Apparatus for the production of biocatalyst beads

An apparatus for the production of biocatalyst beads comprises an immobilization vessel (10) which is provided at its lower end with discharge pipes (23) which extend through a pressure chamber (25) in which a gas pressure prevails. The pressure chamber (25) is terminated by a plate (24) exhibiting holes which coaxially surround the discharge pipes (23). At the lower end of the immobilization vessel (10) is provided a connecting nozzle (30) which is connected in germproof sealing manner to the wall of a collecting vessel (11) which contains a solution of a cross-linking agent or a reprecipitation bath. The collecting vessel (11) may be constructed as a fermenter and provided with an agitator. Both of the vessels may be jointly sterilized.

FIELD OF THE INVENTION 
This invention relates to an apparatus for the production of biocatalyst 
beads, comprising an immobilization vessel which is sealed in a germproof 
manner and exhibits in its upper region at least one filling tube and, in 
its lower region, discharge pipes which extend through a pressure chamber 
which exhibits discharge openings arranged coaxially to the discharge 
pipes; and a collecting vessel or receiver arranged beneath the 
immobilization vessel and containing a solution of a cross-linking agent 
or a reprecipitation bath. 
BACKGROUND OF THE INVENTION 
Immobilized cells are effective catalysts in the enzymatic conversion of 
organic ingredients. It is known to effect the immobilization of cells by 
introducing the cells together with a supporting substance into a polymer 
solution which is capable of gelling and then runs out with formation of 
drops from the immobilization vessel and falls into a collecting vessel 
which contains a solution of a cross-linking agent (J. Klein and K.-D. 
Vorlop (1983) ACS Symposium Series 207, 377-392; American Chemical 
Society, 1983, 17). The drops are formed by supplying sterile compressed 
air coaxially to the discharge pipes coming out of hte immobilization 
vessel, said compressed air sweeping along the discharge pipes and 
facilitating the shaping and detachment of the discharge pipe. While 
freely falling down and being cross linked, the drops assume the shape of 
balls or beads so that biocatalyst beads in the form of balls are present 
in the collecting vessel. 
It is of particular importance when immobilizing cells to maintain them 
sterile before and during the immobilization process. When using the known 
apparatus, it is necessary to separately pre-sterilize all of the parts 
and thereafter use them in a sterile atmosphere. 
OBJECTS AND SUMMARY OF THE INVENTION 
It is an object of the present invention to provide an apparatus of the 
type mentioned above and facilitating the production of biocatalyst beads 
under sterile conditions. 
To accomplish this object, it is provided in accordance with the invention 
that the immobilization vessel exhibits a lower connecting nozzle 
surrounding the entirety of the discharge pipes and that the collecting 
vessel is sealed in a germproof manner and is provided with a cover with 
an opening for the sealed connection of the connecting nozzle. 
The immobilization vessel forms together with the collecting vessel an 
assembly which is capable of being sterilized as a total and, when in use, 
contaminations by the ambient air cannot take place especially in the 
transition region between the immobilization vessel and the collecting 
vessel. However, both of the vessel may yet be manufactured separately and 
also be cleaned or used for other purposes separately. The microorganisms 
or enzymes which are occluded in the biocatalyst beads do not contact the 
atmosphere even during the free fall into the solution of the 
cross-linking agent or into the reprecipitation bath so that the entire 
procedure takes place under germproof conditions. 
The collecting vessel may be constructed as fermenter and provided with an 
agitator and/or a heater. It is possible in this manner to use the 
collecting vessel simultaneously for the further processing of the 
biocatalyst beads without the necessity of still transferring the latter 
into another vessel after having been produced. 
According to a preferred further development or embodiment of the 
invention, it is provided that the discharge pipes are attached in a 
dismountable annular insert of the immobilization vessel, which insert is 
provided with an upper perforated plate having fitted in the holes thereof 
the upper ends of the discharge pipes and with a lower plate which is 
arranged at a distance to the upper perforated plate and in which the 
outflow openings are arranged. The annular insert forms in this case a 
dismountable member which is capable of being removed from the 
immobilization vessel for the purpose of being cleaned and attended. The 
space between the upper perforated plate and the lower plate constitutes 
the pressure chamber into which a sterile gas is introduced which escapes 
through the lower outflow openings coaxially to the discharge pipes and 
assists the detachment of drops at the lower ends of the discharge pipes 
as well as determines the size of the drops. 
The insert is preferably surrounded by an annular space connected to a 
pressure source and exhibits in its wall openings which lead from the 
annular space into the interior of the insert. This ensures uniform action 
of pressure on the pressure chamber while the sterile compressed gas is 
distributed in the annular space outside the insert. Uniform distribution 
of pressure of the flowing compressed gas is particularly important for 
achieving uniform sizes of the biocatalyst beads. 
Preferably the discharge pipes protrude into the interior of the collecting 
vessel to below the cover. Thus, the discharge pipes protrude to beyond 
the lower end of the immobilization vessel so that the detachment of the 
drops takes place in the interior of the collecting vessel. 
The construction of the apparatus for the production of biocatalysts can be 
simplified by the fact that the immobilization vessel exhibits an agitator 
extending from above into its inside space or is provided with a heater. 
It is possible in this manner that the immobilization vessel is used at 
the same time as a mixing vessel into which the supporting substance and 
the enzymes or microorganisms are separately introduced while the mixing 
takes place in the interior of the immobilization vessel. This saves an 
additional mixing vessel for preparing the immobilization. To be able to 
introduce the enzymes or microorganisms into the immobilization vessel 
under sterile conditions, the immobilization vessel is provided at the 
upper end with an innocculating nozzle with a pierceable septum.

DETAILED DESCRIPTION 
According to FIG. 1, the immobilization vessel 10 is fixedly mounted on the 
cover 12 of the collecting vessel 11. The immobilization vessel 10 
consists of a vertical tubular container 13, in the upper end wall of 
which are arranged a closable feed nozzle 14, an inocculating nozzle 15 
provided with a septum which is pierceable with a hollow needle, and a gas 
inlet 16 with a filter 17. The feed nozzle 14 and the gas inlet 16 are 
nozzles which are identically constructed and which may also be exchanged 
with respect to their functions of used for other purposes. 
The peripheral wall of the container 13 is surrounded by a heating device 
18 in the form of a heating jacket which is provided with an inlet nozzle 
19 and an outlet nozzle 20 for a heating medium. The lower end of the 
container 13 is closed by a block 21. The block 21 is provided with a 
horizontal perforated plate 22 which forms the lower bottom wall of the 
container 13 and into the holes of which the upper ends of a great number 
of vertical discharge pipes 23 are fitted. The discharge pipes 23 extend 
through holes of a lower plate 24 downwardly into the interior of the 
collecting vessel 11. Between the plates 22 and 24 is formed a pressure 
chamber 25 into which a sterile gas is introduced through a gas inlet 
nozzle 26 with a filter 27. The gas inlet nozzle 26 extends into the 
annular space 28 in the interior of the block 21. Bores extend from the 
annular space 28 into the pressure chamber 25. 
In the interior of the vessel 13 is arranged an agitator 29 which is either 
connected with an electrical drive mechanism outside the vessel 13 or may 
be rotated manually from the outside at a rotary knob 60. The agitator 
consists of a shaft of agitator and stirring vanes attached to said shaft. 
The lower end of the block 21 of the immobilization vessel 10 is 
constructed as a threaded nozzle 30. The threaded nozzle 30 is inserted by 
threading into a threaded bore of the cover 12. A packing 31 seals the 
passage through the opening 33 of the cover 12. 
The collecting vessel 11 having a volume which is substantially greater 
than that of the immobilization vessel 10 also consists of a tubular 
container which is provided at its outer wall with holders 34 for the 
attachment to a supporting device. Through the bottom wall of the 
collecting vessel 11 extends a shaft 35 of agitator to which stirrer vanes 
36 are attached and which can be driven by a motor 37 arranged outside the 
collecting vessel 11. Additionally, the collecting vessel 11 may be 
heatable. In the present case, it is constructed as a fermenter. 
Superatmospheric pressure may escape from the collecting vessel 11 through 
a vent opening 39 with a filter 40. 
In the mounted state represented, the assembly shown in FIG. 1 and 
comprising the immobilization vessel 10 and collecting vessel 11 can be 
sterilized as a whole. Its use will still be explained in greater detail 
farther below. 
The immobilization vessel shown in FIG. 2 differs only insignificantly from 
that shown in FIG. 1 or outlines the constructional details of the latter 
in greater detail. The pierceable septum 38 consisting of an elastomeric 
material is recognizable in the inocculating nozzle 15 while the locking 
cap has been removed. The shaft of the agitator 29 extends through a 
passage sealed with a packing 32 from the upper end wall of the vessel 13 
to the outside and connected there with a manually rotatable rotary knob 
60. The heater 18' consists of a heater coil wound helically about the 
vessel 13. 
The block 21 which is welded to the lower end of the vessel 13 contains an 
axial bore into which a substantially tubular insert 41 is fitted. The 
perforated plate 22 is formed by a partition wall of the insert 41, and 
the lower plate 24 constitutes the lower conclusion of the insert 41. The 
outflow openings 42 surrounding the discharge pipes can also be seen in 
FIG. 2. The insert 41 is provided with a circular flange 43 which is 
fitted in a step-shaped recess at the lower end of the block 21. From the 
downward side presses against the flange 43 a hasp cap 44 which is 
attached to the block 21 with radial setscrews 45 under axial tension. 
The annular space 28 which surrounds part of the length of the insert 41 
consists of an annular recess at the circumference of the insert 41. The 
sealing of the annular space 28 is effected by annular packings 46. Radial 
bores 47 extend from the annular space 28 into the pressure chamber 25 
through which the discharge pipes 23 are passed paraxially. 
First of all, the technique of the immobilization is described hereinafter 
in general. 
A supporting substance such as a viscous polyelectrolyte solution 
(alginate, pectinate, carboxymethyl cellulose, carrageenane, furcellarane, 
cellulose sulfate, chitosan , etc.), a polymer solution capable of gelling 
(Curdlan, agar, agarose, gelatin etc.) or a non-aqueous polymer solution 
(cellulose acetate, polystyrene, Eudragit, etc.) is mixed with the 
enzymes, cell organella or whole cells. This mixture is then introduced or 
pumped into the temperable immobilization vessel 10 and brought under 
pressure. The pressure is preselected by a pressure gauge, and the gas 
flows through the sterile filter 17. The mixture is added dropwise through 
the thin discharge pipes 23 and into the solution of the cross-linking 
agent or the reprecipitation bath and, in doing so, biocatalyst beads are 
formed momentarily. The laterial lower pressure junction 26 is connected 
through the sterile filter 27 and a pressure gauge to a pressure line. 
This gas stream flows past the steel tubes and, in doing so, prematurely 
tears off the drops having formed at the edges of the discharge pipes 23. 
It is possible to adjust the size of droplets exactly by the selection of 
the pressure in the pressure chamber 25. Beads having a narrow 
distribution of radii are obtained. 
Various working procedures which may be carried out with the use of the 
apparatus are illustrated by FIGS. 3 to 5. 
In case of the working procedure shown in FIG. 3, the immobilization vessel 
10, the collecting vessel 11 and the starile filters 17 and 27 are jointly 
sterilized first of all. The supporting substance is contained in a 
sterile bottle 50 which is equipped with a sterile filter 51 for aeration. 
From the bottle 50 extends a tubing 52 through a pump 53, e.g. a hose 
pump, to the feed nozzle 14 of the immobilization vessel 10. The pump 
pumps the supporting substance into the immobilization vessel, and the 
microorganisms or enzymes are added through the inocculating nozzle 15. 
Both of the substances are mixed by means of the agitator 29. Thus, the 
collection vessel 1 is used at the same time as a fermentation vessel. The 
gas pressure sources connected to the sterile filters 17 and 27 are 
referred to as 54 in FIGS. 3 to 5. 
The working sequence represented in FIG. 4 differs from that shown in FIG. 
3 by the fact that the supporting substance and the enzymes, cell 
organella or whole cells are mixed in an external vessel 55 which is 
provided with a magnetic stirrer 56 and thereafter pumped through the 
tubing 52 and the pump 53 through the feed nozzle 14 into the 
immobilization vessel 10. The vessel 55 is provided with an inlet 
connection 57 for feeding the enzymes or microorganisms. The contents of 
the vessel 55 may be pumped into the immobilization vessel 10 either 
batchwise or continuously. Thus, it is possible to process a volume which 
is greater than that of the immobilization vessel. In case of the 
continuous production of beads, the separate application of pressure 17 
may, if necessary or desired, be dispensed with. 
The immobilization vessel 10 and the collecting vessel 11 which is used as 
a fermenter are jointly sterilized together with the starile filters 17 
and 27 also case of the working procedure represented in FIG. 4. The 
sterilization of the vessel 55 containing the supporting substance and of 
the transfer system 52,53 is effected separately herefrom. 
In case of the working procedure represented in FIG. 5, there is provided a 
separate fermentation vessel 58 into which the immobilized microorganisms 
or enzymes are transferred through a tubing line 59 from the collecting 
vessel 11. For this purpose, the collecting vessel 11 is provided at its 
bottom wall with a connection nozzle for the tubing 59. The supporting 
substance is filled into the feed nozzle 14 of the immobilization vessel 
10, and enzymes, microorganisms or the like are introduced by inocculation 
through the inocculating nozzle 15. The collecting vessel 11 contains the 
solution of the cross-linking agent or the reprecipitation bath. After the 
biocatalyst beads have formed in the collecting vessel 11, the beads are 
sucked off from the bottom of the collecting vessel and transferred 
through the tubing 59 and into the fermentation vessel. 
Two immobilization examples carried out in accordance with the working 
procedure represented in FIG. 4 will be described hereinafter. 
1. In alginate 
To 400 ml of a sterile 3.5% Na alginate solution (Protonal 
LF 20/60) were added 100 ml of an acetobacter sp. cell suspension having a 
wet biomass proportion of 80 g. and well suspended. This suspension was 
thereafter transferred into the immobilization apparatus having been 
described. By applying pressure (1.4 bars) by means of sterile air, the 
suspension was added dropwise through the steel tubes into a 2% CaCl.sub.2 
cross-linking agent solution contained in the collecting vessel. In doing 
so, Ca alginate biocatalyst beads were momentarily formed. The sterile air 
steam used to blow off the beads was selected in such a manner that beads 
having a diameter of 1.2 mm were obtained After a cross-linking time of 30 
minutes, the biocatalyst beads were used to produce gluconic acid from 
oxygen and glucose. The resultant biocatalysts had an activity of 3.88 U/g 
KFM with a residual activity of 48%. 
2. In chitosan 
To 400 ml of a sterile 1.1% chisosan acetate solution (chitosan-hv) were 
added 100 ml of an E. coli cell suspension having a wet biomass proportion 
of 50 g and well suspended. This suspension was thereafter transferred 
into the immobilization apparatus having been described. By applying 
pressure (1.3 bars) by means of sterile air, the suspension was added 
dropwise through the steel tubes into the 2% Na tripolyphosphate 
cross-linking agent solution (pH 8). The sterile air stream used to blow 
off the beads was selected such that beads having a final diameter of 0.6 
mm were obtained. After the complete cross-linking throughout, curing and 
shrinkage of the chitosan biocatalyst beads, they could be used directly 
for the cleavage of Penicillin G to form 6-aminopenicillanic acid and 
phenylacetic acid. The resultant biocatalyst beads had an activity of 60 
U/g KFM with a residual activity of 63%.