Alpha-Amylases

a technology of alpha-amylase and beta-amylase, applied in the field of alpha-amylase variants, can solve the problem of disadvantage of calcium requiremen

Inactive Publication Date: 2012-10-11
NOVOZYMES AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0011]The present invention provides alpha-amylase variants comprising an A-domain of a calcium-sensitive alpha-amylase which has at least 70% and less than 100% sequence identity with any of the A-domains of SEQ ID NOS: 1-12, 29, and 30, a B-domain of a calcium-insensitive alpha-amylase, and a C-domain of a calcium-sensitive alpha-amylase.

Problems solved by technology

This requirement for calcium is a disadvantage in applications where strong chelating compounds are present, such as in detergents or during ethanol production from whole grains, where the plant material comprises a large amount of natural chelators such as phytate.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Preparation of Hybrids

[0438]The following hybrids of the calcium-sensitive alpha-amylase having the sequence shown in SEQ ID NO: 7 and the calcium-insensitive alpha-amylase having the sequence shown in SEQ ID NO: 13 were prepared.

Hybrid 1: the amino acid residues 106-215 of SEQ ID NO: 7 were removed and replaced with the amino acid residues 103-211 of SEQ ID NO: 13, which results in SEQ ID NO: 17, and the following alterations were introduced: E182*, N183*, E188W, N189E and D192T (using SEQ ID NO: 17 numbering), which correspond to E181*, N182*, E187W, N188E and D191T using SEQ ID NO: 27 numbering. The sequence of this hybrid is shown in SEQ ID NO: 18.

Hybrid 2: the amino acid residues 106-214 of SEQ ID NO: 7 were removed and replaced with the amino acid residues 103-210 of SEQ ID NO: 13, which results in SEQ ID NO: 19, and the following alterations were introduced: E182*, N183*, E188W, N189E and D192T (using SEQ ID NO: 19 numbering), which correspond to E181*, N182*, E187W, N188E an...

example 2

Stability in the Presence of Chelator

[0439]Enzyme samples were incubated in buffer pH 8.0 (50 mM EPPS, 0.01% TRITON®X100, pH 8.0) with 1.5% final concentration of DTPA at 49° C. for 1 hour and reference samples were incubated at 4° C. for 1 hour. In addition, enzyme samples were incubated in buffer pH 10.0 (50 mM EPPS, 0.01% TRITON® X100, pH 10.0) with 1.5% final concentration of DTPA at 42° C. for 1 hour and reference samples were incubated at 4° C. for 1 hour.

[0440]For the determination of amylase stability in buffer pH 8 and pH 10 with DTPA the enzymes to be tested were adjusted to 0.25 and 0.5 mg enzyme protein / mL by diluting in 5 mM EPPS, 0.01% TRITON® X100, pH 8.0.

[0441]160 microliters stability buffer (50 mM EPPS, 0.01% TRITON® X100, 1.875% DTPA, pH 8.0 or pH 10.0) and 40 microliters of the amylase solution were transferred to a 96-well PCR microtiter plate in duplicate and the content was mixed for 1 minute. Final concentration of DTPA was 1.5% in each well. 20 microliters f...

example 3

Additional Alpha-Amylases

[0444]The following alpha-amylases were prepared:

Hybrid 4: the amino acid residues 106-212 of SEQ ID NO: 5 were removed and replaced with the amino acid residues 103-208 of SEQ ID NO: 13, which results in SEQ ID NO: 23, and the following alterations were introduced: E182*, N183*, E188W, N189E and D192T (using SEQ ID NO: 23 numbering), which correspond to E181*, N182*, E187W, N188E and D191T in SEQ ID NO: 27 numbering. The sequence of this hybrid is shown in SEQ ID NO: 24.

Hybrid 5: the amino acid residues 106-212 of SEQ ID NO: 8 were removed and replaced with the amino acid residues 103-208 of SEQ ID NO: 13, which results in SEQ ID NO: 25, and the following alterations were introduced: E182*, N183*, E188W, N189E and D192T (using SEQ ID NO: 25 numbering), which correspond to E181*, N182*, E187W, N188E and D191T in SEQ ID NO: 27 numbering. The sequence of this hybrid is shown in SEQ ID NO: 26.

Hybrids 4 and 5 (SEQ ID NOS: 24 and 26), a variant of SEQ ID NO: 5 wi...

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Abstract

The present invention relates to alpha-amylase variants, polynucleotides encoding the variants and nucleic acid constructs, vectors, and host cells comprising the polynucleotides, and methods of using the variant enzymes.

Description

REFERENCE TO SEQUENCE LISTING[0001]This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]The present invention relates to alpha-amylase variants having an improved property, e.g., improved stability, polynucleotides encoding the variants, methods of producing the variants, and methods of using the variants.[0004]2. Description of Related Art[0005]Alpha-amylases (alpha-1,4-glucan-4-glucanohydrolases, E.C. 3.2.1.1) constitute a group of enzymes, which catalyze the hydrolysis of starch and other linear and branched 1,4-glucosidic oligo- and polysaccharides.[0006]Alpha-amylases are used commercially for a variety of purposes such as in the initial stages of starch processing (e.g., liquefaction); in wet milling processes; and in alcohol production from carbohydrate sources. They are also used as cleaning agents or adjuncts in detergent matrices; in the textile indu...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/26C12N9/16C12P19/14C12P7/14C12P7/16C12P7/46C12P7/56C12P7/20C12P7/60C12P13/08C12P21/04C12N1/21C12N5/10C12N1/15C12N1/19C12N15/63C11D3/386C12N9/20
CPCC12N9/2417C12Y302/01001Y02E50/17C12P19/14C12P7/06C12P19/04C11D3/386C12N9/2414Y02E50/10C12P7/14C11D3/38681C12P19/02C12P2203/00C12N15/8257
Inventor ANDERSEN, CARSTENPOULSEN, THOMAS AGERSTEN
Owner NOVOZYMES AS
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