WO2017089408A1

WO2017089408A1 - Method for producing bile acids

Info

Publication number: WO2017089408A1
Application number: PCT/EP2016/078572
Authority: WO
Inventors: ROEPCKE Clarisse Bruning SCHMITT; Cheila CAVANHOLI; Luiza Carneiro Correa GATTO; Paulo Cesar BARJONA
Original assignee: Novozymes A/S
Priority date: 2015-11-24
Filing date: 2016-11-23
Publication date: 2017-06-01

Abstract

The present invention relates to an enzymatic method for producing deconjugated bile acids from bile.

Description

METHOD FOR PRODUCING BILE ACIDS

FIELD OF THE INVENTION

The present invention relates to a method for producing deconjugated bile acids from bile.

BACKGROUND OF THE INVENTION

Bile or gall is a bitter dark green to yellowish brown fluid, produced by the liver of most vertebrates, that aids the digestion of lipids in the small intestine. It is secreted by the liver and stored in the gall bladder. Bovine bile is also known as "cattle bile" or "ox bile".

Bile is a waste product from, e.g., slaughterhouses and has a high content of conjugated bile acids.

Bile acids include cholic acid, chenodeoxycholic acid, deoxycholic acid and lithocholic acid. They are stored in their conjugated form in the gall bladder for secretion into the gastrointestinal tract. Conjugation increases the solubility of their hydrophobic steroid nucleus. The acids are conjugated as an N-acyl amidate with either glycine (glycoconjugated) or taurine (tauroconju- gated).

Bile acids are used in the pharmaceutical industry as high valued products, e.g. as intermediates for ursodeoxycholic acid and corticosteroids production. In the industry, bile acids are used in their deconjugated form. A hydrolysis reaction is needed to cleave the amide bond between the steroid core and the conjugated amino acid (glycine/taurine).

Industrial deconjugation of bile acids applies high temperature, caustics and hazardous acids and many hours of hydrolysis.

It is also known that deconjugation is catalyzed by bile salt hydrolase (BSH) enzymes (EC 3.5.1 .24), which are known to be present in several bacterial species of the gastrointestinal tract. See, e.g., Liong and Shah, International Dairy Journal 15 (2005), 391-398.

Yesair et al., APPLIED MICROBIOLOGY 19(2) (1970), 295-300, has disclosed that conjugated bile acids can be hydrolysed by cell-free extraxcts from aerobic bacteria and mentions that microorganisms have been described that hydrolyze conjugated bile acids, e.g., Aerobacter aero- genes, Bacteroides species, motile gram-negative microorganisms from several sources, Aspergillus oryzae, Clostridium perfringens, Clostridium species, and several species of entero- cocci. And further that cellfree extracts that hydrolyze conjugated bile acids have been obtained from C. perfringens. The enzyme activity responsible for the hydrolysis, the amount of enzyme needed or the extent to which deconjugation is possible in such reaction has not been disclosed. Further, an enzymatic route for industrial production of deconjugated bile acids has not been suggested. The aim of the present invention was to identify a safer and more eco-friendly industrial process for producing deconjugated bile acids from bile. The aim was to use an enzymatic route and add value to bile processing companies by substituting with benefits the existing recipes based on chemical routes using high volumes of hazardous inorganic acids, high temperature and long lasting steps.

SUMMARY OF THE INVENTION

The present inventors have shown that an enzyme preparation having a high aminopeptidase activity was able to release high amounts of free taurine from bovine bile at low temperature and within short time compared to the industrial process currently used. The enzymatic process of the invention thus saves energy, time and chemical reagents and further it is a safer and more eco-friendly process.

The invention thus provides a method for producing deconjugated bile acids which comprises: a) obtaining bile;

b) optionally purifying or partly purifying conjugated bile acids from the bile; and

c) incubating said bile or said purified or partly purified conjugated bile acids with an enzyme preparation having aminopeptidase activity.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method for producing deconjugated bile acids which comprises:

a) obtaining bile;

Preferably, it is a method for industrial production of deconjugated bile acids. More preferably, it is a method for industrial production of deconjugated bile acids to be used in the pharmaceutical industry.

Bile to be used in the method of the invention may be obtained from any suitable animal such as a bird, for example, chicken, turkey, goose, duck, pheasant or owl; a mammal, for example, cow, dog, mouse, goat, sheep, pig, horse, rat, guinea pig, bear or pig; a fish, for example, cod fish, mullet fish, anchovie or harder fish; or a cartilageous fish, for example, shark. In a preferred embodiment, the bile is obtained from any of cow, dog, mouse, goat, sheep, pig, horse, chicken, turkey, goose, duck or shark. In a more preferred embodiment, the bile is bovine bile. In another more preferred embodiment, the bile is obtained from any of chicken, turkey, goose or duck. The bile may, e.g., be obtained from a slaughterhouse. The bile may be a waste product from a slaughterhouse. The bile is subjected to an enzymatic treatment aiming at deconjugating fully or partly the conjugated bile acids in the bile. After the enzymatic treatment, the deconjugated bile acids can be obtained from the bile material.

Prior to the enzymatic treatment, conjugated bile acids may be purified or partly purified from the bile material. The aim of such optional prior purification may be the removal of undesired impurities, such as pigments, lipids and/or protein substances.

Alternatively, the enzymatic treatment may be performed on the bile material more or less as is and the deconjugated bile acids may be purified or partly purified from the bile material after the enzymatic treatment.

Or some purification may take place before the enzymatic treatment and some purification after.

In the context of the present invention, purified or partly purified bile acids cover conjugated and/or deconjugated bile acids (as the case may be) which have been separated from other components originating from the bile using any separation or purification method known in the art. The purified or partly purified bile acids may be essentially free from insoluble components from the bile from which they are obtained. In some embodiments, the bile acids are also separated from some of the soluble components of the bile from which they are obtained. They may be separated by one or more of the unit operations: centrifugation, filtration, precipitation, or chromatography.

The conjugated or the deconjugated bile acids, as the case may be, may be substantially pure, i.e. substantially free from other components from the bile material from which they are obtained.

A large proportion of the bile acids obtained from bile are conjugated with either glycine (gly- coconjugated) or taurine (tauroconjugated). The aim of the present invention is enzymatic de- conjugation of such conjugated bile acids. Bile or purified or partly purified conjugated bile ac- ids, as the case may be, is incubated with an enzyme preparation having aminopeptidase activity- Release of taurine is a measure of the level of deconjugation of tauroconjugated bile acids. The concentration of free taurine may be determined using a method known in the art, e.g., using a method as described in the Examples of the present application.

In one embodiment, incubation of the bile or the purified or partly purified conjugated bile acids, as the case may be, with the enzyme preparation is performed until the concentration of free taurine is at least 100 mM. In another embodiment, incubation is performed until the concentration of free taurine is at least 120 mM, preferably at least 150 mM, more preferably at least 200 mM. In one embodiment, incubation of the bile or the purified or partly purified conjugated bile acids, as the case may be, with the enzyme preparation is performed until the concentration of free taurine is 100-100,000 mM. In another embodiment, incubation is performed until the concentration of free taurine is 120-50,000 mM, preferably 150-30,000 mM, more preferably 200-10,000 mM.

In one embodiment, the concentration of free taurine after incubation with the enzyme preparation is at least 40% higher than the concentration of free taurine obtained in a similar method where incubation is performed without addition of the enzyme preparation. Preferably, the concentration of free taurine after incubation with the enzyme preparation is at least 60% higher, such as at least 80% or at least 100% higher, than the concentration of free taurine obtained in a similar method where incubation is performed without addition of the enzyme preparation. In one preferred embodiment, the concentration of free taurine after incubation with the enzyme preparation is at least 150% higher, such as at least 200% or at at least 300% higher, than the concentration of free taurine obtained in a similar method where incubation is performed without addition of the enzyme preparation.

In one embodiment, the concentration of free taurine after incubation with the enzyme preparation is 40-200,000% higher than the concentration of free taurine obtained in a similar method where incubation is performed without addition of the enzyme preparation. Preferably, the concentration of free taurine after incubation with the enzyme preparation is 60-100,000% higher, such as 80-50,000% or 100-20,000% higher, than the concentration of free taurine obtained in a similar method where incubation is performed without addition of the enzyme preparation. In one preferred embodiment, the concentration of free taurine after incubation with the enzyme preparation is 150-20,000% higher, such as 200-15,000% or 300-10,000% higher, than the concentration of free taurine obtained in a similar method where incubation is performed without addition of the enzyme preparation.

Release of glycine is a measure of the level of deconjugation of glycoconjugated bile acids. The concentration of free glycine may be determined using a method known in the art.

In one embodiment, incubation of the bile or the purified or partly purified conjugated bile acids, as the case may be, with the enzyme preparation is performed until the concentration of free glycine is at least 100 mM. In another embodiment, incubation is performed until the concentration of free glycine is at least 120 mM, preferably at least 150 mM, more preferably at least 200 mM.

In one embodiment, incubation of the bile or the purified or partly purified conjugated bile acids, as the case may be, with the enzyme preparation is performed until the concentration of free glycine is 100-100,000 mM. In another embodiment, incubation is performed until the concen- tration of free glycine is 120-50,000 mM, preferably 150-30,000 mM, more preferably 200- 10,000 mM.

In one embodiment, the concentration of free glycine after incubation with the enzyme preparation is at least 40% higher than the concentration of free glycine obtained in a similar method where incubation is performed without addition of the enzyme preparation. Preferably, the concentration of free glycine after incubation with the enzyme preparation is at least 60% higher, such as at least 80% or at least 100% higher, than the concentration of free glycine obtained in a similar method where incubation is performed without addition of the enzyme preparation. In one preferred embodiment, the concentration of free glycine after incubation with the enzyme preparation is at least 150% higher, such as at least 200% or at at least 300% higher, than the concentration of free glycine obtained in a similar method where incubation is performed without addition of the enzyme preparation.

In one embodiment, the concentration of free glycine after incubation with the enzyme preparation is 40-200,000% higher than the concentration of free glycine obtained in a similar method where incubation is performed without addition of the enzyme preparation. Preferably, the concentration of free glycine after incubation with the enzyme preparation is 60-100,000% higher, such as 80-50,000% or 100-20,000% higher, than the concentration of free glycine obtained in a similar method where incubation is performed without addition of the enzyme preparation. In one preferred embodiment, the concentration of free glycine after incubation with the enzyme preparation is 150-20,000% higher, such as 200-15,000% or 300-10,000% higher, than the concentration of free glycine obtained in a similar method where incubation is performed without addition of the enzyme preparation.

In one embodiment, incubation is performed for at least 10 minutes, preferably at least 30 minutes. In another embodiment, incubation is performed for at least 60 minutes.

In one embodiment, incubation is performed for 10 minutes to 48 hours, preferably for 30 minutes to 36 hours. In another embodiment, incubation is performed for 60 minutes to 12 hours.

Incubation may be performed at any temperature where the enzyme is active. In one embodiment, incubation is performed at a temperature of 30-70°C, preferably 40-60°C.

Incubation may be performed at any pH where the enzyme is active. In one embodiment, no pH adjustment of the bile material is done.

In the method of the present invention, bile or purified or partly purified conjugated bile acids, as the case may be, is incubated with an enzyme preparation having aminopeptidase activity.

The enzyme preparation may have an aminopeptidase activity of at least 50 LAPU/g, preferably at least 100 LAPU/g, more preferably at least 200 LAPU/g. In one embodiment, the enzyme preparation has an aminopeptidase activity of at least 300 LAPU/g, preferably at least 500 LAPU/g, more preferably at least 1 ,000 LAPU/g.

One LAPU (leucine amino peptidase) is defined as the amount that hydrolyzes 1 mmol L-leucine-p- nitroanilide per minute at 37°C, pH 8.0. The absorption increase of the product p-nitroaniline is measured at 405 nm and is proportional to the enzyme activity.

The enzyme preparation may have an aminopeptidase activity of 50-10,000 LAPU/g, preferably 100-5,000 LAPU/g, more preferably 200-3,000 LAPU/g. In one embodiment, the enzyme preparation has an aminopeptidase activity of 300-2,500 LAPU/g, preferably 500-2,000 LAPU/g.

In one embodiment, the enzyme preparation is added to a final concentration of at least 0.1 LAPU/mL, preferably at least 0.5 LAPU/mL, more preferably at least 1 LAPU/mL or at least 2 LAPU/mL. In another embodiment, the enzyme preparation is added to a final concentration of at least 3 LAPU/mL, preferably at least 5 LAPU/mL, more preferably at least 8 LAPU/mL.

In one embodiment, the enzyme preparation is added to a final concentration of 0.1 -100 LAPU/mL, preferably 0.5-75 LAPU/mL, more preferably 1 -50 LAPU/mL or 2-50 LAPU/mL. In another embodiment, the enzyme preparation is added to a final concentration of 3-100 LAPU/mL, preferably 5-75 LAPU/mL, more preferably 8-50 LAPU/mL.

In one embodiment, the enzyme preparation comprises at least five proteolytic components each having an approximate molecular weight, respectively, selected from 23 kD, 27 kD, 31 kD, 32 kD, 35 kD, 38 kD, 42 kD, 47 kD, 53 kD, and 100 kD. In another embodiment, the protease preparation comprises at least five proteolytic components having the approximate molecular weights 23 kD, 31 kD, 35 kD, 38 kD and 53 kD, respectively.

Preferably the enzyme preparation is dervied from a fungus, more preferably a filamentous fungus.

Preferably, the enzyme preparation is dervied from Aspergillus, more preferably from Aspergillus oryzae.

In a preferred embodiment, the enzyme preparation is a fermentation broth supernatant of Aspergillus oryzae with aminopeptidase activity.

In a more preferred embodiment, the enzyme preparation is a fermentation broth supernatant of the Aspergillus oryzae strain ATCC 20386 with aminopeptidase activity.

In another preferred embodiment, the enzyme preparation comprises at least one endopeptidase, at least one aminopeptidase and at least one carboxypeptidase.

In another preferred embodiment, the enzyme preparation comprises endopeptidase in an amount of at least 10%, preferably at least 15%, more preferably at least 20% or at least 25% of the total proteolytic components. In another preferred embodiment, the enzyme preparation comprises aminopeptidase in an amount of at least 10%, preferably at least 15%, more preferably at least 20% or at least 25% of the total proteolytic components.

In another preferred embodiment, the enzyme preparation comprises carboxypeptidase in an amount of at least 5%, preferably at least 7%, more preferably at least 10% or at least 12% of the total proteolytic components.

In another preferred embodiment, the enzyme preparation comprises endopeptidase in an amount of 10-80%, preferably 15-70%, more preferably 20-60% or 25-50% of the total proteolytic components.

In another preferred embodiment, the enzyme preparation comprises aminopeptidase in an amount of 10-80%, preferably 15-70%, more preferably 20-60% or 25-50% of the total proteolytic components.

In another preferred embodiment, the enzyme preparation comprises carboxypeptidase in an amount of 5-40%, preferably 7-35%, more preferably 10-30% or 12-25% of the total proteolytic components.

In a more preferred embodiment, the enzyme preparation comprises (i) endopeptidase in an amount of 10-80%, preferably 15-70%, more preferably 20-60% or 25-50% of the total proteolytic components, (ii) aminopeptidase in an amount of 10-80%, preferably 15-70%, more preferably 20-60% or 25-50% of the total proteolytic components, and (iii) carboxypeptidase in an amount of 5-40%, preferably 7-35%, more preferably 10-30% or 12-25% of the total proteolytic components.

The amount of endopeptidase, aminopeptidase and carboxypeptidase, respectively, may be determined by any method known in the art. Preferably, the respective amounts are determined as described in Example 1.

In another preferred embodiment, the relative ratio of endopeptidase : aminopeptidase : carboxypeptidase is about 2:2:1 based on releative peptide abundance of identified proteins.

EXAMPLES

Enzymes used in the examples

The 13 following enzymes were chosen to be tested:

Enzyme A is a subtilisin endopeptidase from Bacillus clausii having an activity of at least 16 KNPU/g. Enzyme B is a subtilisin endopeptidase from Bacillus clausii having an activity of at least 486 KIPU/g.

Enzyme C is a serine protease from Nocardiopsis prasina having an activity of at least 75000 PROT/g. Enzyme D is a subtilisin endopeptidase from Bacillus licheniformis having an activity of at least 2.5 AU-A/g.

Enzyme E is a blend of a subtilisin endopeptidase from Rhizomucor miehei having an activity of at least 0.22 AU-R/g; and a serine protease from Nocardiopsis prasina having an activity of at least 31400 PROT/g. Enzyme F is a endoprotease that hydrolyzes peptide bonds at the carboxy side of lysine and arginine residues from Fusarium oxysporum having an activity of at least 1 15000 MTU/g.

Enzyme G is a subtilisin endopeptidase from Bacillus amyloliquefaciens having an activity of at least 0.8 AU-N/g.

Enzyme H is a subtilisin endopeptidase from Bacillus amyloliquefaciens having an activity of at least 1 .5 AU-N/g.

Enzyme I is a fermentation broth supernatant of the Aspergillus oryzae strain ATCC20386 having an aminopeptidase activity of 1000 LAPU/g.

Enzyme J is a subtilisin endopeptidase from Rhizomucor miehei having an activity of at least 0.12 AU-R/g. Enzyme K is an experimental carboxypeptidase from Aspergillus oryzae.

Enzyme L is an experimental carboxypeptidase from Aspergillus oryzae.

Enzyme M is a lipase that hydrolyzes esterbonds in glycerides from Candida antarctica having a lipase activity of 5000 LU/g.

Example 1

An ox bile sample from a Brazilian slaughter house was obtained, and stored in a freezer. The initial pH of the sample was 7.0.

30 ml of the ox bile material was collected, put into an incubation tube, the enzyme was dosed (1 % v/v, based on total volume), and the mixture was incubated for 8 hours at 50°C. No pH adjustment was done. After the incubation, the samples were inactivated at 90°C for 10 min. Free Taurine quantification: The reaction was quantified using the taurine release from the deconjugated bile acid molecule, meaning free taurine in the medium. The sample was centrifuged at 4000 RPM for 7 min. 0.2 ml sample was mixed with 1 ml water and 1 ml ninhydrin reagent, vortexed, boiled for 14 min, then cooled, and the absorbance was determined at 570 nm. The final value was compared to a tau- rine standard curve.

Results:

Table 1

Conclusion:

Enzyme I (2% dosage) was able to increase free taurine levels in bovine bile from less than 50 to above 100 mM at 50°C in 8 hours.

Enzyme I is Flavourzyme 1000L which has a declared aminopeptidase activity of 1000 LAPU/g and an approximate density of 1.27 g/ml.

Further identification of the proteolytic components in a Flavourzyme preparation was per- formed. For identification of proteins in solution, a filter assisted sample preparation protocol (FASP) was applied. A sample volume corresponding to 100 μg of protein was reduced and alkylated prior to overnight proteolytic digestion by Trypsin on a 10 kDa cut-off spin-filter device. The resulting peptides were collected and identified by tandem nano-LC/MS using a high resolution LTQ-Orbitrap Velos Pro mass spectrometer (Thermo Scientific, Bremen, Germany). Ac- quired MS/MS spectra were identified by searching against the relevant database using an in- house Mascot server ver 2.4 (Matrix Science, London, UK).

Results were as follows: Percentage Relative ratio

Endopeptidase 38% 2

Aminopeptidase 43% 2

Carboxypeptidase 19% 1

Example 2

Dosage Optimization:

The enzyme applied in this trial was Enzyme I. 30 ml of the ox bile material was collected, put into an incubation tube, the enzyme was dosed in different concentrations (0.75; 1 .00 and 2.00% v/v, based on total volume), and the mixture was incubated for 8 hours at 50°C. No pH adjustment was done. After the incubation, the sample was inactivated at 90°C for 10 min.

Results:

Table 2

Conclusion:

Enzyme I applied at a dosage of 0.75% or 1 % (v/v) was able to increase free taurine levels in bovine bile from less than 50 to above 100 mM at 50°C in 8 hours. At a dosage of 2%, free taurine was increased to almost 200 mM. Example 3

Reaction Kinetics:

1 ^st trial

The enzyme applied in this trial was Enzyme I. 30 ml of the ox bile material was collected, put into an incubation tube, the enzyme was dosed 1 % v/v, based on total volume, and the mixture was incubated for 8 hours at 50°C. Samples were removed each 1 hour. No pH adjustment was done. After the incubation, the sample was inactivated at 90°C for 10 min. 2^nd trial

The enzyme applied in this trial was Enzyme I. 30 ml of the ox bile material was collected, put into an incubation tube, the enzyme was dosed in different concentrations (0.75; 1 .00 and 2.00% v/v, based on total volume), and the mixture was incubated for 3 hours at 50°C. Samples were removed each 1 hour. No pH adjustment was done. After the incubation, the sample was inactivated at 90°C for 10 min.

Results:

Table 3: 1 ^st trial:

Table 4: 2^nd trial:

Conclusion:

At the enzyme dosages applied, after 1 hour of hydrolysis, a plateau of free taurine concentration was reached.

Claims

1 . A method for producing deconjugated bile acids which comprises:

a) obtaining bile;

2. The method of claim 1 wherein the enzyme preparation is added to a final concentration of at least 0.1 LAPU/mL.

3. The method of any of the preceding claims wherein the enzyme preparation is added to a final concentration of 0.1 -100 LAPU/mL.

4. The method of any of the preceding claims wherein the concentration of free taurine after incubation with the enzyme preparation is at least 40% higher than the concentration of free taurine obtained in a similar method where incubation is performed without addition of the enzyme preparation.

5. The method of any of the preceding claims wherein incubation is performed until the concentration of free taurine is at least 100 mM.

6. The method of any of the preceding claims wherein the concentration of free glycine after incubation with the enzyme preparation is at least 40% higher than the concentration of free glycine obtained in a similar method where incubation is performed without addition of the enzyme preparation.

7. The method of any of the preceding claims wherein incubation is performed until the concentration of free glycine is at least 100 mM.

8. The method of any of the preceding claims wherein incubation is performed for at least 10 minutes, preferably at least 30 minutes.

9. The method of any of the preceding claims wherein incubation is performed at a temperature of 30-70°C, preferably 40-60°C.

10. The method of any of the preceding claims wherein the bile is from cow, dog, mouse, goat, sheep, pig, horse, chicken, turkey, goose, duck or shark.

1 1 . The method of any of the preceding claims wherein the bile is bovine bile.

12. The method of any of the preceding claims wherein the enzyme preparation has an aminopeptidase activity of at least 50 LAPU/g, preferably at least 100 LAPU/g, more preferably at least 200 LAPU/g.

13. The method of any of the preceding claims wherein the enzyme preparation is derived from a fungus, such as a filamentous fungus, such as Aspergillus, e.g., Aspergillus oryzae.

14. The method of any of the preceding claims wherein the enzyme preparation is a fermentation broth supernatant of Aspergillus oryzae with aminopeptidase activity.

15. The method of any of the preceding claims wherein the enzyme preparation comprises (i) endopeptidase in an amount of 10-80% of the total proteolytic components, (ii) aminopeptidase in an amount of 10-80% of the total proteolytic components, and (iii) carboxypeptidase in an amount of 5-40% of the total proteolytic components.