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Characterization of 4-androstene-3,17-dione Production through the use of Immobilized
Cells in Minireactors
Lisbon, 08th January 2008
IST – Instituto Superior TécnicoCentre for Biological and Chemical Engineering (CEBQ) BioEngineering Research Group – BERG
Supervisor: Dr. Pedro FernandesCo-Supervisor: Prof. Dr. Joaquim Sampaio Cabral
Institute for Biotechnology and Bioengineering
M.Sc. Thesis
Mauro José Castanho Claudino
1
Aim of this Thesis:
Screening of different immobilization procedures (adsorption, encapsulation and entrapment) for sterol bioconversion and systematically characterize the most suitable immobilization method, using a small scale approach.
Objective
Parameters evaluated: Cell loading capability;
Substrate and product partition effects;
Temperature, pH and hydrodynamic conditions;
Stability (thermal and storage);
Reaction kinetics;
Biocatalyst reusability. 2
Small-scale bioreactors Microtitre plates, test tubes and shaken reactors
Advantages: Parallel and automated experimental set-ups; Cost reduction for media components due to low working volumes used; Less space requirements as compared to the use of conventional
systems (e.g. Erlenmeyer flasks); Wide array of data output with significant time and cost savings; Provide the basis for rational set-up of the evaluating systems.
Disadvantages: Suitable online monitoring of operational parameters; Reduced volumes for conventional evaluation;
Introduction
3
Introduction
Advantages and purposes of immobilization in whole-cellbiocatalysis: Cell retention within the support/bioreactor
High cell concentrations Enhanced volumetric productivities;
Control of biocatalyst microenvironment
Eased separation of biocatalyst from product
Possible biocatalyst reuse;
Contamination avoided;
High dilution rates; Protection against shear forces;
Lower costs in recovery, recycling and downstream processing;
Increased cell stability;
4
Introduction
Potential limitations of cell-immobilized systems:
Increased costs of biocatalyst production
Loss of biocatalytic activity
Empiricism
Immobilization procedure
Matrix nature
Reaction step
pH, temperature extremes;
Toxic reagents;
High shear/mechanical conditions;
Exclusion of molecules;
Local pH shifts;
Mass transfer limitations;
Cell leakage;
Inhibitors build-up;
Need for case specific, multi-parameter optimization;
Difficult process modelling and control.
5
Introduction
Whole-cell immobilization methods:
Adapted from Kourkoutas et al., 2004; Food Microb. (21) 377-397
SiliconeCelite 560
PU-foamScotch-brite®
PVAPVA-LentiKats®
PVA/AlginateAlginate
Alginate/polyurea
Ca-alginate6
Bioconversion System: case study
Selective side-chain cleavage of -sitosterol to 4-androstene-3,17-dione (AD) performed by Mycobacterium sp. NRRL B-3805 resting cells
Main features of biotransformation: Multi-enzymatic oxidative biotransformation requiring cofactors (involves the
use of nine catabolic enzymes in a 14-step metabolic pathway); Mycobacterium sp. is a relatively slow growth microorganism; Oxygen required for reaction; Low solubility of substrate and products in aqueous media (<1.0 mM); 7
HO
O
O
O
O
Pharmaceutical steroids
4-androstene-3,17-dione (AD)
1,4-androstadiene-3,17-dione (ADD)
-Sitosterol
HO
O
O
O
O
Pharmaceutical steroids
4-androstene-3,17-dione (AD)
1,4-androstadiene-3,17-dione (ADD)
-Sitosterol
Materials and Methods
Free-suspended cell growth medium Di-sodium/potassium Phosphate Buffer 0.1 M pH 7.0 Yeast Extract (10 g.l-1) Glycerol (10 g.l-1) NH4Cl (4.0 g.l-1) Tween® 20 (0.8 g.l-1) MgSO47H2O (0.14 g.l-1) Substrate: -sitosterol (activity inducer, 1.0 g.l-1)
Conditions: 30 ºC at 200 rpm for 36-hrs in 2.0 L Erlenmeyer flasks
Cell recovery: Vacuum filtration (wet cell-paste, 60-70% humidity), washed with phosphate buffer and stored at -20ºC
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Biocatalyst Preparation
Surface adsorption (Bio)encapsulationSilicone PU-foam Scotch-brite® fabric Celite 560
Orbital shaking
(30ºC, 200 rpm, 48-hrs)
Harvest
Supports in growth medium + cell inoculum
Storage -20ºC
50 ml Erlenmeyer
Concentrated cell suspension
50 g/L
CaCl2 solution with xanthan gum and
Tween 20
Na-alginate solution
Ca-alginate capsules
Cell suspension with thickener and
surfactant
D = 5-6 mm
Storage -20ºC
180 mg of solid carriers
Interfacial polymerization
reaction
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Biocatalyst Preparation
Entrapment Spherical beads PVA-LentiKats® disks
Plastic Petri-dish Stabilizing bath with activation medium
(Sloughing and re-swelling)
Lenticular shaped particles
(optimum geometry)
gelation
Cell suspension in melted LentiKats®Liquid
(40 g/L)
Cell suspension in Na-alginate or in polyvinyl alcohol
(PVA)
CaCl2 solution or saturated boric-acid
solution
or Ca-alginate beads
PVA beads
Polyurea coating
D = 2.5 mm
Alginate coated with polyurea
(beads more hydrophobic)
D = 3 mm
http://www.geniaLab.de/download/tt-english.pdf
6-hrs evaporation
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Bioconversion Trials
Reaction in aqueous medium Biocatalyst + 1 ml 0.1M Tris-HCl buffer (pH 7.5) + 120 µl of -sitosterol (24 mM) in EtOH 96% (v/v)
Organic-aqueous two-liquid phase reaction Biocatalyst + 0.5 ml 0.1M Tris-HCl buffer (pH 7.5) + 0.5 ml of -sitosterol in BEHP (12 mM)
Reaction in predominantly organic medium Biocatalyst + 1 ml of -sitosterol in BEHP (12 mM)
Analytical methods: HPLC Lichrospher Si-60 column (5 m particle size) Isocratic elution (1 ml.min-1);
Mobile phase: n-heptane/EtOH (92:8, v/v); UV detection (-sitosterol, 220 nm; AD, 254 nm).
Protein determination by Lowry Method
Protein estimation: [Total protein] (mg.l-1) = 304 [Dry biomass] (mg.ml-1) + 0.8
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Incubation conditionsReaction flasks: 15 ml screw-capped vessels (minireactors, 80% headspace);35ºC, 250 rpm, 24-hrs.
Results
I – Screening of several immobilization methods
No biocatalytic activity detected
OverproductionUnderproduction Biocatalyst form
Relative specific AD production (%)(based on biomass weight)
No biocatalytic activity detectedl
100 % ( 7.6)
149 % ( 7.7)119 % ( 8.6)
108 % ( 9.5)
124 % ( 8.2)
27 % ( 10.3)
132 % ( 5.4)
81 % ( 3.0)
24 % ( 8.0)
12 Aqueous media
Silicone 1 mm
Results
II – Adsorption capacity
Celite 560 Silicone 1 mm
Dry biomass content
Protein content
Carrier Partition studies (incubation: 40-hrs, 35ºC, 250 rpm)
Biocatalyst (mg of dry biomass)
Global AD Accumulation(mM)
Global AD specific accumulation (mmolg-1 dry biomass)
AD in aqueous phase (mM)
Sitosterol in aqueous phase (mM)
AD adsorbed onto support (mmolg-1 support)
Sitosterol adsorbed onto support
(mmolg-1 support)
Free cells (0.8) 0.262 0.328 -------- -------- -------- --------
Immobilized cells: Silicone 1mm (1.4) 0.436 0.312 0.321 (76%) 2.7×10-3 2.0×10-3 6.0×10-3
Celite 560 (1.2) 0.220 0.099 0.190 (86%) 0.165 4.0×10-4 1.3×10-3
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Results
III – Temperature, pH and shaking speed
50
Rel
ativ
e sp
ecifi
c ac
tivity
(%)
Temperature (ºC) pH
Shaking speed (rpm)
Rel
ativ
e sp
ecifi
c ac
tivity
(%)
Optimum temperature: 35ºC
6.5 < pH < 8.0
Shaking speed: 150 – 300 rpm
250 rpm, pH 7.5 250 rpm, 35ºC
35ºC, pH 7.5
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Silicone 1 mm
Celite 560
Free cells
IV – Thermal and storage stabilities
Results
Incubation time (days) Storage time (days)
Rel
ativ
e sp
ecifi
c ac
tivity
0
10
E t
B exp t B exp tE
Adapted from Aymard and Belarbi (2000) Enz. Microb. Technol. (27) 612-618
15
Silicone 1 mm
Celite 560
Free cells
Modelling deactivation profiles:
Results
V – Kinetic studies and reusability of the silicone immobilized biocatalyst
Aqueous -sitosterol concentration (mM)
Spe
cific
act
ivity
(m
mol
ADg
-1dr
y bio
mas
sh-1
)R
elat
ive
prod
uct y
ield
, (%
)
Rel
ativ
e am
ount
of b
iom
ass
reta
ined
in s
uppo
rt, (%
)
Batch number #
Michaelis-Menten equation*
*Apparent kinetic parameters obtained using Leonora® software (Cornish -Bowden, 1995):
Vmáx, imm = 0.145 mmol AD.g-1 dry biomass.h-1
Km, imm = 0.14 mM
Silicone 1 mm
Celite 560
16
máx ,imm
m ,imm
v Sv
K S
Silicone 1 mm
Celite 560
Free cells
Conclusions
Sitosterol side-chain cleavage pathway is susceptible to prolonged drying at room temperatures (LentiKats®) and to relatively harsh chemical manipulations (alginate coated with polyurea). Hydrogels provided efficient cell retention but are limited to their hydrophilic nature;
Apparently silicone slabs provide an efficient carrier for cell-surface adsorption displaying catalytic activity for sitosterol side-chain cleavage;
A cell-loading capacity of 6 mg dry biomass per gram of support was achieved;
Hydrophobic nature of silicone favours both cell-adhesion and the substrate partition to the surface, while retaining low quantities of AD formed;
Immobilization provides good stability of biocatalyst preparation under operating conditions up to 300 rpm and 45ºC with an Topt of 35ºC;
The pH/activity profile was not considerably altered as a result of immobilization;
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Michaelis-Menten type kinetics adequately described the bioconversion system in the substrate range evaluated. Low apparent Km, imm value suggests high affinity to -sitosterol;
All biocatalytic systems displayed thermal and storage deactivation. Deactivation profiles can be accurately modelled using a 3 parameter bi-exponential equation;
Repeated batch biotransformations were feasible and simpler to perform when silicone immobilized cells were used. Marked decay of product formed occurred mainly due to loss of cell oxidative potential;
Except for cell-loading capacity, silicone based biocatalysts performed better than Celite immobilized cells, and usually outperformed free cells;
Experiments proved the feasibility of using 15-ml screw-capped shaken bioreactors for screening purposes and system characterization in aqueous medium;
Conclusions
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Biocatalytic activity using bioencapsulation could be improved using more biocompatible hydrophobic matrix and reducing particle size;
Biocapsules could provide a good approach but are limited to high particle size. Reducing membrane thickness along with particle diameter could be the solution while providing a suitable internal microenvironment for bioconversion to occur;
The use of PPG, Ionic Liquids (IL’s) and more hydrophobic materials may help facilitate substrate and oxygen availabilities and partition effects;
For adsorption experiments it is suggested the use of smaller carrier particles (crushed silicone slabs, micronized liquid silicone and/or small latex particles);
Evaluation of silicone hydrophobicity Mycobacteria cell wall, sitosterol and AD;
Assess cell-to-support adsorption profile along fermentation time and correlate to viable biomass and displayed catalytic activity;
Compare results with those to obtain by using 24-well microplates;
Future Work
19
Acknowledgements
Professor Doctor Joaquim Sampaio Cabral
Doctor Pedro Fernandes
Marco Marques
Fellow colleagues of IBB and M.Sc. Course
My outstanding family and friends
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