Process for the preparation of thermoprecipitating affinity...

Details Process for the preparation of thermoprecipitating affinity... Process for the preparation of thermoprecipitating affinity...

2002-03-11

2004-02-10

Zalukaeva, Tatyana (Department: 1713)

Synthetic resins or natural rubbers -- part of the class 520 ser

Synthetic resins

Mixing of two or more solid polymers; mixing of solid...

C525S054110, C521S149000, C521S187000, C435S180000, C435S181000, C435S188000, C435S213000

Reexamination Certificate

active

06689836

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a process for the preparation of thermoprecipitating affinity polymers. More particularly, the present invention relates to a process for the preparation of polymers useful for the separation of enzymes of protease type exemplified by trypsin. Affinity polymers prepared by the process of the present invention exhibit stronger binding with trypsin which is useful in enhancing the recovery of trypsin from dilute aqueous solutions and from a mixture of trypsin and chymotrypsin or a mixture of trypsin and other enzymes.
BACKGROUND OF THE INVENTION
Isolation and purification of biologically active macromolecules such as enzymes, from natural sources is a tedious, multi-step process, which results in very low yields and thus higher costs. As a better alternative to conventional processes, researchers have developed affinity separations based techniques for selective and enhanced separations of enzymes. The basic principle used in these techniques is to form a complex between the active site of an enzyme and inhibitor, selective and high separations are possible. Most of the affinity based operations involved polymers to which inhibitors are chemically linked. The complex formed between polymeric inhibitor and the enzyme is subsequently processed to isolate the enzyme.
Various techniques such as affinity chromatography, affinity partitioning, affinity ultrafiltration, immobilized metal affinity chromatography, affinity imprinting and affinity precipitation have been developed so far. Although all these techniques use the same basic principle of forming an enzyme-inhibitor complex, they suffer from one or the other disadvantages as follows.
Affinity chromatography uses a column containing an inhibitor or a dye or an antibody for a given enzyme for its separation from a mixture of enzymes. The solution of enzymes is poured over the affinity column to retain the desired enzyme on column for subsequent isolation. This technique is efficient only for small capacity columns. With the scale up of columns, the problems of sample pre-treatment and plugging of packed column becomes severe. [Y. Li, G. Kunyu, C. Lubai, Z. Hanfa, Z. Yunkui Sepu, 14, 415 (1996), T. Makriyannis, Y. D. Clonis, Biotech. Bioengg. 53, 49 (1997)].
In case of affinity crossflow ultrafiltration, a mixture of enzymes is filtered through a membrane containing affinity group under pressure. This technique is suitable in the cases where the difference between the molecular weights of the two enzymes is high. Also, with the increase in the filtration time, denaturation of enzymes as well as clogging of membrane takes place due to the pressure applied. [K. Sigmundsen, H. Filippusson, Polymer Int. 41, 335 (1996); T. B. Choe, P. Masse, A. Verdier, Biotech.Lett., 8, 163 (1986)].
Affinity partitioning of two-phase aqueous systems is widely used technique as compared to the methods mentioned above. In this technique, concentrated aqueous solution of poly (ethylene glycol) (PEG) with or without linking affinity group is mixed with enzyme solution containing moderate to high salt concentration. The two phases are mixed well and allowed to separate. The desired enzyme gets predominantly partitioned in one phase, which subsequently can be isolated. Disadvantages of this technique are non-specific extraction of other proteinaceous molecules along with desired enzyme and also poor interactions between enzyme and affinity group due to high ionic strength. [G. Takerkart, E. Segard, M. Monsigny, FEBS Lett., 42, 218 (1972); B. A. Andrews, D. M. Head, P. Dunthorne, J. A. Asenjo, Biotech. Tech., 4, 49 (1990)].
Immobilised metal affinity chromatography is a technique in which the columns of polymeric support containing chelated metal ions are used. These metal ions form coordination complex with histidine, tyrosine, cysteine, etc. present on the surface of the enzyme. Although this technique has advantages like high column capacity, ease in enzyme elution, etc. it is not very selective. [Ehteshami, J. Porath, R. Guzman, G. Ehteshami, J. Mol. Recognit. 9, 733 (1996); A. L. Blomkalns, M. R. Gomez, Prep. Biochem. Biotechnol. 27, 219 (1997)].
Molecular imprinting of matrices containing metal chelates is a recently developed technique, which increases the selectivity [F. H. Arnold, P. Dahl, D. Shnek, S. Plunkett, U.S. Pat. No. 5,310,648 (1994)]. In this technique complex of monomer containing chelated metal ion and enzyme is polymerised with crosslinker in order to imprint the polymer with enzyme. Although this technique exhibits a substantial selectivity, it is not as selective as that of biological antibodies or active site inhibitors of enzymes.
Compared to the techniques described above, affinity precipitation is an attractive technique from the point of view of application. [C. Senstad, B. Mattiasson, Biotech.Bioengg., 33, 216 (1989); M. Schneider, C. Guillot, B. Lamy, Ann. N.Y. Acad Sci. 369, 257 (1981); B. Mattiasson, R. Kaul, “Affinity precipitation”, in Molecular interactions in bioseparations, T. T. Ngo ed., Plenum Press, New “York, p 469-477 (1993); J. P. Chen,
J. Ferment and Bioengg.,
70, 119 (1990); I. Y. Galaev, B. Mattiasson,
Biotech Bioeng.
41, 1101 (1993); M. Pecs, M. Eggert, K. Schnegerl,
New Polymeric Mater.
4, 19 (1993)]. It involves formation of complex between an enzyme and a stimuli sensitive polymeric inhibitor. This complex is precipitated by pH or temperature stimulus and isolated. It is then dissociated, polymer separated by pH or temperature stimulus and the enzyme isolated. Thus, the recovery of the enzyme by this technique is much simpler and the scale up of the process is also easy. Hitherto, affinity precipitation suffers from restrictions on the accessibility of the enzyme towards the polymer bound inhibitor.
The strength of the complex formed between inhibitor and the enzyme decreases 20 to 300 fold when it is bound to the polymer. [K. B. Male, J. H. T. Luong, A. L. Nbuyen,
Enzy. Microb. Tech.,
9, 374 (1987); Yu, I. Galled, B. Matisson,
Biotech. Bioengg.
41, 1101 (1993); J. H. T. Loung, K. B. Male, A. L. Nguyen,
Biotech Bioengg.,
31, 439 (1988); M. Pecs, M. Eggert, K. Schuegerl,
J. Biotech.,
21, 137 (1991)]. This weakening of the complex is attributed mainly to the restrictions on the free access of enzyme to the polymer bound inhibitor. The strength of the complex is expressed in terms of inhibition constant (K
i
). The lower the value of K
I
the higher is the inhibition and stronger is the complex formed. Higher K
i
values of polymeric inhibitors result in poor recovery of enzymes. Also, increased concentration of inhibitors on the high molecular weight polymers results in high K
I
values.
Introduction of spacers between the polymer backbone and the inhibitor is a well-known methodology used in affinity chromatography to enhance the interaction between inhibitor and the enzyme. But in affinity precipitation, the use of spacer containing polymers has not been reported so far, because the complex formation between polymer bound inhibitor and the enzyme takes place in homogeneous solution and it has been suggested that in homogeneous solutions spacers are not required.
OBJECTS OF THE INVENTION
It is therefore an object of the invention to provide a process for the preparation of thermoprecipitating affinity polymers comprising spacers between the polymer backbone and the inhibitor, useful in enhanced recovery process of trypsin by affinity precipitation.
It is another object of the invention to provide a process for the preparation of thermoprecipitating affinity polymers that exhibit enhanced interactions with the enzymes and thereby give high recovery for the desired enzymes.
SUMMARY OF THE INVENTION
Accordingly the present invention provides a process for the preparation of thermoprecipitating affinity polymers useful in the enhanced recovery of enzymes which comprises polymerising a monomer comprising a spacer and a co-monomer with a polymerisation initiator and a polymeri

Affiliated with

Also associated with

Rating

Process for the preparation of thermoprecipitating affinity... does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Process for the preparation of thermoprecipitating affinity..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Process for the preparation of thermoprecipitating affinity... will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3345909