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Title:
AMYLASE
Document Type and Number:
WIPO Patent Application WO/2005/066338
Kind Code:
A1
Abstract:
The inventors have identified amylases in fungal strains of Valsaria and found that the amylase can increase the shelf life of baked products. Particularly, the novel amylase in com-bination with a maltogenic amylase further improves the softness of bread crumb without hav-ing detrimental effects on elasticity.

Inventors:
Liu YE. (14 Zizhuyuan Lu, Hai Dian Distric, Beijing 4, 10004, CN)
Tang, Lan (ZhiChun Road 57-1-707, Hai Dian District, Beijing 0, 10008, CN)
Spendler, Tina (Myrholmen 52, Måløv, DK-2760, DK)
Stringer, Mary Ann (Søborg Hovedgade 39C, 3tv, Søborg, DK-2860, DK)
Application Number:
PCT/DK2004/000896
Publication Date:
July 21, 2005
Filing Date:
December 22, 2004
Export Citation:
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Assignee:
Novozymes, A/s (Krogshøjvej 36, Bagsværd, DK-2880, DK)
Liu YE. (14 Zizhuyuan Lu, Hai Dian Distric, Beijing 4, 10004, CN)
Tang, Lan (ZhiChun Road 57-1-707, Hai Dian District, Beijing 0, 10008, CN)
Spendler, Tina (Myrholmen 52, Måløv, DK-2760, DK)
Stringer, Mary Ann (Søborg Hovedgade 39C, 3tv, Søborg, DK-2860, DK)
International Classes:
A21D8/04; C12N9/30; (IPC1-7): C12N9/30; C12N15/56
Domestic Patent References:
WO2003016535A22003-02-27
WO1991004669A11991-04-18
WO2003083054A22003-10-09
WO1996001323A11996-01-18
Other References:
GUPTA R ET AL: "MICROBIAL ALPHA-AMYLASES: A BIOTECHNOLOGICAL PERSPECTIVE", PROCESS BIOCHEMISTRY, XX, XX, vol. 38, no. 11, 2003, pages 1599 - 1616, XP008043283
Attorney, Agent or Firm:
Novozymes, A/s (Krogshøjvej 36, Bagsværd, DK-2880, DK)
Download PDF:
Claims:
CLAIMS
1. A polypeptide which has amylase activity and has an amino acid sequence which com prises: a) a catalytic core sequence encoded by a DNA sequence present in a plasmid in E. coli DSM 16113 or in Valsaria rubricosa CBS 848.96, b) a sequence as shown in positions 1439 of SEQ ID NO 2 or 19; c) a sequence which has at least 70 % identity to the sequence defined in (a) or (b), or d) a sequence encoded by a nucleic acid sequence which hybridizes at 55 °C with the complementary strand of nucleotides 1461462 of SEQ ID NO: 1 or nucleotides 761392 of SEQ ID NO : 18.
2. The polypeptide of claim 1 wherein the amino acid sequence further comprises a carbohy dratebinding domain.
3. The polypeptide of claim 1 or 2 which has an amino acid sequence comprising: a) a catalytic core sequence and carbohydratebinding module which are encoded by a DNA sequence present in a plasmid in E. coli DSM 16113 b) a sequence as shown in positions 1566 of SEQ ID NO 2 or 19; c) a sequence which has at least 70 % identity to the sequence defined in (a) or (b), d) a sequence encoded by a nucleic acid sequence which hybridizes at 55 °C with the complementary strand of nucleotides 1461843 of SEQ ID NO: 1 or nucleotides 761392 of SEQ ID NO: 18.
4. A polypeptide which has an amino acid sequence comprising a carbohydratebinding mod ule having at least 70 % identity to amino acids 440566 of SEQ ID NO : 2 or 19.
5. A polynucleotide comprising a sequence which encodes the polypeptide of any of claims 1 5.
6. A polynucleotide comprising a sequence which is: a) a DNA sequence present in a plasmid in E. coli DSM 16113 or in Valsaria rubricosa CBS 848.96, which encodes an amylase catalytic core; b) a sequence which has at least 70 % identity to nucleotides 1461462 of SEQ ID NO: 1 or nucleotides 761392 of SEQ ID NO: 18 and encodes an amylase catalytic core; c) a nucleic acid sequence which hybridizes at 55°C with the complementary strand of nucleotides 1461462 of SEQ ID NO : 1 or nucleotides 761392 of SEQ ID NO: 18; or d) the complementary strand of the polynucleotide of a), b), or c).
7. A polynucleotide comprising a sequence which is: a) a DNA sequence present in a plasmid in E. coli DSM 16113 or in Valsaria rubricosa CBS 848.96, which encodes an amylase catalytic core and a carbohydrate binding domain; b) a sequence which has at least 70 % identity to nucleotides 1461843 of SEQ ID NO: 1 or nucleotides 761773 of SEQ ID NO: 18 and encodes an amylase catalytic core and a carbohydrate binding domain; c) a nucleic acid sequence which hybridizes at 55°C with the complementary strand of nucleotides 1461843 of SEQ ID NO: 1 or nucleotides 761773 of SEQ ID NO: 18; or d) the complementary strand of the polynucleotide of a), b), or c).
8. A vector comprising the polynucleotide of any of claims 57 operably linked to one or more control sequences that direct the production of the polypeptide in a suitable host.
9. A transformed host cell comprising the vector of the preceding claim.
10. A method for producing an amylase, which comprises a) cultivating the host cell of the preceding claim under conditions appropriate for ex pression of amylase, and b) recovering the amylase.
11. A dough composition which comprises flour and the polypeptide of any of claims 14.
12. A process for preparing a doughbased product, comprising adding the polypeptide of any of claims 14 to a dough, leavening, and heating the dough.
13. The process of the preceding claim which further comprises adding an exoacting amylase to the dough.
14. The process of the preceding claim wherein the exoacting amylase is a maltogenic alpha amylase.
15. A process for preparing a doughbased product, comprising adding a first and a second amylase to a dough, leavening, and heating the dough, wherein: a) the first amylase retains more than 50% activity after 15 min incubation at 62°C in 50 mM sodium acetate, 1 mM CaCI2, pH 5.7, and has an amino acid sequence comprising a catalytic module and carbohydratebinding module, and b) the second amylase is an exoacting amylase. 16.
16. The process of the preceding claim wherein the first amylase is derived from a fungus.
17. The process of any of claims 15 or 16 wherein the second amylase is a maltogenic alpha amylase.
Description:
AMYLASE FIELD OF THE INVENTION The present invention relates to amylases and their addition to dough to prevent stal- ing of dough-based products.

BACKGROUND OF THE INVENTION Endo-amylases are often added to dough to make the dough more soft and to im- prove the moistness, often in combination with an exo-amylase, e. g. a maltogenic alpha- amylase. However, the addition of endo-amylases of bacterial origin, e. g. B. amyloliquefaciens amylase easily gives a risk of overdosing, giving loss of elasticity and a too gummy crumb.

Fungal amylases are often added to dough, but they only provide little effect on the softness A fungal amylase (Taka amylase) from Aspergillus oryzae is described in Pdb file 6taa (available at www. rcsb. org).

SUMMARY OF THE INVENTION The inventors have identified an amylase in fungal strains of Valsaria and found that the amylase can increase the shelf life of baked products. Particularly, the novel amylase in combination with an exo-amylase further improves the anti-staling effect of the exo-amylase.

The improved anti-staling may be an improved the softness of bread crumb without a detri- mental effect on crumb elasticity or even with an improved elasticity.

The amino acid sequence of the novel amylase was found to include a catalytic do- main and a carbohydrate-binding domain (CBM), each of which can be used separately. Ac- cordingly, the invention provides a polypeptide with a sequence including a catalytic core and a polypeptide with a sequence including a carbohydrate-binding domain (CBM).

The novel amylase is thermostable, and the inventors found that a combination of two amylases can be used for anti-staling, where one is a thermostable amylase (particularly a fungal amylase) which includes both a catalytic core and a CBM, and the other is an exo- amylase.

The invention also provides a polypeptide having an amino acid sequence which can be obtained from the mature polypeptide (particularly the catalytic coreof SEQ ID NO: 2 or 19 by substitution, deletion, and/or insertion of one or more amino acids and a polynucleotide hav- ing a sequence that can be derived from SEQ ID NO: 1 or 18 by substitution, deletion, and/or insertion of one or more nucleotides.

The invention also provides a polynucleotide encoding the amylase, an expression vector comprising the polynucleotide, a transformed host cell comprising the vector, as well as a method of producing the amylase by cultivating the transformant. The invention further pro-

vides a dough composition comprising the amylase, a method of preparing a dough-based product by leavening and heating the dough, e. g. by baking.

DETAILED DESCRIPTION OF THE INVENTION Genomic DNA source A source organism of the amylase of the invention is a fungal strain isolated from soil samples collected from Hainan Province, China, in 2002. The strain was at first classified as Chaetomium sp. and was later re-classified as Valsaria rubricosa belonging to Diaporthales, Ascomycetes, Ascomycota. It was found to harbor an amylase gene shown in SEQ NO: 1. The inventors have cloned the gene into a strain of E. coli and deposited it under the terms of the Budapest Treaty on 16 December 2003 as DSM 16113 with the DSMZ-Deutsche Sammlung von Microorganismen und Zelikulturen GmbH, Mascheroder Weg 1 b, D-38124 Braunschweig DE.

The inventors also found an amylase gene in Valsaria rubricosa CBS 848.96. This strain is available on commercial terms from Centraalbureau voor Schimmelcultures, Uppsala- laan 8, P. O. Box 85167,3584 CT, The Netherlands. Valsaria rubricosa CBS 848.96 was found to harbor an amylase gene shown in SEQ NO : 18 Polypeptide with amylase activity The polypeptide with amylase activity has an amino acid sequence which includes a catalytic core which may be the catalytic core sequence encoded by a DNA sequence in a plasmid present in E. coli DSM 16113, the sequence shown in positions 1-439 of SEQ ID NO 2, or a sequence having at least 70 % identity to said sequences. Also, the catalytic core may be a sequence encoded by a nucleic acid sequence which hybridizes at 55 °C with the com- plementary strand of nucleotides 146-1462 of SEQ ID NO: 1.

Optionally, the amino acid sequence may further comprise both the catalytic domain and a carbohydrate-binding module (CBM), or it may include the catalytic domain without a CBM. The CBM may be that present in the donor strain or an analogue thereof, or an extrinsic CBM may be substituted, or one or more additional CBMs may be inserted.

The polypeptide may have the sequence shown as SEQ ID NO: 19.

Carbohydrate-binding domain (CBM) The carbohydrate-binding module (CBM) or carbohydrate-binding domain (CBD) is a polypeptide which binds preferentially to a poly-or oligosaccharide (carbohydrate), particularly in water-insoluble (e. g. crystalline) form. It may particularly be a starch-binding modules (SBM) or starch-binding domain (SBD). The one or more CBMs may optionally further comprise one

or more polypeptide amino acid sequence regions linking the CBM (s) with the catalytic mod- ule (s), a region of the latter type usually being denoted a"linker".

The invention provides a polypeptide with an amino acid sequence including a CBM with at least 70 % identity to amino acids 440-566 of SEQ ID NO: 2.

Heterologous carbohydrate-binding modules The CBM per se typically consists of more than about 30 and less than about 250 amino acid residues. The CBM may be a"Carbohydrate-Binding Module of Family 20"or a CBM-20 module, typically a sequence of approximately 100 amino acids having at least 45% homology to the Carbohydrate-Binding Module (CBM) of the polypeptide disclosed in figure 1 by Joergensen et al (1997) in Biotechnol. Lett. 19: 1027-1031, where the CBM comprises the last 102 amino acids of the polypeptide, i. e. the subsequence from amino acid 582 to amino acid 683.

The CBM (or SBM) may be derived from a starch degrading enzyme (amylolytic en- zyme), such as a glucoamylase (EC 3.2. 1.3), a cyclodextrin glucanotransferases or CGTase (EC 2.4. 1.19), an alpha-amylase (EC 3.2. 1.1) or a maltogenic alpha-amylase (EC 3.2. 1.133).

The CBM may be derived from fungal, bacterial or plant sources, e. g. derived from Aspergillus <BR> <BR> sp. , Bacillus sp. , Klebsiella sp. , or Rhizopus sp. The CBM may also be in the form of a non- hydrolytic polysaccharide-binding protein, e. g. found in algae, such as red alga Porphyra pur- purea. The CBM may be located at the N or C terminus or at an internal position in a polype- tide (e. g. an enzyme).

Further examples of CBMs are described in PCT/US2004/020499, incorporated herein by reference.

Combination of amylases A combination of two amylases may be added to dough to achieve anti-staling in a product made from the dough.

The first amylase may be a thermostable amylase which includes a CBM, particularly a fungal amylase. It may be an alpha-amylase which retains more than 50% activity after 15 min incubation at 62°C (or 64°C or 66°C) in 50 mM sodium acetate, 1 mM CaC12, pH 5. 7. It may have less than 50% activity at 71 °C (or 69°C) under the same conditions. An example is the Valsaria amylase described above.

The second amylase may be an exo-amylase. It may be capable of hydrolyzing starch by cleaving off linear maltooligosaccharides, e. g. maltose, maltotriose or maltotetraose, from the non-reducing ends of amylopectin. One example is maltogenic alpha-amylase (EC 3.2. 1.133) such as Novamyle or a variant thereof, e. g. having at least 90 % amino acid identity to Novamyl as described in US 6162628, where the Novamyl sequence is shown as SEQ ID NO: 1.

The exo-amylase may hydrolyze amylose (e. g. wheat amylose or synthetic amylose) so that the average molecular weight of the amylose after 0.4-4 % hydrolysis (i. e. between 0.4- 4 % hydrolysis of the total number of bonds) is more than 50 % (particularly more than 75 %) of the value before the hydrolysis. The hydrolysis can be conducted at the conditions de- scribed above, and the molecular weight distribution before and after the hydrolysis can be de- termined by HPLC. The test may be carried out as described in C. Christophersen et al., Starch 50 (1), 39-45 (1998).

Recombinant expression vector The expression vector of the invention typically includes a selectable marker and con- trol sequences encoding a promoter, a 5'untranslated leader and, a transcription terminator.

The vector may be an autonomously replicating vector, or it may be integrated into the host cell genome.

Production by cultivation of transformant The polypeptide of the invention may be produced by transforming a suitable host cell with a DNA sequence encoding the amylase, cultivating the transformed organism under con- ditions permitting the production of the enzyme, and recovering the enzyme from the culture.

The host organism may particularly be a eukaryotic cell, in particular a fungal cell, such as a yeast cell or a filamentous fungal cell, e. g. a strain of Aspergillus, Fusarium, Trichoderma or Saccharomyces, particularly A. niger, A. oryzae, F. graminearum or S. cerevisiae.

Hybridization Suitable conditions for determining hybridization between a nucleotide probe and a homologous DNA or RNA sequence involve presoaking of the filter containing the DNA frag- ments or RNA to hybridize in 5 x SSC (standard saline citrate) for 10 min, and prehybridization of the filter in a solution of 5 x SSC (Sambrook et al. 1989), 5 x Denhardt's solution (Sambrook et al. 1989), 0.5 % SDS and 100 ug/ml of denatured sonicated salmon sperm DNA (Sambrook et al. 1989), followed by hybridization in the same solution containing a random-primed (Feinberg, A. P. and Vogelstein, B. (1983) Anal. Biochem. 132: 6-13), 32P-dCTP-labeled (spe- cific activity > 1 x 109 cpm/ug) probe for 12 hours at approx. 45°C. The filter is then washed two times for 30 minutes in 2 x SSC, 0.5 % SDS at a temperature of at least 55°C, more par- ticularly at least 60°C, more particularly at least 65°C, even more particularly at least 70°C, es- pecially at least 75°C. Molecules to which the oligonucleotide probe hybridizes under these conditions may be detected using a x-ray film.

Alignment and identity The polypeptide and polynucleotide of the invention may have identities to the dis- closed sequences of at least 80 %, particularly at least 85 % or at least 90 %, e. g. at least 95 %.

For purposes of the present invention, alignments of sequences and calculation of identity scores may be done using a Needleman-Wunsch alignment (i. e. global alignment), useful for both protein and DNA alignments. The default scoring matrices BLOSUM50 and the identity matrix are used for protein and DNA alignments respectively. The penalty for the first residue in a gap is-12 for proteins and-16 for DNA, while the penalty for additional residues in a gap is-2 for proteins and-4 for DNA. Alignment is from the FASTA package version v20u6 (W. R. Pearson and D. J. Lipman (1988),"Improved Tools for Biological Sequence Analysis", PNAS 85: 2444-2448, and W. R. Pearson (1990) "Rapid and Sensitive Sequence Comparison with FASTP and FASTA", Methods in Enzymology, 183: 63-98).

Dough The dough of the invention generally comprises flour, particularly wheat flour. The dough may be fresh, frozen or par-baked. It may be a laminated dough.

The dough may also comprise other conventional dough ingredients, e. g.: proteins, such as milk powder and gluten ; eggs (either whole eggs, egg yolks or egg whites); an oxidant such as ascorbic acid, potassium bromate, potassium iodate, azodicarbonamide (ADA) or ammonium persulfate ; an amino acid such as L-cysteine; a sugar; a salt such as sodium chlo- ride, calcium acetate, sodium sulfate or calcium sulfate. The dough may comprise fat (triglyc- eride) such as granulated fat or shortening.

Additional enzyme Optionally, one or more additional enzymes may be added to the dough together with the amylase (s) described above. The additional enzyme may be a lipolytic enzyme (e. g. as described in WO 9953769) or a xylanase.

Dough-based product The invention provides a method for preparing a dough-based product by leavening the dough and heating it, e. g. by baking or steaming. The dough may be leavened e. g. by add- ing chemical leavening agents or yeast, usually Saccharomyces cerevisiae (baker's yeast).

The product may be of a soft or a crisp character, either of a white, light or dark type. Exam- ples are steamed or baked bread (in particular white, whole-meal or rye bread), typically in the form of loaves or rolls.

Amylase Units (AmU) Amylase activity was assayed by incubating with Phadebas amylase test tablets (product of Amersham Pharmacia) suspended in 50 mM sodium acetate + 1 mM CaCI at pH 5.7 and determining OD at 650 nm. The Amylase Unit (AmU) was defined by taking the activity of the commercial product BAN 480L (product of Novozymes A/S) as 480 AmU.

EXAMPLES Materials and methods RNeasy Mini Kit (Qiagen, Cat. #74904).

Taq DNA polymerase (Promega, Cat. # M166A) pGEM-T Vector System f (Promega, Cat. # A3600) Wizard Plus Minipreps DNA Purification System (Promega, Cat. # A7510) 5'Rapid Amplifiction of cDNA End System (Life Technologies, 5'RACE, Cat. # 18374- 041), 3'Rapid Amplifiction of cDNA End System (Life Technologies, 3'RACE, Cat. # 1085805) ElectroMAX DH10B Cells (Life Technologies, Cat. # 18290-015) Example 1: Cultivation of fungal strain for cDNA preparation A fungal strain of Valsaria rubricosa was grown on YG agar plate (4.5 cm diam) for 5 days under 37OC in the darkness and used for inoculating shake flask. The plates with fully grown cultures were stored at 4°C before use.

To obtain the mycelium for cDNA library construction, 4-6 agar plugs with fully grown fungal cultures on the YG agar plates were used to inoculate one shake flask with FG-4 (50 ml in 500 ml Erlenmeyer flask with 2 baffles : 30 g Soymeal, 15 g Maltose, 5 g Peptone, 1000 ml H20, 1% starch, 1 g olive oil (2 drops/flask) ; Autoclave at 121 °C for 30 min) and grown under 370C, 160 rpm for 24 hours. The mycelium was harvested by centrifugation of the culture broth at 8000 rpm and 4OC for 30 minutes. Then mycelium was transferred into a clean plastic bag following by immediately freezing in liquid nitrogen and stored at-800C before total RNA was isolated.

Probe designing : Degenerate primers were designed based on alignment of already known amylase gene sequences: amyD1 (SEQ ID NO: 3) and amyD2R (SEQ ID NO: 4).

The N-terminal amino acid sequence of the purified amylase AM835F was determined as shown in SEQ ID NO: 13. This was used to design four degenerate primers: AM835n-s1

(SEQ ID NO: 5), AM835n-s2a (SEQ ID NO: 6), AM835n-s2b (SEQ ID NO: 7), AM835n-s2c (SEQ ID NO: 8).

Extraction of total RNA: Total RNA was isolated from the frozen mycelium of a strain of Valsaria rubricosa by using RNeasy Mini Kit according to the manufacturer's instructions.

Gene cloning : cDNA was synthesized using 3'RACE kit. The primary PCR was performed by using N-terminal based degenerate primers (primer AM835n-s2 is a mixture of AM835n-s2a, b and c) with AUAP provided by the 3'RACE kit: 10X PCR buffer 5 micro-I 25mM MgCl2 3 micro-I 1 OmM dNTP 1 micro-I 100 micro-M AM835n-s2a 1 micro-I 100 micro-M AM835n-s2b 1 micro-I 100 micro-M AM835n-s2c 1 micro-I AUAP 1 micro-I cDNA 2 micro-I Taq DNA polymerase (5u/micro-l) 1 micro-l H2O 34 micro-I The PCR program was: 94°C for 3 min; 30 cycles of 94 oC for 30s, 50°C for 30s, 72OC for 1.5 min; final extension at 72OC for 10 min.

There was no specific amplification seen when the PCR product was visualized under UV but this product was used for second PCR with degenerate primers designed based on amylase homology. The 2nd PCR was performed by using amylase probes (amyD1 and amyD2R) and using the primary PCR as template: 10X PCR buffer 5 micro-I 25mM MgCl2 3 micro-I 10mM dNTP 1 micro-I 100 micro-M amyD1 1 micro-I 100 micro-M amy D2R 1 micro-I 1''PCR 1 micro-I Taq DNA polymerase (5u/micro-l) 1 micro-I H2O 37 micro-I The PCR program was: 94°C for 3min; 30 cycles of 94 oC for 30s, 50°C for 30s, 72OC for 1min ; final extension at 72OC for 10min. A specific band was amplified at-700bps and this was confirmed to be amylase by sequencing.

Based on the above obtained partial sequence, new primers were designed for 5'and 3'end cloning. For 5'end cloning, after cDNA was synthesized initiated with amy835as1 (SEQ ID NO: 9) by 5'RACE kit, PCR was performed with primer pairs amy835as1 and AAP (provided by the kit). Then nested PCR was performed with primer pair amy835as3 (SEQ ID NO: 14) and AUAP by using primary PCR (amy835as1-AAP) as template. A fragment of-600bp was ob- tained and confirmed by sequencing. For 3'end cloning, PCR was performed by using primer pair of amy835f1 (SEQ ID NO: 15) and AUAP and cDNA as template. The nested PCR was performed by using primer pair amy835f2 (SEQ ID NO: 10) with AUAP and 1 PCR as template.

A fragment at-600 bps was amplified and again confirmed by sequencing.

Then based on the cloned 5'and 3'end sequences, the 5'and 3'end primers for full length cloning was designed and used for full length cloning of the amylase AM835. By using cDNA synthesized by 3'RACE kit as template and probes amy835s00 (SEQ ID NO: 11) and amy835as01 (SEQ ID NO: 12) as primers : 10X PCR buffer 5 micro-I 25mM MgCI2 3 micro- ! 10mM dNTP 1 micro-l 10 micro-M amy835s00 1 micro-I 10 micro-M amy835asO1 1 micro-l cDNA 2 micro-l Taq DNA polymerase (5 u/micro-1) 1 micro-I H20 36 micro-I PCR program was: 94OC for 3 min; 30 cycles of 94 oC for 30s, 50OC for 30s, 72OC for 1.5 min; final extension at 72°C for 10 min.

A specific fragment of-2. 0 kb was PCR-amplified. The fragment was cloned into pGEM-T vector (Promega) which has a 3'-T overhang and transformed into E. coli DH10B (ElectroMAX DH10B Cells, available from Life Technologies, Cat. # 18290-015) and further sequenced.

Example 2: Production of amylase YG and FG-4 media were prepared as follows : YG: Yeast-glucose agar 5.0 g Difco powdered yeast extract; 10.0 g glucose 20.0 g agar; 1000 ml tap water Autoclave at 121 OC for 15-20 min.

FG-4 Media 50 ml/flask : 30 g Soymeal, 15 g Maltose

5 g Peptone, 1000 ml H20 1 g olive oil (2 drops l flask) 50 ml in 500 mi Erlenmeyer flask with 2 baffles. Autoclave at 121 °C for 30 min.

A strain of the thermophilic fungus Valsaria rubricosa was grown on YG agar plate (4.5 cm diam) for 3 days under 37OC in the darkness and used for inoculating shake flask. The plates with fully grown cultures were stored at 4°C before use.

For enzyme production, 4-6 agar plugs with fully grown fungal cultures on the above plates were used to inoculate one shake flask with FG-4 and grown under 37°C, 160 rpm for 72 hours, then harvested by centrifuged the culture broth at 8000 rpm and 4°C for 30 minutes.

The supernatant was collected and used for enzyme purification.

1000 ml supernatant was precipitated with ammonium sulfate (80% saturation) and redissolved in 100 ml 25mM Tris-HCI buffer, pH7. 0, then dialyzed against the same buffer and filtered through a 0.45 mm filter, the final volume was 200 mi. The solution was applied to a 35 ml Source 15Q column (Pharmacia) equilibrated in 25 mM Tris-HCI buffer, pH7.0, and the pro- teins was eluted with a linear NaCI gradient (0-0.3M). Fractions from the column were ana- lyzed for amylase activity on AZCL-amylose at pH 5.5. Fractions with amylase activity were pooled. Then the pooled solution was ultrafiltrated, the concentrated solution was applied to a 180mi Superdex75 column equilibrated with 25 mM Tris-HCI, pH7.0, the proteins was eluted with the same buffer. Amylase containing fractions were analyzed by SDS-PAGE and pure fractions were pooled.

The purified amylase was used for characterization in the following example.

Example 3: Expression of an amylase from Valsaria rubricosa in Aspergillus oryzae The DNA sequence of the Valsaria rubricosa amylase (SEQ ID NO.: 1) was used to design primers for PCR amplification of the amylase encoding-gene from the clone described in Example 1, with appropriate restriction sites added to the primer ends to facilitate sub- cloning of the PCR product (primers AM835.1 and AM835. 2, SEQ ID NO: 16 and 17). PCR amplification was performed using AmpliTaq Gold DNA Polymerase (Applied Biosystems, Fos- ter City, CA, USA) following the manufacturer's instructions and using an annealing tempera- ture of 55°C for the first 5 cycles and 65°C for an additional 25 cycles and an extension time of 2 minutes.

The PCR fragment was restricted with BamHl and Xhol and cloned into the Aspergil- lus expression vector pMStr57 using standard techniques. The expression vector pMStr57 contains the same elements as pCaHj483 (WO 98/00529), with minor modifications made to the Aspergillus NA2 promoter as described for the vector pMT2188 in WO 01/12794, and has sequences for selection and propogation in E. coli, and selection and expression in Aspergil- lus. Specifically, selection in Aspergillus is facilitated by the amdS gene of Aspergillus nidu-

lans, which allows the use of acetamide as a sole nitrogen source. Expression in Aspergillus is mediated by a modified neutral amylase 11 (NA2) promoter from Aspergillus niger which is fused to the 5'leader sequence of the triose phosphate isomerase (tpi) encoding-gene from Aspergillus nidulans, and the terminator from the amyloglucosidase-encoding gene from As- pergillus niger. The amylase-encoding gene of the resulting Aspergillus expression construct, pMStr91, was sequenced and the sequence agreed completely with that determined previ- ously.

The Aspergillus oryzae strain BECh2 (WO 00/39322) was transformed with pMStr91 using standard techniques (Christensen, T. et al., (1988), Biotechnology 6,1419-1422).

Transformants were cultured in YP+2% G medium shaken at 250 RPM at 30°C and expression of amylase was monitored by SDS-PAGE.

Medium YP+2% G 10g yeast extract 20g peptone water to 1 L autoclave at 121°C, 20 minutes add 100m120% sterile glucose solution Example 4: Characterization of amylase The molecular weight of the amylase prepared in a previous example was found to be around 66 kDa as seen on SDS-PAGE. The isoelectric point (pl) was found to be around pH 3. 5, as determined by isoelectric focusing (IEF). pH and temperature profiles were determined with AZCL-amylose (product of Megazyme) as substrate. At 50°C, the amylase was found to be active at pH 4-10 with an op- timum around pH 5-7. At pH 5.5, the amylase was found to be active at 20-70°C with an opti- mum around 60°C. Thus, the Valsaria rubricosa amylase has a wider pH range and a higher temperature optimum than the fungal amylase from Aspergillus oryzae.

Stability of the amylase was determined by incubation at pH 5-7 and 60-80°C for 5-25 minutes. The results showed more than 90 % residual activity after 20 minutes at pH 6-7 and 60°C. At pH 5.0 and 60°C, the amylase was nearly completely inactivated in 15 minutes. At 70°C, the amylase was nearly completely inactivated at in 5-10 minutes at pH 6-7. It was found that at all conditions the Valsaria rubricosa amylase is more stable than the fungal amylase from Aspergillus oryzae.

The amylase showed no activity on the following substrates at pH 7.0 : AZCL- galactomannan, AZCL-beta-glucan, AZCL-dextran, AZCL-xyloglucan, AZCL-potato galactan, AZCL-arabinan, AZCL-pullulan, AZCL-xylan, AZCL-he-cellulose and AZCL-casein.

Example 5: Effect of amylase on freshness of bread Bread were baked according to the sponge & dough method.

Recipes Sponge % on flour basis Soya oil 2,5 Sodium stearyl lactylate (SSL) 0,38 Yeast 5 Wheat flour 60 Water 62 Dough % on flour basis Ascorbic acid optimized for each flour ADA 20 ppm Salt 2 Syrup 7 (dry substance) Water optimized for each flour Wheat flour 40 Calcium propionate 0.25 Enzymes as indicated below Sponge Scaling of ingredients, addition of yeast, water, flour, SSL and oil into mixer bowl Mixing 90 rpm for 1 minutes, 150 rpm for 4 minutes The sponge was weighted, the temperature was measured and the sponge was placed in a bowl-fermentation 3 hours at 27 C, 86 % RH Dough Addition of ingredients and the sponge into the mixer bowl. The sponge and ingredi- ents were mixed together 90 rpm for 9 minutes The temperature was measured, dough characteristics were evaluated, the dough was scaled into smaller pieces of 435 g each.

The dough rests on the table for 10 minutes Doughs were sheeted and molded.

Fermentation for 55 minutes at 42°C and 86% RH.

Bread were baked at 200°C for 22 minutes Enzymes were dosed at 400 MANU/kg of Novamyl together with 0,5 or 20 AmU/kg of the amylase of SEQ ID NO: 2 (prepared as in Example 1).

Bread were stored at room temperature until analysis.

Texture and water migration by NMR were measured on day 7,14 and 21. A small sensory evaluation of softness and moistness was performed on day 21.

Results Firmness of the loaves was measured as described in WO 9953769 The results were as follows : Novamyl dosage Amylase of in-Firmness after 7 Firmness after Firmness after MANU/kg vention days 14 days 21 days AmU/kg g g g 400 0 593 869 1103 400 5 505 814 1000 400 20 480 789 939 Elasticity of the loaves was measured as described in US 6162628. The results were as follows :

Novamyl dosage Amylase of in-Elasticity after 7 Elasticity after Elasticity after MANU/kg vention days 14 days 21 days AmU/kg % % % 400 0 50. 7 46. 5 45. 2 400 5 50. 1 46. 7 44. 7 400 20 50. 7 47. 2 46. 0

The data show that the amylase of the invention has a significant effect on firmness in combination with Novamyl, furthermore the elasticity seems to be comparable to or even better than that of Novamyl after 21 days of storage.

The mobility of free water was determined as described by P. L. Chen, Z. Long, R.

Ruan and T. P. Labuza, Nuclear Magnetic Resonance Studies of water Mobility in Bread dur- ing Storage. Lebensmittel Wissenschaft und Technologie 30,178-183 (1997). The results were as follows : Novamyl dosage Amylase of in-Free water after Free water after Free water after MANU/kg vention 7 days 14 days 21 days AmU/kg Micro-sec Micro-sec Micro-sec 400 0 7498 6921 6198 400 5 7780 6856 6424 400 20 7945 7004 6618

The data show that the amylase of the invention increases the amount of free water.

The amount of free water has been described in literature to correlate to moistness of bread crumb.

The ranking from the small sensory evaluation of softness and moistness on day 21 showed the following ranking (MANU/kg of Novamyl + AmU/kg of amylase of invention): Moistest: 400 MANU + 20 AmU Second: 400 MANU + 5 AmU Lowest (least moist): 400 MANU PCT Original (for SUBMISSION) (This sheet is not part of and does not count as a sheet of the international application) 0-1 Form PCT/RO/134 (SAFE) Indications Relating to Deposited Microorganism (s) or Other Biological Material (PCT Rule 13bis) 0-1-1 Prepared Using PCT-SAFE [EASY mode] Version 3. 50 (Build 0002. 162) 0-2 International Application No. 0-3 Applicant's or agent's file reference 10 583. 204-WO 1 The indications made below relate to the deposited microorganism (s) or other biological material referred to in the description on : 1-1 page 2 2 1-2 line 10-12 1-3 identification of deposit 1-3-1 Name of depositary institution DSMZ DSMZ-Deutsche Sammlung von Mikroor- ganismen und Zellkulturen GmbH 1-3-2 Address of depositary institution Mascheroder Weg lb, D-38124 Braunschweig, Germany 1-3-3 Date of deposit 16 December 2003 (16. 12. 2003) 1-3-4 Accession Number DSMZ 16113 1-5 Designated States for Which all designations Indications are Made FOR RECEIVING OFFICE USE ONLY 0-4 This form was received with the international application : (yes or no) 0-4-1 Authorized officer FOR INTERNATIONAL BUREAU USE ONLY 0-5 This form was received by the international Bureau on : 0-5-1 Authorized officer