CA2323068A1 - Enzymatic preparation of glucose syrup from starch - Google Patents

Abstract

The present invention relates to a process for the preparation of a glucose syrup wherein starch is treated with a Termamyl-like .alpha.-amylase comprising a substitution in Val54 shown in SEQ ID NO: 2 or in the corresponding position in another Termamyl-like .alpha.-amylase. The invention also relates to a glucose syrup obtainable by the process of the invention and the use thereof as ingredient in food products. An object of the invention is also to provide for the use of a Termamyl-like .alpha.-amylase with a substitution in position Val54 using SEQ ID NO: 2 as the backbone or a corresponding position in another Termamyl-like .alpha.-amylase for preparing glucose syrup.

Description

ENZYMATIC PREPARATION OF GLUCOSE SYRUP FROM STARCH
FIELD OF THE INVENTION
The present invention relates to a process for the s preparation of starch-hydrolysate syrups having characteristics which render them particularly attractive for a variety of industrial applications, notably in the food industry. The invention makes it possible using one enzyme, for the first time, to obtain syrups of the above-mentioned kind which io closely match syrups whose preparation previously only feasible using acid hydrolysis (i.e., non-enzymatic hydrolysis) of starch.
BACKGROUND OF THE INVENTION
Glucose syrups with a DE (Dextrose Equivalent) around 42 is widely used in industry as an ingredient in products such as hard boiled candy, toffees, fudge, fondant and the like.
Traditionally 42 DE glucose syrups are produced by standard acid conversion. A starch slurry is initially acidified to pH

2, and is then pumped into a continuous reactor which operates at elevated temperature and pressure. After a period of time the liquor is returned to atmospheric conditions, neutralised, clarified, decolourised and concentrated to the final syrup.
Such acid converted glucose syrup profile shown in Figure 1 reduce the tendency of sucrose to crystallise, they slow down the tendency to shell-Braining and they contribute to the characteristic ~~mouth-feel".
Today also enzymatic conversion of starch into glucose syrup has been suggested. However, such glucose syrups typically have a sugar spectrum which is quite different from..
the traditionally used 42 DE acid converted glucose syrup. .

WO 99!46399 PCT/DK99/00114 SUMMARY OF THE INVENTION
The present invention is based on the finding that a glucose syrup with a DE in the range from 35 to 45 having a sugar spectrum close to that of the traditionally acid converted 42 DE glucose syrup can be obtained by treating starch with a 54W substituted variant of Termamyl° (which is a commercially available Bacillus licheniformis a-amylase).
In the first aspect the invention relates to a process for the preparation of a glucose syrup wherein starch is treated with a Termamyl-like a-amylase comprising a substitution in Va154 shown in SEQ ID NO: 2 or in the corresponding position in s another Termamyl-like a-amylase.
The invention also relates to a glucose syrup obtainable by the process of the invention. Further, an aspect the invention also relates to the use of said glucose syrup obtainable by the process of the invention as ingredient in food products such as hard boiled candy, toffees, fudge, fondant and the like.
Another object of the invention is to provide for the use of a Termamyl-like a-amylase with a substitution in position Va154 using SEQ ID NO: 2 as the backbone (i.e., parent enzyme) or a corresponding position in another Termamyl-like a-amylase io for preparing glucose syrup.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shown the sugar spectrum of a 42 DE acid converted glucose syrup.
Figure 2 shows the sugar spectrum of a Termamyl° (i.e., Bacillus licheniformis a-amylase from Novo Nordisk shown in SEQ ID N0: 2) converted glucose syrup.

Figure 3 shows the sugar spectrum of a V54W substituted Bacillus licheniformis a-amylase variant converted glucose syrup of the invention.
Figure 4 is an alignment of the amino acid sequences of six parent Termamyl-like a-amylases. The numbers on the Extreme left designate the respective amino acid sequences as follows:
1: Bacillus sp. a-amylase, s 2: Kaoamyl a-amylase), 3: Bacillus sp. a-amylase, 4: B. amyloli quefaciens a-amylase(BAN) (SEQ ID N0: 3), 5: Bacillus Iicheniformis a-amylase (SEQ ID N0: 2), 6: a-amylase disclosed in Tsukamoto et al., Biochemical and io Biophysical Research Communications, 151 (1988), pp. 25-31.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is based on the finding that a novel glucose syrup with a DE in the range from 35 to 45 having a sugar spectrum and properties close to that of the traditionally acid converted glucose syrup, often referred to as "42 DE glucose syrup" is obtained by treating starch with a Va154Trp (V54W) substituted variants of the commercially available Bacillus licheniformis a-amylase, sold under the trade name Termamyl° (Novo Nordisk). The DNA and protein sequence of Termamyl° is displayed in SEQ ID N0: 1 and 2, respectively.
Substitution in the Va154 position of Termamyl-like a-amylases, including the B. licheniformis a-amylase, is known from WO 97/41213 (Novo Nordisk). However, it is surprising that a Va154 substituted variant can be used to preparing a syrups from starch having a sugar spectrum which is close to that o-f --an acid converted 42 DE glucose syrup as a glucose syrup prepared from starch treated with parent B. licheniformis a-amylase (SEQ ID NO: 2) has a sugar spectrum quite different therefrom.
In the first aspect the invention relates to a glucose syrup (or speciality syrup) prepared by treating starch with the Bacillus licheniformis a-amylase shown in SEQ ID N0: 2 comprising a substitution in position Va154 or a Termamyl-like a-amylase (as defined below) substituted in a position corresponding to Va154 of SEQ ID NO: 2.
The glucose syrup of the invention has properties close to that of the traditional acid converted 42 DE syrups with regard to its sugar spectrum, i.e., composition of dextrose (DP1), maltose (DP2), maltotriose (DP3), maltotetraose (DP4), maltopentaose (DP5) and a number of higher sugars such as DP10 etc. The rheological properties, such as the viscosity, resembles the traditional acid converted DE 42 syrup much closer than a corresponding syrup prepared under the same conditions by treatment with parent B. licheniformis a-amylase (i.e., SEQ ID NO: 2).
As can been seen clearly by comparing Figure 1 to 3 a glucose syrup prepared by treating starch with the Va154Trp substituted Termamyl variant (Figure 3) has a sugar spectrum closer to that of the acid converted 42 DE syrup (Figure 1) s than that of the glucose syrup prepared using parent B.
licheniformis a-amylase (Figure 2).
By using the Va154 substituted Bacillus licheniformis a-amylase variant for preparing a glucose syrup of the invention it can be seen that especially the DP1 and DP4 sugar content io has been increased to a level closer to that of the traditional 42 DE acid converted glucose syrup and the DP5 sugar content has been decreased to a level closer to that of the 42 DE
glucose syrup in comparison to the corresponding glucose syrup prepared using parent B. licheniformis a-amylase. Further, the content of the higher sugars, as can be seen by comparing the peaks) on the left side of Figures 1 to 3, are also increased to a level closer to that of the acid converted 42 DE glucose s syrup in comparison to corresponding parent B. licheniformis a-amylase converted starch glucose syrup.
According to the invention only one enzyme need to be used for producing the glucose syrup of the invention, i.e. Va154 substituted Termamyl-like a-amylase.
io The glucose syrup of the invention may be prepared by treating starch with a Va154 substituted Termamyl-like a-amylase variant for between 20 and 100 hours, preferably 50-80 hours, especially 60-75 hours at temperature in the range around 80-105°C. The pH should be in the range from pH 4-7, is preferably from pH 4.5-6.5, especially around pH 5.5-6.2. To provide suitable conditions for Termamyl-like a-amylases, which generally seen have a high degree of Calcium dependency, from 20-60 ppm Ca2', preferably around 40 ppm Cap' should be present in the reaction slurry.
2o Enzymatic conversion of starch into a glucose syrup of the acid converted 42 DE syrup type should have a number of advantages including:
~ enzymatic conversion is a mild process, ~ reduction of the formation of colour precursor hydroxymethyl 2s furfural, ~ no formation of anhydroglucose as a by-product, ~ lower ash content because of a reduction in the acid requirements, ~ cheaper downstream processing and refining.
The Termamyl-like a-amylase According to the invention the Termamyl-like variant may be any a-amylases produced by Bacillus spp. with a high degree of homology on the amino acid level to SEQ ID NO. 2 herein, as will be defined below.
s A not exhaustive list of such enzymes are the following Bacillus sp. a-amylases:
B. amyloliquefaciens a-amylase disclosed in SEQ ID N0: 4 of WO
97/41213 which is about 89o homologous with the B. licheniformis a-amylase shown in SEQ ID N0: 2 below; the B. stearothermophilus io a-amylase disclosed in SEQ ID NO: 6 in WO 97/41213. Further, homologous a-amylases include an a-amylase derived from a strain of the Bacillus sp. NCIB 12289, NCIB 12512, NCIB 12513 or DSM
9375, all of which are described in detail in WO 95/26397, and the a-amylase described by Tsukamoto et al., Biochemical and i5 Biophysical Research Communications, 151 (1988), pp. 25-31.
Other Bacillus sp. a-amylases contemplated according to the present invention to be within the definition of Termamyl-like a-amylases are the a-amylases disclosed in SEQ ID NO. 1, 2 , 3 and 7 of WO 96/23873 and variants thereof, including zo specifically the ones described in WO 96/23873.
Variants and hybrids of the above mentioned Termamyl-like a-amylases are also contemplated.
In an embodiment of the invention the parent Termamyl-like a-amylase is a hybrid a-amylase of SEQ ID NO: 2 and SEQ ID N0:
2s 4. Specifically, the parent hybrid Termamyl-like a-amylase may be identical to the Termamyl sequence, i.e., the Bacillus licheni-formis a-amylase shown in SEQ ID NO: 2, except that the N-terminal 35 amino acid residues (of the mature protein) has-3o been replaced by the N-terminal 33 residues of BAN (mature pro-tein), i.e., the Bacillus amyloliquefaciens alpha-amylase shown in SEQ ID NO: 4 (the DNA sequence of the Bacillus amyloliquefa-ciens alpha-amylase is displayed in SEQ ID NO: 3) , which fur-ther may have the following mutations:
H156Y+A181T+N190F+A209V+Q264S (using the numbering in SEQ ID NO:
s 2). The hybrid may be constructed by SOE-PCR (Higuchi et al.
1988, Nucleic Acids Research 16:7351).
Still further Termamyl-like a-amylases include the a-amylase produced by the B. licheniformis strain described in EP
0,252,666 (ATCC 27811), and the a-amylases identified in WO
io 91/00353 and WO 94/18314. Other commercial Termamyl-like a-amylases are OptithermT"' and TakathermTM (available from Sol-vay), MaxamylT"' (available from Gist-Brocades/Genencor), Spezyme AATM and Spezyme Delta AAT"' (available from Genencor) , and Keis-taseTM (available from Daiwa).
is Because of the substantial homology found between these a-amylases, they are considered to belong to the same class of a-amylases, namely the class of "Termamyl-like a-amylases".
Accordingly, in the present context, the term "Termamyl-like a-amylase" is also intended to indicate an a-amylase which, at 2o the amino acid level, exhibits a substantial homology to the B.
licheniformis a-amylase having the amino acid sequence shown in SEQ ID NO: 2 herein. In other words, a "Termamyl-like a-amylase"
is an a-amylase which has the amino acid sequence shown in SEQ
ID N0: 2 herein or any a-amylase which displays at least 60%, 2s such as at least 70%, e.g., at least 75%, or at least 80%, e.g., _ at least 85%, at least 90% or at least 95% homology with SEQ ID
N0; 2.
The "homology" may be determined by use of any conventional algorithm, preferably by use of the GAP progamme from the GCG
3o package version 7.3 (June 1993) using default values for GAP
penalties, which is a GAP creation penalty of 3.0 and GAP

extension penalty of 0.1, (Genetic Computer Group (1991) Programme Manual fcr the GCG Package, version 7, 575 Science Drive, Madison, Wisconsin, USA 53711).
A structural alignment between Termamyl and a Termamyl-like s a-amylase may be used to identify equivalent/corresponding posi tions in other Termamyl-like a-amylases. One method of obtaining said structural alignment 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.
io Other structural alignment methods include the hydrophobic cluster analysis (Gaboriaud et al., (1987), FEBS LETTERS 224, pp. 149-155) and reverse threading (Huber, T ; Torda, AE, PRO-TEIN SCIENCE Vol. 7, No. 1 pp. 142-149 (1998).
In an embodiment of the invention the Termamyl-like a-is amylase variant is one of the following B. Iicheniformis a-amylase variants (the parent B. licheniformis a-amylase is shown in SEQ ID NO: 2):
V54A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y or a Termamyl-like a-amylase or variant (as defined above) with a substitution in a 2o position corresponding to Va154 in SEQ ID N0: 2.
In a preferred embodiment the Termamyl-like a-amylase variant is one of the following substitutions B. licheniformis a-amylase variants with one of the following substitutions:
V54W,Y or F or a Termamyl-like a-amylase variant with a 2s substitution in a corresponding position.
Construction of variants of the invention The Va154 variants may be constructed by standard techniques known in the art, including Site-directed mutagenesis a.ss so described, e.g., by Morinaga et al.,(1984), Biotechnology 2, p.
646-639, and in US patent no. 4,760,025. Another suitable method introducing mutations into a-amylase-encoding DNA sequences is described in Nelson and Long, (1989), Analytical Biochemistry 180, p. 147-151. This method involves a 3-step generation of a PCR fragment containing the desired mutation introduced by using s a chemical synthesized DNA strand as one primer in the PCR
reaction. From the PCR-generated fragment, a DNA fragment carrying the mutation may be isolated by cleavage with restriction endonuclease and reinserted into an expression plasmid.
io A Va154 variant may be expressed by cultivating a microorganism comprising a DNA sequence encoding the variant under conditions which are conducive for producing the variant.
The variant may then subsequently be recovered from the resulting culture broth. Other methods known in the art may also 15 be used. For instance WO 97/41213 discloses a suitable method for providing Va154 variants.
The invention also relates to a glucose syrup obtainable by the process of the invention as described above and illustrated below in the Examples section. Further, an aspect the invention also relates to the use of the glucose syrup obtainable by the process of the invention as ingredient in food products such as hard boiled candy, toffees, fudge, fondant and the like.
In another aspect the invention relates to the use of a Termamyl-like a-amylase with a substitution in position Va154 using SEQ ID N0: 2 as the backbone or a corresponding position 2o in another Termamyl-like a-amylase for preparing a glucose syrup. The Termamyl-like variant may be any of the above mentioned.
MATERIALS AND METHODS . -.
Materials Enzyme:
Termamyl~ from Novo Nordisk shown in SEQ ID NO: 2 substituted in position Va154Trp. The variant may be prepared as described in WO 97/41213.
Other materials:
Waxy maize starch from Cerestar.
Methods DE determination DE (dextrose equivalent is defined as the amount of reduc-ing carbohydrate ( measured as dextrose-equivalents) in a sam-ple expressed as w/w% of the total amount of dissolved dry mat-s ter). It is measured by the neocuproine assay (Dygert, Li Flor-idana(1965) Anal. Biochem. No 368). The principle of the neocuproine assay is that CuS04 is added to the sample, Cu" is reduced by the reducing sugar and the formed neocuproine com-plex is measured at 450 nm.
to General molecular biology methods:
DNA manipulations and transformations were performed using standard methods of molecular biology (Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, F. M. et al. (eds.).
is "Current protocols in Molecular Biology". John Wiley and Sons, 1995; Harwood, C. R., and Cutting, S. M. (eds.) "Molecular Bio-logical Methods for Bacillus". John Wiley and Sons, 1990).
Enzymes for DNA manipulations were used according to the specifications of the suppliers.
EXAMPLES _ Example 1 Preparation of crlucose svrup of the acid converted type enzv maticallv A glucose syrup was prepared by treating a starch slurry s containing 30% DS (30% Dry Solid) waxy maize starch, 40 ppm CaZ" (adding as CaClz) at pH 6.0 with 0.1 mg enzyme protein/g DS
of Va154Trp substituted Bacillus Iicheniformis a-amylase. The temperature was kept at 95°C for one hour and 80°C for 72 hours.
io The sugar profile of the prepared glucose syrup after 20 and 72 hours of treatment is shown in the Table 1 below:
%DPx on DS Time(Hours) _ ~

DP1 7.9 10.1 DP2 19.1 _. 23.2 DP3 14.3 14.0 DP4 8.6 7.6 DP5 8.5 _. 6.6 DP6 2.4 - 2.4 DP7 3,1 4.1 DP8 3.1 3.4 DP9 2.5 2.4 DP10+ 30.5 26.4 ~a~~c 1: Sugar pro=iia azLer ~~ anc~ 72 hours of treatment with V54W substituted Bacillus licheniformis a-amylase. The DE of the obtained syrup is also given.
is Figure 3 shows the sugar spectrum of the glucose syrup ob-tained by treating a pre-cooked 5% Waxy maize starch substrate with a Va154Trp substituted Bacillus licheniformis a-amylase at 60°C for 24 hours. Figure 2 shows the sugar spectrum of a similar substrate treated with the native Bacillus licheni-formis a-amylase under similar conditions.

SEQUENCE LISTING
<110> Novo Nordisk A/S
<120> Enzymatic preparation of glucose syrup from starch <130> 5278 <140>
<141>
<160> 4 <170> PatentIn Ver. 2.0 <210> 1 <211> 1920 <212> DNA
<213> Bacillus licheniformis <220>
<221> CDS
<222> (421)..(1872) <220>
<221> mat_peptide <222> (921)..(1869) <900> 1 cggaagattg gaagtacaaa aataagcaaa agattgtcaa tcatgtcatg agccatgcgg 60 gagacggaaa aatcgtctta atgcacgata tttatgcaac gttcgcagat gctgctgaag 120 agattattaa aaagctgaaa gcaaaaggct atcaattggt aactgtatct cagcttgaag 180 aagtgaagaa gcagagaggc tattgaataa atgagtagaa gcgccatatc ggcgcttttc 240 ttttggaaga aaatataggg aaaatggtac ttgttaaaaa ttcggaatat ttatacaaca 300 tcatatgttt cacattgaaa ggggaggaga atcatgaaac aacaaaaacg gctttacgcc 360 cgattgctga cgctgttatt tgcgctcatc ttcttgctgc ctcattctgc agcagcggcg 420 gca aat ctt aat ggg acg ctg atg cag tat ttt gaa tgg tac atg ccc 968 Ala Asn Leu Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Met Pro aat gac ggc caa cat tgg agg cgt ttg caa aac gac tcg gca tat ttg 516 Asn Asp Gly Gln His Trp Arg Arg Leu Gln Asn Asp Ser Ala Tyr Leu get gaa cac ggt att act gcc gtc tgg att ccc ccg gca tat aag gga 564 Ala Glu His Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly acg agc caa gcg gat gtg ggc tac ggt get tac gac ctt tat gat tta 612 Thr Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu ggg gag ttt cat caa aaa ggg acg gtt cgg aca aag tac ggc aca aaa 660 Gly Glu Phe His Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys gga gag ctg caa tct gcg atc aaa agt ctt cat tcc cgc gac att aac 708 Gly Glu Leu Gln Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn gtt tac ggg gat gtg gtc atc aac cac aaa ggc ggc get gat gcg acc 756 Val Tyr Gly Asp Val Val Ile Asn His Lys Gly Gly Ala Asp Ala Thr gaa gat gta acc gcg gtt gaa gtc gat ccc get gac cgc aac cgc gta 809 Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val att tca gga gaa cac cta att aaa gcc tgg aca cat ttt cat ttt ccg 852 Ile Ser Gly Glu His Leu Ile Lys Ala Trp Thr His Phe His Phe Pro ggg cgc ggc agc aca tac agc gat ttt aaa tgg cat tgg tac cat ttt 900 Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe gac gga acc gat tgg gac gag tcc cga aag ctg aac cgc atc tat aag 998 Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys ttt caa gga aag get tgg gat tgg gaa gtt tcc aat gaa aac ggc aac 996 Phe Gln Gly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn tat gat tat ttg atg tat gcc gac atc gat tat gac cat cct gat gtc 1094 Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val gca gca gaa att aag aga tgg ggc act tgg tat gcc aat gaa ctg caa 1092 Ala Aia G1~: Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln ttg gac ggt ttc cgt ctt gat get gtc aaa cac att aaa ttt tct ttt 1140 Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe ttg cgg gat tgg gtt aat cat gtc agg gaa aaa acg ggg aag gaa atg 1188 Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met ttt acg gta get gaa tat tgg cag aat gac ttg ggc gcg ctg gaa aac 1236 Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu G1u Asn tat ttg aac aaa aca aat ttt aat cat tca gtg ttt gac gtg ccg ctt 1284 Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu cat tat cag ttc cat get gca tcg aca cag gga ggc ggc tat gat atg 1332 His Tyr Gln Phe His Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp Met agg aaa ttg ctg aac ggt acg gtc gtt tcc aag cat ccg ttg aaa tcg 1380 Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser gtt aca ttt gtc gat aac cat gat aca cag ccg ggg caa tcg ctt gag 1428 Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu tcg act gtc caa aca tgg ttt aag ccg. ctt get tac get ttt att ctc 1476 Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu aca agg gaa tct gga tac cct cag gtt ttc tac ggg gat atg tac ggg 1524 Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly acg aaa gga gac tcc cag cgc gaa att cct gcc ttg aaa cac aaa att 1572 Thr Lys Gly Asp Ser Gln Arg Glu Ile Pro Ala Leu Lys His Lys Ile gaa ccg atc tta aaa gcg aga aaa cag tat gcg tac gga gca cag cat 1620 Glu Pro Ile Leu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His gat tat ttc gac cac cat gac att gtc ggc tgg aca agg gaa ggc gac 1668 Asp Tyr Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp agc tcg gtt gca aat tca ggt ttg gcg gca tta ata aca gac gga ccc 1716 Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro ggt ggg gca aag cga atg tat gtc ggc cgg caa aac gcc ggt gag aca 1764 Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr tgg cat gac att acc gga aac cgt tcg gag ccg gtt gtc atc aat tcg 1812 Trp His Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser gaa ggc tgg gga gag ttt cac gta aac ggc ggg tcg gtt tca att tat 1860 Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr gtt caa aga tag aagagcagag aggacggatt tcctgaagga aatccgtttt 1912 Val Gln Arg tttatttt 1920 <210> 2 <211> 483 <212> PRT
<213> Bacillus licheniformis <400> 2 Ala Asn Leu Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Met Pro Asn Asp Gly Gln His Trp Arg Arg Leu Gln Asn Asp Ser Ala Tyr Leu Ala Glu His Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Thr Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu so s5 so Gly Glu Phe His Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Gly Glu Leu Gln Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn _ WO 99/4b399 PCT/DK99/00114 Val Tyr Gly Asp Val Val Ile Asn His Lys Gly Gly Ala Asp Ala Thr Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val Iie Ser Gly Glu His Leu Ile Lys Ala Trp Thr His Phe His Phe Pro Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys Phe Gln Gly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val Ala Ala Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu Glu Asn Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu His Tyr Gln Phe His Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp Met ' Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly Thr Lys Gly Asp Ser Gln Arg Glu Ile Pro Ala Leu Lys His Lys Ile Glu Pro Ile Leu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His Asp Tyr Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr Trp His Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr Val Gln Arg <210> 3 <211> 2609 <212> DNA
<213> Bacillus amyloliquefaciens <220>
<221> -10 signal <222> (707)..(712) <220>
<221> -35_signal <222> (729)..(734) <220>
<221> RBS
<222> (759)..(762) <220> _ <221> sig peptide <222> (770)..(862) <220>
<221> mat peptide <222> (863)..(2314) <220>
<221> terminator <222> (2321)..(2376) <220>
<221> CDS
<222> (770)..(2319) <900> 3 aagcttcaag cggtcaatcg gaatgtgcat ctcgcttcat acttaggttt tcacccgcat 60 attaagcagg cgtttttgaa ccgtgtgaca gaagctgttc gaaaccccgg cgggcggttt 120 gattttaagg ggggacagta tgctgcctct tcacattaat ctcagcggaa aaagaatcat 180 cattgctggc gggggcaatg ttgcattaag aaggctgaaa cggtgtttcc ggaaggcgct 290 gatattaccg tgatcagtct gagcctgcct gaaattaaaa agctggcgga tgaaggacgc 300 atccgctgga ttccccggag aattgaaatg aaagatctca agcccgcttt tttcattatt 360 gccgcgacaa atgaccgagg cgtgaatcag gagatagccg caaacgcttc tgaaacgcag 920 ctggtcaact gtgtaagcaa ggctgaacaa ggcagcgtat atatgccgaa gatcatccgc 480 aaagggcgca ttcaagtatc agtatcaaca agcggggcaa gccccgcaca tacgaaaaga 540 ctggctgaaa acattgagcc'tttgatgact gatgatttgg ctgaagaagt ggatcgattg 600.
tttgagaaaa gaagaagacc ataaaaatac cttgtctgtc atcagacagg gtatttttta 660 tgctgtccag actgtccgct gtgtaaaaaa taggaataaa ggggggttgt tattatttta 720 ctgatatgta aaatataatt tgtataagaa aatgagaggg agaggaaac atg att caa 778 Met Ile Gln aaa cga aag cgg aca gtt tcg ttc aga ctt gtg ctt atg tgc acg ctg 826 Lys Arg Lys Arg Thr Val Ser Phe Arg Leu Val Leu Met Cys Thr Leu tta ttt gtc agt ttg ccg att aca aaa aca tca gcc gta aat ggc acg 879 Leu Phe Val Ser Leu Pro Ile Thr Lys Thr Ser Ala Val Asn Gly Thr _10 _5 _1 1 ctg atg cag tat tLt gaa tgg tat acg ccg aac gac ggc cag cap tgg 922 Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro Asn Asp Gly Gln His Trp aaa cga ttg cag aat gat gcg gaa cat tta tcg gat atc gga atc act 970 Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp Ile G1y Ile Thr gcc gtc tgg att cct ccc gca tac aaa gga ttg agc caa tcc gat aac 1018 Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Leu Ser Gln Ser Asp Asn gga tac gga cct tat gat ttg tat gat tta gga gaa ttc cag caa aaa 1066 Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu Phe Gln Gln Lys ggg acg gtc aga acg aaa tac ggc aca aaa tca gag ctt caa gat gcg 1119 Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser Glu Leu Gln Asp Ala atc ggc tca ctg cat tcc cgg aac gtc caa gta tac gga gat gtg gtt 1162 Ile Gly Ser Leu His Ser Arg Asn Val Gln Val Tyr Gly Asp Val Val ttg aat cat aag get ggt get gat gca aca gaa gat gta act gcc gtc 1210 Leu Asn His Lys Ala Gly Ala Asp Ala Thr Glu Asp Val Thr Ala Val gaa gtc aat ccg gcc aat aga aat cag gaa act tcg gag gaa tat caa 1258 Glu Val Asn Pro Ala Asn Arg Asn Gln Glu Thr Ser Glu Glu Tyr Gln atc aaa gcg tgg acg gat ttt cgt ttt ccg ggc cgt gga aac acg tac 1306 Ile Lys Ala Trp Thr Asp~Phe Arg Phe Pro Gly Arg Gly Asn Thr Tyr agt gat ttt aaa tgg cat tgg tat cat ttc gac gga gcg gac tgg gat 1354 Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly Ala Asp Trp Asp gaa tcc cgg aag atc agc cgc atc ttt aag ttt cgt ggg gaa gga aaa 1902 Glu Ser Arg Lys Ile Ser Arg Ile Phe Lys Phe Arg Gly Glu Gly Lys gcg tgg gat tgg gaa gta tca agt gaa aac ggc aac tat gac tat tta 1950 _ Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn Tyr Asp Tyr Leu atg tat get gat gtt gac tac gac cac cct gat gtc gtg gca gag aca 1498 Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val Val Ala Glu Thr aaa aaa tgg ggt atc tgg tat gcg aat gaa ctg tca tta gac ggc ttc 1546 Lys Lys Trp Gly Ile Trp Tyr Ala Asn Glu Leu Ser Leu Asp Gly Phe cgt att gat gcc gcc aaa cat att aaa ttt tca ttt ctg cgt gat tgg 1594 Arg Ile Asp Ala Ala Lys His Ile Lys Phe Ser Phe Leu Arg Asp Trp gtt cag gcg gtc aga cag gcg acg gga aaa gaa atg ttt acg gtt gcg 1642 Val Gln Ala Val Arg Gln Ala Thr Gly Lys Glu Met Phe Thr Val Ala gag tat tgg cag aat aat gcc ggg aaa ctc gaa aac tac ttg aat aaa 1690 Glu Tyr Trp Gln Asn Asn Ala Gly Lys Leu Glu Asn Tyr Leu Asn Lys aca agc ttt aat caa tcc gtg ttt gat gtt ccg ctt cat ttc aat tta 1738 Thr Ser Phe Asn Gln Ser Val Phe Asp Val Pro Leu His Phe Asn Leu 280 285 29p cag gcg get tcc tca caa gga ggc gga tat gat atg agg cgt ttg ctg 1786 Gln Ala Ala Ser Ser Gln Gly Gly Gly Tyr Asp Met Arg Arg Leu Leu gac ggt acc gtt gtg tcc agg cat ccg gaa aag gcg gtt aca ttt gtt 1834 Asp Gly Thr Val Val Ser Arg His Pro Glu Lys Ala Val Thr Phe Val gaa aat cat gac aca cag ccg gga cag tca ttg gaa tcg aca gtc caa 1882 Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu Ser Thr Val Gln act tgg ttt aaa ccg ctt gca tac gcc ttt att ttg aca aga gaa tcc 1930 Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr Arg Glu Ser ggt tat cct cag gtg ttc tat ggg gat atg tac ggg aca aaa gqg aca 1978 Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly Thr Lys Gly Thr tcg cca aag gaa att ccc tca ctg aaa gat aat ata gag ccg att tta 2026 Ser Pro Lys Glu Ile Pro Ser Leu Lys Asp Asn Ile Glu Pro Ile Leu aaa gcg cgt aag gag tac gca tac ggg ccc cag cac gat tat att gac 2074 Lys Ala Arg Lys Glu Tyr Ala Tyr Gly Pro Gln His Asp Tyr Ile Asp cac ccg gat gtg atc gga tgg acg agg gaa ggt gac agc tcc gcc gcc 2122 His Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp Ser Ser Ala Ala aaa tca ggt ttg gcc get tta atc acg gac gga ccc ggc gga tca aag 2170 Lys Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly Gly Ser Lys cgg atg tat gcc ggc ctg aaa aat gcc ggc gag aca tgg tat gac ata 2218 Arg Met Tyr Ala Gly Leu Lys Asn Ala Gly Glu Thr Trp Tyr Asp Ile acg ggc aac cgt tca gat act gta aaa atc gga tct gac ggc tgg gga 2266 Thr Gly Asn Arg Ser Asp Thr Val Lys Ile Gly Ser Asp Gly Trp Gly gag ttt cat gta aac gat ggg tcc gtc tcc att tat gtt cag aaa taa 2319 Glu Phe His Val Asn Asp Gly Ser Val Ser Ile Tyr Val Gln Lys ggtaataaaa aaacacctcc aagctgagtg cgggtatcag cttggaggtg cgtttatttt 2374 ttcagccgta tgacaaggtc ggcatcaggt gtgacaaata cggtatgctg gctgtcatag 2434 gtgacaaatc cgggttttgc gccgtttggc tttttcacat gtctgatttt tgtataatca 2499 acaggcacgg agccggaatc tttcgccttg gaaaaataag cggcgatcgt agctgcttcc 2554 aatatggatt gttcatcggg atcgctgctt ttaatcacaa cgtgggatcc 2604 <210> 4 <211> 519 <212> PRT
<213> Bacillus amyloliquefaciens <400> 4 Met Ile Gln Lys Arg Lys Arg Thr Val Ser Phe Arg Leu Val Leu Met Cys Thr Leu Leu Phe Val Ser Leu Pro Ile Thr Lys Thr Ser Ala Val _ Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro Asn Asp Gly Gln His Trp Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp Ile ~ Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Leu Ser Gln Ser Asp Asn Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu Phe Gln Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser Glu Leu Gln Asp Ala Ile Gly Ser Leu His Ser Arg Asn Val Gln Val Tyr Gly Asp Val Val Leu Asn His Lys Ala Gly Ala Asp Ala Thr Glu Asp Val Thr Ala Val Glu Val Asn Pro Ala Asn Arg Asn Gln Glu Thr Ser Glu Glu Tyr Gln Ile Lys Ala Trp Thr Asp Phe Arg Phe Pro Gly Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly Ala Asp Trp Asp Glu Ser Arg Lys Ile Ser Arg Ile Phe Lys Phe Arg Gly Glu Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val Val Ala Glu Thr Lys Lys Trp Gly Ile Trp Tyr Ala Asn Glu Leu Ser Leu Asp Gly Phe Arg Ile Asp Ala Ala Lys His Ile Lys Phe Ser Phe Leu Arg Asp Trp Val Gln Ala Val Arg Gln Ala Thr Gly Lys Glu Met Phe Thr Val Ala Glu Tyr Trp Gln Asn Asn Ala Gly Lys Leu Glu Asn Tyr Leu Asn Lys Thr Ser Phe Asn Gln Ser Val Phe Asp Val Pro Leu His Phe Asn Leu Gln Ala Ala Ser Ser Gln Gly Gly Gly Tyr Asp Met Arg Arg Leu Leu Asp Gly Thr Val Val Ser Arg His Pro Glu Lys Ala Val Thr Phe Val Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly Thr Lys Gly Thr Ser Pro Lys Glu Ile Pro Ser Leu Lys Asp Asn Ile Glu Pro Ile Leu Lys Ala Arg Lys Glu Tyr Ala Tyr Gly Pro Gln His Asp Tyr Ile Asp His Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp Ser Ser Ala Ala Lys Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly Gly Ser Lys Arg Met Tyr Ala Gly Leu Lys Asn Ala Gly Glu Thr Trp Tyr Asp Ile Thr Gly Asn Arg Ser Asp Thr Val Lys Ile Gly Ser Asp Gly Trp Gly Glu Phe His Val Asn Asp Gly Ser Val Ser Ile Tyr Val Gln Lys

Claims (9)

1. A process for the preparation of a glucose syrup wherein starch is treated with a Termamyl-like alpha-amylase comprising a substitution in Va154 shown in SEQ ID NO: 2 or in the corresponding position in another Termamyl-like alpha-amylase until a glucose syrup having a dextrose equivalent in the range from 35 to 45 is obtained.

2. The process according to claim 1, wherein the variant is one of the following variants:
V54A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y using the B.
licheniformis alpha-amylase as the backbone or another Termamyl-like alpha-amylase variant with a corresponding substitution.

3. The process according to claim 2, wherein the Termamyl-like alpha-amylase variant as one of the following substitutions:
V54W, Y or F or a corresponding variant in another Termamyl-like alpha-amylase.

4. The process according to claims 1-3, wherein the starch is treated with an alpha-amylase variant of any of claims 2 to 4 for from 20 to 100 hours, preferably 50-80 hours, especially 60-75 hours.

5. A glucose syrup obtainable by the process according to any of claims 1 to 4.

6. Use of a glucose syrup according to claim 5 as ingredient in food products.

7. Use of a Termamyl-like alpha-amylase for producing a glucose syrup with a dextrose equivalent in the range from 35 to 45, characterized in that the Termamyl-like alpha-amylase has [with] a substitution in position Va154 using SEQ ID NO: 2 as the backbone or a corresponding position in another Termamyl-like alpha-amylase [for preparing glucose syrup].

8. The use according to claim 7, wherein the B. licheniformis .alpha.-amylase shown in SEQ ID NO: 2 is the backbone if the variant.

9. The use according to claim 8, wherein the variant is one of the following: V54A,R;D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y using the B. licheniformis .alpha.-amylase as the backbone or another Termamyl-like variant with a corresponding substitution.