WO2001096537A2

WO2001096537A2 - Pre-oxidized alpha-amylase

Info

Publication number: WO2001096537A2
Application number: PCT/DK2001/000404
Authority: WO
Inventors: Bjarne Rønfeldt Nielsen; Mads Weibye
Original assignee: Novozymes A/S
Priority date: 2000-06-14
Filing date: 2001-06-13
Publication date: 2001-12-20
Also published as: WO2001096537A3; AU2001273880A1

Abstract

The present invention relates to a modified alpha-amylase suitable for producing a maltodextrin or glucose syrup; a composition comprising the modified alpha-amylase of the invention; a method of producing a syrup; the obtained syrup and side product; and the use of the modified syrup and side product as ingredient in foods or pharmaceuticals.

Description

Title: Pre-oxidized Alpha-Amylase

FIELD OF THE INVENTION

The present invention relates to a modified alpha-amylase suitable for maltodextrin or glucose syrup production; a process for the production of a maltodextrin or glucose syrup; a composition comprising a modified alpha-amylase of the invention suitable for producing a maltodextrin or glucose syrup; and finally the use of said product .

BACKGROUND OF THE INVENTION

Enzymes are today used for the production of maltodextrin (DE around 5-20) and glucose syrups (DE from 20-100), as a substitution for acids and other chemicals. In certain case it is desired to produce a syrup, which has a similar sugar profile as the, e.g., traditionally acid converted syrup product. In other case it is desirable to produce a new product with a sugar profile tailored to the application for which the syrup is used. WO 99/46399 (Novozymes A/S) concerns a process of producing speciality syrups with a DE around 42 resembling the traditionally acid converted 42 DE glucose syrup used as an ingredient in products such as hard boiled candy, toffees, fudge, fondant and the like.

Short Description of the Drawing

Figure 1 shows alignment of a number of Termamyl-like alpha- amylases .

Figure 2 shows oxidation of a Methionine residue with H₂0₂ DETAILED DESCRIPTION OF THE INVENTION

The modified enzyme of the invention

The invention relates to a modified alpha-amylase, which provides a desired sugar profile when used for treating starch for the production of maltodextrin or glucose syrups.

In the first aspect the invention relates to a modified alpha-amylase, wherein the alpha-amylase is derived from the genus Bacillus and/or is a Termamyl-like alpha-amylase, wherein the alpha-amylase has been pre-oxidized.

According to the invention the pre-oxidation may be carried out in any way known in the art using any suitable oxidation agent. Example of suitable oxidation agents include: H₂0₂, KMn0₄, urea peroxide (carbamide peroxide) , perborates, bro- mates, chlorites, iodates, bromates, persulphates, tetra- thionates, and other H₂0₂ generating agents.

Termamyl-like alpha-amylases

According to the invention the alpha-amylase include all of the below listed.

A number of alpha-amylases produced by Bacillus spp. are highly homologous (identical) on the amino acid level. The identity of a number of Bacillus alpha-amylases can be found below in Table 1 :

Table 1

Percent identity

707 AP1378 BAN BSG SP690 SP722 AA560 Term. 707 100.0 86.4 66.9 66.5 87.6 86.2 95.5 68.1 AP1378 86.4 100.0 67.1 68.1 95.1 86.6 86.0 69.4 BAN 66 . 9 67.1 100.0 65.6 67.1 68.8 66 . 9 80.7 BSG 66.5 68.1 65.6 100.0 67.9 67.1 66.3 65.4 SP690 87.6 95.1 67.1 67.9 100.0 87.2 87.0 69.2 SP722 86,.2 86.6 68 . 8 67 . 1 87 . 2 100 . 0 86 . 8 70 . 8

AA560 95, .5 86.0 66 . 9 66 . 3 87 . 0 86 . 8 100 . 0 68 . 3

Term. 68, .1 69.4 80 . 7 65 . 4 69 . 2 70 . 8 68 . 3 100 . 0

For instance, the B . licheniformis alpha-amylase compris- ing the amino acid sequence shown in SEQ ID NO: 8 (commercially available as Termamyl^®) has been found to be about 81% homologous with the B . amyloliquefaciens alpha-amylase comprising the amino acid sequence shown in SEQ ID NO: 10 and about 65% homologous with the B. stearothermophilus alpha- amylase comprising the amino acid sequence shown in SEQ ID NO: 6. Further homologous alpha-amylases include SP690 and SP722 disclosed in WO 95/26397 and further depicted in SEQ ID NO : 2 and SEQ ID NO: 4, respectively, herein. Other amylases are the AA560 alpha-amylase derived from Bacillus sp. and shown in SEQ ID NO: 12, and the #707 alpha-amylase derived from Bacillus sp. #707, shown in SEQ ID NO: 13 and described by Tsukamoto et al . , Bio-chemical and Biophysical Research Communications, 151 (1988), pp. 25-31. The KSM AP1378 alpha-amylase is disclosed in WO 97/00324 (from KAO Corporation) . Still further homologous alpha-amylases include the alpha-amylase produced by the B. licheniformis strain described in EP 0252666 (ATCC 27811) , and the alpha-amylases identified in WO 91/00353 and WO 94/18314. Other commercial Termamyl-like alpha-amylases are comprised in the products sold under the following tradenames: Optitherm^® and Takatherm^® (available from Solvay) ; Maxamyl^® (available from Gist- Brocades/Genencor) , Spezym AA^® and Spezyme Delta AA™ (available from Genencor) , and Keistase^® (available from Daiwa) .

Because of the substantial homology found between these alpha-amylases, they are considered to belong to the same class of alpha-amylases, namely the class of "Termamyl-like alpha-amylases" . Accordingly, in the present context, the term "Termamyl- like alpha-amylase" is intended to indicate an alpha-amylase, which, at the amino acid level, exhibits a substantial identity to Termamyl^®, i.e., the B. licheniformis alpha-amylase having the amino acid sequence shown in SEQ ID NO: 8 herein.

In other words, all the following alpha-amylases, which has the amino acid sequences shown in SEQ ID NOS : 2, 4, 6, 8, 10, 12 and 13 herein are considered to be "Termamyl-like alpha-amylase" . Other Termamyl-like alpha-amylases are alpha- amylases i) which displays at least 60%, such as at least 70%, e.g., at least 75%, or at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% homology with at least one of said amino acid sequences shown in SEQ ID NOS : 2,4,6,8,10,12, or 13, and/or ii) is encoded by a DNA sequence which hybridizes to the DNA sequences encoding the above- specified alpha-amylases which are apparent from SEQ ID NOS: 1,3,5,7,9, or 11 of the present specification (which encoding sequences encode the amino acid sequences shown in SEQ ID NOS: 2, 4, 6, 8, 10 or 12 herein, respectively) In connection with property i) , the homology may be determined as the degree of identity between the two sequences indicating a derivation of the first sequence from the second. The homology may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG pro- gram package (described above) . Thus, Gap GCGvδ may be used with the following default parameters: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, default scoring matrix. GAP uses the method of Needleman/Wunsch/Sellers to make alignments . A structural alignment between Termamyl (SEQ ID NO : 8) and a Termamyl-like alpha-amylase may be used to identify equivalent/corresponding positions in other Termamyl-like alpha-amylases. One method of obtaining said structural align- ment is to use the Pile Up programme from the GCG package using default values of gap penalties, i.e., a gap creation penalty of 3.0 and gap extension penalty of 0.1. Other structural alignment methods include the hydrophobic cluster analysis (Gaboriaud et al . , (1987), FEBS LETTERS 224, pp. 149-155) and reverse thread-ing (Huber, T; Torda, AE, PROTEIN SCIENCE Vol. 7, No. 1 pp. 142-149 (1998) .

Hybridisation The oligonucleotide probe used in the characterization of the Termamyl-like alpha-amylase in accordance with property ii) above may suitably be prepared on the basis of the full or partial nucleotide or amino acid sequence of the alpha-amylase in question. Suitable conditions for testing hybridisation involve pre-soaking in 5xSSC and prehybridizing for 1 hour at ~40°C in a solution of 20% formamide, 5xDenhardt ' s solution, 50mM sodium phosphate, pH 6.8, and 50mg of denatured sonicated calf thymus DNA, followed by hybridisation in the same solution supplemented with lOOmM ATP for 18 hours at ~40°C, followed by three times washing of the filter in 2xSSC, 0.2% SDS at 40°C for 30 minutes (low stringency) , preferred at 50°C (medium stringency) , more preferably at 65°C (high stringency) , even more preferably at ~75°C (very high stringency) . More details about the hybridisation method can be found in Sambrook et al . , Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989.

In the present context, "derived from" is intended not only to indicate an alpha-amylase produced or producible by a strain of the organism in question, but also an alpha-amylase encoded by a DNA sequence isolated from such strain and produced in a host organism transformed with said DNA sequence. Finally, the term is intended to indicate an alpha-amylase, which is encoded by a DNA sequence of synthetic and/or cDNA origin and which has the identifying characteristics of the alpha-amylase in question. The term is also intended to indicate that the parent alpha-amylase may be a variant of a natu- rally occurring alpha-amylase, i.e., a variant, which is the result of a modification (insertion, substitution, deletion) of one or more amino acid residues of the naturally occurring alpha-amylase .

Parent Unmodified Alpha-Amylases

According to the invention all Termamy-like alpha- amylases, as defined above, may be used as the parent (i.e., backbone) alpha-amylase. In a preferred embodiment of the invention the parent alpha-amylase is derived from B . licheni - formis, e.g., one of those referred to above, such as the B . licheniformis alpha-amylase having the amino acid sequence shown in SEQ ID NO : 8. Other preferred parent alpha-amylases are the SP722 alpha-amylase and the AA560 alpha-amylases. Parent Unmodified Hybrid Alpha-Amylases The parent unmodified alpha-amylase may also be a hybrid alpha-amylase, i.e., an alpha-amylase, which comprises a combination of partial amino acid sequences derived from at least two alpha-amylases.

The parent hybrid alpha-amylase may be one, which on the basis of amino acid homology (identity) and/or DNA hybridization (as defined above) can be determined to belong to the Termamyl-like alpha-amylase family. In this case, the hybrid alpha-amylase is typically composed of at least one part of a Termamyl-like alpha-amylase and part (s) of one or more other alpha-amylases selected from Termamyl-like alpha-amylases or non-Termamyl-like alpha-amylases of microbial (bacterial or fungal) and/or mammalian origin. Thus, the parent hybrid alpha-amylase may comprise a combination of partial amino acid sequences deriving from at least two Termamyl-like alpha-amylases, or from at least one Termamyl-like and at least one non-Termamyl-like bacterial al- pha-amylase, or from at least one Termamyl-like and at least one fungal alpha-amylase. The Termamyl-like alpha-amylase from which a partial amino acid sequence derives, may be any of those specific Termamyl-like alpha-amylase referred to herein. For instance, the parent alpha-amylase may comprise a C- terminal part of an alpha-amylase derived from a strain of B. licheniformis, and a N-terminal part of an alpha-amylase derived from a strain of B . amyloliquefaciens or from a strain of B. stearothermophilus . For instance, the parent alpha- amylase may comprise at least 430 amino acid residues of the C-terminal part of the B . licheniformis alpha-amylase, and may, e.g., comprise a) an amino acid segment corresponding to the 37 N-terminal amino acid residues of the B. amyloliquefaciens alpha-amylase having the amino acid sequence shown in SEQ ID NO: 10 and an amino acid segment corresponding to the 445 C-terminal amino acid residues of the B. licheniformis alpha-amylase having the amino acid sequence shown in SEQ ID NO: 8, or a hybrid Termamyl-like alpha-amylase being identical to the Termamyl sequence, i.e., the Bacillus licheniformis alpha- amylase shown in SEQ ID NO: 8, except that the N-terminal 35 amino acid residues (of the mature protein) has been replaced by the N-terminal 33 residues of BAN (mature protein), i.e., the Bacillus amyloliquefaciens alpha-amylase shown in SEQ ID NO: 10; or b) an amino acid segment corresponding to the 68 N- terminal amino acid residues of the B . stearothermophilus al- pha-amylase having the amino acid sequence shown in SEQ ID NO: 6 and an amino acid segment corresponding to the 415 C- terminal amino acid residues of the B . licheniformis alpha- amylase having the amino acid sequence shown in SEQ ID NO: 8. Another suitable parent hybrid alpha-amylase is the one previously described in WO 96/23874 (from Novo Nordisk) constituting the N-terminus of BAN, Bacillus amyloliquefaciens alpha-amylase (amino acids 1-300 of the mature protein) and the C-terminus from Termamyl (amino acids 301-483 of the mature protein) .

In a preferred embodiment the alpha-amylase has a Me- thionine residue in position 197, using SEQ ID No : 8 for numbering, or a corresponding position in another Termamyl-like alpha-amylase has been oxidized. It is believed that also the Methionine in position 15 (B . licheniformis alpha-amylase numbering) and other Methionines, in corresponding positions will be oxidized.

In a preferred embodiment the alpha-amylase has a Me- thionine residue in the active site cleft or close thereto or on the surface of the enzyme structure .

A parent B. licheniformis alpha-amylase has of about 7,500 NU(T) /mg.

The B . licheniformis alpha-amylase is commercially avail- able as Termamyl^® and is sold with difference activity levels. For instance, Termamyl 120L has an activity of 120 KNU(T)/g and Termamyl^® 300L has an activity of 300 KNU(T)/g.

An oxidized alpha-amylase of the invention has, calculated relatively to the unmodified parent alpha-amylase (i.e., having 100% relative activity) a relative residual activity in the range from 10-70%, preferably from 20-50%, especially around 25-35% activity.

The level of oxidation - and the residual activity level (e.g., in KNU(T)/g) - can be adjusted to a predetermined de- sired level . In the "Material & Methods" section one method of oxidating alpha-amylase have been described.

Specifically - using Termamyl 120L (120 KNU(T)/g)- an oxidized B . licheniformis alpha-amylase of the invention has an activity in the range from 5-100 KNU(T)/g, preferably in the range from 15-80 KNU(T)/g, more preferably in the range from 30-65, especially around 40 KNU(T)/g, and/or a specific activity in the range from 500-7,000 NU/mg, preferably in the range from 1,000-5,000 NU/mg, more preferably in the ranger from 2,000-4,000 NU/mg, especially around 2,500 NU/mg.

In a specific embodiment the Termamyl-like alpha-amylase is the Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 8, which has a Methionine residue in positions M197. According to the invention the unmodified alpha-amylase is oxidized using any suitable oxidation agent. This oxidation results in that one or more of the Methionine residues, preferably at or close to the active site, especially in position M197 or corresponding positions of Termamyl-like alpha- amylases (i.e., M202 in SP722 (SEQ ID NO: 4) or AA560 (SEQ ID NO: 12) are oxidized providing a modified Methionine residue (see Fig. 2)

Thio-ethers are easily oxidized into sulfoxides by H₂0₂ or H₂0₂ generating agents (see below) . The oxidation to sul- fones may also be mediated by H₂0₂, but has in general a lower rate. Other oxidation agents include KMn0₄.

It is believed that the activity of the modified alpha- amylase of the invention is reduced by the introduction of one or more oxygen, which increases the size of the Methionine residue (s) in or close to the active site (cleft) and gives the modified enzyme altered specificity. Position M15 is considered to be close to the active site. Oxidation of one or more other Methionines, e.g., one or more of the Methionines in positions 8, 256, 304, 366 or 438 (SEQ ID NO : 8 numbering) may also be at least partly responsible for the changed specificity and altered sugar profile of the maltodextrin or glucose syrup . The Process and Product of the invention

The invention also relates to the product (i.e., maltodextrin or glucose syrup) obtainable by the process of the invention. To produce a maltodextrin or a glucose syrup of the invention with a desired sugar profile a modified alpha-amylase of the invention may be used.

According to the invention starch is treated (standard conditions well known to the skilled person) with a pre- oxidized alpha-amylase, preferably a modified alpha-amylase of the invention, until a product with a DE of between 5 and 45 has been provided and/or a product with a molecular weight in the range from 5-30 kda has been provided.

The product of the invention may in an embodiment be a product directly obtained by treating starch until a desired

DE (Dextrose Equivalent) or Molecular Weight (Mw) . The product may also be a fraction of such directly obtained product, which has been separated there from.

Thus, in an embodiment the product is a maltodextrin or glucose syrup with a DE in the range from 5-45. Especially contemplated are maltodextrin or glucose syrups with a DE of 5, 6.5, 9, 14, 17, 18.5, 21.5, 28, 34, 37.5, 42 and 45.

In another embodiment it may be a maltodextrin or glucose syrup with a molecular weight in the range from 5-30 kda, preferably 8-25 kda, especially in the range from 10-20 kda, such as 14-16 kda. Expressed as Degree of Polymerization (DP) this equals to 30-190, preferably 50-155, especially 60-125, such as 85-100.

Use of a maltodextrin or Glucose Syrup of the invention

Maltodextrin or glucose syrups of the invention may be used for a number of applications, e.g., within the food or pharmaceutical industry. Examples of applications of the product of the invention are:

Confectionery, such as candies, gelatine gum, tablets, panned goods, chewy candies; Beverages, such as isotonic drinks, low alcohol beers; Bakery, such as Cereal bars, fat filling glazing, extruded snacks, icing mixes; dairy and ice cream, such as coffee whiteners, imitation cheese, spreads, ice creams; conventional food, such as salad dressings, Soups and sauce mixes, beverage mixes, baby and health food, frozen food; food ingredients and preparations, such as cured meat, fermented meat, spices and seasonings encapsulated flavors. Also the use as carrier or coating in pharmaceuticals is contemplated.

The composition of the invention The invention also relates to a composition comprising a modified alpha-amylase of the invention. The composition may further comprise one or more enzymes having one or more of the following activities: alpha-amylase, glucoamylase, protease, such as acidic protease, especially derived from A . niger, pullulanases, isoamylases, or beta-amylase .

Glucoamylases

The glucoamylase in question may be derived from a microorganism or a plant. Preferred are glucoamylases of fungal or bac- terial origin selected from the group consisting of Aspergil - lus glucoamylases, in particular A . niger Gl or G2 glucoamylase (Boel et al . (1984), EMBO J. 3 (5), p. 1097-1102), or variants thereof, such as disclosed in WO 92/00381 and WO 00/04136; the A . awamori glucoamylase (WO 84/02921), A . oryzae (Agric. Biol. Chem. (1991), 55 (4), p. 941-949), or variants or fragments thereof.

Other contemplated Aspergillus glucoamylase variants include variants to enhance the thermal stability: G137A and G139A (Chen et al . (1996), Prot . Engng. 9, 499-505); D257E and D293E/Q (Chen et al . (1995), Prot. Engng. 8, 575-582); N182 (Chen et al . (1994), Bioche . J. 301, 275-281); disulphide bonds, A246C (Fierobe et al . (1996), Biochemistry, 35, 8698- 8704; and introduction of Pro residues in position A435 and S436 (Li et al . (1997), Protein Engng. 10, 1199-1204. Furthermore Clark Ford presented a paper on Oct 17, 1997, ENZYME ENGINEERING 14, Beijing/China Oct 12-17, 97, Abstract number: Abstract book p. 0-61. The abstract suggests mutations in po- sitions G137A, N20C/A27C, and S3OP in an Aspergillus awamori glucoamylase to improve the thermal stability. Other glucoamylases include Talaromyces glucoamylases, in particular derived from Talaromyces emersonii (WO 99/28448) , Talaromyces leycet- tanus (US patent no. Re. 32,153), Talaromyces duponti , Talaro- myces thermophilus (US patent no. 4,587,215). Bacterial glucoamylases contemplated include glucoamylases from the genus Clostridium, in particular C. thermoamylolyticum (EP 135,138), and C. thermohydrosulfuricum (WO 86/01831) . Commercial products include SAN™ Super and AMG™ E. from Novo Nordisk

Protease

Suitable proteases include fungal and bacterial proteases .

Preferred proteases are acidic proteases, i.e., proteases characterized by the ability to hydrolyze proteins under acidic conditions below pH 7.

Suitable acid fungal proteases include fungal proteases derived from Aspergillus, Mucor, Rhizopus, Candida, Coriolus, Endothia, Enthomophtra , Irpex, Penicillium, Sclerotium and To- rulopsis . Especially contemplated are proteases derived from

Aspergillus niger (see, e.g., Koaze et al . , (1964), Agr. Biol.

Chem. Japan, 28, 216), Aspergillus sai toi (see, e.g., Yoshida,

(1954) J. Agr. Chem. Soc . Japan, 28, 66), Aspergillus awamori (Hayashida et al . , (1977) Agric. Biol. Chem., 42(5), 927-933,

Aspergillus aculeatus (WO 95/02044) , or Aspergillus oryzae; and acidic proteases from Mucor pusillus or Mucor miehei .

Bacterial proteases, which are not acidic proteases, include the commercially available products Alcalase^® and Neutrase^®

(available from Novo Nordisk) .

Pullanase

Examples of pullulanases include, but are not limited to a thermostable pullulanase from, e.g., Pyrococcus or a protein engineered pullulanase from, e.g., a Bacillus strain such as Bacillus acidopullulyticus (e.g., Promozyme^® available from Novo Nordiks) or Bacillus deramificans (Promozyme™ D) ; or the Bacillus deramificans pullulanase with GeneBank accession num- ber Q68699) .

Examples of commercially available pullulanases include Promozyme™, Promozyme D and Optimax from Novo Nordisk.

MATERIALS & METHODS Enzyme:

Bacillus licheniformis alpha-amylase available under the tradename Termamyl^® 120L from Novo Nordisk, Denmark)

Catalase (available from Novo Nordsk under the tradename Ter- minox^® ultra) .

METHODS

Fermentation and purification of Alpha-Amylase

Fermentation and purification may be performed by methods well known in the art. Specific activity determination

The specific activity is determined using the method described under "Assay for Alpha-amylase Activity) . A Termamyl standard is used to calculate the activity in KNU(T)/g.

Assays for Alpha-Amylase Activity

Alpha-amylase activity is determined by a method employing the PNP-G7 substrate. PNP-G7 which is a abbreviation for p-nitrophenyl-alpha, D-maltoheptaoside is a blocked oligosac- charide which can be cleaved by an endo-amylase. Following the cleavage, the alpha-Glucosidase included in the kit digest the substrate to liberate a free PNP molecule which has a yellow colour and thus can be measured by visible spectophometry at l=405nm. (400-420 nm.). Kits containing PNP-G7 substrate and alpha-Glucosidase is manufactured by Boehringer-Mannheim (cat. No. 1054635) .

To prepare the substrate one bottle of substrate (BM 1442309) is added to 5 ml buffer (BM1442309) . To prepare the alpha-Glucosidase one bottle of alpha-Glucosidase (BM 1462309) is added to 45 ml buffer (BM1442309) . The working solution is made by mixing 5 ml alpha-Glucosidase solution with 0.5 ml substrate .

The assay is performed by transforming 20 micro 1 enzyme solution to a 96 well microtitre plate and incubating at 25°C. 200 micro 1 working solution, 25°C is added. The solution is mixed and preincubated 1 minute and absorption is measured every 15 sec. over 3 minutes at OD 405 nm.

The slope of the time dependent absorption-curve is directly proportional to the specific activity (activity per mg enzyme) of the alpha-amylase in question under the given set of conditions. Method of Pre-Oxidizing Alpha-Amylase

The alpha-amylase in question is pre-oxidized with 0.1 M H₂0₂ at pH 7 and 40°C. Samples of pre-oxidized alpha-amylase (10 ml) are taken after selected time intervals of, e.g., 5, 10, 20, 30 or more minutes, or when desired activity levels are reached (determined as KNU(T)/g). The oxidation reaction is stopped by adding 20 micro litre catalase (2 mg/ml) while mixing the solution carefully.

EXAMPLES

Example 1

Pre-oxidation of Termamyl^® alpha-amylase

Bacillus licheniformis alpha-amylase (Termamyl 120L) was oxi- dized using 0.1 M H₂0₂ at pH 7 and 40°C until an activity around 40 KNU(T)/g was reached.

Claims

1. A modified alpha-amylase, wherein i) the alpha-amylase is derived from the genus Bacillus, and/or ii) is a Termamyl-like alpha-amylase, wherein said alpha-amylase has been pre-oxidized.

2. The modified alpha-amylase of claim 1, wherein the one or more Methionines in positions 8, 15, 197, 256, 304, 366, and 438, using SEQ ID No: 8 for numbering, or a corresponding position in another Termamyl-like alpha-amylase has been oxidized.

3. The modified alpha-amylase of claims 1 or 2 , the alpha- amylase at least has a Methionine in position 197, using SEQ

ID No: 8 for numbering, or a corresponding position in another Termamyl-like alpha-amylase has been oxidized.

4. The modified alpha-amylase of any of claims 1-4, wherein the parent Termamyl-like alpha-amylase is derived from a strain of B. licheniformis, B . amyloliquefaciens, B. stearothermophilus, Bacillus sp. NCIB 12289, NCIB 12512, NCIB 12513 or DSM 9375, or DSMZ no. 12649, KSM AP1378 or Bacillus sp. #707.

5. The modified alpha-amylase of claims 1-4, wherein the alpha-amylase is any of the alpha-amylases shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, or 13.

6. The modified alpha-amylase of any of claims 1-5, wherein the alpha-amylase has an amino acid sequence which has a degree of identity to SEQ ID NO: 2, 4, 6, 8, 10, 12, or 13 of at least 60%, preferably 70%, more preferably at least 80%, even more preferably at least about 90%, even more preferably at least 95%, even more preferably at least 97%, and even more preferably at least 99%.

7. The modified alpha-amylase of any of claims 1-6, wherein the alpha-amylase is encoded by a nucleic acid sequence, which hybridizes under low, preferably medium, preferred high stringency conditions, with the nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 9 or 11.

8. The modified alpha-amylase of claims 1-7, wherein the oxidized alpha-amylase has a specific activity in the range from 500-7,000 NU(T) /mg.

9. The modified alpha-amylase of claims 1-8, wherein the alpha-amylase, relative to a starting point of 120 KMU(T)/g, has an activity in the range from 5-100 KNU(T)/g, preferably in the range from 15-80 KNU(T)/g, more preferably in the range from 30-65 KNU(T)/g, especially around 40 KNU(T)/g.

10. The modified alpha-amylase of claims 1-9, wherein the alpha-amylase has an activity in the range from 1,000-5,000 NU/mg, preferably, in the ranger from 2,000-4,000 NU/mg, especially around 2,500 NU/mg.

11. A process of producing a maltodextrin or glucose syrup, wherein starch is treated with a pre-oxidized alpha-amylase until a product with DE between 5 and 45 has been provided and/or until a product with a molecular weight of between 5 and 30 kda has been has been provided.

12. The process of claim 11, wherein the pre-oxidized alpha- amylase is any of the modified alpha-amylases of claims 1-10.

13. The process of claims 11 or 12, wherein the product comprises a maltodextrin with a DE of 18.5 and/or a maltodextrin with a molecular weight of 14- 16 kda.

14. A maltodextrin or glucose syrup obtainable by the process of any of claims 11 to 13.

15. A composition comprising a modified alpha-amylase of claims 1-10.

16. The composition of claim 15, wherein the composition further comprises one or more of enzymes selected from the group of amylases, glucoamylases, proteases, such as acidic prote- ases, especially derived from A . niger, pullulanases, isoamy- lases, or beta-amylases .

17. Use of the modified alpha-amylase of claims 1-10 or the composition of claims 15 or 16 for producing a maltodextrin or glucose syrup

18. Use of a syrup of claim 17 as ingredient in food, feed or pharmaceuticals .