IMPROVEMENT OF BIOCHEMICAL PROCESS EFFICIENCY AND ...sschi.chtf.stuba.sk/permea/After/Papers/Files/Trusek.pdf · particularly, when catalysis is carried out in a two-phase organic

Lecture presented at the conference PERMEA 2003, Tatranské Matliare, Slovakia, September 7-11, 2003

IMPROVEMENT OF BIOCHEMICAL PROCESS EFFICIENCY AND

PRODUCTIVITY, AND FACILITATION OF REACTANTS SEPARATION BY

THE APPLICATION OF A MEMBRANE PHASE CONTACTOR

A. Trusek-Holownia

Wroclaw University of Technology, Institute of Chemical Engineering,

Norwida 4/6, 50-373 Wroclaw, Poland

Tel. +48 71 3202653, Fax +48 71 3281318, E-mail: [email protected]

Abstract

By applying a porous membrane that keeps interface and protects two adjacent

phases against dispersion, a fully controllable equipment to carry out (bio)chemical

reactions is obtained. The membrane phase contactor with expanded interfacial area and

almost arbitrary phase ratio and broad range of phase flows enables an intensification of

the process of reagent mass transport that could have an influence on the rate and

conversion degree in biochemical transformations. This results in obtaining high

process efficiency [mol Product /mol Substrate] at high yield [mol s-1] and purity of the

product isolated from one of the phases separated by the membrane.

Additionally, the (bio)catalyst can be concentrated in the region of increased

substrate concentration through an oriented immobilization of the (bio)catalyst on the

surface of membrane in the form of a hydrated gel layer. Such process solution enables

full integration of substrate mass transport rate with the rate of its transformation, which

1


ensures exceptionally high purity of the reaction products in the receiving phase free of

the substrate and catalyst.

Keywords: membrane contactor; reactant separation; two-phase system; enzymatic

conversion; enzymatic gel layer

1. Introduction

In biotechnological processes where enzymes or microorganism cells are

reaction catalysts, both the biochemical transformation and separation processes of a

bioproduct are of special importance. The first process is intensified because of the need

to use to the maximum an activity of biocatalyst which is frequently the most expensive

component of the system. The other process is related to the required high purity of

products that are often applied in pharmaceutical and food industry. Hence, it is

desirable to attain the maximum conversion degree of the substrate which facilitates

remarkably the isolation of pure product from the reaction mixture. It is not easy to

increase both process productivity [mol s-1] and efficiency [mol Product/mol Substrate],

particularly, when catalysis is carried out in a two-phase organic solvent-water system.

Such reaction systems are recommended to carry out reactions in which at least one of

the reagents (substrate or/and product) has a hydrophobic character, i.e. dissolves to a

limited extent in water being a reaction medium for most biochemical transformations.

The organic phase is then a reservoir of this hydrophobic component which gradually

diffuses during the process to/from the reaction zone. So, in the system, two unit

processes take place at the same time: biochemical transformation in the place where

the enzyme is located (usually the whole volume of the water phase or its part) and

2


mass transport of the hydrophobic reagent [1]. From the point of view of control of both

processes, it is advantageous that the two phases could be fully monitored during the

process.

In the membrane phase contactor, contrary to other equipments used in the

extraction processes, both phases flowing along two sides of the membrane are totally

separated. The role of a porous membrane is to keep interface in the pores which

protects the system against dispersion.

In practice, it is no problem to keep the phases in the state of separation when

breakthrough pressure ∆Pb defined by the Laplace-Young is applied on the side of the

opposite phase to the one that wets the membrane pores (Eq.1) [2].

θσ=∆ wm

wnb cos .

r2. P (1)

Pores of the hydrophilic membrane are usually wetted with water phase, while

those of the hydrophobic membrane with organic phase. Hence, breakthrough pressure

is applied to organic and water phase, respectively.

During the process, phase separation has a big effect on parameter controllability

in the whole range concerning both phases and much facilitates product isolation during

the process or after its completion. The separation of components from the reaction

medium is easier when reagents substrates/products differ significantly in their

hydrophilic-hydrophobic character, i.e. their partition coefficients assume extreme

values << 1 and >>1. In fact, these coefficients are often in the range from 0.1 to 10,

i.e. important part of the component appears also in the second phase. Hence, product

purity depends greatly on the reaction degree.

As it will be shown further in this paper, both the rate of a biochemical process

and the degree of reaction strongly depend on the effective mass transport through the

3


interface. In the membrane phase contactor its rate can be fully controlled and

intensified due to applicability of a broad range of parameters, i.e. specific surface,

phase streams, phase ratio. These properties of the membrane phase contactor

distinguish it positively from other equipments used in extraction processes (mixer-

settler, extraction column), where particular parameters are strictly limited [3]. The

mixer, however, enables development of the interfacial area but the system dispersion

often results in the formation of stable emulsions that are stabilized additionally by

enzymatic protein. This is the reason of problems with an on-line monitoring of the

process in the mixer, and with settling of the emulsion and separation of reagents after

completing the process. In the case of a membrane contactor, the interfacial area

depends on the density of membrane packing and porosity and is many times bigger in

comparison with an extraction column [4]. For a capillary module it could be expressed

as:

εΠ= m.l.n.d. A (2)

Other advantages of the membrane contactor include a possibility of phase flow

adjustment; in the extraction column these values are limited by a column flooding

phenomenon.

In the two-phase organic solvent-water systems, usually such reactions are

carried out where water is one of the reaction substrates (hydrolysis reactions [5,6]) or is

a product of the reaction (e.g. esterification, synthesis reaction [7,8,9]). From among

numerous reaction networks that may occur in the two-phase systems, two were

selected as particularly interesting because of product purity dependent on substrate

reaction degree.

4


2. A membrane phase contactor application – cases study

The control of processes carried out in two-phase organic solvent-water systems

covers a preliminary identification of certain system properties that are not considered

in homogeneous catalysis.

When applying an organic solvent, it is important to consider its effect on a

catalyst. Only a few organic solvents do not cause the loss of the enzyme activity, and

they should be selected for each catalyst separately as there is no universal solvent.

Since the catalyst is usually dissolved/suspended in the water phase, it can be

inactivated by the organic solvent on the interfacial surface or inside the water phase

[10], where the organic solvent concentration is on the level of its solubility in water.

At the next stage of research, the solubility of individual reagents in each phase

of the system applied (f(T, pH)) should be identified and extraction equilibrium denoted

usually as partition coefficient P should be determined. Next, by values of mass transfer

coefficients determined experimentally or calculated on the basis of correlations

available in the literature, the rate of mass transport as a function of both phase flows is

described. These coefficients refer to a device used during the process and to a specified

phase flow.

At the last stage of the preliminary research the process equilibrium (f(T, pH))

and the rate of enzymatic transformation in the water phase saturated with an organic

solvent as a function of substrate and catalyst concentrations at specified temperature

and pH are determined.

The data obtained allow us to analyze the process and to optimize it according to

the previously stated priorities.

5


The aim of considerations presented in this paper is to show how through a

controlled interfacial mass transport rate in the membrane phase contactor, it is possible

to increase the rate and conversion degree in biotechnological processes, and

consequently, to increase the purity of a product separated from one of the phases

separated by the membrane.

2.1. Equilibrium synthesis reaction

S1(hydrophobic) + S2(hydrophilic) = P (hydrophobic) + H20

An example of such a reaction are, among the others, the reactions of synthesis

of peptide compounds carried out in the presence of proteases. Depending on the

composition of amino acids, the substrates (single amino acids, blocked amino acids or

oligopeptides) are either hydrophobic or hydrophilic. The product is usually poorly

soluble in water, therefore it is received from the organic phase or crystallizes from the

water phase after exceeding supersaturation concentration.

Specially interesting is the case when one of the substrates reveals also a

hydrophobic character due to which it is supplied to the reaction zone (water phase) by

extraction from the organic phase. Owing to reduced concentration in the water phase, it

is this substrate which is limiting and the enzymatic transformation rate depends mainly

on its concentration [11].

The problem is considered in detail on the basis of ZAlaPheOMe dipeptide

synthesis catalyzed by thermolysine in the ethyl acetate-polypropylene membrane-water

system. Two partly blocked amino acids ZAlaOH (P = 3.2) and PheOMe (P = 0.4) take

6


part in the reaction. The product being formed reveals a strongly hydrophobic character

(P = 273) and hardly dissolves in water (csol. = 0.76 mol m-3).

Process productivity – the rate of product formation in the water phase

The rate of product formation is determined by enzyme activity and its

concentration and the concentration of substrates in the reaction zone (i.e. in the water

phase). To carry out the process at a maximum rate which is desired for a full use of the

catalyst, a state close to the extraction equilibrium should be ensured in the system

during the process. Then, the concentration of a hydrophobic substrate (ZAlaOH) taken

from the organic phase, will be the highest possible in the water phase in given

conditions. Whether such equilibrium will occur depends on the relative extraction rate

referred to the rate of enzymatic transformation. To compare the values of both

processes, the Hatta number is often used [11,12]. As it is not advisable to decrease the

enzymatic transformation rate (this causes a decrease of process productivity), attempts

are made to intensify the mass transfer process which could be easy made in the

membrane contactor by changing parameters in a wide range.

In the reaction being considered, this intensification of mass transport aims not

only at the transport of hydrophobic substrate but also at the diffusion of product from

the reaction zone. As the reaction product is characterized by very poor solubility in the

water phase, it is not difficult to exceed the supersaturation concentration and cause

product precipitation from the water phase. Due to the multiphase system complexity,

crystals of the formed product, will present an additional problem during separation,

particularly when the case of co-crystallization of the substrate or precipitation of

7


catalytic protein takes place. To avoid this situation, the product must be very quickly

submitted to the organic phase.

The performed analysis (Fig. 1) shows that due to a strongly hydrophobic

character of the product, it is this compound and not the substrate that determines the

value of interfacial area affecting the mass transport rate. The Figure 1 shows how the

distance from interfacial equilibrium (ϕ) changes during the process. In the whole tested

period, the two substrates have concentrations similar to the concentrations

corresponding to the interfacial equilibrium, which is an evidence of over-dimensioning

of the interfacial surface in relation to these components. The analysis of the relation for

the reaction product – ZAlaPheOMe confirms its strong influence on the value of the

interfacial area that is especially remarked at the initial stages of the process. Then, the

driving force of the extraction process is small and the reaction rate is the highest owing

to high substrate concentrations.

Process productivity – equilibrium shift

The process of ZAlaPheOMe synthesis carried out in a single-phase system

takes place at low productivity because a hydrophobic substrate dissolves in water at a

limited degree and is used in the reaction at low concentrations and because the reaction

equilibrium is shifted towards substrates [7]. Quick extraction of the product to the

region beyond the reaction, dependent on the applied process equipment and

parameters, allows the product to be removed from the place of catalysis where, because

of this fact, the equilibrium state could be not reached. Hence, in the two-phase system a

full reaction of the substrate is possible. The final process yield is determined by the

concentration of both substrates and volumetric phase ratio – Eq. 3 [7].

8


) β P + 1 (β

) β P + 1 ( ) β P + 1 ( ) α - M ( ) α - 1 (N

cαV =K

P

BA*A

*AA0

O2H*AO

⋅⋅⋅⋅⋅

⋅⋅⋅

⋅⋅ (3)

Product purity – separation from the organic phase

A very high value of the partition coefficient for the product (P = 273) allows

this compound to be isolated only from the organic phase because its concentration in

the water phase is close to zero. Due to phase separation during the process carried out

in the membrane phase contactor, the reaction product can be isolated from the organic

phase immediately after completing the synthesis process.

Hence, it is important that the organic phase be free from unreacted substrates to

the maximum. Due to partition coefficients of the reaction substrates, mainly

hydrophobic reagent (in this case ZAlaOH) constitutes a contaminant in this phase. It

often happens that just this reagent is a limiting substrate, hence in the reaction close to

unity, which is possible due to shifting of the reaction equilibrium, its presence is not a

problem. Taking into account biochemical transformation kinetics [7], the hydrophilic

substrate is used in excess and it is present in the system at the end of the process. As it

prefers water environment, most part of its mass is concentrated in this phase, however,

the organic phase is not free from this compound, in considered case PPheOMe = 0.4. To

accumulate (and use in the next charge) most of mass of this compound in the water

phase, its volume should be several times higher than that of the organic phase (miO/miW

= VW/(VO .Pi)) – Fig. 2.

As the membrane contactor enables extraction at nearly arbitrary volumetric

ratio of phases, after finishing the process of synthesis, the water phase volume can be

increased in order to receive the hydrophilic substrate from the organic phase. A new

9


state of extraction equilibrium at high phase circulation is reached relatively quickly.

Naturally, an additional volume of the water phase will extract an additional mass of the

product, nevertheless its strongly hydrophobic character causes that these losses will be

small.

2.2 Hydrolysis reaction

S (hydrophobic) + H2O = P1 (hydrophilic) + P2 (hydrophilic)

The reaction of hydrolysis takes place in the water phase from which a substrate

present mainly in the organic phase, to undergo a reaction must diffuse to the zone

where the catalyst occurs. The lower is the substrate partition coefficient, the higher is

the substrate concentration in the reaction region, which will have a positive effect on

the process productivity. However, at the same time, with the lack of complete reaction

of the substrate, this will cause contamination of products that due to their hydrophilic

character are present in the water phase. In turn, when PSubstrate >>1, the process will

take place at very low productivity because of low substrate concentration in the water

phase (i.e. reactive phase).

Membrane contactor provides a possibility to keep the water phase completely

free of the reaction substrate, and at the same time enables an increase of the reaction

rate by a local increase of the substrate concentration in the reaction region. This can be

achieved by forming an active catalyst layer near or just on the interface. By controlling

the rate of transport of substrate mass to the catalyst layer and its residence time in the

protein layer that depends on the layer thickness, the parameters can be selected in such

10


a way that the whole transported substrate mass reacts in the layer and the water phase

is free of a hydrophobic substrate. To carry out such catalysis, one should prepare a

stable and active protein layer on the membrane surface of thickness depending on its

reactivity and diffusivity.

The above problem was considered taking as an example the hydrolysis of

glycidyl butyrate catalyzed by a lipase from Candida antarctica and a lipase from

pancreatic porcine in the hexane-polyamide membrane-catalyst gel layer-water system.

The partition coefficient in hexane-water phase system of glycidyl butyrate is 3.45 and

solubility of this compound in the water phase is 120 mM. Hence, a reservoir of this

compound is the organic phase. The reaction product is glycidol and butyric acid. Both

products have a strongly hydrophilic character (P<<1).

Preparation and determination of the protein layer thickness

The enzymes were immobilized on polyamide membrane surface by the

chemical binding by the use glutaraldehyde method [13]. The amount of protein bound

to the membrane was determined on the basis of mass balance, at the known protein

concentration, checked by the use Lowry method [14], in the solution before and after

the immobilization.

The amount of protein bound to the membrane suggested that more than one

protein layer was formed on the membrane. Probably, only the first protein layer was

bound to the membrane by a chemical bond, while the other layers, whose number reach

up some hundreds, as estimated taking a constant diameter of hydrated protein particle

equal to 5.10-9 m, were bound to the subsequent protein layers by sorption. In the

11


calculation of the protein layer thickness, stratified packing of molecules in the layer

was assumed and the following formula was received:

Md . No . C

sp

3p= (4)

For the used membranes with surface area 4.9 . 10-4 m2, the amount of the bound mass

corresponded to the following layer thickness -Table 1:

Gel layer productivity

The rate of enzymatic transformation in the gel layer is affected by catalytic

properties of the immobilized enzyme and substrate concentration profile in the layer.

The profile depends on the substrate concentration in the organic phase, as a partition

coefficient and relation of the enzymatic transformation rate to the compound

diffusivity in the layer. The distribution of concentrations in the layer depends also on

hydrodynamic conditions in adjacent phases.

High substrate concentration may occur particularly at the enter to the layer and

in its first cross-sections – in this region it can be much higher than it would be in the

equilibrium state in the water phase. This has a positive impact on process productivity,

which is also affected by a usually high activity of the immobilized catalyst and its very

high stability [13]. Hence, efficiency of processes that take place in the catalytic layer

expressed on the enzyme mass can be several or more times higher as compared to the

process catalyzed by a native enzyme – Fig. 3. In presented case the process efficiency

is 0.058 µmol Product /µg native enzyme and 0.665 µmol Product /µg immobilized enzyme after 20 h.

12


Reaction degree in the gel layer – product purity

The considered process is illustrated in Fig. 4. The values of streams n1 and n2

can be expressed as [15] :

) . sinh( . ) . . . ( ) . cosh( . ) . ( . ) . sinh( . . . . ] . - ) . cosh( . [ . . .

ww

owow1 sPkkDskP

skkDccPsckn

1r1

1r1λβλαλβα

++++

=β

λ (5)

) . sinh( . ) . . . ( ) . cosh( . ) . ( . ) . sinh( . . . . - ] ) . cosh( . . - [ . . .

ww

wwwow2 sPkkDskP

skDcscPckn

1r1

r1λβλβαλβλβα

+++=

(6)

where:

kD r . =α (7)

Dkr =λ (8)

Separation of a product from the water phase is much easier when the substrate

completely reacts in the layer, i.e. the stream leaving the layer has a value close to zero.

From this point of view of separation, the value of the parameter ζ = 1 − n2/n1 is

interesting. It is interpreted as the conversion degree in the layer and exactly is

expressed by:

P . cc - sinh . A cosh

)sinh . A cosh( .P . cc -1

-1

o

w1

2o

w

φ+φ

φ+φ=ζ (9)

where:

s.Dk r=φ (10)

13


βα

=w

1 A (11)

P . k A

12

α= (12)

When the concentration in the water phase is equal to 0, the relation (9) is simplified to

the form:

φ + φ=ζ

1 sinh.Acosh1-1 (13)

The most important parameter determining the conversion in the layer, in the

considered case, is the value of Thiele module (φ). Figure 5 shows how the conversion

degree changes with the change in Thiele module for several different values of

parameter A1.

The conversion degree in the layer increases with an increase of Thiele module,

which in this case corresponds either to the growth of layer thickness or its reactivity, or

to the decrease of diffusion coefficient in the layer. It is significant that the amount of

protein used for the preparation of the catalytic layer should be not in excess. For given

process conditions there can be such a layer thickness above which part of the catalytic

protein does not participate in the process because of the lack of contact with the

substrate (the concentration profile in the layer drops down to zero). Owing to high

costs of the biocatalyst, this problem should be considered extensively when preparing

the protein layer.

The problem was considered in detail on the basis of glycidyl butyrate

hydrolysis for which the reaction constant kr assumes values ranging from 0.16 to

1.4 s-1 [16] depending on the applied enzyme and substrate enantiomer, and the

determined diffusion coefficient in the gel layer is 3.83 . 10-12 m2 s-1 [17].

14


Figure 6 illustrates how the conversion degree changes with the layer thickness

at different values of βW (that is a component of parameter A1), when the reaction rate

kr is 0.05 s-1 (the process slower than in experiments carried out), 0.1, 1.0 and 5 s-1 (the

process faster than that in the experiment).

It was observed that for highly active protein (kr =1-5 s-1) already a very thin gel

layer (10-7-10-6 m) ensured high (complete or almost complete) reaction of the substrate

and only slightly depending on transfer coefficient in the water phase. This dependence

becomes much stronger when gel reactivity is much lower (kr = 0.05-0.1 s-1). Then, to

obtain high product purity in the water phase, βW should be significantly decreased by

decreasing the system turbulence on the side of water phase or by eliminating

circulation of this phase completely, or finally the gel should be extended to reach the

value 10-5 m.

Conclusions

Problems discussed in this paper show very significant advantages of the

application of a membrane phase contactor in biotechnological processes. Particularly

important is its use in two-phase systems, where a porous membrane that keeps up the

interface, enables retaining the phases in non-dispersed state. This method of contacting

the phases allows for a complete monitoring of the process in both phases and a control

of parameters that have an influence on the rate of processes and much facilitates

product separation from the phase in which it is accumulated. Purity of this compound

depends on the hydrophobic character of substrates and the conversion degree. It is

important that when carrying out the process in the membrane contactor, this conversion

15


degree for equilibrium reactions can be increased by a quick interfacial diffusion

transport of the product from the reaction zone.

Intensification of the interfacial mass transport in the membrane contactor can

also affect the growth of enzymatic transformation rate by increasing substrate

concentrations in the zone where the enzyme is accumulated, i.e. in the whole volume

of the water phase or in the near-membrane zone on which the enzyme is deposited in

the form of a gel layer. This form of enzyme immobilization offers the advantages of

growing catalyst activity and stability, and additionally enables a complete separation of

products from the catalyst and often also from the reaction substrates. It also provides a

possibility of multiple use of the biocatalyst.

The discussed high process productivity and efficiency as well as product purity

are obtained after integrating the biochemical transformation rate dependent on the

applied biocatalyst with the controlled mass transport rate in the membrane module.

Symbols

A - surface, m2

C - concentration on surface, g m-2

c - concentration, g m-3, mol m-3

csol - solubility concentration, mol m-3

c* - equilibrium concentration, mol m-3

D - diffusion coefficient in gel layer, m2 s-1

d - membrane capillary diameter, m

dp - diameter of hydrated molecule, m

K - reaction equilibrium constant

16


kr - enzymatic reaction rate constant, s-1

k1 - mass transfer coefficient to gel layer, m s-1

l - membrane capillary length, m

M - substrate molar ratio, M=NB0/NA0

Mp - particle molecular mass, g mol-1

m - substrate mass, g

N - amount of substrate, mol

No - Avogadro number, mol-1

n - numbers of membrane capillary

n1 - stream density of entering substrate mass, mol m-2 s-1

n2 - stream density of nonconverted substrate mass, mol m-2 s-1

P - partition coefficient, P=c*iO/c*iW

r - pore radius at the line of contact with interface

s - protein gel layer thickness, m

V - volume, m3

α - parameter, kD r . =α

α∗ - equilibrium conversion degree

β - phases volume ratio, β=VO/VW

βW - mass transfer coefficient in water phase, m s-1

εm - membrane porosity

φ - Thiele module

ϕ - distance from interfacial equilibrium

17


λ - parameter, Dkr =λ

θwm - angle of contact between wetting liquid and membrane

σwn - interfacial tension at the liquid-liquid interface, N m-1

ζ - parameter, ζ = 1 − n2/n1

subscripts

A - limiting (hydrophobic) substrate

B - hydrophilic substrate

i - compound

O - organic phase

P - product

p - particle

W - water phase

0 - initial time

References

[1] A. Noworyta, Inz. Chem. Proc. 20 (1999), 629-648.

[2] E. Drioli, L. Giorno, Biocatalytic membrane reactors, Taylor&Francis Ltd., London

1999.

[3] A. Trusek-Holownia, Membrane contactors, in: A. Noworyta, A. Trusek-

Holownia, eds., Membrane separations, Argi, Wroclaw, 2000, pp. 215-235.

18


[4] R. Rautenbach, Membrane processes, WNT, Warszawa, 1996.

[5] A. Ismail, A. Soultani, M. Ghoul, J. Biotechnol. 69 (1999), 145-149

[6] M. Venu Madhav, C. Ching, J. Chem. Technol. Biotechnol. 76 (2001), 941-948

[7] A.Trusek-Hołownia, A. Noworyta, J. Biotechnol. 102 (2003) 153-163.

[8] E. Vulfson, Trends Food Sci. Technol. 4 (1993), 209-215.

[9] D. Eggers, H. Blanch, J. Prausnitz, Enzyme Microb. Technol. 11 (1989), 84-89.

[10] P. Halling, Enzyme Microb Technol. 16 (1994), 178-206.

[11] A. Trusek-Holownia, Biochem. Eng. J. 16 (2003), 69-77.

[12] L. Doraiswamy, M. Sharma, Heterogeneous Reactions, Wiley&Sons, 1984.

[13] A. Trusek-Holownia, A. Noworyta, Desalination 144 (2002), 427-432.

[14] O. Lowry, N. Rosebrough, A. Farr, R. Randall, Biol. Chem. 193 (1951) 265-270.

[15] A. Trusek-Holownia, A. Noworyta, J. Membrane Sci. (in revision).

[16] A. Trusek-Holownia, Proceedings of First International Symposium on

Process Intensification & Miniaturisation, Newcastle 2003, p.14

[17] A. Trusek-Holownia, A. Noworyta, Proceedings of the conference PERMEA,

Slovakia 2003, p.79

19


Table 1. Protein mass immobilised on membrane [µg]

Concentration on surface [g m-2]

Layer thickness [m]

260 0.53 5.3 . 10-7 280 0.57 5.7 . 10-7 450 0.92 9.2 . 10-7 1540 3.14 3,1 . 10-6 1920 3.92 3.9 . 10-6 2220 4.53 4.5 . 10-6

20


0

0,2

0,4

0,6

0,8

1

1,2

0 20 40 60 80time [h]

100

=c O

i /(P i

. cW

i)

ZAlaOH

PheOMe

ZAlaPheOMe

Fig.1

0

10

20

30

40

50

0 2 4 6 8VW/VO

miW

/miO

10

Pi=0.2

Pi=0.4

Pi=0.6

Fig. 2

21


0

20

40

60

80

100

0 5 10 15 20

time [h]

native enzyme

immobilised enzyme

Fig.3

Fig. 4

22


0

0,2

0,4

0,6

0,8

1

0 1 2 3 4 5

Thiele module

2520100

Fig.5

23


kr=5.0 s-1

0

0,2

0,4

0,6

0,8

1

0,E+00 2,E-07 4,E-07 6,E-07 8,E-07 1,E-06

s [m]

ζ 6,00E-073,00E-076,00E-083,00E-08

kr=1.0 s-1

0

0,2

0,4

0,6

0,8

1

0,E+00 2,E-07 4,E-07 6,E-07 8,E-07 1,E-06

s [m]

ζ

6,00E-07

3,00E-07

6,00E-08

3,00E-08

kr=0.1 s-1

0

0,2

0,4

0,6

0,8

1

0,E+00 2,E-06 4,E-06 6,E-06 8,E-06 1,E-05

s [m]

ζ

6,00E-07

3,00E-07

6,00E-08

3,00E-08

kr=0.05 s-1

0

0,2

0,4

0,6

0,8

1

0,E+00 2,E-06 4,E-06 6,E-06 8,E-06 1,E-05

s [m]

ζ

6,00E-07

3,00E-07

6,00E-08

3,00E-08

Fig.6

24


Table 1. The layer thickness calculated on the base of enzyme mass immobilised on

membrane surface.

Fig. 1. Extraction process equilibrium during enzymatic synthesis of ZAlaPheOMe in

membrane contactor with volume of water phase equal to 1.10-3 m3 and organic phase

equal to 2.7.10-3 m3 and streams of both phases equal to 2.10-5 m3s-1 by A=0.28 m2.

Fig. 2. Influence phases ratio on hydrophilic substrates mass concentration in water

phase by few different value of their partition coefficient.

Fig. 3. Efficiency of hydrolysis process of glycidyl butyrate carried out with lipase from

Candida antarctica in native and immobilized form (cSubstrate=20 mol m-3, VO=1.10-5 m3,

0.3 mg native or immobilized enzyme).

Fig. 4. Concentration profile of hydrophobic substrate in membrane contactor with

enzyme gel layer immobilized on membrane surface.

Fig. 5. Conversion degree in gel layer changes with the change in Thiele module for

several different values of parameter A1.

Fig. 6. Conversion degree in gel layer changes with the enzyme layer thickness for several different values of bW.

25

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IMPROVEMENT OF BIOCHEMICAL PROCESS EFFICIENCY AND ...sschi.chtf.stuba.sk/permea/After/Papers/Files/Trusek.pdf · particularly, when catalysis is carried out in a two-phase organic