JP2003526314A - Expression of starch binding domain (SBD) - Google Patents

Abstract

  (57) [Summary] The present invention relates to the construction and expression of stable starch binding domains from maltogenic amylase and related enzymes.

Description

Detailed Description of the Invention

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

BACKGROUND OF THE INVENTION EP Patent No. 120,693 discloses maltogenic amylase derived from Bacillus stearothermophilus C599.

[0003] The starch binding domain or later, "starch binding domain" ( "S tarch B inding D omain ) is" SBD "
Is abbreviated, and is meant to define all polypeptides or peptide sequences that have a binding affinity for starch.

[0004] Today the most well-known SBD is the CGT _ase, that is, a cyclodextrin glucanotransferase (E.C.2.4.1.19) and glucoamylase (E.C.3.2.1.3 ) Can be found. See also Chen et al. (1991), Gene 991, p. 121-126, which publishes starch-binding domain hybrids.

Specifically, SBD can be found in the commercially available enzyme AMG ™ (glucoamylase) from A. niger (Belshaw et al.
1993) "Specificity of the binding domain of glycoamylase I" Eur. J. Bi
ochem, 211: 717-724). In addition, C derived from B. macerans
SBD from _GTase has also been announced.

Dalmia et al. (1995) Biotech, Bioeng., Vol. 47, pp. 576-584 is an E. Kori (E.co
li) and glucoamylase I from Aspergillus awamori (A. awamori)
And it has announced the expression of Bacillus macerans starch binding domain of the CGT _ase derived from (E- domain).

E. Expression of SBD in E. coli is not true extracellular expression and results in unsatisfactory yields that are too low for industrial scale production of SBD. Furthermore, E. E. coli expression results in the aggregation of insoluble proteins (ie inclusion bodies) within the cell.

Therefore, there is a need for a method of producing SBD in high yield and / or via conventional fermentation techniques involving extracellular production of SBD, and also economically achieving industrial applicability of SBD.

SUMMARY OF THE INVENTION The object of the present invention is to provide a single SBD (or isolated SBD) and to express this single SBD in Bacillus host cells.

Single Starch Binding Domain (Single SBD) The term "single SBD" may also mean "isolated SBD" or "separated SBD".

In the context of the present invention, a “single SBD” is essentially free of a catalytic domain, but up to the entire amino acid sequence of an SBD-containing enzyme, such as a polysaccharide hydrolase, which carries an SBD unit. including.

Thus, in the context of the present invention, the entire catalytic amino acid sequence of a starch degrading enzyme (eg glucoamylase) or other enzyme comprising one or more SBD units is not meant to be a single SBD.

[0013] Typically, a single SBD is one or more SBD units of a polysaccharide hydrolase, one or more SBD units of a starch-binding protein, or a protein designed and / or engineered to bind to starch. Make up.

The sole SBD is at least the size of the minimum number of amino acids in the amino acid sequence required for binding to starch.

A single SBD can also be an amino acid sequence in which the binding and catalytic domains are identical.

Isolation of Starch Binding Domains Several genetic approaches are available for the isolation of starch binding domains such as glucoamylase. One method utilizes restriction enzymes to remove a portion of the gene and then fuse the remaining gene-vector fragment in-frame to obtain a mutant gene encoding a truncated protein for the particular gene fragment. Especially. Another method exonuclease example <br/> using B al 31 eg to outside direction from the 5 'and 3' ends of the DNA, or systematically deleted the nucleotides inwardly from the restriction gap within the gene Including that. Such a gene deletion method results in a mutant gene encoding a shortened gene molecule, which can be evaluated for starch binding capacity.

Once the DNA sequence encoding the starch binding domain of interest , either the enzyme-SBD hybrid construct cDNA or chromosomal DNA, has been identified, it is then manipulated in various ways to obtain the DNA sequence encoding the enzyme of interest. May be fused. The starch binding domain coding fragment and the DNA encoding the enzyme of interest are then ligated with or without a linker. The resulting ligated DNA may be manipulated in various ways to provide for expression. A microbial host, such as Aspergillus, such as A. Niger and A. Oryza (A. oryzae), Bacillus, E. Coli or S. S. cerevisiae is preferred.

In a first aspect, the present invention relates to a maltogenic amylase E produced by Bacillus stearothermophilus C599 and having no enzymatic activity of the maltogenic amylase produced by Bacillus stearothermophilus C599. -Related to an isolated DNA sequence comprising a DNA sequence encoding the domain (SEQ ID NO: 2).

Bacillus stearothermophilus C599 is described in EP 120,693.

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

In a third aspect, the invention provides a D encoding the starch-binding domain of the invention.
Related to a Bacillus host transformed with a vector comprising the NA sequence and capable of expressing the sequence.

In a fourth aspect, the invention provides an insert DNA encoding a starch binding domain.
It relates to a Bacillus expression vector carrying sequences, in particular the isolated DNA sequences of the invention described above.

Further, the present invention provides a method for producing a single starch binding domain polypeptide in a Bacillus host cell comprising: an operably linked component of: a) a transcription and translation initiation regulatory region; b) A DNA sequence encoding the starch-binding domain polypeptide; c) transcription and translation termination regulatory regions, wherein the regulatory regions are functional in the host; and d) selectable markers for selecting transformed host cells. A Bacillus host cell transformed with an expression cassette containing a gene; is grown in a nutrient medium under conditions that overproduce the starch binding domain; and the starch binding domain polypeptide is recovered. Related to the method of becoming.

The present invention further provides a method for optimizing SBD expression in a Bacillus host: a. Expression of SBD fused to a reporter molecule in said host; b. Monitoring the concentration of expressed SBD in the supernatant of the fermentation host by measuring one or more characteristic properties of said reporter molecule.

Finally, the present invention is a method for producing a hybrid expressed in a Bacillus host, which comprises growing the transformed host under conditions in which the transformed nutrient is substantially free of untransformed cells. A method of incubating the transformed nutrient in a nutrient medium, thus overproducing the hybrid; and recovering the hybrid.

DETAILED DESCRIPTION OF THE INVENTION The inventor of the present invention has found that by alignment of the amino acid sequence (within the D and E domains) of Bacillus stearothermophilus C599 maltogenic amylase,
It CTG _ase, especially Bacillus circulans (B. circulans) strain _{251 CTG ase (Lawson, CL,} Montfort, RV, Strokopytov, B., Rozeboom, HJ
., Kalk, KH, Vries, GEd, Penninga, D., Dijkhuizen, L. and Dijkstra,
B. 1994; J. Mol. Biol. 236, p. 590-600).

Smith-Waterman method and BLOSUM 45 table (Henikoff S, Henikoff JG, 1992,
Proc. Natl. Acad. Sci. USA 89: 10915-10919). The identity for the domain was found to be 48%, and the identity for the DE-domain was 45%.

B. complexed with maltose The structure of circulance strain 251 CTG _ase is Laws
on CL et al. (1994), supra, and Code 1 CDG. It can be found under pdb.

[0029] CGT _ase structure consists of five domains, A of which, B and C domains are similar to domains that Miidase in α- amylase. Each of the other domains D and E is unique in _CTGase .

The function of the D-domain is believed to form a stable linkage between the E-domain and the catalytically active domain. Maltose binding site 1 in the E-domain
Two maltose binding sites, designated (MBS1) and maltose binding site 2 (MBS2), were identified within this structure. W616 and W662 overlap the glucose ring, and the side chains of K651 and N667 form a hydrogen bond to the -OH group on the glucose ring in MBS1.

As shown in the alignment (see Figure 1), all four residues can also be found in the maltogenic amylase. MBS2 overlaps with Y633 as well as residue T5
It is defined by H-bonds to the side chains of 98, N603, N627 and Q628. The maltogenic amylase also has positions T598, Q628 and Y633, while position N603 is replaced by K (Lys), which also has the ability to form an H-bond to the -OH group. There is nothing similar to N627.

[0032] Alignment, and by the presence of residues similar to MBS determining residues in CGT _ase, maltose binding sites seems that Miidase in the maltogenic amylase. The high homology between _CTGase and _maltogenic amylase suggests the same structure overall, and thus the structure of _CTGase is available for the determination of different domains within the _maltogenic amylase. Utilizing the alignment and CGTase structure, the _{origin of} the D-domain was determined to be amino acid residue 494 and the E-domain was determined to begin at amino acid residue 576. Therefore, D-E
The theoretical sizes of the and E-domains are 193 and 111 amino acids, respectively, corresponding to 20 and 12 kDa.

As mentioned above, the inventor of the present invention is EP Patent No. 120,693 (Novo Industri A / S).
It has been found that the maltogenic amylase product produced by Bacillus stearothermophilus C599 disclosed in U.S.A. comprises an SBD at the C-terminal portion of the protein sequence.

In a first aspect, the present invention provides a maltogenic amylase E produced by Bacillus stearothermophilus C599 and having no enzymatic activity of the maltogenic amylase produced by Bacillus stearothermophilus C599. Related to an isolated DNA sequence comprising a DNA sequence encoding a domain. This E domain-encoding DNA sequence is the sequence shown in SEQ ID NO: 1.
The corresponding protein sequence is shown in SEQ ID NO: 2.

In one embodiment, the isolated DNA sequence of the invention is produced by Bacillus stearothermophilus C599 and is Bacillus stearothermophilus C
It further comprises a D-domain of maltogenic amylase which does not have the enzymatic activity of maltogenic amylase produced by 599. In other words, the isolated DNA sequence contains the DE-domain coding DNA sequence. This DE-domain coding sequence is shown in SEQ ID NO: 3. This DE domain protein sequence is SE
Q ID NO: Shown in 4.

In a second aspect, the invention relates to a single SBD having a starch binding affinity encoded by the above isolated DNA sequence of the invention. This single SBD with starch binding affinity has SEQ ID NO: 2 (E-domain) and SEQ ID NO
: 4 (DE-domain).

Example 1 describes the construction of an expression vector and expression of a single SDB from a Bacillus stearothermophilus C599-derived maltogenic amylase in a Bacillus host.

Further, in a 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: -operably linked. The following components are included: a) transcription and translation initiation regulatory region; b) DNA sequence encoding the starch binding domain polypeptide; c) transcription and translation termination regulatory region, wherein the region is functional in the host. And d) a Bacillus host cell transformed with an expression cassette comprising a selectable marker gene for selecting transformed host cells; is grown in a nutrient medium under conditions in which the starch binding domain is overproduced; And-recovering said starch binding domain polypeptide.

Several types of SBD are E. Although it is expressed in E. coli, it has not been reported to be expressed or secreted from Bacillus sp. E. coli as an expression host for heterologous proteins. E. coli has several advantages over Bacillus spp. E. coli has a periplasmic space, which allows proper folding of heterologous expressed proteins (for example, Hockney, RC (1994) TIBTECH, vol. 12, p. 456-463).
checking). In particular, the oxidative capacity and the presence of disulfide oxytriductase within the periplasm are necessary for the expression of proteins with functional dependence on properly arranged disulfide bonds (Emmanuel Brun et al. (1995)).

Furthermore, E. E. coli periplasm also acts to protect the heterologously expressed protein against the action of proteases present in the supernatant and cytoplasm.

It is also known that when a secreted protein having a disulfide bond is expressed in Bacillus subtilis, the level of expression is significantly reduced (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 heterologous proteins is the proteolysis of the expressed protein. Bacillus subtilis is known to express at least seven extracellular proteases (Eds. AL Sonenshein, JA Hoch and Richard Losick (1993).
Bacillus subtilis and other Gram-Positive Bacteria, American Society for
microbiology, p. 939).

In particular for the very hydrophobic protein SBD, the translocation of this protein when expressed in Bacillus subtilis is significantly hindered and when the protein becomes anchored in the cell membrane due to its hydrophobicity It may even lead to cell death due to adverse effects.

In a fourth aspect, the invention relates to a Bacillus host cell transformed with a vector comprising a DNA sequence coding for SBD and capable of expressing this sequence.

In a preferred embodiment, the expressed single SBD or SBD-containing polypeptide has a molecular weight (Mw) of about 4 kD or higher. Preferably, this Mw is about 35 kD or less, more preferably about 32 kD or less, even more preferably about 30 kD or less, especially about 25 kD or less.

As mentioned above, the D-domain of Bacillus stearothermophilus C599 has an Mw of approximately 12 kD and the DE-domain has an Mw of approximately 20 kD.

The SBD may be expressed in the form of a single SBD, as described above, ie in the form of a polypeptide comprising one SBD. SBDs, on the other hand, are in the form of dimers or trimers, or multimers, ie two, three, or even more than three identical SBD “units”.
Can be expressed in the form of a polypeptide or protein.

The SBD can also be expressed as part of a multi-domain polypeptide, the non-SBD part of such a polypeptide being, for example, one, two or more non-catalytic domains.

Most SBDs can be expressed according to the invention, ie by a transformed Bacillus host. In a preferred embodiment, the SBD obtainable from a microorganism or plant, more preferably a bacterium or fungus is expressed.

Examples of SBDs of bacterial and fungal origin include SBDs obtainable from the species mentioned above in the “Background” chapter, in particular bacteria belonging to the genus Bacillus and fungi of the genus Aspergillus.

The Bacillus host cell of the present invention is a neutrophilic or alkalophilic, psychrophilic or thermophilic host cell.

Examples of hosts useful in the context of the present invention are hosts derived from the species Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus and Bacillus amyloliquefaciens. However, it is contemplated that other Bacillus species may also be useful hosts for the expression of SBD.

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

In another preferred embodiment of the invention, this vector is present as an expression plasmid, preferably a multicopy plasmid.

In a fifth aspect, the present invention relates to a Bacillus expression vector carrying an inserted SBD-encoding DNA sequence. Preferably, the expression cassette of this vector comprises a regulatory region from Bacillus sp., More preferably such regulatory region is endogenous to the host.

In a sixth aspect, the invention relates to a method for producing an SBD polypeptide, which method comprises the steps of: -as an operably linked component: a) transcription And a translation initiation regulatory region; b) a DNA sequence encoding the starch binding domain polypeptide; c) a transcription and translation termination regulatory region, wherein the region is functional in the host; and d) a transformed host cell. A Bacillus host cell transformed with an expression cassette comprising: a selectable marker gene for selecting in a nutrient medium is grown under conditions in which the starch binding domain is overproduced; and-the starch binding domain polypeptide Collect.

In a seventh aspect, the present invention is a method for the optimization of SBD expression in a Bacillus host: expression of said SBD fused to a reporter molecule in said host; and of said reporter molecule. Monitoring the concentration of expressed SBD in the supernatant of the fermentation host by measuring one or more characteristic properties of the fermentation host.

In a preferred embodiment, the reporter molecule is green fluorescent protein and the unique property is fluorescence.

In an eighth and ninth aspect, the invention relates to a polypeptide hybrid consisting essentially of one or more starch binding domains fused to a green fluorescent protein, and expression in a Bacillus host. A method of producing a hybrid comprising growing a transformed host under conditions in which the transformed culture is substantially free of untransformed cells; incubating the transformed culture in a nutrient medium, Thus overproducing the hybrid; and a method of recovering the hybrid.

Expression of a Single SBD Recombinant Expression Vector The recombinant vector comprising the DNA construct encoding the single SBD of the present invention may be any vector which can be readily subjected to recombinant DNA procedures, and The choice of vector will often depend on the host cell into which it is introduced. Introduction of a vector into a host cell is often referred to as transformation of the host cell. Such transformation may be carried out by transforming DNA into host cells, eg protoplasts, naturally competent cells,
By transfection, conjugation, electroporation or any equivalent method of introduction is meant. Thus, the vector may be a self-replicating vector, ie a vector that resides in the extrachromosomal matrix and whose replication is independent of chromosomal replication, eg a plasmid. On the other hand, the vector may be one which, when introduced into a host cell, integrates into part or the whole of the host cell genome and replicates with the chromosome in which it is integrated.

This vector preferably contains a DNA sequence encoding the sole SBD of the present invention DN
Is an expression vector operably linked to another segment required for transcription of A. Generally, the expression vector is derived from plasmid or viral DNA, or may contain elements of both. "Operably linked" means that the segments are aligned so that they function in concert to function for their intended purpose, for example, initiation of transcription within the promoter and passage of the SBD-encoding DNA sequence. means.

The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from any gene encoding a protein homologous or heterologous to this host cell.

[0063]   Examples of suitable promoters for use in bacterial host cells include Bacillus
Stearothermophilus maltogenic amylase gene, Bacillus lysii
Eniformis α-amylase gene, Bacillus amyloliquefaciens α-a
Millase gene, Bacillus subtilis alkaline protease gene, or Bacillus subtilis
Promoter of the B. pumilus xylosidase gene, or
Vage Lambda P_ROr P_LA promoter, or E. Stiffnesslac，trp Or tac  A promoter is mentioned. On the other hand, the DNA encoding SBD is the host genome
After being properly integrated into, for example, downstream of the resident promoter,
It is possible to design an integrative vector to be functional in the first place.

The SBD-encoding DNA sequence of the present invention may, if desired, be operably linked to a suitable terminator. The recombinant vector of the present invention may further comprise a DNA sequence enabling the vector to replicate in the host cell of interest.

This vector may be a selectable marker, for example a gene whose product complements a defect in the host cell, or resistance or heavy metals to antibiotics such as kanamycin, chloramphenicol, erythromycin, tetracycline, spectinomycin or the like. It may comprise a gene encoding resistance to a herbicide.

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

DNA sequences encoding each of the SBD, promoter and / or optionally terminator and / or secretory signal sequence are ligated, or these sequences are assembled by a suitable PCR amplification scheme, and they are replicated or The procedures utilized to insert an appropriate vector containing the necessary information for recombination are well known to those of skill in the art (see, eg, Sambrook et al., Supra).
.

Green Fluorescent Protein (GFP) is beginning to become a widely used reporter molecule for gene expression monitors, cell lineage tracers, and fusion tags for proteins. (Andreas Crameri et al. (1996) Im
proved Green Fluorescent Protein by molecular evolution using DNA shuffl
ing, Nature Biotechnology, Vol. 14, p. 315-319; Andrew B. Cubitt et al. (19
95), Understanding, improving and using fluorescent proteins, TIBS, vol.
20, p. 448-455).

GFP can be fused to SBD to make a fusion protein with starch binding and fluorescent properties. Expression of this fusion protein was found in SB within Bacillus sp.
It can be used to monitor the expression of D and therefore optimize the expression level of a given SBD.

Examples Materials and Methods Strain: Bacillus stearothermophilus C599 (EP120,683) comprises a maltogenic amylase.

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

Plasmid: pDN1981 (PL Jorgensen, CKHansen, GBPoulsen and B. Dideric
hsen, (1990), In vivo genetic engineering: homologues recombination as
a tool for plasmid construction, Gene, 96, p. 37-41).

General purpose molecular biology methods: DNA manipulations and transformations were performed using standard methods of molecular biology (Sa
mbrook et al. (1989), Molecular cloning: A laboratory manual, Cold Spring Ha
rbor lab., Cold Spring Harbor, NY; Ausubel, FM et al. (ed.) `` Current
protocols in Molecular Biology. "John Wiley and Sons, 1995; Harwood, C
.R., And Cutting, SM (eds.) `` Molecular Biological Methods for Bacillu
S .. John Wiley and Sons, 1990).

[0074]   Enzymes for DNA manipulations were utilized according to the supplier's specifications.

EXAMPLES Example 1 Construction of an Expression Vector Encoding a Single SBD Oligonucleotides to express only the E-domain and the D + E domain part of the AmyM protein (ie, the maltogenic amylase of Bacillus stearothermophilus C599). PCR primers were designed. The theory is that Bacillus licheniformis α-amylase (AmyL, Termamyl ™, PL Jor
gensen et al. (1990), Gene, 96, p. 37-41).
Place it in front of the yM fragment to obtain the protein secreted by Bacillus.

# 110755: PstI 5'-GATGCTGCAGCAGCGGCG TCCGCTTCAGCGCCGC -3 '(SEQ ID NO: 5) (The underlined portion corresponds to the 1783-1798 position of the amyM sequence; Genbank Accession nb.
(M36539)

# 110756: PstI 5′-GATGCTGCAGCAGCGGCG AGTGGAACGCAGACATCG -3 ′ (SEQ ID NO: 6) (The underlined portion corresponds to positions 2032-2049 of the amyM sequence; Genbank Accession nb.
(M36539)

# 110757: EcoRI BamHI 5'-GATGGAATTCGGATCC TCCATATGTACTACTCC -3 '(SEQ ID NO: 7) (The underlined portion corresponds to the amyM sequence at positions 2569-2553; Genbank Accession nb.
(M36539)

The template for the PCR reaction was a sample of plasmid pDN1413. This is essentially the plasmid pUB110 which contains the amyM gene fragment derived from the deposit NCIB11837 via the plasmid pDN452 described in EP120,693.

The conditions for PCR amplification were as follows: 94 ° C. for 2 minutes, then 49 ° C. for 30 seconds, 43 ° C. for 1 minute, 72 ° C. for 2 minutes, 20 cycles, then 72 ° C. One cycle of 15 minutes.

PCR fragments of the correct size were obtained by amplification. Primer # 110755 is
Together with # 110757, it provides a fragment of 820 base pairs, primer # 11075
6 together with # 110757 provided a fragment of 571 base pairs. The PCR fragment was purified using the QIAquick PCR Purification Kit Cat. No. 28106 from Qiagen and digested with EcoRI + PstI.

Plasmid pDN1981 (PL Jorgensen et al. (1990), Gene, 96, p. 37-41)
Was used as a cloning vector. pDN1981 was digested with EcoRI + PstI and the 3.9 kb fragment was purified from an agarose gel. This vector fragment was ligated with each digested PCR fragment and the ligation mixture was transformed into competent cells of Bacillus subtilis DN1885 (Diderichsen et al., Journal of Bacteriology, vol. 172, p.
.4315-4321, 1990), kanamycin resistance (10 μg / ml) was selected. Four colonies from each transformation were reisolated and liquid TY for plasmid preparation.
Grow in medium. All the extracted plasmids had a proper structure as judged by restriction digestion.

Two different transformants were preserved: SJ4302 and JS4303 both contained a plasmid carrying the # 110755 + # 110757 PCR fragment, ie the fragment encoding the D + E domain. Both SJ4304 and JS4305 contained a plasmid carrying the # 110756 + # 110757 PCR fragment, ie the fragment encoding only the E-domain.

Domain Expression: Strains SJ4302-SJ4305 were inoculated into 10 ml TY broth containing 0.4% glucose and 10 μg / ml kanamycin and incubated at 37 ° C. for 2 days with shaking. Strain DN1885 (B. subtilis host strain) containing 0.4% glucose 10
ml of TY broth was inoculated and incubated at 37 ° C for 2 days with shaking.

[0085]   The supernatant was analyzed by SDS-polyacrylamide gel electrophoresis.

A protein with an apparent molecular weight of approximately 25 kDa was observed in the supernatant from strain SJ4302. SJ4303 did not appear to produce a similar protein. This protein was not found in the other three samples.

The difference between SJ4302 and SJ4303 may be due to the fact that they carry PCR amplification constructs that have not been confirmed by DNA sequencing.
It is possible that an error was introduced into the SJ4303 clone).

A protein having an apparent molecular weight of about 10 kDa was observed in the supernatants from the strains SJ4304 and SJ4305. This protein was not observed in the other three samples.

In summary, the expected protein was produced from the D + E domain clone SJ4302, and the expected protein was the E-domain clone SJ4304.
+ Produced from SJ4305.

These strains were simply propagated as described above or they were among the above broths but from the Boehringer Mannheim protease inhibitor Comple.
te (Complete ™ Protease Inhibitor Cocktail Tablets Cat. No. 1697498
1 tablet was dissolved in 2 ml of water and 160 μl of this solution was added to each 10 ml of culture) and no difference in expression level was observed (amount of accumulating domain). . This concentration of protease inhibitor allows growth, but almost completely inhibits the extracellular protease present in DN1885 broth, as judged by spotting the broth on casein-containing agar plates.

Example 2 Expression of single SBD (E-domain) and expression of purified E-domain PSJ4305 (E-domain, above) was transformed into B-subtilis ToC46 competent cells (Diderichsen et al., Journal of Bacteriology, vol. 172). , p. 4315-4
321, 1990) and the transformant was stored as SJ4547.

50 shake flasks with a total volume of 500 ml containing 200 ml TY broth each containing 0.4% glucose and 10 mg / ml kanamycin were inoculated with SJ4547 and for 45 hours at 37 ° C. with shaking at 300 rpm. Incubated and the supernatant was separated from the cells by centrifugation.

The E-domain purified supernatant was centrifuged using a Sorvall RC-3B centrifuge equipped with a 4600A rotor head (4500 rpm, 15 min, 8 ° C.) and then filtered through a 0.7 mm glass microfiber filter. did. The supernatant was added to 25 mM sodium acetate, 1 mM
Α-Cyclodextrin agarose (1.6 x 5 cm) in CaCl ₂ , 0.5 M NaCl, pH 5.0 at a flow rate of 300 ml-k ^-1 . This column is 25 mM
Of sodium acetate, 1 mM CaCl ₂ , 0.5 M NaCl, pH 5.0 (10 column volumes), and SBD alone in the same buffer containing 2% (w / v) α-cyclodextrin. It was eluted. The eluted SBD was pooled and dialyzed against 50 mM sodium acetate, 1 mM CaCl ₂ , pH 5.0. The purified SBD was homogeneous as determined by SDS-PAGE (see below). N-terminal sequencing revealed SGTQTSVVF and confirmed that this purified E-domain was identical to residue Ser 576 of the Bacillus stearothermophilus C599 maltogenic amylase.

Approximately 21 mg of homogeneous SBD was purified from 1 L of culture filtrate by running 10 consecutive runs over α-cyclodextrin agarose. SDS-PAGE of purified SBD
The analysis is shown in FIG.

Example 3 Starch-Bound Granules of Alone SBD The adsorption of a single SBD on starch varies with the amount of SBD (0-3 mg / ml).
Granules of corn starch (10 mg / ml) in 5 mM sodium acetate, pH 3.6 for 16 hours at 4 ° C, essentially as described (Belshaw & Wiliamson, 1990).
It was determined by incubating. The reaction was stopped by centrifugation and the protein concentration in the supernatant was measured and subtracted from the total protein amount to determine the amount of protein bound to starch.

[Brief description of drawings]

FIG. 1 B. Stearothermophilus C599 and B. Circulans
) Strain 251 CGTase-derived _maltogenic amylase (D and E-
Domain) sequence alignment. The "bold" typeface is the residues involved in maltose binding through its side chains, and the "italic" typeface is the residues having side chains involved in maltose binding in MBS2. S-sequence no .; 1 =
B. Stearothermophilus C599 maltogenic amylase sequence; 2 = B
． Circulence strain 251 _CTGase sequence; D = domain.

FIG. 2 shows an SDS-PAGE gel of purified E-domain alone SBD. Lanes 1 and 3
: Molecular weight standard (from top): 94, 67, 43, 30, 20, 14; lane 2:
SBD alone (E-domain).

[Sequence list]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12R 1:07) C12R 1: 125 (C12N 15/09 ZNA 1:11 C12R 1:10) C12N 15/00 ZNAA (C12N 15/09 ZNA C12R 1: 125) (C12N 15/09 ZNA C12R 1:11) (C12N 1/21 C12R 1:07) (C12P 21/02 C12R 1:07) (81) Designated Country EP ( AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (A , AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU , LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, U Z, VN, YU, ZW (72) Inventor Andersen, Karsten Denmark, Deko-2880 Bagsbaelt, Novo Are, Novo Nordisk Actisel Scab F Term (reference) 4B024 AA05 BA13 CA04 DA05 EA04 GA11 HA01 HA20 4B050 CC03 DD02 FF09E L L05 4B064 AG01 CA02 CA19 CC24 DA10 4B065 AA18Y AA19X AB01 BA02 CA32 CA41

Claims (26)

[Claims] 1. Bacillus stearothermo
philus) C599 produced by Bacillus stearothermophilus C5
An isolated DNA sequence comprising a DNA sequence encoding the E-domain of a maltogenic amylase having no enzymatic activity of the maltogenic amylase produced by 99. 2. The isolated DNA sequence of claim 1, wherein the E domain sequence is the sequence set forth in SEQ ID NO: 1. 3. A D-domain of a maltogenic amylase produced by Bacillus stearothermophilus C599 and having no enzymatic activity of the maltogenic amylase produced by Bacillus stearothermophilus C599. 3. The isolated DNA sequence according to claim 1 or 2. 4. The isolated DNA sequence of claim 3, wherein the D + E domain sequence is the sequence shown in SEQ ID NO: 3. 5. A single SBD having a starch binding affinity encoded by the DNA sequence according to any one of claims 1 to 4. 6. The single SBD according to claim 5, which has 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 encoding a single starch binding domain and capable of expressing said DNA sequence. 8. The host cell of claim 7, wherein the DNA sequence is from a source other than Bacillus sp. 9. The host cell according to claim 7, which is capable of expressing the starch-binding domain as a single polypeptide main. 10. A DNA in which the vector encodes a single starch domain.
A host cell according to any one of claims 7 to 9 which comprises a sequence. 11. A host cell according to any one of claims 7-10, wherein said vector comprises a DNA sequence encoding a starch binding domain linked to at least one other non-catalytically active domain. 12. A host cell according to any one of claims 7 to 11, wherein the starch binding domain is obtainable from a microorganism or plant, preferably a bacterium or a fungus. 13. The bacterium is selected from the group consisting of the genus Bacillus.
The host cell according to 2. 14. The host cell of claim 13, wherein the fungus is selected from the group consisting of the genus Aspergillus. 15. The host cell of the genus Bacillus according to claim 7, which is neutrophilic, alkaliphilic, room temperature or thermophilic. 16. The following species: B. subtilis, B. licheniformis, B. megaterium.
aterium), Bacillus stearothermophilus and Bacillus amyloliquefaciens (B. amyloliquefaciens). 17. The host cell according to claim 7, wherein the vector is integrated in the genome of an untransformed host. 18. The method of claim 7, wherein the vector exists as an expression plasmid.
The host cell according to any one of claims 1 to 17. 19. The host cell according to claim 7, wherein the vector is amplified on the genome or the expression plasmid is a multicopy plasmid. 20. Inserted DNA encoding a single starch binding domain
A Bacillus expression vector carrying a sequence. 21. The vector of claim 20, wherein the expression cassette comprises regulatory regions derived from Bacillus spp. 22. The vector of claim 21, wherein the Bacillus sp. Regulatory region is endogenous to the host. 23. A method of producing a single starch binding domain polypeptide in a Bacillus host cell comprising: operably linked the following components: a) a transcription and translation initiation regulatory region; b) the starch binding. A DNA sequence encoding a domain polypeptide; c) a transcriptional and translational termination regulatory region, wherein the regulatory region is functional in the host; and d) a selectable marker gene for selecting transformed host cells. Growing a Bacillus host cell transformed with an expression cassette comprising in an nutrient medium under conditions that overproduce the starch binding domain; and recovering the starch binding domain polypeptide. 24. A method for optimizing SBD expression in a Bacillus host comprising: a. Expression of SBD fused to a reporter molecule in said host; b. Monitoring the concentration of expressed SBD in the supernatant of the fermentation host by measuring one or more properties characteristic of the reporter molecule. 25. The method of claim 24, wherein the reporter molecule is green fluorescent protein and the unique property is fluorescence. 26. A method for producing a hybrid according to claim 24 or 25, which is expressed in a Bacillus host, wherein the transformed culture comprises transforming untransformed cells into substantially untransformed cells. Propagating under conditions that are free of ;; incubating the transformed culture in nutrient medium, thus overproducing the hybrid; and recovering primarily the hybrid.