WO1999015636A1

WO1999015636A1 - Expression of starch-binding domain (sbd)

Info

Publication number: WO1999015636A1
Application number: PCT/DK1998/000411
Authority: WO
Inventors: Steen Troels JØRGENSEN; Torben Peter Frandsen; Carsten Andersen
Original assignee: Novo Nordisk A/S
Priority date: 1997-09-24
Filing date: 1998-09-23
Publication date: 1999-04-01
Also published as: JP2003526314A; AU9254198A; EP1023438A1

Abstract

The present invention relates to construction and expression of stable starch-binding domain from maltogenic amylases and related enzymes.

Description

Expression of starch-binding domain (SBD)

THE FIELD OF THE INVENTION The present invention relates to a DNA construct encoding a starch-binding domain being a region in the maltogenic amylase enzyme product produced by Bacillus stearothβrmophilus C599 disclosed in EP patent no. 120,693 (Novo Industri A/S), the starch-binding domain itself, an expression vector comprising the starch-binding domain of the invention, a host cell transformed with said expression vector of the invention, a method for expressing the starch-binding domain of the invention in a Bacillus sp. host strain transformed with the expression vector of the invention.

BACKGROUND OF THE INVENTION

EP patent no. 120,693 discloses a maltogenic amylase derived from Bacillus stearothermophilus C599.

Starch-binding domain

In the following "Starch Binding Domain" will be abbreviated as "SBD" and is meant to define all polypeptide sequences or pep- tide sequences having affinity for binding to starch.

Most known SBDs today are found in CGTases, i.e. cyclodextrin glucanotransferases (E.C. 2.4.1.19), and glucoamylases (E.C. 3.2.1.3). See also Chen et al. (1991), Gene 991, p. 121-126, describing Starch Binding Domain hybrids.

Specifically, a SBD has been found in the commercially available enzyme AMG™ (a glucoamylase) from Aspergillus niger (Belshaw et al. (1993), "Specificity of the binding doamin of glycoamylase I" Eur. J. Biochem, 211:717-724). Furthermore, a SBD from a CGTase derived from Bacillus macerans has also been described. Dalmia et al. (1995), Biothech. Bioeng., Vol 47, pp. 576-584 describes expression of the starch-binding domain (E-domain) of glucoamylase I from Aspergillus awamori and of a CGTase from Ba cillus macerans in E. coli .

Expression of SBDs in E. coli is not a true extracellular expression and results in an unsatisfactory yield which is too low for industrial scale production of SBD. Further, E. coli expression results in intracellular none-soluble protein aggregate (i.e. inclusion bodies).

Accordingly, there is a need for a method for producing SBDs in a high yield and/or by means of a conventional fermentation technique involving extracellular production of the SBDs which makes the use of SBDs in industrial applications economically feasible.

SUMMERY OF THE INVENTION

It is the object of the present invention to provide single SBDs (or isolated SBDs) and to express the single SBDs in a Bacillus host cell.

Single starch-binding domain (single SBD)

The term "single SBD" may also be referred to as "isolated SBD" or "separate SBD".

In the context of the present invention a "single SBD" includes up to the entire part of the amino acid sequence of a SBD- containing enzyme, e . g. a polysaccharide hydrolyzing enzyme, being essentially free of the catalytic domain, but retaining the SBD unit (s) .

Thus, in the context of the invention, the entire catalytic amino acid sequence of a starch degrading enzyme (e.g. a glu- coamylase) or other enzymes comprising one or more SBD units is not to be regarded as a single SBD.

Typically a single SBD constitutes one or more SBD units of a polysaccharide hydrolyzing enzyme, one or more SBD units of a starch binding protein or a protein designed and/or engineered to be capable of binding to starch.

The single SBD is at least as large as the minimum number of amino acids in the amino acid sequence required to bind to starch.

A single SBD may also be an amino acid sequence, in which the binding and catalytic domain are one and the same.

Isolation of a starch-binding domain In order to isolate the starch-binding domain of e.g. a glucoamylase, several genetic approaches may be used. One method uses restriction enzymes to remove a portion of the gene and then to fuse the remaining gene-vector fragment in frame to obtain a mutated gene that encodes a protein truncated for a particular gene fragment. Another method involves the use of exonucleases such as Bal31 to systematically delete nucleotides either externally from the 5' and the 3' ends of the DNA or internally from a restricted gap within the gene. These gene deletion methods result in a mutated gene encoding a shortened gene molecule which may then be evaluated for starch binding ability.

Enzyme-SBD hybrid construction

Once a DNA sequence encoding the starch binding domain has been identified, either as cDNA or chromosomal DNA, it may then be manipulated in a variety of ways to fuse it to a DNA sequence encoding an enzyme of interest. The starch-binding domain encoding fragment and the DNA encoding the enzyme of interest are then ligated with or without a linker. The resulting ligated DNA may then be manipulated in a variety of ways to provide for expression. Microbial hosts such as Aspergillus, e . g. , A. niger and A. oryzae, Bacillus, E. coli or S . cerevisiae are preferred.

In the first aspect the invention relates to an isolated DNA sequence comprising a DNA sequence encoding the E-domain (SEQ ID NO: 2) of the maltogenic amylase produced by Bacillus stea- rothermophilus C599, without having the enzymatic activity of the maltogenic amylase produced by Bacillus stearothermophilus C599. The Bacillus stearothermophilus C599 is described in EP 120,693.

In the second aspect the invention relates to a single SBD polypeptide encoded by a DNA sequence of the invention encoding a SBD of the invention, in particular the ones shown in SEQ ID NO: 2 (E domain) or SEQ ID NO: 4 (D+E domain) .

In the third aspect the invention relates to a Bacillus host transformed with a vector comprising a DNA sequence coding for a starch-binding domain of the invention and which is capable of expressing said sequence. In the forth aspect the invention relates to a Bacillus expression vector which carries an inserted DNA sequence encoding for a starch-binding domain, in particular an isolated DNA sequence of the invention as described above.

Further, the invention relates to a method of producing a single starch-binding domain polypeptide in a Bacillus host cell, the method comprising the steps of:

- growing under conditions to overproduce starch-binding domains in a nutrient medium a Bacillus host cell which has been transformed with an expression cassette which includes, as operably joined components: a) a transcriptional and translational initiation regulatory region, b) a DNA sequence encoding the starch-binding domain polypeptide, C) a transcriptional and translational termination regulatory region, wherein the regulatory regions are functional in the host, and d) a selection marker gene for selecting transformed host cells; and

- recovering the starch-binding domain polypeptide.

The invention also relates to a method for optimization of SBD expression in a Bacillus host, the method comprising the steps of: a. expression in the host of a SBD fused to a reporter molecule; b. monitoring the concentration of expressed SBD in the supernatant of the fermented host by measuring the intrinsic property or properties of the reporter molecule.

Finally the invention relates to a method of producing a hybrid, wherein the hybrid is expressed in a Bacillus host, growing the transformed host under conditions whereby the transformed culture is substantially free of un-transformed cells; incubating the transformed culture in a nutrient medium, whereby the hybrid is overproduced; and recovering the hybrid.

BRIEF DESCRIPTION OF THE DRAWING

Figure 1 shows the sequence alignment (of the D and E-domain) of the maltogenic amylase from B . stearothermophilus C599 and B . circulans strain 251 CGTase. Written in "Bold" are residues in^¬ volved in maltose binding via their side-chain and written in " Italic" are residues with side-chains involved in maltose binding in MBS 2. S=sequence no.; 1= The B. stearothermophilus C599 maltogenic amylase sequence; 2= The B . circulans strain 251 CGTase sequence; D=domain.

Figure 2 shows the SDS-PAGE gel of the purified E-domain single SBD: Lane 1 and 3: Molecular weight standards (from above): 94, 67, 43, 30, 20, 14 kDa; lane 2: single SBD (E-domain).

DETAILED DESCRIPTION OF THE INVENTION

The inventors of the present invention found by alignment of the amino acid sequence (in the D and E domains) of the Bacillus stearothermophilus C599 maltogenic amylase that it had a high identity to CGTases, in particular to a Bacillus circulans strain 251 CGTase (Lawson, C.L., Montfort, R.v., Strokopytov, B., Rozeboom, H.J., Kalk, K.H., Vries, G.E.d., Penninga, D. , Dijkhuizen, L. and Dijkstra, B. 1994; J. Mol. Biol. 236, p.590- 600) .

The identity for the E-domains was found to be 48% and the identity for the D-E-domains 45% using the "align" program based on the Smith-Waterman method and BLOSUM45 table (Henikoff S, Henikoff JG, 1992, Proc. Natl. Acad. Sci. USA89: 10915-10919) . The structure of the B . circulans strain 251 CGTase in complex with maltose was published by Lawson C.L. et al. (1994), supra) and can be found in Brookhaven data-bank under the code lCDG.pdb. The CGTase structure consists of five domains of which the A, B and C domains are similar to the domains found in α-amylases. The additional domains named D and E, respectively, are unique for CGTases.

The function of the D-domain is believed to form a stable linkages between the E-domain and the catalytic active domains. In the E-domain two maltose-binding sites, denoted maltose binding site 1 (MBS 1) and maltose binding site 2 (MBS 2) have been identified in the structure. W616 and W662 are stacking with the glucose rings and the side-chains of K651 and N667 are forming hydrogen bonds to -OH groups at the glucose rings in MBS 1.

As indicated in the alignment (see Figure 1) all four residues are also found in the maltogenic amylase. The MBS 2 is defined by stacking with Y633 and H-bonds to the side-chain of residue T598, N603, N627 and Q628. Also in the maltogenic a y- lase the positions T598, Q628 and Y633 are present, whereas the N603 position is substituted by K (Lys) which is also potential to form H-bond to -OH groups. No analogue to N627 is present. Based on the alignment and the present of the residues analogue to MBS defining residues in the CGTase it is most likely that the maltose binding sites are also found in the maltogenic amylase. The high homology between the CGTase and the maltogenic amylase indicates the same overall structure, and the structure of the CGTase can therefore be used for determining the different domains in the maltogenic amylase. Using the alignment and the CGTase structure the starting point of the D-domain was determined to amino residue 494 and the E-domain determined to start with amino residue 576. The theoretical sizes of D-E and E-domains are therefore 193 amino acids and 111 amino acids respectively, corresponding to 20 kDa and 12 kDa.

As described above the inventors have found that the maltogenic amylase product produced by Bacillus stearothermophilus C599 disclosed in EP patent no. 120,693 (Novo Industri A/S) comprises a SBD in the C-terminal part of the protein sequence.

In the first aspect the invention relates to an isolated DNA sequence comprising a DNA sequence encoding the E domain of the maltogenic amylase produced by Bacillus stearothermophilus C599 without having the enzymatic activity of the maltogenic amylase produced by Bacillus stearothermophilus C599. The E domain coding DNA sequence is the sequence shown in SEQ ID NO: 1. The corresponding protein sequence is shown in SEQ ID NO: 2. In an embodiment the isolated DNA sequence of the invention further comprising the D-domain of the maltogenic amylase produced by Bacillus stearothermophilus C599 without having the enzymatic activity of the maltogenic amylase produced by Bacillus stearothermophilus C599. In other words, the isolated DNA se- quence contains the DE-domain coding DNA sequence. The DE-domain coding sequence is shown in SEQ ID NO: 3. The DE domain protein sequence is shown in SEQ ID NO: 4. In a second embodiment the invention relates to a single SBD with starch-binding affinity encoded by the above-mentioned iso^¬ lated DNA sequences of the invention.

The single SBD with starch-binding affinity is shown in SEQ ID NO: 2 (E-domain) and SEQ ID NO: 4 (DE-domain) .

Example 1 below describes the construction of an expression vector and expression of the single SDB from the maltogenic amylase from Bacillus stearothermophilus C599 in a Bacillus host . Further, in the third aspect the invention relates to a method of producing a single starch-binding domain polypeptide in a Bacillus host cell, the method comprising the steps of:

- growing under conditions to overproduce the starch-binding do^¬ main, in a nutrient medium, a Bacillus host cell which has been transformed with an expression cassette which includes, as op- erably joined components: a) a transcriptional and translational initiation regulatory region, b) a DNA sequence encoding the starch-binding domain polypep- tide, c) a transcriptional and translational termination regulatory region, wherein the regulatory regions are functional in the host, and d) a selection marker gene for selecting transformed host cells; and

- recovering the starch-binding domain polypeptide.

Several SBDs have been expressed in E . coli , however, none have been reported to be expressed and secreted from Bacill us sp. E . coli , as an expression host for heterologues proteins has several advantages over Bacillus spp. , firstly because E . coli has a periplasmic space where proper folding of heterologues expressed genes is possible (for review see for example Hockney, R.C. (1994) TIBTECH, vol.12, p. 56-463). Especially the oxidiz- ing potential and the existence of disulfide oxidoreductases in the periplasma are necessary when expressing proteins with a functionality dependent on properly arranged disulfide bridges (Emmanuel Brun et al. (1995) . Furthermore, the periplasma of E. coli also acts in protecting the heterelogously expressed protein towards the action of proteases present in the supernatant as well as the cytoplasm. It is also known that when expressing secreted proteins with disulfide bridges in Bacillus subtilis the level of expression drops significantly (Bertus van den Berg et al., (1993), Introduction of disulfide bonds into Bacillus subtilis neutral protease. Protein Engineering, vol.6 no.5, p. 521-527).

Another problem with heterologue expression is the prote- olytic degradation of the expressed protein. Bacillus subtilis is known to express at least 7 different extracellular proteases (Eds. A.L. Sonenshein, J.A. Hoch and Richard Losick (1993) Bacillus subtilis and other Gram-Positive Bacteria, American Society for microbiology, p.939).

Especially for SBDs, which are highly hydrophobic proteins, the translocation of the protein when expressed in Bacillus subtilis could be severely hampered and even cause cell death due to deleterious effects if the protein gets anchored to the cell membrane because of its hydrophobicity.

In the forth aspect the present invention relates to a Bacil- lus host cell transformed with a vector comprising a DNA sequence coding for a SBD and capable of expressing the sequence.

In a preferred embodiment, the expressed single SBD or SBD- containing polypeptide has a molecular weight (M_w) which is equal to or higher than about 4 kD. Preferably, the M_w is equal to or below about 35 kD, more preferably about 32 kD, even more preferably about 30 kD, especially about 25 kD. As mentioned above the D-domain of Bacillus stearothermophi lus C599 has a M_w around 12 kD and the DE-domain a M_w around 20 kD.

The SBD may be expressed in the form of a single SBD as de- fined above, i . e . , a polypeptide comprising one SBD. Alternatively, the SBD may be expressed in the form of a dimer or trimer or even a polymer, i . e . a polypeptide or protein comprising two, three, or even more than three identical SBD "units". The SBD can also be expressed as a part of a multi-domain polypeptide, the non-SBD part of such a polypeptide being for example one, two or even more domains without catalytic activity.

Most SBDs can be expressed according to the present invention, i.e. by means of a transformed Bacillus host. In a pre- ferred embodiment SBDs, obtainable from a microorganism or a plant, more preferably from a bacterium or from a fungus, are expressed.

Examples of SBDs from bacteria and fungus include SBDs obtainable from species mentioned above in the "Background" sec- tion, in particular the ones belonging to bacteria genus Bacil^¬ lus and the fungus genus Aspergillus .

The Bacillus host cell of the invention is a neutralo- philic or an alkalophilic or a mesophilic or a thermophilic host cell. EX Examples of hosts which are useful in the context of the present invention are hosts from the species Bacillus subtilis , Bacillus licheniformis, Bacillus megaterium, Bacillus stea- rothermophilos , and Bacillus amyloliquefaciens . However, it is contemplated that other Bacillus species may also be useful hosts for expression of SBDs.

As described in further detail below, the host cell of the invention is transformed with a vector comprising a SBD encoding DNA sequence. Preferably, the vector is integrated into the genome of the host, more preferably it has been amplified on the genome .

In another preferred embodiment of the invention, the vector is present as an expression plasmid, preferably as a multi- copy plasmid.

In the fifth aspect the present invention relates to a Bacillus expression vector which carries an inserted SBD-encoding DNA sequence. Preferably, the expression cassette of the vector comprises regulatory regions from a Bacillus sp. , more preferably are such regulatory regions endogenous to the host.

In a sixth aspect, the present invention relates to a method for producing a SBD polypeptide, the method comprising the steps of:

- growing under conditions to overproduce the starch-binding do- main, in a nutrient medium, Bacillus host cells which have been transformed with an expression cassette which includes, as op- erably joined components, a) a transcriptional and translational initiation regulatory region, b) a DNA sequence encoding the starch-binding domain polypeptide, c) a transcriptional and translational termination regulatory region, wherein the regulatory regions are functional in the host, and d) a selection marker gene for selecting transformed host cell; and

- recovering the starch-binding domain polypeptide.

In its seventh aspect, the present invention relates to a method for optimization of SBD expression in a Bacillus host, the method comprising the steps of expression in the host of a SBD fused to a reporter molecule; and monitoring the concentration of expressed SBD in the supernatant of the fermented host by measuring the intrinsic property or properties of the reporter molecule.

In a preferred embodiment, the reporter molecule is a Green Fluorescent Protein, and the intrinsic property is fluorescence emission.

In its eighth and ninth aspect, the invention relates to a polypeptide hybrid consisting essentially of one or more starch- binding domain (s) fused to a green fluorescent protein, and to a method of producing such a hybrid by expression in a Bacillus host, growth of the transformed host under conditions whereby the transformed culture is substantially free of untransformed cells; incubation of the transformed culture in a nutrient medium, whereby the hybrid is overproduced; and recovery of the hybrid.

EXPRESSION OF A SINGLE SBD

Recombinant expression vectors

A recombinant vector comprising a DNA construct encoding the single SBD of the invention may be any vector which conveniently may be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell, into which it is to be introduced. This introduction of vector into the host cell is often referred to as the transformation of the host cell. Such transformation indicates introduction of DNA into a host cell by using, e.g. protoplasts, natural competent cells, transfection, conjugation, electroporation, or any equivalent method. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome in part or in its entirety and replicated together with the chromosome (s) , into which it has been integrated.

The vector is preferably an expression vector in which the DNA sequence encoding the single SBD of the invention is operably linked to additional segments required for transcription of the DNA. In general, the expression vector is derived from plasmid or viral DNA, or may contain elements of both. The term, "operably linked" indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in a promoter and proceeds through the DNA sequence coding for the SBD.

The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.

Examples of suitable promoters for use in bacterial host cells include the promoter of the Bacillus stearothermophilus maltogenic amylase gene, the Bacillus licheniformis -amylase gene, the Bacillus amyloliquefaciens α-amylase gene, the Bacillus subtilis alkaline protease gene, or the Bacillus pumilus xylosidase gene, or the phage Lambda P_R or P_L promoters or the E. coli lac, trp or tac promoters. Alternatively, it is possible to design integration vectors such that the DNA encoding the SBD will only become functionally expressed once it is properly integrated into the host genome, e.g. downstream from a resident promoter.

The DNA sequence encoding the SBD of the invention may also, if necessary, be operably connected to a suitable terminator. The recombinant vector of the invention may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.

The vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, or a gene encoding resistance to e.g. antibiotics like kanamycin, chloramphenicol, erythromycin, tetracycline, spectinomycine, or the like, or resistance to heavy metals or herbicides.

To direct a SBD of the present invention into the secretory pathway of the host cells, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) may be provided in the recombinant vector. The secretory signal sequence is joined to the DNA sequence encoding the SBD in the correct reading frame. Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the. The secretory signal sequence may be that normally associated with the SBD or may be from a gene encoding another secreted protein.

The procedures used to ligate the DNA sequences coding for the present SBD, the promoter and optionally the terminator and/or secretory signal sequence, respectively, or to assemble these sequences by suitable PCR amplification schemes, and to insert them into suitable vectors containing the information necessary for replication or integration, are well known to persons skilled in the art (cf., for instance, Sambrook et al., op.cit. ) .

Green Fluorescent Protein (GFP) has become a widely used reporter molecule for monitoring gene expression, tracers of cell lineage and as fusion tags for proteins. (Andreas Crameri et al. (1996) Improved Green Fluorescent Pro- tein by molecular evolution using DNA shuffling, Nature Biotechnology, Vol. 14, p. 315-319; Andrew B. Cubitt et al. (1995), Understanding, improving and using fluorescent proteins, TIBS, Vol. 20, p. 448-455.

GFP could be fused to SBD' s creating a fusion protein having the starch-binding property as well as the fluorescent properties. The expression of this fusion protein could be used to monitor the expressing of SBD' s in Bacillus species and hereby be used to optimize expression levels of given SBD's. EXAMPLES

MATERIALS AND METHODS Strains : Bacillus stearothermophilus C599 (EP 120,683) comprises the maltogenic amylase.

Bacillus subtilis DN1885 and Toc46 Diderichsen et al., (1990), Journal of Bacteriology, Vol. 172, p. 4315-4321)

Plasmids: pDN1981 (P.L. Jørgensen, C.K.Hansen, G.B.Poulsen and B. Diderichsen, (1990), In vivo genetic engineering: homologues recombination as a tool for plasmid construction, Gene, 96, p. 37- 41.)

General molecular biology methods :

DNA manipulations and transformations were performed using standard methods of molecular biology (Sambrook et al., (1989), Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, F. M. et al. (eds.) "Current protocols in Molecular Biology". John Wiley and Sons, 1995; Harwood, C. R., and Cutting, S. M. (eds.) "Molecular Biological Methods for Bacillus". John Wiley and Sons, 1990).

Enzymes for DNA manipulations were used according to the specifications of the suppliers.

EXAMPLES

EXAMPLE 1 Construction of an expression vector encoding a single SBD

Oligonucleotide PCR primers were designed to express the E- domain alone and the D+E domain part of the AmyM protein (i.e. maltogenic amylase of Bacillus stearothermophilus C599) . The ra- tionale was to add the signal sequence of the Bacillus licheni - formis α-amylase (AmyL, Termamyl™, see P.L. Jørgensen et al. (1990), Gene, 96, p. 37-41) in front of these AmyM fragments in attempts to have the proteins secreted from Bacillus . The following primers were used: #110755:

Pstl 5'-GATGCTGCAGCAGCGGCGTCCGCTTCAGCGCCGC-3' (SEQ ID NO: 5) (the underscored region corresponds to pos. 1783-1798 in the amyM sequence, Genbank Accession nb. M36539)

#110756:

Pstl 5' -GATGCTGCAGCAGCGGCGAGTGGAACGCAGACATCG-3' (SEQ ID NO: 6) (the underscored region corresponds to pos. 2032-2049 in the amyM sequence, Genbank Accession nb. M36539)

#110757:

EcoRI BamHI 5' -GATGGAATTCGGATCCTCCATATGTACTACTCC-3' (SEQ ID NO: 7)

(the underscored region corresponds to pos. 2569-2553 in the amyM sequence, Genbank Accession nb. M36539)

Template for the PCR reaction was a sample of plasmid pDN1413. This is essentially plasmid pUBllO containing the amyM gene fragment, derived from the deposited strain NCIB 11837 via plasmid pDN452 which is described in EP 120,693.

Conditions for the PCR amplification were the following: 94°C for 2 minutes, then 20 cycles of 49°C for 30 seconds, 43°C for 1 minute, 72°C for 2 minutes, then one cycle of 72°C for 5 minutes.

Correctly sized PCR fragments were obtained upon amplifica^¬ tion. Primer #110755 together with #110757 resulted in a 820 basepair fragment, primer #110756 together with #110757 resulted in a 571 basepair fragment.

PCR fragments were purified using a QIAquick PCR Purification Kit Cat. No. 28106 from Qiagen, and digested with EcoRI + Pstl. Plasmid pDN1981 (P.L. Jørgensen et al. (1990), Gene, 96, p. 37-41) was used as cloning vector. pDN 1981 was digested with EcoRI + Pstl, and the 3.9 kb fragment purified from an agarose gel. The vector fragment was ligated with each of the digested PCR fragments, and the ligation mixtures transformed into compe- tent cells of Bacillus subtilis DN1885 (Diderichsen et al.,

Journal of Bacteriology, vol. 172, p. 4315-4321, 1990), selecting kanamycin resistance (10 μg/ml) . Four colonies from each transformation were reisolated and grown in liquid TY cultures for plasmid preparation. The extracted plasmids all had the correct structure, as judged by restriction digests. Two transformants of each kind were kept: SJ4302 and SJ4303 both contained plasmids harboring the #110755 + #110757 PCR fragment, i.e. encoding the D+E domain. SJ4304 and SJ4305 both contained plasmids harboring the #110756 + #110757 PCR fragment, i.e. encoding the E-domain only.

Expression of domains:

Strains SJ4302-SJ4305 were inoculated in 10 ml TY broth containing 0.4% glucose and lOμg/ml kanamycin, and incubated at 37°C with shaking for two days.

Strain DN1885 (the B . subtilis host strain) was inoculated in 10 ml TY broth with 0.4% glucose, and incubated at 37°C with shaking for two days.

Supernatants were analyzed by SDS-Polyacrylamide gel electro- phoresis.

In the supernatant from strain SJ4302, a protein with an apparent molecular weight of approximately 25 kDa was observed. It was less obvious that SJ4303 produced a similar protein. This protein was not seen in the other 3 samples.

The difference between SJ4302 and SJ4303 may be due to these clones harboring PCR amplified constructs, that were not veri- fied by DNA sequencing - an error might thus have been introduced into the SJ4303 clone.

In the supernatant from strains SJ4304 and SJ4305, a protein with an apparent molecular weight of approximately 10 kDa was observed. This protein was not observed in the other three sam- pies.

In conclusion, a protein as expected was produced from the D+E domain clone SJ4302, and a protein as expected was produced from the E-domain clones SJ4304-SJ4305.

No difference in expression level (amount of accumulated do- main) was observed when the strains were simply propagated as above, or when the strains were propagated in broth as above, but supplemented with the protease inhibitor Complete from Boe- hringer Mannheim (CompleteTM Protease inhibitor cocktail tablets Cat. No. 1697498; One tablet was dissolved in 2 ml water, and 160 microliters of this solution added to each 10 ml culture) . This concentration of protease inhibitor allowed growth, but almost totally inhibited the extracellular proteases present in the DN1885 broth, as judged from spotting broth on agar plates with casein.

EXAMPLE 2

Expression and purification of single SBD (E-domain) .

Expression of the E-domain

PSJ4305 (E-domain clone) was transformed into competent cells of B . subtilis ToC46 (Diderichsen et al., Journal of Bacteriology, vol. 172, p. 4315-4321, 1990), selecting kanamycin resistance (10 mg/ml) , and a transformant was kept as SJ4547. 50 shake flasks of 500 ml total volume, each containing 200 ml of TY broth supplemented with 0.4% glucose and 10 mg/ml kanamycin, were inoculated with SJ4547 and incubated with shaking at 300 rpm at 37 °C for 45 hours, and the supernatant separated from the cells by centrifugation.

Purification of the E-domain

The supernatant was centrifuged (4500 rpm, 15 min, 8°C) using a Sorvall RC-3B centrifuge, equipped with a 4600 A rotor head, followed by subsequent filtration through a 0.7 mm glass mi- crofibre filter. The supernatant was applied to α-cyclodextrin- agarose (1.6 x 5 cm) in 25 mM sodium acetate, 1 mM CaCl₂, 0.5 M NaCl, pH 5.0, at a flow rate of 300 ml h^"1. The column was washed (10 column volumes) using 25 mM sodium acetate, 1 mM CaCl₂, 0.5 M NaCl, pH 5.0, and the single SBD was eluted in the same buffer containing 2% (w/v) α-cyclodextrin. The eluted SBD was pooled and dialyzed against 50 mM sodium acetate, 1 mM CaCl₂, pH 5.0. The purified single SBD was homogeneous as determined using SDS- PAGE (see below) . N-terminal sequencing showed SGTQTSVVF and confirmed that the purified E-domain is identical to residue

Ser576 of the maltogenic amylase of Bacillus stearothermophilus C599.

Approximately 21 mg homogeneous SBD was purified from 1 L culture filtrate in ten subsequent runs on α-cyclodextrin- agarose. SDS-PAGE analysis of the purified SBD is shown in Figure 2.

Example 3

Starch binding of single SBD Adsorption of single SBD onto granular starch is determined by incubating increasing amounts of SBD (0-3 mg/ml) with granular corn starch (10 mg/ml) in 5 mM sodium acetate, pH 3.6 at 4°C for 16 hours, essentially as described (Belshaw & Wil- liamson, 1990) . The reaction is terminated by centrifugation and the protein concentration in the supernatant is subsequently determined and subtracted from the total protein to give the amount of starch bound protein.

Claims

1. An isolated DNA sequence comprising a DNA sequence encoding the E-domain of the maltogenic amylase produced by Bacillus stearothermophilus C599 without having the enzymatic activity of the maltogenic amylase produced by Bacillus stearothermophilus C599.

2 . The isolated DNA sequence according to claims 1, wherein the E domain sequence is the sequence shown in SEQ ID NO: 1.

3. The isolated DNA sequence according to claims 1 or 2 further comprising the D-domain of the maltogenic amylase produced by Bacillus stearothermophilus C599 without having the enzymatic activity of the maltogenic amylase produced by Bacillus stea - rothermophilus C599.

4. The isolated DNA sequence according to claims 3, wherein the D+E domain sequence is the sequence shown in SEQ ID NO: 3.

5. A single SBD with starch-binding affinity encoded by a DNA sequence according to any of claims 1 to .

6. The single SBD according to claim 5 having the sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4.

7. A Bacillus host cell transformed with a vector comprising a DNA sequence coding for a single starch-binding domain and which is capable of expressing said sequence.

8. The host cell according to claim 7, wherein the DNA sequence is of another origin than Bacillus ssp.

9. The host cell according to any of claims 7 or 8 which is capable of expressing the starch-binding domain as a single polypeptide domain.

10. The host cell according to any of the claims 7-9, wherein the vector comprises a DNA sequence encoding for a single starch-binding domain.

11. The host cell according to any of the claims 7-10, wherein the vector comprises a DNA sequence encoding for a starch- binding domain which is linked to at least one other non- catalytically active domain.

12. The host cell according to any of the claims 7-11, wherein the starch-binding domain is obtainable from a microorganism or from a plant, preferably a bacterium or a fungus.

13. The host cell according to claim 12, wherein the bacterium is selected from the group consisting of the genus Bacillus .

14. The host cell according to claim 13, wherein the fungus is selected from the group consisting of the genus Aspergillus .

15. The Bacillus host cell according to any of the claims 7-14 which is neutralophilic, alkalophilic, mesophilic or thermophilic.

16. The Bacillus host cell according to claim 15 which is selected from the group comprising the following species: Bacillus sujbtilis, Bacillus licheniformis , Bacillus mega terium, Bacillus stearothermophilos , and Bacillus amyloliquefaciens .

17. The host cell according to any of the claims 7-16, wherein the vector is integrated into the genome of the untransformed host.

18. The host cell according to any of the claims 7-17, wherein the vector is present as an expression plasmid.

19. The host cell according to any of the claims 7-18, wherein the vector has been amplified on the genome or the expression plasmid is a multicopy plasmid.

20. A Bacillus expression vector which carries an inserted DNA sequence encoding a single starch-binding domain.

21. The vector according to claim 20 in which the expression cassette comprises regulatory regions from a Bacillus sp.

22. The vector according to claim 21, wherein the Bacillus sp. regulatory regions are endogeneous to the host.

23. A method for producing a single starch-binding domain polypeptide in a Bacillus host cell, the method comprising the steps of

Γûá growing under conditions to overproduce starch-binding domain in a nutrient medium, a Bacillus host cell which has been transformed with an expression cassette which includes, as operably joined components, a) a transcriptional and translational initiation regulatory region, b) a DNA sequence encoding the starch-binding domain polypeptide, c) a transcriptional and translational termination regulatory region, wherein the regulatory regions are functional in the host, and d) a selection marker gene for selecting transformed host cells; and

- recovering the starch-binding domain polypeptide.

24. A method for optimization of SBD expression in a Bacillus host, the method comprising the steps of a. expression in the host of a SBD fused to a reporter molecule; b. monitoring the concentration of expressed SBD in the supernatant of the fermented host by measuring the intrinsic property or properties of the reporter molecule.

25. The method according to claim 24, wherein the reporter molecule is a Green Fluorescent Protein, and the intrinsic property is fluorescence emission.

26. A method of producing the hybrid according to claim 24 or 25, wherein the hybrid is expressed in a Bacill us host, growing the transformed host under conditions whereby the transformed culture is substantially free of un-transformed cells; incubating the transformed culture in a nutrient medium, whereby the hybrid is overproduced; and recovering the hybrid.