DD242822A1 - PROCESS FOR IMMOBILIZING BIOLOGICAL MATERIAL - Google Patents

Abstract

A method for the fixation of biological material is presented in which some disadvantages of fixation, such as the action of aggressive agents, of crosslinkers u. ae. be avoided on biological material. The method is characterized in that as a carrier material for fixing bacterial cellulose is used, which can be prepared so that a network is formed. In this biological material can be stored without further measures and is available for the implementation of enzymatic reactions (product formation) available.

Description

Characteristic of the known technical solutions

Specific enzyme-catalyzed reactions with microbial cells or with immobilized enzymes have long been carried out particularly in the pharmaceutical industry (eg steroid transformations). The immobilization is the practical imitation of a phenomenon that can also be observed in nature as well as in some industrial processes. The spontaneous formation of "biological films" by microbial growth on surfaces of sand, minerals, polymers, wood, fibers, etc. has long been known (wastewater treatment, acetic acid recovery) .In addition to the advantages of fixed enzymes, the immobilization of microbial cells has additional advantages:

- The enzymes contained in the cells do not have to be isolated and purified first.

- Multi-enzyme reactions and cofactor- or coenzyme-enhancing reactions with in situ regeneration are easier to carry out.

- The enzymes are in their natural micromilieu and therefore have higher stability.

- Immobilized cells have z.T. a higher temperature or pH stability than free cells.

Immobilized cells often tolerate higher substrate and product concentrations (BERGER, R., 1981, Acta Biotechnolica 1/1, 73-102, BERGER, R., 1982, Acta Biotechnologica 2 / 4,343-358).

The following methods of immobilization are known:

1) Inclusion of the cells

For the immobilization of cells by inclusion in mostly offset gels, membrane (film) -like structures, fiber cavities, micro-chips, etc. are used:

Gels of polyacrylamide, of polymethacrylamide, of other acrylate polymers, of ionic gels, etc. (Jap. Pat. 70884, Jap. Pat. 120185, Jap. Pat.7425189, Jap. Pat.7475782, Jap. Pat. 7481590, US Pat Pat., 3791926, DE Offenlegungsschrift 2414128, DE Offenlegungsschrift 2420102.

2) Adsorption of the cells

Microbial cells are adsorbed to numerous organic and inorganic materials or polymers such as filter paper, silica gel, nylon netting, sand, activated carbon, etc.

Pat., No. 3,799,393, Japanese Patent No. 75160475, Japanese Patent No. 7686142, DE Offenlegungsschrift No. 2413694, US Pat. No. 3,769,175, US Pat. No. 4,032,407). 3) Intercellular crosslinking

(DE Offenleg 2345185, DE Offenlegungsschrift 2345186, DE Offenlegungsschrift 1915970, US Pat. 3939041, U. S. Patent 3821086, Japanese Patent 7703891,

Jap. Pat. 7703889)

 4) Covalent fixation

(DE Offenleg.2752499, DE Offenlegungsschrift 1915970, DE Offenlegungsschrift 2513929, US Pat.3957580, KLEIN, J. and WAGNER, F., 1978 DECHEMA-Monogr.82, 142-164, KLEIN, J. and WAGNER, F., 1979DECHEMA monogr.84, 265-335).

All of these methods have the disadvantage that the fixation with the aid of one or more chemical reactions (crosslinking, functionalization, formation of covalent bonds) takes place, usually emanating from the reaction conditions chemischoxische or physical side effects that affect the cell metabolism or enzyme activity.

Object of the invention

The aim of the invention is to develop a method for the immobilization of biological material, with which one can carry out the previously individually running partial steps simultaneously or successively in the same reaction chamber in discontinuous or continuous operation.

Essence of the invention

The invention is therefore based on the object to select a suitable carrier which is chemically inert and thermally stable and has such a structure that can store the biological material to be fixed therein and that the mass transfer between the cell and medium can proceed at high speed without to be hampered by the immobilization.

According to the invention, the object is achieved by using bacterial cellulose as the carrier material.

The bacterial cellulose must have a network-like structure for use as a carrier material and is synthesized in a known manner.

It is expedient to carry out the immobilization in the same apparatus in which the synthesis of the bacterial cellulose takes place. This is done by terminating the cellulose synthesis by removal of the synthesis solution or by exhaustion of the carbohydrate source after formation of a sufficient cellulosic layer. If necessary, the Acetobacter cells can be killed by heat treatment. Subsequently, bringing the material to be fixed in the same apparatus in contact with the cellulose support and filling the necessary for the desired performance of the fixed material reaction solution. The tracking of spent reaction solution or the discharge of the products formed are carried out according to the invention continuously or batchwise.

The following examples are intended to explain the invention in more detail:

Examples Example 1:

A non-woven of bacterial cellulose, which was produced in a known manner, is heated at 95 ° C for 15 minutes, whereby the cellulosic bacteria of Acetobacter xylinum are killed. After cooling to 3O ⁰ C, the spent culture fluid is removed and replaced by an aqueous nutrient medium has the following composition: 0.5% yeast extract 0.3% KH ₂ PO ₄

The carrier layer produced must be brought into contact with the nutrient medium. This is mixed with 80 ml per liter of culture medium filled a suspension of Gluconobacter oxydans CCM1783, which contains about 10 ⁴ cells per ml, and kept for 30 min at 3O ⁰ C (pH 6.2) with the entire carrier in contact. Thereafter, glycerol is added to a final concentration of 100 g / l of nutrient medium. Throughout the procedure, aseptic operations are maintained. The glycerol is converted within 3 days with a yield of 87% in dihydroxyacetone.

Example 2:

The synthesis of a nonwoven from bacterial cellulose is initiated in a known manner. Already after 48 hours, when the cellulose matrix is still poorly developed, the nutrient solution is aseptically added 40 ml per liter of a suspension of Glukonobacter oxydans CCM1783 containing approximately 10 ⁴ cells per ml.

These bacteria are fixed in the capillary spaces of the fleece and additionally secured and covered by newly formed cellulose fibrils, without the need for special measures are required. The described addition of Gluconobacter oxydans suspension is repeated after a further 48 hours. After a further 48 hours, the suture medium is removed and replaced by a sterile aqueous medium of the following composition:

0.5% yeast extract

0.3% KH ₂ PO ₄

6.0% glycerol

12 hours later, continuous operation starts with D = 0.1 h ~ \

Example 3:

The procedure is as in Example 2. Instead of the viable cells of a product-forming agent, a preparation is added to the medium, which consists of biomass of Acetobacter methanolicus MB 58, which was made incapable of reproduction by pretreatment with a 20% aqueous ethanol solution. The preparation is added in an amount of 6 g / l, based on the bacterial dry matter. The addition is made in three equal parts at intervals of 48 hours. The components of the preparation are, without further action required, fixed in the cellulose fleece and woven through newly formed cellulose fibrils. After 96 hours, the nutrient medium for the cellulose synthesis is exchanged for a product-forming medium consisting of a 2% aqueous ethanol solution. 8 hours later, the continuous operation begins with D = 0.1 h ^-1 and a 95% conversion of ethanol to acetic acid.

Claims (1)

Claim: Process for the immobilization of biological material, characterized in that bacterial cellulose is used as carrier material. Field of application of the invention The invention relates to a method for the immobilization of biological material, such as cells, enzymes or bioactive molecules, and belongs to the class C 12 N. The field of application of the invention is the biotechnological industry.