AU729928B2 - Immobilized esterases from crude extract and their use - Google Patents

Description

WO 98/26055 PCT/EP97/06894 IMMOBILIZED ESTERASES FROM CRUDE EXTRACT AND THEIR USE This invention relates to immobilized proteins from crude extract and their use for fermentation in bioreactors.

In biochemical and biotechnological processes, very frequently several reactions proceed simultaneously, of which generally only one yields the desired product. All reactions that proceed in this way are referred to as simultaneous reactions follow-on and parallel reactions). In production that is done on an industrial scale, it is important to obtain the highest possible yield of end products with simultaneously high purity and low costs.

Important enzymes, which are used in fermenters or bioreactors in the case of biochemical or biotechnological reactions, are esterases.

Carboxylesterases are very common in nature. They catalyze the hydrolysis of carboxylic acid esters in free acid anions and alcohols.

A process for the production of benzodicarboxylic acid monomers and their derivatives using pig liver carboxylesterase is already known (JP 2-199616).

It is also known that enzymes that are suitable for fermentation can be immobilized.

Immobilization is always carried out with the isolated and purified enzymes, however.

It would therefore be desirable to carry out immobilization of the enzymes for fermentation in such a way that the enzymes must be neither isolated nor purified beforehand, which reduces costs and which results in considerably fewer losses in activity and thus in a better yield of products.

It has now been found that esterases directly from the crude extract of tissues and cells of animals or plants can be immobilized by fastening to resins without prior isolation and purification.

Thus according to a first aspect of the present invention there is provided an immobilised esterase prepared by covalently bonding a non-isolated and non-purified esterase obtained from the crude extract of tissues and cells of animals or plants to a resin.

•According to a second aspect of the present invention there is provided a use of an immobilised esterase according to the first aspect for cleavage of a carboxylic acid ester to a free acid anion and alcohol.

th According to a third aspect of the present invention there is provided a process for the preparation of an immobilised resin-bound esterase including the step of covalently bonding a non-isolated and non-purified esterase.

0 obtained from the crude extract of tissues and cells of animals or plants to a resin, and esterases prepared by said process. Other aspects will become apparent from the description which follows.

The subject of this invention is therefore esterases, covalently bonded to resins and thus immobilized, from crude extracts of animal or plant cells and animal or plant tissue.

-2A- Suitable resins for this purpose are, for example, epoxide resins, whilst esterase from crude pig liver extract has proven especially valuable.

The subject of this invention is thus especially esterase, bonded to resins, from the crude pig liver extract.

A resin that is suitable for this purpose is, for example, the epoxide resin eupergit C.

The esterase, bonded to resins, from crude pig liver extract can be used for cleavage of carboxylic acid esters into free acid anions and alcohols in bioreactors with high yield and high activity.

The esterase that is immobilized according to the invention can preferably be used for cleavage of nitroisophthalic acid dimethyl ester (NIPA-DME) to nitroisophthalic acid monomethyl.

3 ester (NIPA-MME), whereby the undesired reaction to nitroisophthalic acid (NIPA) does not take place.

The esterase that is immobilized according to the invention can also be used preferably for saponification of 2-fluoro-araadenine-triacetate to 2-F-ara-adenine (F-araA), a precursor of Fludara.

Another preferable use of the esterase that is immobilized according to the invention is the use in the partial saponification of Reichstein's substance S-17,21-diacetate into Reichstein's substance S-17-monoacetate.

In the case of fermentation, it was possible to achieve a high yield of end product, surprisingly enough, with the esterase that is immobilized according to the invention.

This could not be predicted, since it is generally known that in the case of enzymatic reactions, isolated and purified enzymes are used to achieve the largest possible conversion or S: large yields. Contamination of the enzyme generally is considered to be activity-inhibiting. It can generally also be assumed from it that other enzymes that are present in the crude extract disturb the reaction or even catalyze other enzymatic reactions.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

WO 98/26055 PCT/EP97/06894 Description of the Figures Fig. 1 shows the SDS-gel electrophoresis (12.5% acrylamide gel) with purified esterase from pig liver and pig liver esterase from the crude extract.

1, 7 mean marker proteins (26.6 kd of triosephosphatisomerase, 55.56 kd of glutamate dehydrogenase) 2, 3 mean purified pig liver esterase 4-6, 8-9 mean crude pig liver extract in various dilutions Fig. 2 shows the structure required for fermentation In the figure: PC computer NH3 aqueous ammonia solution 718Stat Titrino titration device T temperature, measured via temperature sensor Fig. 3a shows the specific loss of activity of the immobilizate during long-term use.

Fig. 3b shows the standardized loss of activity during longterm use.

WO 98/26055 PCT/EP97/06894 The embodiments below explain the invention without limiting the latter to the examples.

Example 1 Extraction of Esterase from Pig Liver Fresh pig liver is decomposed in a homogenizer with potassium phosphate buffer at pH 7.5 at a mass ratio of 1:4 at room temperature for 3 minutes. The homogenizate is centrifuged.

The supernatant is decanted. The sediment is discarded. The amount of supernatant corresponds to the buffer that is used.

The supernatant that is thus obtained is the crude pig liver extract, which has a high catalytic activity. 66 mg of total protein/ml of extract was obtained.

The volume activity of the esterase is about 6.5 U/ml; the specific activity is about 0.1 U/mg.

Example 2 Study of Esterase from Pig Liver The quaternary structure of the esterase from pig liver is in most cases a trimeric protein with three identical subunits and in each case an active center with a molecular weight of about 60 kd. In the case of gel electrophoresis, therefore, only a band at 60 kd must occur. To study the esterase, a pig liver extract, which was produced as described in Example 1, was therefore applied in various dilutions to an SDS gel in addition to commercially available pig liver esterase and marker proteins.

The result of the gel electrophoresis is depicted in Fig. 1.

Example 3 Production of the Immobilized Esterase from Crude Pig Liver Extract 1 g of eupergit C resin is added to 10 ml of the crude extract that is obtained in Example 1 and it is slowly shaken for 72 hours at room temperature. The crude extract-esterase that is immobilized on eupergit C is separated from mucus and blood via a nutsch filter, and it is washed three times with 20 ml of 0.1 M potassium phosphate buffer in each case. The immobilized crude extract-esterase is stored with 0.1 M of potassium phosphate buffer at a 1:1 ratio.

1 g of eupergit C binds about 0.7 g of protein covalently.

The volume activity of the immobilized esterase is about 8.5 U/g with NIPA-DME as a substrate.

Isolated and purified pig liver esterase is treated by a similar method for purposes of comparison.

Example 4 Execution of Fermentation in the Reactor The immobilizates of the crude extracts from Example 3 are introduced into the reactor. The product can be suctioned off with a filter without the reactor being completely emptied.

Then, the reactor can be re-coated with substrates. This process can be repeated until the enzyme has lost its activity. It has been shown in practice that the process can be repeated more than 100 times without loss of activity.

4.1 Results of Kinetics Studies on the Immobilizate For the immobilizate, kinetics studies were performed with starting concentration with 10 g/l and 50 g/l of NIPA-DME. The kinetics studies show that the kinetics parameters for the substrate affinity and the inhibition constants for methanol of the free esterase are identical to those of the immobilized esterase. The bonded enzyme in its kinetic properties thus behaves like the free enzyme.

The specific activity of the moist immobilized esterase is about 8.5 U/g or 0.51 mol of substrate/hour and kg of immobilizate.

The total activity of the immobilizate compared to that of the pig liver extract that is used is reduced by the immobilization process to about 4.2 Long-term Stability Study of Immobilized Esterase on Eupergit Decisive for the use of the immobilizate on an industrial scale is the stability of the immobilized enzyme with repeated use.

For immobilization, the liver was decomposed at a 1:4 ratio with 1 M phosphate buffer (pH g of eupergit was used in 500 ml of crude pig liver extract with a specific activity of 6.5 U/ml and a total activity of 3250 U, and it was shaken for 80 hours at 25 0 C. After the eupergit was separated from the extract, a specific activity of the immobilizate of 35.75 U/g, relative to the dry weight, was noted. This corresponds to a total activity of 1787.5 U and an activity loss by the immobilization of 45%. During immobilization, the immobilizate swells to about 4x the value of the dry solid (200 Since the immobilizate is stored and used in the moist state, the activity of the immobilizate for the swollen state is 8.94 U/g, compared to the moist weight. A longterm study was performed with this immobilizate, whereby the use of NIPA-DME per batch was 10 g/l. The reaction was performed at 38 0 C, and a pH of 7.5 was executed over a period of 24 hours. As a result, the separation of the immobilizate from the solution and the renewed use of the immobilizate were carried out. The reduction over time of the specific activity and the standardized activity of the enzyme are depicted in Fig. 3a and Fig. 3b.

After about 200 operating hours, the immobilized esterase from the crude pig liver extract still has 65% remaining activity.

The half-life of the immobilizate is about 250 hours and is thus extremely well suited for technical. use in the fermenter. When used for 500 hours, at least 100 batches with a mean reaction time of 5 hours can be run at 10% concentration of the immobilizate with 10 g/l of NIPA-DME.

Example Comparison of the Activity of Isolated and Purified Esterase with Esterase from Crude Pig Liver Extract in the Fermentation Process The execution of controlled fermentation was carried out in a Biostat ED fermenter according to the arrangement that is depicted in Fig. 2.

The comparison was made with 10 g of NIPA/DME/1.

Commercially available, purified pig liver esterase (103 U/mg of protein) and esterase from crude pig liver extracts were treated analogously. First, the enzymatic activity of the unbonded esterases (purified and from crude extract) was studied in each case.

Then, the purified esterase and the crude extract, which contains the unpurified esterase, were reacted with eupergit C according to Example 3.

5.1 Comparison of Activities of Unbonded Esterases During comparison of the unbonded pig liver esterases, it follows that only about 1/30 of the amount of immobilized crude extract must be used for a comparable conversion, in contrast to the purified esterase. In this case, 2 g of crude protein extract corresponds to about 70 mg of purified, commercially available esterase.

5.2 Comparison of Activities of the Esterases that are Bonded to Eupergit C During comparison of the pig liver esterases that are bonded to eupergit C, it follows that to achieve comparable results in contrast to purified esterase, only about 1/5 of the amount of immobilized crude extract must be applied.

In this case, 4 g of immobilized crude extract corresponds to about 0.75 g of immobilized purified esterase.

It can be seen from the results that the immobilizate from the crude extract of the pig liver is significantly more active than the immobilizate with commercially available pig liver esterase. In addition to the significantly higher activity, the immobilizate from the crude extract compared to the immobilizate from the purified esterase is far more inexpensive in production, since the upstream expensive purification processes are no longer necessary.

Claims (11)

1. An immobilised esterase prepared by covalently bonding a non-isolated and non-purified esterase obtained from the crude extract of tissues and cells of animals or plants to a resin.

2. An immobilised esterase according to claim 1, characterized in that the esterase is obtained from the crude extract of pig liver.

3. An immobilised esterase according to claims 1 and 2, wherein the resin is an epoxide resin.

4. Use of an immobilised esterase according to any of claims 1-3 for cleavage of a carboxylic acid ester to a free acid anion and alcohol.

5. Use of an immobilised esterase according to any of *9 claims 1-3 for cleavage of a carboxylic acid ester to a free acid anion and alcohol in a bioreactor.

6. Use of an immobilised esterase according to any of claims 1-3 for cleavage of nitroisophthalic acid dimethyl ester (NIPA-DME) to nitroisophthalic acid mono-methyl ester (NIPA-MME).

7. Use of an immobilised esterase according to any of claims 1-3 for saponification of 2-fluoro-ara- triacetate to 2-F-ara-adenine.

8. Use of an immobilised esterase according to any of claims 1-3 for partial saponification of Reichstein's substance S-17,21-diacetate into Reichstein's substance S-17-monoacetate.

12- 9. A process for the preparation of an immobilised resin- bound esterase including the step of covalently bonding a non-isolated and non-purified esterase obtained from the crude extract of tissues and cells of animals or plants to a resin. A process of claim 9 wherein the esterase is obtained from the crude extract of pig liver. 11. A process of claim 9 or 10 wherein the resin is an epoxide resin. 12. A process for the preparation of an immobilised resin- bound esterase substantially as herein described especially with reference to the Examples.

13. An immobilised esterase prepared by a process of any of claims 9-12.

14. Use of an immobilised esterase substantially as herein described especially with reference to the Examples. S DATED this 20th day of October 2000 Schering Aktiengesellschaft By its Patent Attorneys DAVIES COLLISON CAVE