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AND DEHYDROXYLATION OF CHOLIC ACID
BY CLOSTRIDIUM BIFERMENTANS
A thes i s presented in partial ful filment o f the requi rements for the degree of Doctor of Phi losophy in Biotechnology
at Mas sey Unive rsity
RICHARD HAMILTON ARCHER 19 80
ABSTRACT
The transformation of bile acids by
Clos tridium bifermen tans
was studied with a view to developing a.process whereby the bile acid conjugates of New Zealand mutton and beef gall may be converted to deoxycholic acid.
Statistically designed experiments were employed to maximise 7a-dehydroxylation of cholic acid to deoxycholic acid and to minimise the ?a-dehydrogenation of cholic acid to 7-ketodeoxycholic acid. Both transformations showed optima near pH 7. High deoxycholate yields were associated with conditions less favourable to strong growth and with relatively high electrode potentials. 7-ketodeoxycholic acid production was not as sensitive to environmental factors as was 7a-de
hydroxylation and could not be eliminated merely by manipulating fermentation variables.
Studies on the ?a-dehydrogenation of cholic acid with washed resting-stage cells of
Cl. bifermentans
indicated several means of manipulating 7-ketodeoxycholate yields which were then tested using batch fermentation. In the presence of zn++ions, 7-ketodeoxycholate yields were reduced but dehydroxylation was completely inhibited. In the presence of EDTA, ?a-dehydrogenation was almost quantitative but deoxycholate yields were again nil. Both transformations were enhanced during aerobic incubation. The highest deoxycholate yield observed during the work (50 molar %) was obtained by sweeping the fermenter headspace with air.Growing cells of
Cl. bifermentans
effected the near-quantitative hydrolysis of glycodeoxycholate, taurodeoxycholate and taurocholate within 48 h whilst glycbcholate was 90% deconjugated. At substrate concentrations greater than 0.1% w/v however, taurine conjugates were less well hydrolysed.i
l
ACKNOWLEDGEMENTS
I wish to acknowledge the following:
Dr I.S. Maddox and Dr R. Chong, for their supervision and assistance.
The Department of Scientific and Industrial Research, for financial assistance.
Dr R.P. Garland and New Zealand Pharmaceuticals Ltd., for advice, the,provision of bile acids and the use of analytical equipment.
Professor Annamarie Ferrari of Milan University, for a culture of
Clos tridiwn bife rmentans
SD 10 .Mr J.T. Alger and Mr D.W. Couling, for their assistance in building and maintaining equipment.
Mr L.E. O' Brien and Dr I.F. Boag, for advice on the statistical design of experiments.
My mother, for typing, and my father, for critical advice.
My wife, Deborah 1 for support and for draft typing and proof
reading.
TABLE OF CONTENTS
ABSTRACT
ACKNOWLEDGEMENTS TABLE OF CONTENTS TABLE OF FIGURES
TABLE OF TABLES
BILE ACID NOMENCLATURE ABBREVIATIONS
CHAPTER 1 PRELUDE
CHAPTER 2 INTRODUCTION AND LITERATURE REVIEW 2.1
2.2
2.3 2.4
2.5 2.6 2.7
Dehydroxylation Dehydrogenation
2.2.1 7a-Hydroxycholanoyl Dehydrogenases 2.2.2 3a-Hydroxycholanoyl Dehydrogenases 2.2.3 12a-Hydroxycholanoyl Dehydrogenases Cholanoylglycine and Cholanoyltaurine Hydrolases Occurrence of Multiple Transformation Abilities
in Single Organisms Choice of Organism
Clostridium bifermentans
Inhibition of Bacterial Growth by Bile Acids
CHAPTER 3 METHODS 3.1
3.2
3.3 3.4 3.5 3.6
Melting Points Materials 3.2.1 Media
3.2.2 Chromatography 3.2.3 Bile Acids
Materials
3.2.3.1 3.2.3.2
7-Ketodeoxycholic Acid 3a,7a-dihydroxy-5S-chol-ll-
3.2.4 3.2.5
3.2.3.3 Solvents
en-24-oic-acid Conjugates
Gases and Other Chemicals Organisms
Sterilisation of Media Cleaning of Glassware Analytical Methods 3.6.1 pH Measurement
3.6.2 Dry Weight Determination 3.6.3 Cell Counts
3.6.4 3.6.5
3.6.6
Thin-layer Chromatography
High Performance Liquid Chromatography 3.6.5.1 Mobile Phase
3.6.5.2 3.6.5.3 Infra-red
Resolution Operation
Spectrophotometry
iii
i ii iii vi ix xi xii 1
3 4 9 10 12 13 15 18
21 22 23
25 25 25 25 25 25 26 26
26 28 28 29 29 30 30 30 30 30 30 31 31 31 33 33
3 . 7
3 . 8
3 . 9 3 . 1 0
3 . 1 1
3 . 12
Culture Conditions
3 . 7 . 1 Small Scale Experiments 3 . 7 . 2
Washed 3 . 8 . 1 3 . 8 . 2 3 . 8 . 3
Fermentation
3 . 7 . 2 . 1 Equipment
3 . 7 . 2 . 2 Instrumentation 3 . 7 . 2 . 3 Sterilisation
3 . 7 . 2 . 4 Inoculum Preparation 3 . 7 . 2 . 5 Operation
3 . 7 . 2 . 6 Sampling Technique Cell Methodology
Cell Production Equipment
Harvesting and Washing 3 . 8 . 4 Incubation
Extraction and H.P.L.C. Sample Preparation 3 . 9 . 1 Solvent Extraction
3 . 9 . 2 Freeze Dry Extraction Product Characterisation
3 . 10 . 1 Action of Strain ATCC 9 7 14 on Cholic Acid 3 . 10 . 2 Action of Strain SD 10 on Conjugates 3 . 10 . 3 Action of Strain ATCC 9 7 14 on Conjugates Calculations
3 . 1 1 . 1 H .P.L.C. Data Analysis
3 . 1 1 . 2 Calculation of Molar Compositions 3 . 1 1 . 3 Mass Balance Calculations
Discussion of Methods
CHAPTER 4 EXPERIMENT 1 : EFFECT OF pH AND ATMOSPHERIC COMPOSITION ON CHOLIC ACID TRANSFORMATION 4 . 1
4 . 2 4 . 3 4 . 4 4 . 5 4 . 6
Introduction Experiment Design
Fermentation Conditions Results
4 . 4 . 1 Statistical Analysis Discussion of Experiment 1 Conclusions
CHAPTER 5 EXPERIMENT 2 : IDENTIFICATION OF FERMENTATION VARIABLES IMPORTANT TO DEHYDROXYLATION AND DEHYDROGENATION OF CHOLIC ACID
5 . 1 5 . 2 5 . 3 5 . 4 5 . 5 5 .6
Introduction Experiment Design
Fermentation Conditions Results
Discussion of Experiment 2 Conclusions
CHAPTER 6 CHOLIC ACID TRANSFORMATION USING WASHED RESTING CELLS OF Cl. BIFERMENTANS ATCC 9 7 14 6 . 1
6 . 2 6 . 3
6 . 4
Introduction
Fermentation Conditions and Cell Preparation Results and Discussion
6 . 3 . 1 Time Scale and Reproducibility of the Reaction 6 . 3 . 2 Effect of Aerobic Incubation
6 . 3 . 3 Effect of Atmosphere, pH, Incubation Time
6 . 3 . 4
and Temperature
Effects of Common Enzyme Inhibitors and Other Miscellaneous Chemicals Conclusions
3 3 3 3 3 3 3 3 3 8 4 1 4 1 42 42 4 3 4 3 4 3 4 3 4 3 4 7 4 7 4 7 4 8 4 8 49 51 5 2 5 1 52 52 5 3
56 56 56 5 7 5 8 5 8 76 8 3
8 5
8 5 85 90 90 112 117
1 19 119 120 120 1 2 1 12 1 124 1 2 7 1 3 1
CHAPTER 7 EFFECT OF AEROBIC INCUBATION AND
MISCELLANEOUS COMPOUNDS ON CHOLIC ACID TRANSFORMATION DURING BATCH FERMENTATION 7 . 1
7 . 2 7 . 3
Introduction
Fermentation Conditions Results and Discussion
7 . 3 . 1 Effect of Very Late Substrate Addition 7 . 3 . 2 Effect of zn++ ion
7 . 3 . 3 Effect of High Thioglycollate Levels
7 . 3 . 4 Effect of EDTA on Cholic Acid Transformation 7 . 3 . 5 Effect of Air on Cholic Acid Transformation 7 . 4 Conclusions
CHAPTER 8 HYDROLYSIS OF BILE ACID CONJUGATES BY CLOSTRIDIUM BIFERMENTANS
8 . 1 Introduction
8 . 2 Fermentation Conditions 8 . 3 Results
8. 4 Discussion 8 . 5 Conclusions CHAPTER 9
CHAPTER 10 REFERENCES
APPENDICES
Appendix 1 Appendix 2 Appendix 3 Appendix 4 Appendix 5
GENERAL DISCUSSION
GENERAL CONCLUSIONS
Infra-red Spectra
Parsimonious Models and Regression Statistics for Experiment 1
Parsimonious Models and Regression Statistics for Experiment 2
Electrode Potentials of Cell Production Fermentation
Raw Data for Deconjugation Experiments Conducted on the Small Scale
V
1 3 3 1 3 3 1 3 3 1 34 1 34 1 3 7 1 3 7 1-39 144 1 5 1
15 2 1 52 1 52 154 155 167
169 1 7 8 1 80
189 195 2 0 1 209 2 10
3 . 1 3 . 2 3 . 3 3 . 4 3 . 5 3 . 6 3 . 7 3 . 8 3 . 9 3 . 10 4 . 1 4 . 2 4 . 3 4 . 4 4 . 5 4 . 6 4 . 7 4 . 8 4 . 9 4 . 10 4 . 1 1 4 . 12
4 . 1 3 4 . 14 4 . 15 4 . 16
5 . 1 5 . 2 5 . 3 5 . 4 5 . 5 5 . 6
TABLE OF F I GURES
The As sembled Fe rmente r
The As semble d Fermente r - Key to Figure 3. 1 The 2- l i t re Fermenter Vess e l Head
Plan o f the 2-litre Fermenter Ve s sel He ad The Gas-Mixe r/Flow- Regulator
S chematic of Gas-Mixe r/Flow-Regul ator Ce l l Washing Equipment
Washe d Ce ll In cubation Rack Ce l l Washing Equipment - Di agram
Di f fe renti al Re fractometer Li ne arity Characte ri stic Course o f Growth an d Transfo rmation fo r Exp . l , Run l
Run 2 Run 3 Run 4 Run 5 Run 6 Run 7 Run 8 Run 9 Run 10 Run 11 Res i dual Plot Number 2 fo r the P ars imonious Mode l for
De oxycholate Yield
Pre dicted Deoxy cholate Yield ( vi a log- trans form mode l ) P re di cted 7-Ke todeoxycholate Yield
Pre di cted Cel l Yield ( as L . M. C . , log o f maximum obs erve d numbe r of cells pe r mi lli litre of culture )
Course o f Growth and Trans fo rmation for Expe riment 1 , Run l Repeate d
Cours e o f Growth and Trans formation for Exp . 2 , Run l Run 2 Run 3 Run 4 Run 5 Run 6
34 35 36 37 39 40 44 45 46 54 59 60 61 62 6 3 64 65 66 67 6 8 69
72 7 3 74 75
78 9 1 92 9 3 94 9 5 96
5 . 7 5 . 8 5 . 9 5 . 10 5 . 11 5 . 12 5 . 1 3 5 . 14 5 . 15 5 . 16 5 . 17 5 . 18
6 . 1 6 . 2
6 . 3 6 . 4 7 . 1 7 . 2 7 . 3
7 . 4 7 . 5 7 . 6 7 . 7 7 . 8
8 . 1 8 . 2 8 . 3 8 . 4
Course o f Growth and Trans formation for E xp . 2 , Run 7 Run 8 Run 9 Run 10 Run l l Run 12 Run 1 3 Run 14 Run 15 Run 16 H al f-Normal Plot for Deoxycho late Yield from Exp . 2 Re s i dual Plot Number 2 for the Pars imonious Mode l for
7-Ketode oxycholate Yie l d f rom E xpe riment 2
�ime S c ale o f Dehydrogenation by Whole Res ting Cel l s E f fect of Aerob ic Incubation o n Dehy drogenation by
Whole Re sting Ce lls
E f fe ct of pH and Atmosphe re on Dehydrogenation E f fe ct of pH , Atmosphere an d Incubati on Time on
Dehydrogenation by Whole Res ting Ce lls
Course of Growth and Trans formation for Fe rmentation with 18-hour S ubstrate Addi tion
Cours e pf Growth and Trans formation for Fermentation with 6-hour Addi tion o f zn++ Ion to 5mM
Cours e of Growth and Tran s formation for Fe rmentation with 6-hour Addition of Sodium Thioglycol late to 0 . 15% w/v
Course of Growth and Trans format ion for Fe rmentation wi th 6-hour Addi tion of EDTA. ( a)
Cours e o f Growth and Trans formation for Fe rmentation wi th 6-hour Addition o f EDTA. (b)
Course of Growth and Trans formation for Fe rmentation with Air- Sweeping . ( a)
Course o f Growth and Trans formati on for Fermentation with Ai r-Sweeping . ( b )
Course o f Growth an d Trans formati on for Fermentation with Air-Sweeping . ( c)
E f fe ct o f Sub strate Con centration on Deconj ugation Yields E f fe ct o f Substrate Con centration on Total Extent o f
Decon j ugation
Course o f Growth o f S train S D 10 and Trans fo rmation o f Glycocholic Aci d i n Batch Fermentation
Course of Growth o f S train S D 10 and Transformation of Glycodeoxycholi c Aci d in Batch Fermentation
vii
9 7 9 8 9 9 lOO 10 1 102 10 3 104 105 106 l l l
l l l 1 2 2
12 3 12 5 125 135 1 3 8
140 142 143 146 147
148 156
1 5 7
158 159
8 . 5 8 . 6 8 . 7
8 . 8
A2 . 1 A2 . 2 A4 . 1
Course of Growth of Strain so 10 and Trans formation of Sodium Taurocholate in Batch Fe rmentation
Course o f Growth of Strain so 10 and Trans formation of Sodi um Taurodeoxycholate in B atch Fe rmentation Course
Cours e
o f Growth o f St rain SD 10 and Trans formation Glycocholic Acid and Sodium Taurocholate in B at ch Fermentation with 6-hour Substrate Addi tion
o f Growth of Strain so 10 and Trans fo rmation Glycocholic Ac id and Sodi um Taurocholate in Batch Fermentation with Ai r-Sweeping from 7 . 5 to 19 hours
o f
o f
Res idual Plot Numbe r 1 for the Pars imonious Mode l for 7-Ke todeoxycholate Yie ld
Residual Plot Numbe r 3 for the Pars imonious Mode l for L.M.C. ( i . e . log of maximum observed ce ll count) Ele ctrode Potential Curves and Dry Weights of Ce ll
S uspensions for Washe d Ce l l Production Runs
160 16 1
16 2
16 3 200 200 209
TABLE OF TABLES
2 . 1 7a- Dehydroxylation Yields Previ ous Pumli shed in the
3 . 1 3 . 2a 3 . 2b 3 . 3 4 . 1
Lite rature
Mel ting Points of Synthetic Conj ugates Retention Volumes of Bile Aci ds
Retention Volumes of Bile Acids
Me lting Points of De con j ugation P roducts Des ign and Run Orde r , Expe riment 1
4 . 2 The E f fe ct o f pH and Gas on Ce ll Growth and Cholic Aci d Trans formation
ix
7 2 7
32 32 5 1 5 7
5 8
4 . 3 Full Re gres s ion Models for Expe ri ment 1 70
4 . 4 Matrix o f'Corre lation Coe ffici ents for the Pairs of Re sponse
Vari ables in Experiment l 76
5 . 1 De sign Matrix and Variables for Experiment 2 89
5 . 2 a 5 . 2b 5 . 3
Raw Trans formati on and Growth Data for Experiment 2 10 7 Raw Elect rode Potential Data for E xpe riment 2 108 Coe f fi cients from the Ful l Re gre s si on Mode ls of Expe riment 2 109 5 . 4 Matrix o f Corre lation Coe f ficients for the P aris o f Re spon se
Vari ab les in Experiment 2 llO
6 . 1 Effe cts o f Mi s ce llaneous Compounds on Cholate Trans formation 8 . 1
A2 . 1 A2 . 2 A2 . 3 A2 . 4 A2 . 5
A 3 . 1 A 3 . 2 A3 . 3 A3 . 4 A 3 . 5 A3 . 6
by Whole Ce lls 129
Yield o f De conjugation after 4 8 h
Pars imoni ous Mode ls and Regre ss ion Statistics for Experiment l :
For 7-Ketodeoxycholate Yield For Cholic Acid Remaining For Deoxycholic Acid Yield
For the Natural Logari thm o f De oxycholi c Acid Yie ld For the Base Ten Logarithm o f the Maximum Numbe r o f
Ce lls Observed. ( L . M. C . )
Pars imonious Mode ls and Regress ion S tatistics for Experi ment 2 :
For 7-Ketodeoxy cholate Yield
154
19 5 196 197 19 8 199
2 0 1 For the Natural Logari thm o f 7-Ketodeoxy cholate Yield 2 0 2 F o r the Cholic Acid Remaining at 48 Hours
For Deoxycholate Yield
For the Base 10 Logarithm of Maximum Obs erve d Ce l l Counts ( L . M. C . )
For the Maximum Rate o f E lectrode Potential De c l ine ( l�Ec ) max lit
2 0 3 2 0 4 2 0 5
2 0 6
A3 . 7 A3 . 8
AS . l A5 . 2
Pars imonious Mode ls and Regression Statistics for Experiment 2 ( cont inued) :
For Minimum Elect rode Potential For Ini tial Electrode Potential ( Eci )
Raw Data for De conj ugati on Experiments Conducted on the Small S cale :
Extent of Transfo rmation o f Conj ugate ove r 4 8 h Effect o f Subs trate Concentration on T rans fo rmation
o f Conj ugates Afte r 48 h
2 0 7 2 0 8
2 10 2 1 1
B I LE ACI D NOMENCLATURE
Throughout this work , b i le acids wi l l be re fe rre d to by the t ri vi a l names emp loyed in " The Bile Acids " ( Mats chine r , 19 7 1 ) . These are listed be low , together with the appropriate I . U . P . A. C . systematic chemical name s ( I . U . P . A . C . - I . U . B . , 1969 ) .
cholic aci d 3 a , 7a, l2a-trihydroxy- S S- cholan- 2 4-oic acid deoxycholi c aci d 3 a , l2a- dihydroxy-S S- cholan- 2 4-oic acid chenodeoxycho li c acid - 3 a , 7 a- dihydroxy-S S-cholan- 2 4-oic acid ursodeoxycholic acid 3 a , 7 S- dihydroxy-S S- cholan- 2 4-oi c acid l i thocholi c acid 3a-hydroxy-SS- cholan- 2 4-oi c acid 7-ketodeoxycholic acid
3 a , l2a- dihydroxy-7-oxo- S S- cholan- 24-oi c acid 7-ke tolithocholic acid
- 3a-hydroxy- 7-oxo-S S- cho lan- 24-oic aci d
glycochol i c acid 3a , 7a , l2a- trihydroxy- S 8-cholan- 2 4- oylglycine glycode oxycholi c aci d - 3 a , l2a- dihydroxy-SS-cholan- 24-oylglyc ine taurocholic ac id 3a , 7 a , l2a-trihydroxy- S S- cholan-2 4-oyltaurine taurodeoxycholi c aci d - 3a , l2 a- di hydroxy- S S- cholan-2 4-oyltaurine
Aci ds will be re fe rred to by the s uffi xes " - ate " and " - oi c acid"
interchangeab ly , for examp le 7-ketodeoxycholic aci d and 7-ketodeoxycholate .
xi
ABBREVIATIONS
Abb re viations o f bile aci d n ames ( from Eneroth an d Sjovall, 19 7 1) :
c cholic aci d
D deoxycholic acid
7KD 7-ketodeoxycholic acid
GC glycocholic aci d
GD glycode oxycholic acid
TC taurocholic acid
TD taurodeoxycholic acid
Abbrevi ations of uni ts :
g h l m M min Pa rev tonne s V
gramme hour litre metre
moles per litre minutes
Pas cal ( Newton per square metre ) revo lution
tonne ( 1 , 000 Kg ) se cond
volt
Othe r abb re vi ati ons :
A ATCC B
DMSO Ec
code d val ue of pH in Experiment l (A Ame ri can Type Culture Colle ction
pH - 7 . 0 )
code d val ue o f gas i n Experiment l ( B = l for H2 , B = 0 for N2 , B = - 1 for N 2 - co2 , 9: 1)
dime thylsulph oxi de
e le ctrode potential re lative to the s aturated calome l e le ctrode
Ec . nun Ec.
l.
(6Ec/6t) max EDTA
Eh
F
H.P.T . . C'.
Km L.M.C.
ln
log log K1
MSlof MS pe
N NAD NADP NCIB
T.L.C.
Tr1s
minimum Ec observed during a fermentation Ec prevailing at inoculation
maximum rate of decline of Ec during a fermentation
ethylenediaminetetraacetic acid
electrode potential relative to the standard hydrogen electrode
ratio of mean sums of squares (i.e. F ratio) high performance liquid chromatography
the Michaelis constant
base 10 logarithm of the maximum cell count observed
during a fermentation natural logarithm
base 10 logarithm
first formation constant of chelate complex mean sum of squares due to lack of fit
mean sum of squares due to pure error
number of independent variables in an experiment design
nicotinamide adenine dinucleotide
nicotinamide adenine dinucleotide phosphate National Collection of Industrial Bacteria parachloromercuribenzoate
T.L.C. mobility relative to solvent front mobility
thin-layer chromatography tris(hydroxymethyl)methylamine
estimated value of response variable being modelled
xiii
