|
| |
Applications:
AFFINITY CHROMATOGRAPHY
- SIZE-EXCLUSION EFFECT OF A SUBSTRATE UPON KINETICS OF TRYPSIN IMMOBILIZED ON
POROUS BEAD CELLULOSE. 1. INFLUENCE OF DISTRIBUTION COEFFICIENT OF A SUBSTRATE
(Iontosorb AV)
- Gemeiner P., Polak C., Breier A., Petrus L., Benes
M.J.: Enzyme Microb. Technol., 8
(1986) 109-114
A model of heterogeneous biocatalysis, in which kinetics and partition effects are
connected via the size-exclusion principle, was worked up experimentally and
theoretically. The present paper shows that the maximum relative activity of trypsin (EC
3.4.21.4) immobilized on porous bead (spherical) cellulose is directly proportional to the
available distribution coefficient of the substrate. Providing that the excess of
substrate is not sufficient (e.g. S/Km ~ 1) to safeguard saturated enzyme
kinetics, the originally linear relationship of Ra versus Kav
turns to an exponential one, without any dependence upon the manner of enzyme
immobilization. It is suggested that the above may be a result of partition resistance and
that the main fractrs determining the shape of the Ra versus Kav,
relation in conditions of substrate shortage are the size and geometry of the matrix. The
physical characteristics of the porous carrier as well as the manner of covalent
immobilization of the enzyme are all reflected in the constants applied in the derived
equations.
- SIZE-EXCLUSION EFFECT OF A SUBSTRATE UPON KINETICS OF TRYPSIN IMMOBILIZED ON
POROUS BEAD CELLULOSE. 1. INFLUENCE OF HYDRODYNAMIC DIAMETER OF SUBSTRATE
(Iontosorb AV)
- Gemeiner P., Barteltova L., Soltes L., Breier A.:
Enzyme Microb. Technol., 9 (1987)
44-46
The present paper deals with the dependence of the relative activity ( Ra )of
the immobilized enzyme on the hydrodynamic diameter h of the substrate un
reaction-controlled conditions. The partition effect follows the size-exclusion principle.
This could be confirmed by the fact that the immobilized enzyme failed to become saturated
(e.g. S/Km ~ 1). The exponential form of the relation Ra versus h is
determined by the beometry of the matrix and/or posre and may be expressed by the model
describing an isotropic network of random planes in which all plane orientations are
equally represented. The constants of the exponential equation depend on the physical
properties of the matrix as well as on the mode of covalent immobilization, i.e. chemical
modification of the enzyme.
- BEADED CELLULOSE AND ITS DERIVATIVES IN ENZYME ENGINEERING. RECENT
DEVELOPMENT (Iontosorb TS, Iontosorb DETA, Iontosorb DTTA)
- Gemeiner P., Breier A., Benes M.J.: "Inter
Biotech ´87" - Enzyme Technologies, Proceedings of the
International Symposium on Biotechnology, Bratislava, Czechoslovakia, June
25-26, 1987, Elsevier Sci. Publ., Amsterdam 1987
The chemistry of beaded cellulose used in enzyme engineering is reviewed.
Immobilization of enzymes and affinity techniques are mentioned as examples of solid-phase
tehniques. Quantitative relationships between the structure of the solid oarrier and the
catalytic activity of immobilized enzyme (QSAR) and a time-concentration model of
adsorption in solid-liquid systems were developed for the description of these systems.
- STUDY OF POROUS CELLULOSE BEADS AS AN ENZYME CARRIER VIA SIMPLE MATHEMATICAL
MODELS FOR THE HYDROLYSIS OF SACCHAROSE USING IMMOBILISED INVERTASE REACTORS
(Iontosorb AV, Iontosorb CM)
- Stefuca V., Gemeiner P., Bales V.: Enzyme
Microb. Technol., 10 (1988) 306-311
Among four conjugates of invertase differentlyimmobilized on porous cellulose beads,
the chlorotriazinyl cellulose-invertase (stable catalytic activity in the time interval
between 10th and 80th day of storage) was chosen for studies of reaction kinetics in a
stirred tank reactor and a packed bed reactor. In the stirred tank reactor, due to
localization of invertase on external surface of the beads, the external diffusion does
not exert any inffuence for reaction kinetics. The spherical shape of cellulose beads was
partly destroyed particularly when the rate of stirring exceeded 200 rev min-1
. In the packed bed reactor, owing to the Sherwood number kept to below 2 (flow rates 0.5
- 4 ml min-1 ), deformation of the ideal spherical shape of beads was observed.
When the preservation of original shape of beads is required, other types of reactors or
reinforcement of beads is recommended.
- EFFECT OF THE CONCENTRATION OF 5,5´DITHIOBIS(2-NITROBENZOIC ACID) ON PARAMETERS
OF THE KINETICS OF ITS CHEMISORPTION ON THIOL DERIVATIVES OF CELLULOSE
(Iontosorb AV)
- Mislovicova D., Gemeiner P., Durisova V.: Collect.
Czech. Chem. Commun., 51 (1986) 545-552
Equations describing the dependence of parameters of sorption kinetics on the sorbate
concentration have been determined. The validity of the equations has been verified for
the chemisorption of 5,5'-dithiobis(2-nitrobenzoic acid) on bead O-(2-mercaptoethyl)-,
O-(3-mercapto-2-hydroxypropyl)- and O-[2-(4-mercaptophenylsulfonyl)- ethyl]cellulose.
Isothermic constants obtained from the equations can be calculated also under experimental
conditions unfavourable for their determination. These constants may be utilized for
characterizing relations between the chemical structure of cellulose derivatives and the
sorption process. The equation which provides a complete time-concentration description of
sorption is suggested.
- APPLICATION OF THE ENZYME THERMISTOR TO THE DIRECT ESTIMATION OF INTRINSIC
KINETICS USING THE SACCHAROSE-IMMOBILISED INVERTASE SYSTEM
(Iontosorb AV)
- Stefuca V., Gemeiner P., Kurillova L., Danielsson
B., Bales V.: Enzyme Microb. Technol., 12
(1990) 830-835
The possibility of using the enzyme thermistor (ET) for the direct determination of
kinetic parameters (Km, Ki, Vm) of immobilized enzyme (IME) was evaluated using different
preparations of invertase conjugated to bead cellulose. Two different ET columns packed
with IME were operated in the mode of a differential enzyme reactor (short length, low
subtrate conversion). Kinetic parameters of the above IME reactor were computed by a
nonlinear curve-fitting procedure. The obtained kinetic parameters were superverified by
means of an independent differential reactor (DR) system. This system utilized an indirect
postcolumn analytical method based on determination of glucose concentration in the
stirred reservoir. Best agreement between the data acquired by direct (ET) and indirect
(DR) methods was obtained if the ET column was operated at flow rates within the range of
1.0 - 1.5 ml/min using invertase-cellulose chlorotriazine conjugate. Influence of heat
loss and flow nonideality is discussed. The proposed ET method offers a rapid, convenient,
and general approach to determination of kinetic constants of IME preparations by omitting
postcolumn analytical methods.
- SCREENING OF CONCANAVALIN A - BEAD CELLULOSE CONJUGATES USING AN ENZYME
THERMISTOR WITH IMMOBILISED INVERTASE AS THE REPORTER CATALYST
(Iontosorb AV)
- Docolomansky P., Gemeiner P., Mislovicova D.,
Stefuca V., Danielsson B.: Biotechnology and Bioengineering,
43 (1994) 286-292
Screening and design of immobilizes biocatalysts (IMBs) is time-consuming process. An
ideal process should be universal, fast, convenient, precise and reproducible. Many of
these requirements are met by enzymatic flow microcalorimeters, also known as enzyme
thermistors (ETs) or thermal assay probes (TAPs). Adaption of ETs to real measurements of
reaction rates requires coupling of the mathematical description of the reaction -
diffusion phenomena in the ET column with heat balance and, subsequently, experimental
verification of the mathematical model. This article presents such a process developed as
an adaptation of ETs for the characterization of the microkinetic properties of IMBs and
their further application for screening of IMBs. The IMBs characterized were the
preparations of invertase, biospecificaly adsorbed on concavalin A conjugated to activated
bead cellulose.
- NEW APPROACHES FOR VERIFICATION OF KINETIC PARAMETERS OF IMMOBILISED
CONCANAVALIN A: INVERTASE PREPARATIONS INVESTIGATED BY FLOW MICROCALORIMETRY
(Iontosorb AV)
- Gemeiner P., Docolomansky P., Nahalka J., Stefuca
V., Danielsson B.: Biotechnology and Bioengineering, 49
(1996) 26-35
In our preceding article, we demonstrated a procedure based upon enzymic flow
microcalorimetry using an enzyme termistor (ET) to characterize the microkinetic
properties of an immobilized enzyme (IME) and its further application in the screening of
IMEs. To consider the ET method (single ET unit, ET systém 1) as standard, it was
necessary to show that the estimated relative kinetic parameter (Tmax) calorimetrically corresponds with the
absolute value for the reaction rate within the whole measurement range. This article
present theree experimental verification procedures. Two procedures arebased on adaptation
of the flow-through ET column to a mini-defferential-reactor (DR) system with substrate
recirculation and post-ET-column methods for determination of the concentration change of
the product (spectrophotometrically in ET systém 2) or the substrate (calorimetrically in
ET system 3) with the IME-catalyzed enzymatic hydrolysis. The third procedure is an
independently operating DR systém with spectrophotometrically estimates the concentration
change of the product. The results obtained exhibited good correlation (r = 0.921) betweem
the relative kinetic parameter Tmax , as
determined calorimetrically by ET systém 1, and the absolute value for the reaction rate
(rmax ) as determined by ET systems 2 and 3. These data proved that, within the
whole range of experimental conditions applied in this study,
the parameter Tmax instead of the true reaction rate could be employed
for the IME screening. Moreover, the generality of the detection principle and the
standardized configuration of the ET favor ET systems 2 and 3 for normal screening of IMEs
and as miniaturized DR systems allowing dual measurements of kinetic parameters.
- PURIFICATION AND IMMOBILIZATION OF SOYBEAN (GLYCINE MAX) UREASE
(Iontosorb A-UREA, Iontosorb DETA)
- Kminkova M., Kucera J.: Czech J. Food
Sci., 17 (5) 171-175 (1999)
Urease (urea amidohydrolase, EC 3.5.1.5) was isolated from soybean with 58 % purity as
judged by gel chromatography on Superose 12HR 10/30. The purification was done using
affinity sorbent - phenylurea bound to bead celulose (Iontosorb A-UREA). The results were
compared the commonly used chromatography on urea bond to epoxy-Sepharose and other new
affinity sorbent - urea bond to cyanurchloride - activated bead cellulose (Iontosorb
C-UREA - Iontosorb CNC). Resulting purified enzyme was immobilized on diethylene-triamino
- substituted bead cellulose (Iontosorb DETA) by the glutaraldehyde method. The activity
yield was as high as 7.36 %, specific activity 35230 per g of immobilized enzyme (DM
basic) and 2467 per mg of bound protein (DM basic). The immobilized enzyme was stable in
the presence of pancreatin. This implies that the urease purified and immobilized by this
way can be proposed for avoiding urea from physiological fluids.
|