Hybrid enzymes

a technology of enzymes and hybrids, applied in the field of hybrid enzymes, can solve the problems of unfavorable loss of elasticity in baked products, and achieve the effects of reducing activity, increasing baking performance, and increasing activity towards raw starch

Inactive Publication Date: 2006-07-06
NOVOZYMES AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0005] The present inventors have now surprisingly discovered that by addition of a carbohydrate binding module (CBM) to an endo-amylase the catalytic activity of the endo-amylase can be modified thereby resulting in an increased baking performance compared to the wild type enzyme. There is no significant change in the taste or smell of the baked product. Without being bound by theory it is suggested that the effect is due to an increased activity towards raw starch in the dough conferred by the CBM, and / or a reduced activity towards the heated starch in the baking bread conferred by the CBM. The endo-amylase with a CBM can be used as a baking enzyme with less risk of overdosing compared to the enzyme without a CBM. Such hybrids consisting of a polypeptide having endo-amylase activity and a carbohydrate binding module, primarily having affinity for starch like e.g. the CBM20, have the advantage over existing endo-amylases that by selecting a catalytic domain with desire properties e.g. the pH profile, the temperature profile, the oxidation resistance, the calcium stability, the substrate affinity or the product profile can be combined with a carbohydrate binding module with stronger or weaker binding affinities, e.g., specific affinities for amylose, specific affinities for amylopectin or affinities for specific structure in the carbohydrate. The hybrid may be used as a baking additive, e.g., as an anti-staling enzyme.
[0006] The present inventors have further surprisingly discovered that by adding a carbohydrate-binding module (CBM) to an endo-amylase the activity and specificity can be altered thereby increasing the efficacy of various starch degrading processes, e.g., comprising degradation of raw, e.g., ungelatinized starch as well as gelatinized starch. Due to the superior hydrolysis activity of these endo-amylases having a CBM the overall starch conversion process can be performed without having to gelatinize the starch, i.e. the endo-amylases having a CBM hydrolyses granular starch in a raw starch process as well as fully or partially gelatinized starch in a traditional starch process.

Problems solved by technology

However, the endo-amylases are easy to overdose which may results in gumminess and an undesirable loss in elasticity in the baked product.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Construction of Hybrids Between an Endo-Amylase and the CBM from AMY1048

[0165] The amylase AMY1048 is a wild type Bacillus amylase made up of a catalytic fragment of 484 amino acid and in addition a CBM20 fragment of 101 aa. The DNA sequence coding the AMY1048 is included as SEQ ID NO:1 and the mature AMY1048 sequence is included as SEQ ID NO:2. In SEQ ID NO:1 the CBM is defined as amino acid residues 485 to 586 which correspond to nucleotides 1540-1845 in SEQ ID NO:2. The amylase including the CBM can be expressed from a construction similar to what have been described for other amylases i.e. e.g., inserted into a vector under the control of a constitutive active promoter and flanked by the signal sequence (SEQ ID NO: 15) and the terminator sequence of B. licheniformis endo-amylase.

[0166] Replacing the catalytic fragment of the AMY1048 endo-amylase with a catalytic domain of another endo-amylase, thus creating a hybrid of the CBM from AMY1048 and a new endo-amylase, is made by am...

example 2

Construction of a Hybrid Amylase with Carbohydrate Binding Domain

[0178] The catalytic fragment of the B. flavothermus endo-amylase, AMY1048 can further be divided into the central AB-domain harboring the catalytic center and a C domain c-terminal to the catalytic domain but prior to the CBM. In SEQ ID NO:2 the catalytic core domain consist of the first 397 amino acid residues, the C domain is defined as the amino acid residues from 398 to 484 and the CBM is defined as amino acid residues 485 to 586. In SEQ ID NO:1 the signal sequence is encoded by nucleotide 1 to 87, the catalytic core domain is encoded by nucleotide 88-1278, the C domain is encoded by the nucleotides 1279-1539, and the CBM is encoded by nucleotide 1540-1845.

[0179] The amylase including the CBM can be expressed from a vector construction similar to what have been described in WO0060060A2 in example 4—i.e. the amylase gene is inserted into a vector under the control of a amylase promoter and flanked by the signal s...

example 3

Determination of Exo-Endo Improvement Factor (EIF)

[0189] EIF is the measure of an increment of the exo / endo ratio relative to a parent enzyme i.e. EIF=(exo / endo of variant) / (exo / endo of parent enzyme). An enzyme has an increase in exo / endo ratio compared to its parent enzyme if EIF>1. EIF may be based on one of the following methods.

[0190] EIF1 Endo activity assay: The Phadebas Amylase Test (Pharmacia Diagnostics) is run according to the suppliers recommenda-tions and the endo units calculated from the supplied formula where the natural logarithm to the activ-ity equals N, where N=A+square root [B+C*ln(Abs)]. Abs is the absorbance at 620 nm, A=−13.3235, B=243.3293, and C=26.73797

[0191] Exo activity assay: 50 microL of 50 mM sodium citrate, 5 mM CaCl2, pH 6.5 is mixed with 25 microL of enzyme in the same buffer and 25 microL Betamyl substrate (Betamyl Method, Megazyme) dissolved according to suppliers recommendations. The assay mix is incubated for 30 min. at 40° C. and the reacti...

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Abstract

The present invention relates to a polypeptide and the use thereof.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims, under 35 U.S.C. 119, priority of Danish application nos. PA 2004 01976, filed Dec. 22, 2004, PA 2005 01261, filed Sep. 9, 2005, and the benefit of U.S. provisional application Nos. 60 / 639,181, filed Dec. 22, 2004 and 60 / 717,274, filed Sep. 14, 2005, the contents of which are fully incorporated herein by reference.SEQUENCE LISTING [0002] This application contains a sequence listing. A computer readable form containing the sequence listing accompanies this application, and the computer readable form of the sequence listing is hereby incorporated by reference. FIELD OF THE INVENTION [0003] The present invention relates, inter alia, to hybrid enzymes comprising a carbohydrate binding module and having endo-amylase activity. The enzymes may be applied in processes comprising starch modification and / or degradation, or in dough making processes. BACKGROUND OF THE INVENTION [0004] Bacterial endo-amylases are used in a l...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A21D8/02C07H21/04C12P21/06C12N9/32C12N15/74
CPCA21D8/042Y02E50/17Y02E50/10
Inventor SVENDSEN, ALLANANDERSEN, CARSTENSPENDLER, TINANIELSEN, ANDERSOESTDAL, HENRIK
Owner NOVOZYMES AS
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