DE19948408A1 - New immobilizable amylosucrase and its use and process for the production of poly (1,4-alpha-glucan) - Google Patents

Abstract

The invention relates to a novel biotechnical continuous method for producing linear poly(1,4-alpha-glucan), whereby the poly(1,4-glucan) is produced as a solid in a modified biotransformation by using immobilized fusion proteins which have an amylosucrase activity.

Description

The invention relates to a new immobilizable amylosucrase and its Ver application and a process for the production of poly (1,4-alpha-glucan).

Amylosucrase (enzyme class EC 2.4.1.4) is available from microorganisms such as B. Neisseria polysaccharea, which sucrose undergoes a biotransformation in Po ly (1,4-alpha-glucan) and fructose. This enzyme activity has long been known (MacKenzie et al., 1977), the corresponding gene, is first in Pa tent registration WO 95/31553 and PCT / EP 98/05573. On the Offenba The content of these registrations is expressly referred to below.

Poly (1,4-alpha-glucan) has a number of advantageous properties, which for example for processing into foils, as an additive in the food industry, as a raw material for the production of cyclodextrin and as an auxiliary in the pharmaceutical industry processing and assembly can be used. An overview of various applications Possible uses of polysaccharides can be found in Bucke (1998). As well the applicant refers to your own registrations with a special application of poly (1,4-alpha-glucan), as described, for example, in PCT / EP / 98/03960, PCT / EP / 98/03920, PCT / EP / 98/05297, PCT / EP / 99/00473, PCT / EP / 99/02386, PCT / EP / 99/02385, DE 198 52 826.4.

The technical extraction of poly (1,4-alpha-glucan) in the context of a process is known Biotransformation using the recombinantly obtained amylosucrase, as in WO 95/31553. For the biotechnological extraction of amylosucrase A wide variety of heterologous host organisms can be used, but is preferred the gut bacterium Escherichia coli with the best protein yields. The ge  The amylosucrase obtained is then used for the enzymatic conversion (bio transformation) from sucrose to fructose and poly (1,4-alpha-glucan).

The previously used production process of poly (1,4-alpha-glucan) using re Combined amylosucrase (short: AmSu) is enriched using AmSu solutions implemented. Obtaining pure AmSu using conventional, chromatographic cleaning is currently very complex and costly.

The biotransformation itself is carried out in the prior art in a batch process, i. H. in a static process. The disadvantage here is that the used After the product has been separated off, the enzyme is discarded with the reaction broth that must. Process management in a batch approach also means that or removal of starting materials or products is not operated. This leads to problems men, because a product inhibition of AmSu is suspected by fructose.

The object of the present invention is therefore an immobilized protein with the To provide activity of an amylosucrase. The invention further relates to also an efficient and inexpensive process for continuous production position of poly (1,4-alpha-glucan).

The object is achieved in that a nucleic acid coding for an amylo sucrase, preferably with a nucleic acid according to SEQ ID No. 4, especially be preferably a nucleic acid according to SEQ ID No. 5 is ligated with an anchor sequence, whose expression product with the activity of an amylosucrase - a fusion pro tein - immobilization on a solid phase allowed (hereinafter fiction appropriate fusion protein called).

Such fusion proteins according to the invention are particularly preferred holding an N-terminal glutathione-S-transferase fusion day (short: "GST- AmSu ") - obtained for the purpose of bioaffective immobilization on glutathione-Sepharose.

For the purposes of the invention, fusion protein means a protein which is produced by compound the protein-coding parts of two or more nucleic acids are obtained  can be connected to each other in the correct reading frame and as a hybrid or Fusion gene expressed in a suitable host cell - here: preferably E. coli become.

The E. coli strains DH5, for example, are suitable as further host cells, HB101 or BL21, the yeast strain Saccharomyces cerevisiae, the insect cell line Lepidopteran, e.g. B. from Spodoptera Frugiperda, or animal cells such as COS, Vero, 293 and HeLa, all of which are generally available.

Another object of the invention is therefore the expression of a fusion protein the construction of suitable cloning vectors, so-called fusion vectors according to SEQ ID No. 6 or particularly preferably SEQ ID No. 7, which out pGEX-4T-1 (Pharmacia) can be obtained (see Example 3) and in one Host cell - preferably E. coli intracellularly the desired product syntheti siert.

Further examples of possible fusion proteins are those with epitopes, which are from spe specific antibodies can be recognized (HA, FLAG, c-myc and others), with short Histidine oligomers (HIS tag) and enzymes or other proteins that are a star have no affinity for a binding partner (protein A, ConA, glutathione-S- Transferase), all of which are commercially available. Especially for GST-AmSu could be shown that the amylosucrase activity is not inactivated (de Montalk et al., 1999).

A special embodiment is preferred, according to the invention SEQ ID No. 5 and the associated fusion vector according to SEQ ID No. Containing 7 a nucleic acid coding for an amylosucrase function and a GST Anchor sequence, including thrombin interface, its expression product against was truncated above the native protein N-terminally, and surprisingly shows an amylosucrase activity that is above the native protein (see example 4). The N-terminal truncations affect some of the N-terminal amino acids of the native protein found in Neisseria polysaccharea, the original organism of the selected AmSu act as part of a putative secretion signal.  

The increased activity of the AmSu was not to be expected because the product of the biocatalysis poly (1,4-alpha-glucan) is insoluble in water and there is a risk of blocking. Biocatalysts with a solid, polymeric and water-insoluble reaction product have not yet been carried out in the prior art with enzymes mobilized according to the invention. In addition, "GST-AmSu-Sepharose beads" (see FIG. 6) are obtained which, in multiple rounds of biocatalysis, enable stable, continuous reactions for days and ensure particular advantages in the process according to the invention below.

The inventions therefore preferably relate to such functional variants of inventions nucleic acids according to the invention as in SEQ ID No. 5 running in conjunction with a suitable anchor sequence via an expression vector - as in SEQ ID. No 7 represents in nucleotides 258-2825 - for a fusi according to the invention for example according to SEQ ID No. Encode 8; with the activity of an mobilizable amylosucrase.

According to the present Er, the term “functional variant” is understood finding a nucleic acid functionally related to an amylosucrase, such as disclosed in WO 95/31553 and PCT / EP / 98/05573 or in part or in these Whole can be selected (as given in SEQ ID No. 4 and SEQ ID No. 5). Examples of related nucleic acids are also nucleic acids from different Chen cells or tissues (animal, plant, microorganism and corresponding transgenic representatives) or allelic variants. The present Er also includes Finding variants of nucleic acids from different individuals en / representatives.

In a broader sense, the term "variants" is understood to mean the the invention nucleic acids which have a homology, in particular a sequence tity of about 60%, preferably of about 75%, in particular of about 90% and above all of about 95%.  

The parts of the nucleic acids according to the invention can be used, for example Positioning of individual epitopes, as probes for the identification of further functional Vari anten or as antisense nucleic acids can be used. For example a nucleic acid composed of at least approx. 8 nucleotides as antisense Nucleic acid, a nucleic acid of at least approx. 15 nucleotides as a primer in the PCR method, a nucleic acid of at least about 20 nucleotides for the ides tification of further variants and a nucleic acid from at least approx. 100 nu kleotides as a probe, preferably used in gene libraries.

Another object of the present invention is also the Invention appropriate fusion protein as such with a preferred amino acid sequence according to SEQ ID No. 8 consisting of the functional units of amino acid synthesis sequences 1-220 for the preferred anchor sequence glutathione-S- Transferase (GST) and amino acids 221-226 for a thromb chosen here bin interface including a multiple cloning site (227-229) and 230-856 with one Amylosucrase activity or a functional variant thereof, and parts thereof at least six amino acids, preferably with at least 12 amino acids, in particular with at least 65 amino acids and especially with 626 and 856 ami nonacids. For example, an approximately 6-12, preferably approximately 8 amino acids long polypeptide contain an epitope, which after coupling to a carrier for Production of specific poly- or monoclonal antibodies is used (see here e.g. B. US 5,656,435). Polypeptides with a length of at least approx. 65 amino acids Ren can also be used directly without a carrier for the production of poly- or monoclonal Antibodies serve.

The invention therefore relates to an immobilizable amylosucrase consisting of the functional units of an amylosucrase and an anchor sequence and, if necessary, wide re help sequences.

Under the term "functional variant" in the sense of the present invention ver one stands polypeptides that are functional with the fusion protein according to the invention are related, d. H. have immobilizable amylosucrase activity - included tend the functional units mentioned, as preferably on the SEQ ID. No 8  explained. Variants are also understood to mean allelic variants or polypeptides, that of other cells or tissues (animal, plant, microorganism and transgenic representatives). It also means polypep tide that come from different individuals / representatives.

In a broader sense, this also includes polypeptides that contain a sequence ho mology, in particular a sequence identity of approx. 70%, preferably of approx. 80%, in particular of about 90%, especially of about 95% of the polypeptide with the Amino acid sequence according to SEQ ID No. Have 8. This also includes Deleti on the polypeptide, preferably in the range of amino acids 230-856 (Amylosu crase unit) of SEQ ID. No 8, in the range of about 1-60, preferably about 1- 30, in particular from about 1-15, especially from about 1-5 amino acids.

An essential feature of the present invention is the repeated use of the Fusion protein obtained according to the invention by binding to a matrix and a commitment to continuous biotransformation. It is already known that En zymes by immobilization on solid, inert, insoluble matrices for biocatalysts can be used (Nelson and Griddin, 1916). However, there is always the problem that the immobilized enzyme due to the covalent coupling to the carrier material for steric reasons inactivated in whole or in part can (Saleemuddin, 1999). In addition, covalent bonding is usually irreversible and consequently cannot be used for purification of the soluble protein.

Another object of the invention relates to a process for the preparation of poly (1,4-alpha-glucan), wherein the fusion proteins of the invention are immobilized on a fe most phase. Such a solid phase is, for example, Sepha rose (primarily glutathione-Sepharose when using SEQ ID No. 8). In the context of the method according to the invention, the resulting loaded solid phase beads (see FIG. 6) of the active fusion proteins according to the invention have the particular advantage of not permanently blocking with poly (1,4-alpha-glucan), that is to say covered by the product of biocatalysis and thus to be "inactivated". The process comprises the following steps: Conversion of sucrose to poly (1,4-alpha glucan) by means of immobilized fusion protein according to the invention with vigorous shaking, particularly preferably GST-AmSu, on a solid phase. Separation of the solid phase beads from the reaction broth by means of filtration (e.g. glass frit) when the reaction conversion is reached. Rinsing the solid phase beads with DMSO, the product poly (1,4-alpha-glucan) going into solution. Subsequently, the solid phase bead is washed with PBS free of DMSO and reloaded with (according to) Fusi on protein according to the invention. The solid phase loaded in this way can be used again for a new round of bio-formation.

The fusion proteins according to the invention and the ste in this context The existing process leads to a number of, sometimes unexpected, advantages in the course of Biocatalysis. The specificity of biocatalysis is improved, which increases in an shows product yield and less by-product. The by-product palatino It is formed to less than 15% of the expected total amount of product.

The conventional batch process, on the other hand, shows a product yield of deut Lich less than 80% of the theoretically possible amount.

Another crucial advantage is the acceleration of the reaction. You need takes less than 24 h until all of the substrate from the biosynthesis batch is used needs is. In comparison, the batch process takes at least 48-72 h needed. This is probably because unlike the conventional one Process the reaction mixture can be mixed vigorously during the reaction. The batch process is carried out with an almost non-mixed approach leads. Experiments with amylosucrase showed that the enzyme can be found both in the na door form, as well as a fusion protein when mixed vigorously foams and is thereby inactivated. Only the binding of the fusion protein to one Solid phase enables strong mixing. No foam is created here and the activity remains.

The resulting increase in the space-time yield and the decrease By-products is a decisive improvement of the invention Process compared to the prior art.  

Another object of the invention is therefore a composite stable catalyst containing the fusion protein according to the invention, preferably according to SEQ ID No. 8, immobilized on a solid phase such as Sepharose and possibly other auxiliaries and additives. A possible embodiment (so-called "Sepharose bead") is shown in FIG. 6.

Another object of the invention relates to the invention Process for the production of microparticles from poly (1,4-alpha-glucan), which in different size and distribution in particle form - up to about 5 µm - obtained become. Such particles are in the application WO 99/11695 by the applicant already described. However, the microparticles are in the context of the Invention received procedures in such a quality that they are now for those there described applications can be used particularly advantageously.  

The following examples serve to illustrate the invention without the Er to limit the finding to these examples.

Examples example 1 Production of the truncated amylosucrase gene

Various forms of the Amylosucra se gene were produced by a standard PCR method. The nucleic acid sequence of Amylosucra se from WO 95/31553 served as a template. The PCR was carried out using the "forward" primer AmSu (Seq. ID No. 1) or AmSu5 (Seq. ID No. 2) and the "reverse" primer AmSu Xhol rev (Seq. ID No. 3). The primers generated an EcoRI and an Xhol interface at the 3 'end. The amplificates were given the names AmSu (Seq. ID No. 4) and AmSu5 (Seq. ID No. 5). The following PCR conditions were used for the amplification: about 1 ng template DNA, 1 µl AmSu or AmSu5 and AmSu Xhol rev primer (100 µmol / µl each), 4 µl dNTP's (2.5 mM each), 5 µl 10x incubation buffer with MgSO ₄ (Boehringer Mannheim), 5 U Pwol DNA polymerase (Boehringer Mannheim), ad 50 µl water. The following temperature program was used: 5 min 94 ° C then 30 cycles 30 s 94 ° C, 30 s 55 ° C, 3 min 72 ° C, followed by 20 min 72 ° C. AmSu represents the complete amylosucrase gene, AmSu5 denotes the shortened version.

Example 2 Cloning of the AmSu variants in an expression plasmid

The amplificates AmSu and AmSu5 were purified by ethanol precipitation and turned on finally cut with EcoRI and Xhol. Following the precipitated DNA were the following Volumes pipetted: 4 µl 10x restriction buffer 2 (NEB), 0.41 µl BSA (NEB), 2 µl EcoRI (NEB, 20 U), 4 µl Xhol (NEB, 20 U) and 31.6 <M water. The restriction takes place at 37 ° C for 2 h. The enzymes were then deactivated at 65 ° C. for 10 min. 1 µg plasmid pGEX-4T-1 (Pharmacia) was cut with EcoRI and Xhol. Fol The following conditions were set: 3 µl 10x restriction buffer 2 (NEB), 0.3 µl BSA (NEB), 2 µl EcoRI (NEB, 20 U), 2 µl Xhol (NEB, 20 U) ad 30 µl water. The Re  striction took place at 37 ° C for 2 h. The enzymes were then at 65 ° C for Deactivated for 10 min.

Cut plasmid and cut AmSu versions were ligated according to the following scheme: 100 ng cut plasmid are combined with about 100 ng cut PCR products (AmSu and AmSu5). In addition there were 2 µl 10x ligation buffer (NEB) and 2 µl T4 DNA ligase (NEB); made up to 20 µl with water. The ligation was carried out at room temperature for 2 h. The pGEX-4T-1 vector carrying the Am-Su gene is shown in Fig. 1 (Seq. ID No. 6); the vector - carrying the AmSu5 fragment is shown in Fig. 2 (Seq. ID No. 7).

The entire ligation batches were transformed according to E.coli TOP10 using the CaCl ₂ method. The transformants were selected for ampicillin resistance and cultured with ampicillin for miniprep analysis in LB medium. The clones selected were checked by DNA sequencing.

The stock must be kept in glycerin at -80 ° C.

Example 3 Expression of the amylosucrase variants and binding to glutathione Sepharose

20 ml LB medium with 100 µg / ml ampicillin were in a 100 ml flask with 100 µl Freeze culture inoculated. The culture was poured overnight at 37 ° C. and 240 rpm incubated.

A main culture with 250 ml LB medium with 100 ug / ml ampicillin (or a correspondingly larger culture volume) was inoculated with the preculture in a 1000 ml flask so that the optical density 600 nm (OD ₆₀₀ ) of the culture after 3 h growth is about 1. The TAC promoter was then induced by adding IPTG (final concentration 1 mM). The culture was incubated for a further 3 h. The cultivations were carried out at 37 ° C and 240 rpm shaking frequency. The cells were then harvested by centrifugation at 2000 × g for 10 min. The cell pellet was taken up in 10 ml PBS buffer with 0.1 g lysozyme (Sigma) and 250 U benzonase (Merck) and incubated at 37 ° C for 60 min. Then 100 ul PMSF (100 mM in isopropanol) (Sigma) and 100 ul Triton X-100 (Sigma) were added. After the cells were lysed, the cell residues were centrifuged off at 10,000 × g for 10 min and the supernatant was mixed with 2 ml of 50% glutathione-Sepharose (Pharmacia) in PBS. The mixture is shaken gently for 1 h. The Sepharose was then centrifuged at 500 × g for 10 min and the Sepharose pellet was washed three times in 10 ml of PBS. The washed pellet is taken up in PBS such that the total volume was again 2 ml. 200 ul glycerol were added and the Sepharose loaded with amylosucrase is stored at -20 ° C. Fig. 3 clearly shows the induction of GST-AMSU and the effectiveness of the purification.

Example 4 Determination of the specific activity of the GST-AmSu

Glutathione-Sepharose - described according to Example 3 - was loaded either with a 10 mM glutathione solution with 50 mM Tris / HCl pH 8 or with thrombin (150 mM NaCl, 50 mM Tris / HCl pH 8, 2.5 mM CaCl ₂ , 0.1% mercaptoethanol and 5.5 mg / ml thrombin). In the case of glutathione elution, very pure GST-AmSu protein was eluted from the column. In the case of thrombin elution, the Amylosu crase fusion part was eluted since there is a thrombin cleavage site between the GST part and the AmSu ( FIG. 4). The specific activity of the AmSu versions could be determined by determining the AmSu activity and the protein concentration of the eluates. The fusion GST-AmSu5 (glutathione eluate) and the thrombin eluate (corresponds to AmSu5) both have a specific amylosucrase activity of 0.2 U / µg. The genetically unchanged AmSu only has a specific activity of 0.17 U / µl.

Example 5 Simple elution of the GST-AmSu

The manufacturers of glutathione-Sepharose (Pharmacia) recommend dissolved glutathione for the elution of bound fusion proteins. However, this eluent is too expensive for large-scale production of cleaned GST-AmSu. In the search for a cheap eluent, HCl solutions with different pH values were tested. Surprisingly, a relatively small drop in pH led to the elution of functional GST-AmSu. For this experiment, 2 ml bed volume (Pharmacia) glutathione-Sepharose columns were loaded with an E. coli digestion containing GST-AmSu (see above). HCl solutions with 140 mM NaCl and the pH values 6, 5, 4, 3 and 2 were prepared. Each 4 ml of pH was added to the column and 1 ml fractions were collected. An amylosucrase activity test was carried out and it became clear that the protein was already detached from the column when the pH was lowered to pH 6 ( FIG. 5). This elution method is gentle and cheap at the same time. Only then is the industrial use of affinity purification possible in this special case.

Example 6 Biocatalysis with GST-AmSu immobilized on Sepharose

About 50 U GST-AmSu were mixed with 5 ml Biotrafo buffer (20% sucrose, 50 mM Na citrate pH 6.5, 0.02% NaN ₃ ). The mixture was incubated at 37 ° C. with shaking (180 rpm). The course of the reaction - and thus the polymer formation - was followed by the sucrose consumption and the fructose formation.

Fig. 6 shows the typical course of a biocatalysis with the bound enzyme (GST-AMSU to Sepharose). It was found that the entire saccharose was consumed after about 48 hours. The fructose concentration rose accordingly from 0% to 7.6% within this time. During this time, a water-insoluble amylose precipitate was formed ( Fig. 7).

Example 7 Quality of those produced by biocatalysis with immobilized GST-AmSu Amylose

A biocatalysis approach according to Example 6 was freeze-dried after the end of the reaction and taken up in DMSO. The distribution of the molecular sizes of the amylose molecules was determined by means of a GPC analysis. Fig. 8 shows the size distribution of the poly (1,4-alpha-glucan).

Example 8 Production of microparticles from the process product

The poly (1,4-alpha-glucan) obtained can be converted into very small, water-insoluble microparticles, as in the application WO 99/11695 a special quality of amylose is necessary. The amylose obtained in biocatalysis with an immobilized enzyme has this special quality standard. In FIG. 8, microparticles consist of amylose to see. Poly (1,4-alpha glucan) according to Example 6 was used as an approach. The microparticles were prepared as follows: 1 g of poly (1,4-alpha-glucan) was dissolved in 5 ml of DMSO at 60 ° C. The poly (1,4-alpha-glucan) in solution was added to 100 ml of water with stirring. The microparticles were precipitated at 5 ° C. overnight and then washed, frozen and freeze-dried.

Example 9 Multiple use of the loaded glutathione-Sepharose as a catalyst

Due to their different sizes (see FIG. 6), the Sepharose beads and the microparticles formed during the catalysis can be easily separated from one another by filtration with a glass frit. The separated Sepharose beads can be used for new biotransformations. Fig. 10 shows the results of multiple operations AMSU GST-loaded glutathione sepharose. The multiple approaches are comparable to the approach described in Example 6.

Example 10 Regeneration of the glutathione-Sepharose beads

Biocatalysis forms an amylose layer around the catalysts that Mass transfer between the catalyst and the Biotrafo buffer hampers and ultimately even prevented. To regenerate the glutathione-Sepharose beads, the biocatalysts are washed with DMSO and can be rinsed with PBS again with GST-AmSu - e.g. B. from a digestion of recombinant E. coli's - be loaded.

Example 11 Recycle approach

GST-AmSu is eluted from a loaded glutathione-Sepharose using pH shift (see example 5) and used for a biotransformation analogous to example 6. After complete conversion, the product poly (1,4-alpha-glucan) by centrifuge  gation separated from the reaction supernatant with the GST-AmSu. The GST-AmSu was then bound again to glutathione-Sepharose and consequently isolated. To the amylosucrase was eluted in a washing step (pH shift) and with new bio transformer buffer offset.  

literature

Bucke, C. (1998) Polysaccharide biotechnology - a Cinderella subject. Trends in bio technol. 16: 50-53.
De Montalk, GP, Remaud-Simeon, M., Willemot, RM, Planchot, V. and Monsan, P. (1999) Seuquence analysis of the gene encoding amylosucrase from Neisseria polysaccharea and characterization of the recombinant enzyme. J. Bacteriol. 181, 375-381.
MacKenzie, CR, Perry, MB, McDonald., IJ and Johnson, KG (1977) Glycogen synthesis by amylosucrase from Neisseria perflava. Can. J. Microbiol. 23: 1303-1307.
Nelson, JM and Griffin, EG (1916) Adsorption of invertase. J. Am. Chem. Soc. 38: 1109-1115.
Saleemuddin, M. (1999) Bioaffinity based immobilization of enzymes. Adv. Biochem. Closely. Biotechnol. 64: 203-226.

Description of the figures

Fig. 1: Plasmid map of pGEX-4T-1-AmSu

Figure 2: Plasmid map of pGEX-4T-1-AmSu5

Fig. 3: A) SDS-PAGE stained with Coomassie. Lanes 1 and 2: GST-AmSu eluted with thrombin; Lane 3: Disruption of E. coli cells carrying the plasmid pGEX-4T-1 AmSu after the IPTG induction; Lane 4: Disruption of E. coli cells carrying the plasmid pGEX-4T-1-AmSu before the IPTG induction. The arrows point to the GST-AmSu and to the smaller form that results from the thrombin cleavage.

B) SDS-PAGE stained with Coomassie. Lanes 1 and 2 show this with glutathione eluted GST-AmSu protein. Lane 3 shows a solid sample of GST-AmSu bela which glutathione-Sepharose.

Fig. 4: Alternative elution of the GST-AmSu from the glutathione-Sepharose matrix with different pH values. The eluate was collected in 1 ml volume and tested for amylosucrase activity. Fraction No. 5 contains about 50% of the total activity.

FIG. 5 shows a course of biocatalysis with immobilized GST-AMSU.

Fig. 6: Microscopic image of a biocatalysis approach with immobilized GST-AmSu (magnification 100 times). In the center of the picture is a Sepharose bead loaded with GST-AmSu. The catalyst is surrounded by NEO amylose beads.

Fig. 7: Size distribution of amylose in the amylose-NEO according to GPC analysis. The average molecular weight is 4350 g / mol.

Fig. 8: Electron micrograph of microparticles that were made from NEO amylose.

Fig. 9: Course of biocatalysts with multiple catalyst.

Sequences

Claims (13)

1. Immobilizable amylosucrase on a solid phase containing a fusion protein consisting of the functional units of an amylosucrase and one Anchor sequence and possibly further auxiliary sequences. 2. Fusion protein according to claim 1 with an amino acid sequence according to SEQ ID No. 8 or a functional variant or parts thereof with at least 6 Ami non-acids, whereby SEQ ID No. 8 is part of the claim. 3. Nucleic acid coding for an immobilizable amylosucrase according to claim 1 and 2, characterized in that the nucleic acid is a DNA with a nu Small acid sequence according to SEQ ID No. 5, available from SEQ 1D No. 4, ligated with an anchor sequence or a functional variant or parts thereof with minde at least 8 nucleotides, whereby SEQ ID No. 5 is part of the claim. 4. Expression vector containing a nucleic acid according to claim 3, characterized ge indicates that in a host cell the fusion protein according to claim 1 or 2 is expressed. 5. Host cell according to claim 4 selected from E. coli. 6. Expression vector according to claim 4 and 5, characterized in that the Expression vector from a nucleic acid sequence according to SEQ ID No. 7 contains. 7. anchor sequence according to claim 3, characterized in that the nucleic acid sequence encoded for an epitope. 8. anchor sequence according to claim 3, characterized in that the nucleic acid sequence encoded for a binding partner with high affinity. 9. anchor sequence according to claim 3, characterized in that the nucleic acid sequence encoded for glutathione-S-transferase.   10. solid phase according to claim 1 and 2 and claim 9, characterized in that it consists of glutathione-Sepharose. 11. Compound stable catalyst containing an immobilized amylo sucrase on Sepharose according to claims 1 and 2 and claims 9 and 10 for Production of poly (1,4-alpha-glucan). 12. A process for the biocatalytic production of poly (1,4-alpha-glucan), characterized in that an immobilized fusion protein on a solid phase with the enzymatic activity of an amylosucrase according to claim 1 or 2:

• a) converting sucrose to poly (1,4-alpha-glucan) with vigorous shaking;
• b) and after separation from the reaction broth with DMSO with solution of poly (1,4-alpha-glucan) rinsed and washed with PBS DMSO-free
• c) and if necessary (after) loading.

13. Use of the catalyst according to claim 11 and the method according to An Proverb 12 for the production of microparticles.