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

Ji, Qingzhi; Wang, Bochu; Chou, Li-Der; Hao, Jinglan; Feng, Wenjing

doi:10.1039/c8ra06798h

Cited by 14 publications

(8 citation statements)

References 32 publications

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“…Furthermore, from the change of peak area, we can see that there was almost no conversion of TCDCA when the concentration of bilirubin in buffer was 0.8 mM. Except for bilirubin, the experiments showed that Cu 2+ and Ca 2+ also have some inhibition to 7α-and 7β-HSDH [22].…”

Section: The Effect Of Bilirubin On Enzymatic Reactionmentioning

confidence: 87%

See 1 more Smart Citation

The Impact of Bilirubin on 7α- and 7β- Hydroxysteroid Dehydrogenases: Spectra and Docking Analysis

Ji¹,

Chen²,

Zhu³

et al. 2023

Preprint

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7α- and 7β-hydroxysteroid dehydrogenases (HSDH) are a pair of enzymes that can catalyze the isomerization of hydroxyl group at site 7 of bile acids. In previous study, we found that the activities of 7α- and 7β-HSDH can be inhibited by bilirubin. In order to clarify the impact, the effects of bilirubin on enzymes were studied by kinetics, spectrum and docking analysis. Relative activity of 7α-HSDH remained less than 40 % under 1 mM bilirubin, for 7β-HSDH, only 18 % activity left at the same condition. Using taurochenodeoxycholic acid (TCDCA) as substrate, the Km of 7α-HSDH was up to 0.63 mM from 0.24 mM after binding with bilirubin, the Km of 7β-HSDH rose from 1.14 mM to 1.87 mM for the catalysis of tauroursodeoxycholic acid (TUDCA). The affinity of 7α- and 7β-HSDH to substrate decreased with the effect of bilirubin. The binding of bilirubin with 7α- or 7β-HSDH were analyzed by UV–Vis spectra, Fluorescence spectra and Circular dichroism (CD) spectra. The results reflected that bilirubin caused a slight change in the secondary structure of 7α- or 7β-HSDH, and the changes were correlated with the ratio of bilirubin to enzyme. 10 candidate molecular docking results were presented to reflect the binding of bilirubin with 7α- or 7β-HSDH and to explore the inhibition mechanism. This research not only provides the more in-depth understanding of 7α- and 7β-HSDH, but also reminds us to avoid the inhibition of bilirubin on hydroxysteroid dehydrogenases.

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Section: The Effect Of Bilirubin On Enzymatic Reactionmentioning

confidence: 87%

“…Furthermore, both free enzymes and immobilized enzymes are suitable for the two steps catalytic reactions. Besides, the waste chicken bile is rich in TCDCA, the sufficient source of substrate can be ensured [22]. Bilirubin is one of the main components of waste chicken bile, from which substrate TCDCA is obtained.…”

Section: Introductionmentioning

confidence: 99%

The Impact of Bilirubin on 7α- and 7β- Hydroxysteroid Dehydrogenases: Spectra and Docking Analysis

Ji¹,

Chen²,

Zhu³

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…In previous study, 7α-and 7β-HSDHs were co-immobilized on modified chitosan microspheres and used as recyclable biocatalyst for the catalysis of chicken bile [22]. We found that the activities of 7α-and 7β-HSDHs were affected by bilirubin and Cu 2+ .…”

Section: Introductionmentioning

confidence: 88%

“…Furthermore, both free enzymes and immobilized enzymes are suitable for two-step catalytic reactions. The waste chicken bile is rich in TCDCA and a sufficient source of substrate can be ensured [22]. Bilirubin is one of the main components of waste chicken bile, from which substrate TCDCA is obtained.…”

Section: Introductionmentioning

confidence: 99%

The Impact of Bilirubin on 7α- and 7β-Hydroxysteroid Dehydrogenases: Spectra and Docking Analysis

Chen

Zhu

et al. 2023

Catalysts

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7α- and 7β-hydroxysteroid dehydrogenases (HSDHs) are enzymes that can catalyze the isomerization of hydroxyl groups at site seven of bile acids. In a previous study, we found that the activities of 7α- and 7β-HSDHs can be inhibited by bilirubin. In order to clarify the impact, the effects of bilirubin on enzymes were studied by kinetics, spectrum, and docking analysis. The relative activity of 7α-HSDH remained less than 40% under 1 mM bilirubin, and only 18% activity of 7β-HSDH kept in the same condition. Using taurochenodeoxycholic acid (TCDCA) as substrate, the Km of 7α-HSDH was up to 0.63 mM from 0.24 mM after binding with bilirubin and the Km of 7β-HSDH rose from 1.14 mM to 1.87 mM for the catalysis of tauroursodeoxycholic acid (TUDCA). The affinity of 7α- and 7β-HSDHs to substrates decreased with the effect of bilirubin. The binding of bilirubin with 7α- or 7β-HSDHs was analyzed by UV–vis, fluorescence, and circular dichroism (CD) spectroscopy. The results reflected that bilirubin caused a slight change in the secondary structure of 7α- or 7β-HSDHs, and the changes were correlated with the ratio of bilirubin to enzymes. Ten candidate molecular docking results were presented to reflect the binding of bilirubin with 7α- or 7β-HSDHs and to explore the inhibition mechanism. This research provides a more in-depth understanding of the effect of bilirubin on 7α- and 7β-HSDHs.

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“…7α-HSDHs can catalyze the C-7 α-position hydroxyl dehydroxylation reaction of CA or CDCA and their taurine or glycine conjugates, such as TCA, TCDCA, glycocholic acid (GCA), and glycochenodeoxycholic acid (GCDCA) to form keto groups, then 7β-HSDHs catalyze the reduction of the keto group to β-OH, realizing the epimerization of 7-OH configuration inversion, and the coenzyme NADP(H) is added to realize the reaction cycle (Scheme 1) [10]. Ji et al pointed out that chicken bile powder could be selected as the substrate for biosynthesis of TUDCA owing to its high content of TCDCA (about 42.58%), turning waste into treasure [11,12]. However, chicken bile powder contains 4.74% TCA, and a new by-product TUCA could be produced, resulting in purification difficulties and lower production of TUDCA.…”

Section: Introductionmentioning

confidence: 99%

A Novel NADP(H)-Dependent 7alpha-HSDH: Discovery and Construction of Substrate Selectivity Mutant by C-Terminal Truncation

et al. 2022

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7α-Hydroxysteroid dehydrogenase (7α-HSDH) plays an important role in the biosynthesis of tauroursodeoxycholic acid (TUDCA) using complex substrate chicken bile powder as raw material. However, chicken bile powder contains 4.74% taurocholic acid (TCA), and a new by-product tauroursocholic acid (TUCA) will be produced, having the risk of causing colorectal cancer. Here, we obtained a novel NADP(H)-dependent 7α-HSDH with good thermostability from Ursus thibetanus gut microbiota (named St-2-2). St-2-2 could catalyze taurochenodeoxycholic acid (TCDCA) and TCA with the catalytic activity of 128.13 and 269.39 U/mg, respectively. Interestingly, by a structure-based C-terminal truncation strategy, St-2-2△C10 only remained catalytic activity on TCDCA (14.19 U/mg) and had no activity on TCA. As a result, it can selectively catalyze TCDCA in waste chicken bile powder. MD simulation and structural analysis indicated that enhanced surface hydrophilicity and improved C-terminal rigidity affected the entry and exit of substrates. Hydrogen bond interactions between different subunits and interaction changes in Phe249 of the C-terminal loop inverted the substrate catalytic activity. This is the first report on substrate selectivity of 7α-HSDH by C-terminal truncation strategy and it can be extended to other 7α-HSDHs (J-1-1, S1-a-1).

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

Cited by 14 publications

References 32 publications

The Impact of Bilirubin on 7α- and 7β- Hydroxysteroid Dehydrogenases: Spectra and Docking Analysis

The Impact of Bilirubin on 7α- and 7β- Hydroxysteroid Dehydrogenases: Spectra and Docking Analysis

The Impact of Bilirubin on 7α- and 7β-Hydroxysteroid Dehydrogenases: Spectra and Docking Analysis

A Novel NADP(H)-Dependent 7alpha-HSDH: Discovery and Construction of Substrate Selectivity Mutant by C-Terminal Truncation

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