WO1999046399A1

WO1999046399A1 - Enzymatic preparation of glucose syrup from starch

Info

Publication number: WO1999046399A1
Application number: PCT/DK1999/000114
Authority: WO
Inventors: Barrie Edmund Norman; Hanne Vang Hendriksen
Original assignee: Novo Nordisk A/S
Priority date: 1998-03-09
Filing date: 1999-03-08
Publication date: 1999-09-16
Also published as: AR020058A1; CA2323068A1; EP1062359A1; KR20010041617A; AU2612499A; JP2002505885A

Abstract

The present invention relates to a process for the preparation of a glucose syrup wherein starch is treated with a Termamyl-like α-amylase comprising a substitution in Val54 shown in SEQ ID NO: 2 or in the corresponding position in another Termamyl-like α-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 α-amylase with a substitution in position Val54 using SEQ ID NO: 2 as the backbone or a corresponding position in another Termamyl-like α-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 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 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 -graining 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. 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 54 substituted variant of Termamyl^® (which is a commercially available Bacillus lichenifor is α-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 α-amylase comprising a substitution in

Val54 shown in SEQ ID NO: 2 or in the corresponding position in another Termamyl-like α-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 α-amylase with a substitution in position Val54 using SEQ ID NO: 2 as the backbone (i.e., parent enzyme) or a corresponding position in another Termamyl-like α-amylase 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 α-amylase from Novo Nordisk shown in SEQ ID NO: 2) converted glucose syrup. Figure 3 shows the sugar spectrum of a V54 substituted Bacillus licheniformis α-amylase variant converted glucose syrup of the invention.

Figure 4 is an alignment of the amino acid sequences of six parent Termamyl-like α-amylases. The numbers on the Extreme left designate the respective amino acid sequences as follows: 1: Bacillus sp . α-amylase, 2: Kaoamyl α-amylase), 3: Bacillus sp . α-amylase,

4: B . amyloliquefaciens α-amylase (BAN) (SEQ ID NO: 3), 5: Bacillus licheniformis α-amylase (SEQ ID NO: 2), 6: α-amylase disclosed in Tsukamoto et al . , Biochemical and 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 Val54Trp (V54 ) substituted variants of the commercially available Bacillus licheniformis α-amylase, sold under the trade name Termamyl^® (Novo Nordisk) . The DNA and protein sequence of Termamyl^® is displayed in SEQ ID NO: 1 and 2, respectively.

Substitution in the Val54 position of Termamyl-like α- amylases, including the B . licheniformis α-amylase, is known from WO 97/41213 (Novo Nordisk) . However, it is surprising that a Val54 substituted variant can be used to preparing a syrup^~- from starch having a sugar spectrum which is close to that of an acid converted 42 DE glucose syrup as a glucose syrup prepared from starch treated with parent B . li cheniformis α- 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 α-amylase shown in SEQ ID NO: 2 comprising a substitution in position Val54 or a Termamyl-like α-amylase (as defined below) substituted in a position corresponding to Val54 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 (DPI), 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 α-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 Val54Trp substituted Termamyl variant (Figure 3) has a sugar spectrum closer to that of the acid converted 42 DE syrup (Figure 1) than that of the glucose syrup prepared using parent B . licheniformis α-amylase (Figure 2) .

By using the Val54 substituted Bacillus licheniformis α- amylase variant for preparing a glucose syrup of the invention it can be seen that especially the DPI and DP4 sugar content 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 α-amylase. Further, the content of the higher sugars, as can be seen by comparing the peak(s) 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 syrup in comparison to corresponding parent B . licheniformis α-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. Val54 substituted Termamyl-like α-amylase. The glucose syrup of the invention may be prepared by treating starch with a Val54 substituted Termamyl-like α- 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, preferably from pH 4.5-6.5, especially around pH 5.5-6.2. To provide suitable conditions for Termamyl-like α-amylases, which generally seen have a high degree of Calcium dependency, from 20-60 ppm Ca²⁺, preferably around 40 ppm Ca²⁺ should be present in the reaction slurry. 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 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 α-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. A not exhaustive list of such enzymes are the following Bacillus sp. α-amylases :

B . amyloliquefaciens a-amylase disclosed in SEQ ID NO: 4 of WO 97/41213 which is about 89% homologous with the B . licheniformis α-amylase shown in SEQ ID NO: 2 below; the B . stearother ophilus 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 Biophysical Research Communications, 151 (1988), pp. 25-31.

Other Bacillus s . α-amylases contemplated according to the present invention to be within the definition of Termamyl-like α-amylases are the α-amylases disclosed in SEQ ID NO. 1, 2, 3 and 7 of WO 96/23873 and variants thereof, including specifically the ones described in WO 96/23873.

Variants and hybrids of the above mentioned Termamyl-like α- amylases are also contemplated.

In an embodiment of the invention the parent Termamyl-like α-amylase is a hybrid α-amylase of SEQ ID NO: 2 and SEQ ID NO: 4. Specifically, the parent hybrid Termamyl-like α-amylase may be identical to the Termamyl sequence, i.e., the Bacillus licheni formis α-amylase shown in SEQ ID NO : 2, 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: 4 (the DNA sequence of the Bacillus amyloliquefa ciens alpha-amylase is displayed in SEQ ID NO: 3), which further may have the following mutations: H156Y+A181T+N190F+A209V+Q264S (using the numbering in SEQ ID NO: 5 2) . The hybrid may be constructed by SOE-PCR (Higuchi et al . 1988, Nucleic Acids Research 16:7351).

Still further Termamyl-like α-amylases include the α- amylase produced by the B . licheniformis strain described in EP 0,252,666 (ATCC 27811), and the α-amylases identified in WO

10 91/00353 and WO 94/18314. Other commercial Termamyl-like α-amylases are Optitherm™ and Takatherm™ (available from Sol- vay) , Maxamyl™ (available from Gist-Brocades/Genencor) , Spezyme AA™ and Spezyme Delta AA™ (available from Genencor) , and Keis- tase™ (available from Daiwa) .

15 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

20 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 NO: 2 herein or any α-amylase which displays at least 60%,

25 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 NO; 2.

The "homology" may be determined by use of any conventional algorithm, preferably by use of the GAP progamme from the GCG

30 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 for the GCG Package, version 7, 575 Science Drive, Madison, Wisconsin, USA 53711) .

A structural alignment between Termamyl and a Termamyl-like α-amylase may be used to identify equivalent/corresponding positions in other Termamyl-like α-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. 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, PROTEIN SCIENCE Vol. 7, No. 1 pp. 142-149 (1998).

In an embodiment of the invention the Termamyl-like α- amylase variant is one of the following B . licheniformis α- amylase variants (the parent B . li cheniformi s α-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 α- amylase or variant (as defined above) with a substitution in a position corresponding to Val54 in SEQ ID NO: 2.

In a preferred embodiment the Termamyl-like α-amylase variant is one of the following substitutions B . licheniformis α-amylase variants with one of the following substitutions: V54W,Y or F or a Termamyl-like α-amylase variant with a substitution in a corresponding position.

Construction of variants of the invention

The Val54 variants may be constructed by standard techniques known in the art, including Site-directed mutagenesis a. 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 α-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 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. A Val54 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 be used. For instance WO 97/41213 discloses a suitable method for providing Val54 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 α-amylase with a substitution in position Val54 using SEQ ID NO: 2 as the backbone or a corresponding position in another Termamyl-like α-amylase for preparing a glucose syrup. The Termamyl-like variant may be any of the above mentioned.

MATERIALS AND METHODS Materials 10

Enzyme :

Termamyl^® from Novo Nordisk shown in SEQ ID NO: 2 substituted in position Val54Trp. 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 reducing carbohydrate ( measured as dextrose-equivalents) in a sample expressed as w/w% of the total amount of dissolved dry mat- 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 complex is measured at 450 nm.

General molecular biology methods:

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

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

EXAMPLES 11

Example 1

Preparation of glucose syrup of the acid converted-type enzy- maticallv

A glucose syrup was prepared by treating a starch slurry containing 30% DS (30% Dry Solid) waxy maize starch, 40 ppm Ca²⁺ (adding as CaCl₂) at pH 6.0 with 0.1 mg enzyme protein/g DS of Val54Trp substituted Bacillus licheniformis α-amylase. The temperature was kept at 95°C for one hour and 80°C for 72 hours . 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)

20 72

DPI 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

DE 35 43

Table 1: Sugar profile after 20 and 72 hours of treatment with V54W substituted Bacillus licheniformis α-amylase. The DE of the obtained syrup is also given. Figure 3 shows the sugar spectrum of the glucose syrup obtained by treating a pre-cooked 5% Waxy maize starch substrate with a Val54Trp substituted Bacillus licheniformis α-amylase at 60°C for 24 hours. Figure 2 shows the sugar spectrum of a 12

similar substrate treated with the native Bacillus licheni formis α-amylase under similar conditions

Claims

13CLAIMS

1. A process for the preparation of a glucose syrup wherein starch is treated with a Termamyl-like ╬▒-amylase comprising a substitution in Val54 shown in SEQ ID NO: 2 or in the corresponding position in another Termamyl-like ╬▒-amylase.

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 ╬▒-amylase as the backbone or another Termamyl- like variant with a corresponding substitution.

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

4. The process according to claims 1-3, wherein the starch is treated with an ╬▒-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 ╬▒-amylase with a substitution in position Val54 using SEQ ID NO: 2 as the backbone or a 14

corresponding position in another Termamyl-like ╬▒-amylase for preparing glucose syrup.

8. The use according to claim 7, wherein the B . licheniformis ╬▒-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 ╬▒-amylase as the backbone or another Termamyl-like variant with a corresponding substitution.