Variant alpha amylases with enhanced activity on starch polymers

A technology of amylase and starch binding tank, which is applied in the preparation of detergent mixture compositions, detergent compositions, enzymes, etc., and can solve problems such as unsolved accessibility.

Active Publication Date: 2018-10-23
DANISCO US INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the use of α-amylases with enhanced specific activity can only marginally improve starch hydrolysis since it does not address the issue of accessibility

Method used

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  • Variant alpha amylases with enhanced activity on starch polymers
  • Variant alpha amylases with enhanced activity on starch polymers
  • Variant alpha amylases with enhanced activity on starch polymers

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0259] Example 1: Purification of uPWA

[0260] Variant alpha-amylase uPWA (SEQ ID NO: 2 in US Patent No. 7,273,740) derived from Pyrococcus worriii was purchased from Verenium Corporation (San Diego, CA, USA). uPWA contains 58 amino acid substitutions relative to wild type P. The amino acid sequences of uPWA and PWA are shown in SEQ ID NO:5 and SEQ ID NO:1 below, respectively.

[0261] Variant Pyrococcus worrieli amylase uPWA (SEQ ID NO: 5)

[0262]

[0263] Wild-type Pyrococcus wausii amylase (SEQ ID NO: 1)

[0264]

[0265] The uPWA was purified by hydrophobic interaction chromatography and transferred to a mixture of 50 mM glycine (pH 10) and 2 mM CaCl 2 The composed high pH buffer was then mixed for twenty minutes. Ammonium sulfate was added to achieve a final concentration of 1 M and the solution was mixed for an additional 30 minutes before being applied to a 30 mL phenyl sepharose column equilibrated with the same buffer. The column was washed with the sam...

example 2

[0267] Example 2: Crystallization of uPWA

[0268] Crystallization experiments were performed using 24-well sitting drop in Cryschem plates (Hampton Research) at room temperature. Initial screening was performed using commercially available screening protocols (Hampton and Qiagen). 2 μl of protein sample was mixed with an equal amount of reservoir solution to form a sitting drop and then left to equilibrate against 250 μl of reservoir solution. Crystals appeared after 5-7 days in which stock solution consisted of 0.1M Tris-HCl pH 8.5 and 20% (v / v) ethanol or 0.2M NaCl, 0.1M HEPES pH 7.5 and 10% (v / v) isopropanol composition. Crystals were cryoprotected by adding (dissolving) glucose crystals to sitting drops prior to X-ray diffraction analysis. The crystals were housed in nylon sheaths and quenched in liquid nitrogen.

[0269] Data were collected using synchrotron radiation at the Stanford Synchrotron Radiation Lightsource (SSRL beamline BL12-2). The complete data set w...

example 3

[0270] Example 3. uPWA structure determination and refinement

[0271] The structure of uPWA was determined by molecular replacement using Phaser (McCoy, A.J. et al. (2007) J. Appl. Crystallogr. 40:658-674 (McCoy, A.J. et al., 2007, Journal of Applied Crystallography, pp. 40, pp. 658-674)), wherein the wild-type Pyrococcus wausii amylase (PDB access code: 1MWO; Linden, A. et al. (2003) J. Biol. Chem. 278: 9875-9884 ( The structure of Linden, A. et al., 2003, Journal of Biochemistry, Vol. 278, pp. 9875-9884)) was used as the search model. Using PHENIX (Adams, P.D.et al. (2002) ActaCrystallogr.D Biol.Crystallogr.58:1948-1954 (Adams, P.D. et al., 2002, Acta Crystallographica Series D: Biological Crystallography, Vol. 58, No. 1948-1954 pages)) and COOT (Emsley, P. and Cowtan, K. (2004) ActaCrystallogr.D Biol.Crystallogr.60:2126-2132 (Emsley, P. and Cowtan, K., 2004, Acta Crystallogr. Series D: Biological Crystallography, Volume 60, Pages 2126-2132)) carried out model refinement,...

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PUM

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Abstract

The present invention describes variants of alpha-amylases useful in industrial processes such as starch liquefaction. The α-amylase variants have enhanced specific activity, allowing for faster reduction of peak viscosity during liquefaction. The α-amylase is modified by introducing a mutation into the amino acid sequence of a parental Family 13 α-amylase polypeptide at an amino acid residue in the starch-binding groove; wherein the starch-binding groove is modified by the first β in the A domain Amino acid residues in the α-helix preceding the ‑strand, the loop between the sixth α‑helix and the seventh β‑strand in the A domain, the seventh α‑helix and the eighth β in the A domain - the loop between the chains, and the loop formation connecting the A domain and the C domain; and wherein the mutation changes the binding of starch to the variant alpha amylase polypeptide compared to the parent alpha amylase polypeptide.

Description

[0001] priority [0002] This patent application claims priority to US Provisional Patent Application No. 61 / 657,501, filed June 8, 2012, which is hereby incorporated by reference in its entirety. technical field [0003] The present invention describes compositions and methods involving variant alpha-amylases for use in industrial processes such as starch liquefaction. The alpha-amylase variants have enhanced specific activity, allowing for faster reduction of peak viscosity during liquefaction. Background technique [0004] Alpha-amylases are used in a variety of industrial and commercial processes, including starch liquefaction, textile desizing, food manufacturing, laundry cleaning, and dishwashing. In such applications, alpha-amylase breaks down starch to release smaller carbohydrates. However, starch bundles are resistant to alpha-amylase hydrolysis because the structured starch polymers exclude enzymes. Therefore, the use of alpha-amylases with enhanced specific ac...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/26
CPCC12N9/2414C12Y302/01001C11D3/386C12P19/02C12P19/14C12N9/2408C12N9/242C12N9/2417A23L2/382A23V2002/00C11D11/0017C11D11/0023
Inventor R·R·博特L·G·卡斯康-佩雷拉D·A·埃斯特尔M·科尔克曼D·E·维尔德斯
Owner DANISCO US INC
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