a study with enzymatic membrane reactor for conversion of lactose in to galacto-oligosaccharides....
TRANSCRIPT
A study with enzymatic membrane reactor for conversion of lactose into
galacto-oligosaccharides
Tapas Palai, Pallavi Kumari, Prashant K. BhattacharyaDepartment of Chemical EngineeringIndian Institute of Technology Kanpur
Kanpur-208016, India
1 May 2023ICChE-2011, Dhaka
Broad objectives of the work Enzymatic synthesis of GOS from lactose Mode of synthesis
- Under free enzyme condition- Under immobilized enzyme on a suitable support
Membrane as a choice of support for immobilizing enzyme (why?):- High surface area of reaction
As membrane modules are available to provide large surface area per unit volume of the module, like utilizing hollow fiber membrane contactor
Enzymatic membrane reactor: a combination of reactor (Part-I) and separation unit (Part-II)
Part-I: Present work is an attempt to carry out synthesis of GOS from lactose by immobilizing enzyme on a hydrophobic porous membrane in a three compartment test cell
1 May 2023 2ICChE-2011, Dhaka
Galacto-oligosaccharides (GOS)
HH
HO
H OH
HO HO
CH2OH
H
H
OHH
H OH
HO HO
CH2OH
HO
n
A nutraceutical & recognized prebiotics
Mixture of tri-, tetra-, penta-saccharides, etc.
Consists of galactose chain with a terminal glucose residue i.e. (galactose)n- glucose
Galactose
Glucose
General structure of GOS
GOS from lactose The enzyme catalyzes two major reactions simultaneously
Trans-galactosylation reaction
Hydrolysis reaction
1 May 2023 3ICChE-2011, Dhaka
Trans-galactosylation reaction Leads to the formation of GOS
Predominates at higher lactose concentration
Lactose Tri-saccharide
Trans-galactosylation reaction
β-Galactosidase
HH
HO
H OH
HO HO
CH2OH
H
H
OHH
H OH
HO HO
CH2OH
HO
HH
H OH
HO HO
CH2OH
H
H
OHH
H OH
HO HO
CH2OH
HO
HH
HO
H OH
HO HO
CH2OH
H
O
1 May 2023 4ICChE-2011, Dhaka
Hydrolysis reaction Leads to hydrolysis of β-1-4 glycosidic linkage in lactose releasing glucose and galactose Predominates at low lactose concentration
LactoseGalactose GlucoseHH
HO
H OH
HO HO
CH2OH
H
H
OHH
H OH
HO HO
CH2OH
HO
HH
HO
H OH
HO HO
CH2OH
H
H
OHH
H OH
HO HO
CH2OH
H
OH
HO
β-Galactosidase
Hydrolysis reaction
+
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Experimental
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Immobilization of enzymeo Immobilization techniques
Adsorption Entrapment Cross-linking Covalent binding
Hydrolysis of lactose
Reaction mixture analysis by HPLC
Immobilization of enzymeEquilibration of membrane in
phosphate buffer (1h)
Washing with phosphate buffer
Washing with brine
Activation with 4% glutaraldehyde at 20oC (4h)
Incubation with enzyme solution at 20oC (18h)
Enzymeβ-Galactosidase (Biolacta FN5) from Bacillus circulans (Daiwa Kasei K.K., Japan)
Activity: 1.2 Unit/mg (using ONPG substrate)
Membrane specificationMaterial : PVDFNature : HydrophobicPore size : 0.22 µmThickness: 125 µmDiameter : 47 mmPorosity : 75%
Scheme of enzyme immobilization
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Reaction mixture analysis: HPLC
Operating conditions Column : Sugar-Pak-I (Waters)
Detector : RID-10A
Mobile phase : 50 mg/L Ca-EDTA
di-sodium solution
Mobile phase flow rate : 0.5 mL/min
Injection volume : 20 μL
Temperature
Column : 75oC
Detector : 35oC
a bc d e
f
HPLC chromatogram
a : Bufferb : Tetra-saccharidec : Tri-saccharide
d : Lactosee : Glucosef : Galactose
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Lactose hydrolysis
Reaction cell for immobilized condition
Operating conditions
Immobilized condition
Free condition
Initial lactose concentration (g/L) 50-320 50-525
Enzyme concentration (g/L) 5.0 -15.0 0.05 –1.575
Temperature (oC) 40 40
pH 6.0 6.0
Mode Batch Batch
Reaction volume (mL) 25 50
Reaction time (h) 30 8
1 May 2023 9ICChE-2011, Dhaka
Results & discussionEnzyme loading
Enzyme loading at different enzyme concentration
O NE
O NE
O NE
O N E
O N E
O N E
PVD
F M
embr
ane
Glutaraldehyde Enzyme
Enzyme immobilized on membrane Maximum 51 mg enzyme loading
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Effect of initial lactose concentration
Effect of initial lactose concentration on products formation
GOS : 7 → 28% Glucose : 2.5 → 18% Galactose : 0.5 → 5.7%
Substrate inhibition beyond 200 g/L lactose concentration
Plot of initial reaction rate vs. substrate concentration
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Effect of enzyme concentration
Effect of enzyme concentration on GOS formation at 100 g/L ILC
GOS formation increases initially and then remains constant
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Reusability: Enzyme and membrane
Reusability of immobilized enzyme. Maximum GOS formation (inset)
Reuse of membrane for enzyme loading
ILC – 100 g/LEnzyme – 0.015 g/mL
Amount of enzyme loading remains almost same
Membrane can be reused for 3 times
Maximum GOS formation & activity of immobilized enzyme decreases with repeated use
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GOS formation
a) Product composition and b) GOS composition
Time course product composition for lactose hydrolysis at 200 g/L ILC. GOS formation (inset)
Steady state after ~ 20 h
Maximum GOS formation – 28%
Product composition
Lactose – 50%
Tri-saccharide – 19%
Tetra-saccharide – 7.5%
Penta-saccharide – 1.5%
Glucose – 16%
Galactose – 6%
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Development of kinetic model
1
1
2
3
3
4
2 2
2
2
k
k
k
k
k
k
E G EG
EG EG G
EG G E GOS
EG GOS E GOS
Reaction mechanism
E : EnzymeG : Monosaccharide (glucose & galactose)G2 : LactoseGOS : Galacto-oligosaccharides (tri- & tetra-saccharides)k : Rate constant
Assumptions Follows Michaelis-Menten kinetics
Negligible enzyme inhibition
Negligible penta-saccharide formation
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Parameter estimation Mathematical equations
ODE solver → COPASI 4.7 (Evolutionary Programming Algorithm)
1 2 1 2 3 2 3 4
21 2 1 2 2 2
2 2 3 2 3 4
21 2 1 2 3 2 3
[ ] – [ ][ ] [ ] [ ][ ] [ ][ ] [ ][ ]
[ ] [ ][ ] [ ] [ ]
[ ] [ ] [ ][ ] [ ][ ] [ ][ ]
[ ] [ ][ ] [ ] [ ][ ] [ ][ ]
[ ]
d E k E G k EG k EG G k E GOS k EG GOSdtd EG k E G k EG k EGdt
d EG k EG k EG G k E GOS k EG GOSdt
d G k E G k EG k EG G k E GOSdtd G
2 2
3 2 3
[ ]
[ ] [ ][ ] [ ][ ]
k EGdtd GOS k EG G k E GOSdt
Rate constants
Average values
k1 (mM-1h-1) 1023.63
k-1 (h-1) 0.435
k2 (h-1) 1.19 x 1011
k3 (mM-1h-1) 5703.08
k-3 (mM-1h-1) 1.4 x 106
k4 (mM-1h-1) 17.758
Estimated parameters
Conditions:Enzyme : 1.5725 g/LLactose : 525 g/L
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Model simulation: Free enzyme
2
max maxexp, ,1
dN
i sim ii
d p
C CC C
SDN N
Lactose
Monosaccharides
GOS
Enzyme – 1.4595 g/LLactose – 417 g/L
Simulated result (solid line) and experimental data (symbols)
C : ConcentrationNd : No. of data pointsNp : No. of parameters
Components SDGOS 0.0313
Lactose 0.0155
Monosaccharides 0.0371
Standard deviation
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Comparison: Free and immobilized enzyme
Comparative GOS yield between free and immobilized conditions
Maximum GOS formation
Free condition – 31%
Immobilized condition – 28%
Enzyme activity does not change
Same types of GOS formed
Substrate inhibition
Free enzyme – No
Immobilized enzyme - Yes
1 May 2023 18ICChE-2011, Dhaka
Conclusion PVDF membrane was successfully utilized for immobilization.
Hence, paving the way to utilize hollow fiber membrane contactor for immobilizing enzyme
Maximum GOS formation Immobilized enzyme - 28% (at 200 g/L ILC) as compared to 39% (at 525 g/L
ILC) under free enzyme condition
Increased lactose concentration (200 g/L) favours reaction GOS and enzyme activity do not suffer under immobilization Developed model matches well; however, tested under free
enzyme condition
1 May 2023 19ICChE-2011, Dhaka
Thank You