean way for the preparation of TUDCA.
Received 13th August 2018
Accepted 26th September 2018
DOI: 10.1039/c8ra06798h
rsc.li/rsc-advances
are similar, such as TUDCA and taurochenodeoxycholic acid
(TCDCA), UDCA and CDCA, both of them are epimers with the
1. Introduction
difference of –OH at 7 site as Fig. 1. Chicken is a kind of deli-
cious food eaten around the world. However, chicken gall-
bladder is oen abandoned, which is rich in TCDCA. If the
chicken bile can be used reasonably, it will become an abun-
dant bioresource. The value of chicken bile will be greatly
increased once TCDCA is transformed into TUDCA. Further
exploitation of such a bioresource will be promoted, as well. The
biotransformation of chicken bile not only provides a new way
to produce TUDCA, but also provides benet for environment
protection. Torres et al. had reported one similar example.
Soybean peroxidase was immobilized onto activated carbon
obtained from agricultural waste with a total utilization of
biomass and minimal waste generation.13
The epimerization of TCDCA to TUDCA can be catalysed by
7a- and 7b-hydroxysteroid dehydrogenases (7a- and 7b-
HSDH).14,15 7a- and 7b-HSDH belong to short chain
dehydrogenase/reductase (SDR) family and exist in some gut
microbes, such as Clostridium absonum,15 Collinsella aero-
faciens16 and Bacteroides fragilis.17 The transformation of CDCA
to UDCA, TCDCA to TUDCA have been proved to be feasible.18,19
The reactions catalyzed by both free enzymes and immobilised
enzymes have been studied. However, in all the former related
studies, CDCA or TCDCA was used as the catalytic substrate. We
recognize that there has not been any reports on the biotrans-
formation of TCDCA in complex substrate to get high-value
TUDCA. In nature, most of the substance exist in the form of
mixtures. It is more meaningful to obtain TUDCA using chicken
bile as substrate.
Tauroursodeoxycholic acid (TUDCA) and its hydrolysate urso-
deoxycholic acid (UDCA) are secondary bile acids, which are
synthesized from primary bile acid chenodeoxycholic acid
(CDCA) in enterohepatic circulation.1 These are candidate
medicines commonly used for the treatment of hepatobiliary
disease.2–4 Furthermore, TUDCA can prevent hearing and vision
loss, and slow down retinal degeneration.5,6 Recent papers have
reported that TUDCA and UDCA are neuroprotective in Par-
kinson's, Huntington's and Alzheimer's diseases, and also in
humans with amyotrophic lateral sclerosis.7–9 There are many
additional ancillary features of TUDCA, such as promoting
blood vessel repair,10 inhibiting obesity11 and enhancing islet
function.12 Even TUDCA and UDCA can be prepared through
a series of chemical processes; it is therefore very meaningful to
obtain TUDCA in a clean and high-performance way.
There are few reports about the acquisition of TUDCA
through the transformation of bile acids in vitro, especially
using a complex as substrate. The structure of some bile acids
aSchool of Pharmacy, Yancheng Teachers' University, No. 2, Hope Avenue, Yancheng,
Jiangsu 224051, P. R. China. E-mail: jiqingzhi202@126.com; Fax: +86-0515-
88258773; Tel: +86-0515-88258773
bKey Laboratory of Biorheological Science and Technology (Chongqing University),
Ministry of Education, College of Bioengineering, Chongqing University, Chongqing
400030, P. R. China
cCollege of Marine and Bio-engineering, Yancheng Teachers' University, Yancheng,
Jiangsu 224051, P. R. China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c8ra06798h
34192 | RSC Adv., 2018, 8, 34192–34201
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
Fig. 1 The reaction process of TCDCA to TUDCA catalysed by 7a- and 7b-HSDH. TCDCA and TUDCA are the isomeride at 7 site. 7a- and 7b-
HSDH can isomerize the –OH of 7 site for TCDCA or TUDCA.
This research was focus on the transformation of TCDCA in for 12 h and freeze drying for 48 h. Aer the sample was
chicken bile to obtain high valuable TUDCA with the catalysis of completely dry, it was stored in desiccator for the following
co-immobilised 7a-, 7b-HSDH. The co-immobilised 7a-, 7b- experiments.
HSDH was prepared using modied chitosan microspheres as
The content determination of bile acid in poultry bile used
carrier. Both the stability of enzymes and the effects of complex high performance liquid chromatography coupled with evapo-
substrate were carefully studied. The reaction time and the rative light scattering detector (HPLC-ELSD). The content of
concentration of coenzyme were also optimized. The possible cholesterol and bilirubin was detected using appropriate kits
compositions that affected enzyme activity in complicated according to instructions.
substrate were conrmed. Molecular docking was used to
explain the possible mechanism of bilirubin or complex
2.3 Enzyme immobilisation and activity analysis
substrate on enzyme activity.
The process of immobilisation between chitosan and enzymes
was shown in Fig. 2. The chitosan microspheres were prepared
according to ref. 14 and 20, with slight modications: a chitosan
solution was prepared by dissolving 0.5 g of chitosan akes into
50 mL of 2% (v/v) acetic acid solution. The resulting solution
was dropped into 3% (w/v) sodium tripolyphosphate (STPP) and
incubated for 3 h. Then the chitosan microspheres were acti-
vated with 0.125% glutaraldehyde (GA) solution at 25 ꢁC and
130 rpm for 3 h. The carrier was separated by ltration and
washed three times with water to remove redundant GA.
The activated microspheres (approximately 3 g) were incu-
bated with 6 mL (1 mg mLꢀ1) of 7a-HSDH or 7b-HSDH solution
in phosphate buffer (50 mM, pH 7.2–7.4) for 4 h at 16 ꢁC.
Finally, the chitosan microspheres with immobilised enzymes
were ltered and washed with phosphate buffered saline solu-
tion (pH, 7.2). The amount of immobilised protein was deter-
mined by testing the concentration of initial protein solution
(mg mLꢀ1) and the protein remaining in the supernatant (mg
mLꢀ1) with BCA Protein Assay Kit. Bovine serum albumin was
used as the standard.
2. Experimental section
2.1 Materials
Fresh poultry gallbladder was obtained from supermarket
(Chongqing, China). The chitosan (particle size, 40–100 mesh)
from Sangon Biotech (Shanghai, China) was used to prepare the
carrier of enzymes. BCA Protein Assay Reagent from Beyotime
(Shanghai, China) was used to detect the protein concentration.
TCDCA and TUDCA standards were purchased from the
National Institutes for Food and Drug Control (Beijing, China).
NADP-Na2 and NADPH-Na4 (purity $ 97%) produced by Roche
(Switzerland). Total Cholesterol Assay Reagent Kit (F0021-1),
and Bilirubin Assay Reagent Kit (C018) were purchased from
Jiancheng Bioengineering Institute (Nanjing, China). All the
other chemicals used in this study were of analytical grade and
used without further purication. Double-distilled water was
used in all experiments.
2.2 Preparation and component analysis of poultry bile
Fresh poultry gallbladder was cut and excluded membrane aer
To determine the activity of immobilised 7a-HSDH (or 7b-
sterilized by 75% ethanol. The mixture solution was ltrated by HSDH), the enzyme assay mixture in a total volume of 3 mL
a lter with 100 mm pore size. The ltrate was freezed at ꢀ80 ꢁC contained 0.5 mM TCDCA (or TUDCA), 0.2 mM NADP+ and 1.5 g
Fig. 2 The process of enzyme immobilisation between chitosan and enzymes. Chitosan microspheres were firstly activated by glutaraldehyde.
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 34192–34201 | 34193

View Article Online
RSC Advances
Paper
Table 1 Immobilization and activity of 7a- and 7b-HSDHa
Protein loading
(mg-protein per g-support)
Protein loading
yield (%)
Immobilized enzyme
Activity
yield (%)
Activity (U mgꢀ1
)
activity (Ugꢀ1
)
7a-HSDH
7b-HSDH
12.49 ꢃ 4.62
10.54 ꢃ 2.74
1.87 ꢃ 0.08
1.69 ꢃ 0.14
93.50 ꢃ 4.00
84.50 ꢃ 7.00
9.87 ꢃ 1.89
8.75 ꢃ 3.41
42.26 ꢃ 8.09
49.12 ꢃ 19.14
a
Conditions for immobilization: GA-modied chitosan microspheres (3 g) were reacted with 7a- or 7b-HSDH (6 mL, 1.0 mg mLꢀ1) in phosphate
buffer (50 mM, pH 7.2) at 16 ꢁC for 4 h. Conditions for enzyme detection: TCDCA or T-7-KLCA (0.5 mM) catalysed by immobilized 7a- or 7b-HSDH (3
g) with NADP(H) (0.5 mM) in Tris–HCl (50 mM, pH 8.5) at 25 ꢁC for 1 min.
immobilised 7a-HSDH (or 7b-HSDH). When free enzymes was
used as catalyst, the nal concentration was 1 mg mLꢀ1. The
amount of NADPH was proportional to the ultraviolet absorp-
tion at 340 nm. So change of ultraviolet absorbance at 340 nm
was recorded to measure enzyme activity. One unit of enzyme
activity was dened as the amount of enzyme needed to convert
1 mM substrate (TCDCA/TUDCA) within 1 minute at 25 ꢁC in
a buffer containing 50 mM Tris–HCl (pH 8.5).
2.4 Enzymatic reaction
ꢁ
The reaction was carried out on the condition of 25 C, pH 8.5
Tris–HCl (50 mM), using TCDCA (8 mM) and chicken bile (equal
TCDCA amount) as substrate, respectively. The reaction was
continued for 5 hours with the catalysis of co-immobilised 7a-,
7b-HSDH, six consecutive batch reactions were conducted.
TCDCA conversion and TUDCA yield were analyzed. The cata-
lytic efficiency were also studied with different concentration of
TCDCA (2–24 mM) and NADP+ (0.1–4 mM). The other reaction
conditions were consistent.
2.5 The effect of complex substrate on the activity of
enzymes
The effects of chicken bile on the activity of free 7a-HSDH and
immobilised 7a-HSDH were conducted using TCDCA and
chicken bile (containing equal TCDCA). The chicken bile
powder was dissolved by 50 mM Tris–HCl (pH 8.5) and soni-
cated for 30 min. The solution was centrifuged for 10 min at
8000 rpm. The supernatant was used as substrate. The detection
of enzyme activity was according to 2.3.
The effect of bilirubin on the activity of 7a-HSDH and 7b-
HSDH was studied according to the following methods. Bili-
rubin was added to the solution of TCDCA or TUDCA (8 mM),
and insure that the nal concentration of bilirubin was 0.457%
(mass ratio) equal to the amount of chicken bile. Then the
substrate was catalyzed by free enzymes or immobilised
enzymes. The activity of enzymes was detected according to 2.3.
The metal ions usually affected the activity of enzymes.
According to the content of chicken bile, each metal ions were
added to TCDCA solution, respectively, and the effect on
enzyme activity was detected according to 2.3.
Fig. 3 (a) The variation of TUDCA yield with time. TCDCA and chicken
bile that with equal TCDCA were used as substrates. (b) The effect of
NADP+ concentration on the conversion of TCDCA and yield of
TUDCA. The reaction was catalysed by co-immobilized 7a- and 7b-
2.6 High performance liquid chromatography (HPLC)
HSDH and the concentration of substrate TCDCA was
TUDCA generated
8 mM
TCDCA conversion and TUDCA yield were detected using HPLC-
ELSD. The HPLC analysis was performed with a Waters Acquity
SQD (Santa Clara, CA, USA) equipped with a 150 ꢂ 2.1 mm, 3 mm
TUDCA yield ¼
ꢂ 100%.
TCDCA in substrate
34194 | RSC Adv., 2018, 8, 34192–34201
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
Welch Ultimate XB-C18 column. The injection volume was 5 mL. binding modes with the lowest binding free energy and the
Two solvents (A: 100% methanol; B: 50 mM aqueous ammonium most cluster members were chosen for the optimum docking
acetate solution) were used at a constant ow rate of 0.8 conformation. The binding results were illustrated using the
mL minꢀ1. Elution was performed using a linear gradient from PyMOL Molecular Graphics System Version 1.5.
60 to 90% A for 15 min, from 90 to 60% A immediately, and then
held constant for 10 min. The ELSD detection temperature was
80 ꢁC. The detection was carried out in nitrogen environment and
3. Results and discussion
3.1 The activity of immobilised enzymes
the speed of nitrogen was 3.0 L minꢀ1
.
21
The results of immobilisation and activity for the 7a-HSDH and
7b-HSDH were shown in Table 1. Aer 4 h immobilisation at
16 ꢁC, the protein loading yields of 7a-HSDH and 7b-HSDH.
were about 93.50% and 84.50%, respectively. The activity of
immobilised 7a-HSDH was about 9.87 U gꢀ1-support, which
2.7 Docking of bilirubin and enzymes
The molecular docking of bilirubin to 7a-HSDH or 7b-HSDH
was performed using the 3D crystal structure of these proteins
(PDB codes 5EPO and 5GT9) obtained from the Protein Data
Bank.22,23 The ionizable residues were set to their pH 7.4
protonation states; the His, Arg and Lys were protonated while
those of Asp and Glu were deprotonated. The Autodock 4.2
plugin was used for all dockings in this study. The docking
parameters for AutoDock 4.2 were kept to their default values.
The docking results were ranked by the binding free energy. The
was higher than immobilised 7b-HSDH (about 8.75 U gꢀ1
support). However, the specic activity yields of 7a-HSDH and
-
Fig. 5 The effect of cholesterol and bilirubin on the activity of
Fig. 4 The effect of reaction times on the transformation of TCDCA in enzymes. TCDCA was used as the substrate of free 7a-HSDH and
six continuous reactions, (a) TCDCA (8 mM) as substrate; (b) chicken immobilised 7a-HSDH as (a) TUDCA was used as the substrate of free
bile with equal TCDCA (8 mM) as substrate.
7b-HSDH and immobilised 7b-HSDH as (b) *p < 0.05, **p < 0.01.
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 34192–34201 | 34195

View Article Online
RSC Advances
Paper
7b-HSDH were only 42.26% and 49.12%, which demonstrated of NADP+ was less than 2 mM, TCDCA conversion and TUDCA
that more than half activity of two enzymes lost in the process of yield gradually increased with NADP+. The highest TCDCA
immobilisation. This phenomenon is so common in enzyme conversion was about 82%, and the highest TUDCA yield was
immobilisation.
about 58%. When the concentration of NADP+ was more than
2 mM, the changes were minimal for both TCDCA conversion
and TUDCA yield. The effect of NADP+ concentration on TCDCA
conversion and TUDCA yield may due to the reaction is
reversible. The catalytic equilibrium can be regulated by the
concentration of NADP+ to some extent.
3.2 The effect of time and NADP+ concentration on
transformation
At the equilibrium, TUDCA yields were about 70% both TCDCA
and chicken bile as substrates with the catalysis of co-
immobilised 7a-, 7b-HSDH as Fig. 3a. However, it took longer
time to reach equilibrium using chicken bile solution as
3.3 Operation stability of catalysis
substrate compared with TCDCA. It was found that the catalytic In order to examine the catalytic stability of co-immobilised 7a-
reaction of TCDCA reached equilibrium within 60 min, while and 7b-HSDH, both TCDCA and chicken bile were used as
the catalysis of chicken bile was as long as 240 min. The longer substrates for experiments. In six continuous batch reactions,
reaction time may due to the low activity of 7a- and 7b-HSDH to the changes of TCDCA conversion and TUDCA yield were not
TCDCA in chicken bile. 7a- or 7b-HSDH may be inhibited by signicant using TCDCA as substrate. TCDCA conversion was
some components in complex substrate.
more than 85% and TUDCA yield was about 50–60%. However,
As shown in Fig. 3b, the optimum concentration of NADP+ the catalysis of chicken bile solution was not as stable as
was 2 mM for the catalysis of TCDCA. When the concentration TCDCA. Aer four continuous reactions, TCDCA conversion was
Fig. 6 The effect of metal ions on the activity of immobilised 7a- and 7b-HSDH. The concentration of each metal ions was determined
according to the content of chicken bile when the concentration of TCDCA defined as 8 mM. The study of Li et al. was referred.27 TCDCA and
TUDCA were used as the substrates for the activity detection of 7a- and 7b-HSDH as (a) and (b), respectively. *p < 0.05, **p < 0.01.
34196 | RSC Adv., 2018, 8, 34192–34201
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
lower than 40% and TUDCA yield was about 15%. In the h bile solution was less than catalysis of TCDCA. It may be in
and sixth reactions, the TUDCA generated could not detected by relation to the complexity of the substrate chicken bile. Using
HPLC-ELSD as described in Fig. 4b.
continuous stirred tank membrane reactors, the stability of b-
The operation stability of immobilised enzymes is depen- glucosidase immobilised on chitosan pellets was studied by
dent on many factors such as carrier, immobilisation methods Gallifuoco et al.26 Fructose did not decrease biocatalyst stability,
and reaction medium, in addition to the structure of free while alcohol affected enzyme half-life from 2586 h at 3% (w/v)
enzymes. The projects to improve the stability of immobilised ethanol to 1378 h at 12% (w/v). The addition of terpenes to
enzymes had been reviewed by Bommarius et al.24 and Ste- solution containing 10% (w/v) alcohol reduced the half-life by
pankova et al.25 The number of recycling times of co- a further 10%. So further exploration was conducted in order to
immobilised 7a- and 7b-HSDH for the catalysis of chicken explain which composition affected the activity of enzymes.
Fig. 7 The docking simulation of bilirubin and 7a-HSDH. (a) the overview of combination between bilirubin and 7a-HSDH; (b) the specific amino
acid residues of 7a-HSDH combined with bilirubin. Molecular docking was performed on autodock 4.2 and the results were analysed by PyMOL
1.5.
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 34192–34201 | 34197

View Article Online
RSC Advances
Paper
HSDH, the inhibition rate of bilirubin was about 25%, and
about 19% to immobilised 7b-HSDH. The effect of cholesterol
was not as signicant as bilirubin, the inhibition rate was about
5–10%. Most of metal ions in chicken bile had no effect on the
activity of 7a-HSDH, however, Cu2+ and Ca2+ had signicant
effect on the activity of 7a-HSDH, especially, Cu2+ to free 7a-
HSDH. The inhibition rate of Cu2+ to free 7a-HSDH was about
29%, and about 20% to immobilised 7a-HSDH as Fig. 6a. The
inhibition to 7b-HSDH even more. The inhibition rate of Cu2+ to
free 7b-HSDH was about 30%, and about 24% to immobilised
7b-HSDH (Fig. 6b). The different inhibition of metal ions to free
3.4 The effect of complex substrate on activity of enzymes
The different activity of 7a-HSDH for the catalysis of TCDCA and
chicken bile revealed that some compositions in complex
substrate have negative effect on enzymes activity. Except bile
acid, the other four main components of chicken bile are
protein, amino acid, cholesterol and bilirubin. Due to the
species diversity of protein and amino acid, we focused on the
effect of cholesterol and bilirubin. From Fig. 5, we found that
the activity of enzymes were signicantly affected by bilirubin (p
< 0.1). Inhibition rate of bilirubin was about 21% to free 7a-
HSDH, and about 15% to immobilised 7a-HSDH. To free 7b-
Fig. 8 The docking simulation of bilirubin and 7b-HSDH. (a) the overview of combination between bilirubin and 7b-HSDH; (b) the specific amino
acid residues of 7b-HSDH combined with bilirubin. Molecular docking was performed on autodock 4.2 and the results were analysed by PyMOL
1.5.
34198 | RSC Adv., 2018, 8, 34192–34201
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
enzymes and immobilised enzymes may attribute to the partly Chryseobacterium sp. CA49 by Tang.31 The experiments
adsorption of modied chitosan carrier.
demonstrated that Ag+, Cu2+, or Fe3+ negatively impacted the
The effect of bilirubin on 7a-HSDH activity may attribute to activity of ChKRED20. Some of metal ions showed clear stimu-
the binding of bilirubin with protein. Bilirubin can be lative effect, such as Ni2+, Mg2+, Zn2+, K+, Co2+ and Rb+.
combined with a variety of proteins according to references.
Orlov et al. reported that bilirubin can bind to different binding
sites of bovine serum albumins and human serum albumins.28
3.5 Exploration of inhibition mechanism for 7a- and 7b-
HSDH
Furthermore, bilirubin can also bind to hepatic Y-Protein and Z-
Protein.29 So it was no wonder that 7a- and 7b-HSDH were partly
inhibited by bilirubin.
Aer random docking of bilirubin to 7a- or 7b-HSDH, the
combination with high probability was analyzed as Fig. 7. Bili-
rubin was most probably combined with Arg (16), Arg (20) and
Arg (194) of 7a-HSDH as Fig. 6b. Arg (16) and Arg (194) are the
important residues of catalytic process which had been
demonstrated by Lou et al.22 It was no wonder that the activity of
7a-HSDH was effected by the combination of bilirubin. For 7b-
HSDH, the binding site of bilirubin to 7b-HSDH were Arg (40),
Met (97) and Lys (107) which were located at the exit of substrate
and product (Fig. 8). The activity decrease of 7b-HSDH may
attributed to the access blocking of substrates and products.
In this paper, inhibition mechanism for 7a- and 7b-HSDH
was simply studied by docking. The docking results were ranked
7a- and 7b-HSDH are both belong to short-chain
dehydrogenase/reductase (SDR). Some metal ions have an
impact on the activity of SDR enzymes, such as, Cu2+, Ca2+ and
Ni2+. Chen et al. reported a novel 3a-HSDH from Pseudomonas
aeruginosa, which inhibited by Cu2+, Ca2+ and Co2+. The stron-
gest inhibiting ion Cu2+ decreased the enzyme activity to only
67% of the control. 3a-HSDH can be activated by Zn2+, K+, Mn2+,
Fe3+ and Na+, among which Zn2+ exhibited the strongest acti-
vating effect by increasing the enzyme activity 1.75-fold. Some
other metal ions Mg2+, Ni2+ and Al3+ have no effect on 3a-
HSDH.30 ChKRED20 one SDR, was cloned from
Fig. 9 Chicken bile and reaction product in methanol were analyzed by HPLC-ELSD. (a) The concentrations of chicken bile powder was 1.0 mg
mLꢀ1, and the injection volume was 10 mL. (b) The concentrations of reaction product is 0.5 mg mLꢀ1, and the injection volume was 5 mL. (c) The
optical photographs of chicken bile powder. (d) The optical photographs of reaction product.
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 34192–34201 | 34199

View Article Online
RSC Advances
Paper
by binding free energy. Further detailed studies are under way. packed bed reactors, the yield of UDCA reached nearly 100%. In
It is well known that both ligands and receptors are exible, this study, the TUDCA was prepared within 4 h with the catalysis
molecular dynamics simulations is a good way for conforma- of co-immobilised 7a- and 7b-HSDH using chicken bile as
tional changes and virtual screening studies. In order to better substrate. The TUDCA yield was approximately 62% and the
investigate the mechanism of human butyryl cholinesterase content of TUDCA in reaction product was about 33.16%. This
inhibited by cyclosarin and sarin, docking and mixed quantum research provided a novel approach for the exploitation of waste
and molecular mechanics combined with principal compo- chicken bile, which complies with green chemistry principles.
nents analysis were performed to evaluate the docking results.32
Theoretical strategy combining docking (MM), chemometric
analysis and QM calculations was employed by de Lima et al. to
4. Conclusions
check out the association and kinetic reactivation coefficients
associated to oximes. The docking results were selected by
means of the principal components analysis and submitted to
QM calculations.33
In conclusion, 7a- and 7b-HSDH from Clostridium absonum
were co-immobilised on glutaraldehyde modied chitosan
microspheres. We are sure high-value TUDCA was the rst time
prepared using waste chicken bile as substrate. The catalytic
reaction reached equilibrium within 4 h. TUDCA yield was
3.6 The analysis of product composition
approximately 62% aer equilibrium and the content of TUDCA
in reaction product was about 33.16%. TUDCA was prepared
high-performance with the catalysis of recyclable biocatalyst.
The effect of complex substrate on the activity of enzymes
demonstrated that 7a-HSDH is signicantly inhibited by bili-
rubin, Cu2+ and Ca2+. The inhibition mechanism of bilirubin
was explored by freedom docking of moleculars. Further
research on chicken bile treatment and bioreactor design will
be gradual promoted.
The composition of reaction product was analysed by HPLC-
ELSD. The peak positions of TCDCA (about 13.8 min) and
TUDCA (about 9.5 min) were presented in Fig. 9a and b. Most of
TCDCA in chicken bile was transformed and high-value TUDCA
generated successfully. The analysis of bile acid in reaction
product showed that the proportion of TUDCA was as high as
33.16% and attained to 57.21% to the total bile acid in product
(as Table 2). The nature chicken bile is dark green as Fig. 9c,
aer transformation by co-immobilised 7a- and 7b-HSDH, the
color of sample changed into reddish-brown as Fig. 9d. This
result may be attributed to the generation of TUDCA and partly
adsorption of bilirubin.
Conflicts of interest
There are no conicts of interest to declare.
According to the literature, 7a- and 7b-HSDH from Xantho-
monas maltophilia has been puried and immobilised on resin
by Pedrini et al. and used for UDCA preparation.18 It took 7 days
to obtain 75% UDCA through epimerization based on immo-
bilised 7a- and 7b-HSDH. Zheng et al. adopted a two-step
reaction strategy for the synthesis of UDCA from CDCA,19
where 7a-HSDH from Clostridium absonum, 7b-HSDH from
Ruminococcus torques. 7a-HSDH, lactate dehydrogenase (LDH),
7b-HSDH and glucose dehydrogenase (GDH) were co-
immobilised onto an epoxy-functionalized resin by Zheng
et al.34 Continuous production of UDCA was achieved with the
catalysis of two cascade reactors. Lasted for at least 12 h in the
Acknowledgements
This work was supported by Supported by Visiting Scholar
Foundation of Key Laboratory of Biorheological Science and
Technology (Chongqing University), Ministry of Education
(CQKLBST-2018-018), National Science and Technology Major
Projects for “Major New Drugs Innovation and Development”
(2014ZX09301306-007).
References
1 J. M. Ridlon, D.-J. Kang and P. B. Hylemon, J. Lipid Res., 2006,
47, 241–259.
2 G. Alpini, L. Baiocchi, S. Glaser, Y. Ueno, M. Marzioni,
H. Francis, J. L. Phinizy, M. Angelico and G. LeSage,
Hepatology, 2002, 35, 1041–1052.
Table
2 The composition analysis of chicken bile and reaction
producta
Content (%)
Chicken bile
Reaction product
3 L. Baiocchi, G. Tisone, M. A. Russo, C. Longhi, G. Palmieri,
A. Volpe, C. Almerighi, C. Telesca, M. Carbone, L. Toti,
F. De Leonardis and M. Angelico, Transplant Int., 2008, 21,
792–800.
4 W. Wang, J. Zhao, W. Gui, D. Sun, H. Dai, L. Xiao, H. Chu,
F. Du, Q. Zhu and B. Schnabl, Br. J. Pharmacol., 2018, 175,
469–484.
5 J. Hu, M. Xu, J. Yuan, B. Li, S. Entenman, H. Yu and
Q. Y. Zheng, Neuroscience, 2016, 316, 311–320.
6 L. Fernandez-Sanchez, P. Lax, A. Noailles, A. Angulo,
V. Maneu and N. Cuenca, Molecules, 2015, 20, 13875–13893.
TUDCA
TCDCA
—
33.61 ꢃ 0.17
12.40 ꢃ 0.10
57.21 ꢃ 0.44
3.24 ꢃ 0.16
19.20 ꢃ 3.33
0.19 ꢃ 0.01
0.25 ꢃ 0.05
42.57 ꢃ 0.93
42.57 ꢃ 0.93
7.76 ꢃ 0.14
11.00 ꢃ 0.57
0.50 ꢃ 0.07
0.46 ꢃ 0.02
Total bile acid
Protein
Amino acids
Cholesterol
Bilirubin
a
TUDCA: tauroursodeoxycholic acid. TCDCA: taurochenodeoxycholic
acid. The content of bile acid in chicken bile and reaction product
were detected by HPLC-ELSD. Protein, amino acids, cholesterol and
bilirubin content are quantied by test kits. “—”stands for this
composition detected but not quantied.
34200 | RSC Adv., 2018, 8, 34192–34201
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
7 A. E. Elia, S. Lalli, M. R. Monsurro, A. Sagnelli, A. C. Taiello, 20 S. A. Cetinus and H. N. Oztop, Enzyme Microb. Technol., 2003,
B. Reggiori, V. La Bella, G. Tedeschi and A. Albanese, Eur. J.
Neurol., 2016, 23, 45–52.
8 L. M. Cortez, J. Campeau, G. Norman, M. Kalayil, J. Van der
32, 889–894.
21 Y. Zhao, L. X. Zan and W. J. Sun, Chin. J. Pharm. Anal., 2006,
26, 127–129.
Merwe, D. McKenzie and V. L. Sim, J. Virol., 2015, 89, 7660– 22 D. S. Lou, B. C. Wang, J. Tan, L. C. Zhu, X. X. Cen, Q. Z. Ji and
7672. Y. Wang, Sci. Rep., 2016, 6, 22885.
9 A. I. Rosa, S. Duarte-Silva, A. Silva-Fernandes, M. J. Nunes, 23 R. Wang, J. Wu, D. K. Jin, Y. Chen, Z. Lv, Q. Chen, Q. Miao,
A. N. Carvalho, E. Rodrigues, M. J. Gama, R. Cmp, X. Huo and F. Wang, Acta Crystallogr., 2017, 73, 246–252.
P. Maciel and M. Castro-Caldas, Mol. Neurobiol., 2018, 1–17. 24 A. S. Bommarius and M. F. Paye, Chem. Soc. Rev., 2013, 42,
10 J. G. Cho, J. H. Lee, S. H. Hong, H. N. Lee, C. M. Kim, 6534–6565.
S. Y. Kim, K. J. Yoon, B. J. Oh, J. H. Kim, S. Y. Jung, 25 V. Stepankova, S. Bidmanova, T. Koudelakova, Z. Prokop,
T. Asahara, S. M. Kwon and S. G. Park, Stem Cells, 2015,
33, 792–805.
R. Chaloupkova and J. Damborsky, ACS Catal., 2013, 3,
2823–2836.
11 Q. Y. Guo, Q. D. Shi, H. X. Li, J. L. Liu, S. F. Wu, H. Z. Sun and 26 A. Gallifuoco, F. Alfani, M. Cantarella, G. Spagna and
B. Zhou, Int. J. Endocrinol., 2015, 687938, DOI: 10.1155/2015/
687938.
12 Y. Y. Lee, S. H. Hong, Y. J. Lee, S. S. Chung, H. S. Jung,
P. G. Pifferi, Process Biochem., 1999, 35, 179–185.
27 F. Li, B. C. Wang and L. C. Zhu, Chin. Tradit. Pat. Med., 2015,
37, 2555–2558.
S. G. Park and K. S. Park, Biochem. Biophys. Res. Commun., 28 S. Orlov, I. Goncharova and M. Urbanova, Spectrochim. Acta,
2010, 397, 735–739. Part A, 2014, 126, 68–75.
13 J. A. Torres, F. G. E. Nogueira, M. C. Silva, J. H. Lopes, 29 D. R. Davis and R. A. Yeary, Proc. Soc. Exp. Biol. Med., 1975,
ˆ
T. S. Tavares, T. C. Ramalho and A. D. Correa, RSC Adv.,
148, 9–13.
2017, 7, 16460–16466.
14 Q. Z. Ji, J. Tan, L. C. Zhu, D. S. Lou and B. C. Wang, Biochem.
Eng. J., 2016, 105, 1–9.
30 J. M. Chen, X. F. Gao, L. Hong, L. T. Ma and Y. S. Li, Protein
Expression Purif., 2015, 115, 102–108.
31 T. X. Tang, Y. Liu and Z. L. Wu, J. Mol. Catal. B: Enzym., 2014,
105, 82–88.
15 E. E. Ferrandi, G. M. Bertolesi, F. Polentini, A. Negri, S. Riva
and D. Monti, Appl. Microbiol. Biotechnol., 2012, 95, 1221– 32 T. C. Ramalho, E. F. F. D. Cunha, A. A. D. Castro, A. F. Pereira
1233.
and W. E. A. D. Lima, Lett. Drug Des. Discovery, 2016, 13, 360–
371.
33 W. E. A. D. Lima, A. Francisco, E. F. F. D. Cunha, Z. Radic,
P. Taylor, T. C. C. França and T. C. Ramalho, J. Biomol.
Struct. Dyn., 2017, 35, 1272–1282.
16 T. Akao, T. Akao and K. Kobashi, J. Biochem., 1987, 102, 613–
619.
17 M. J. Bennett, S. L. McKnight and J. P. Coleman, Curr.
Microbiol., 2003, 47, 475–484.
18 P. Pedrini, E. Andreotti, A. Guerrini, M. Dean, G. Fantin and 34 M. M. Zheng, F. F. Chen, H. Li, C. X. Li and J. H. Xu,
P. P. Giovannini, Steroids, 2006, 71, 189–198.
19 M. M. Zheng, R. F. Wang, C. X. Li and J. H. Xu, Process
Biochem., 2015, 50, 598–604.
ChemBioChem, 2017, 19, 347–353.
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 34192–34201 | 34201