A study with enzymatic membrane reactor for conversion of lactose in to galacto-oligosaccharides. -Tapas Palai, Pallavi Kumari and Prashant K. Bhattacharya. -Tapas Palai, Pallavi Kumari

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


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


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

+


Experimental


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


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


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


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


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


Effect of enzyme concentration

Effect of enzyme concentration on GOS formation at 100 g/L ILC

GOS formation increases initially and then remains constant


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


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%

1 May 2023 141 May 2023 14ICChE-2011, Dhaka

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


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


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


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


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


Thank You