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New chimeric alpha-amylase variants

A technology of amylase and starch slurry, applied in the directions of enzymes, enzyme stabilization, detergent compounding agents, etc., can solve problems such as poor thermal stability

Inactive Publication Date: 2011-04-13
DANISCO US INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is also known in the art that AmyQ, an alpha-amylase from Bacillus amyloliquefaciens highly homologous to both AmyS and AmyL, is less thermostable than AmyS or AmyL

Method used

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  • New chimeric alpha-amylase variants
  • New chimeric alpha-amylase variants
  • New chimeric alpha-amylase variants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0195] Creation of novel chimeric amylases from AmyL and AmyS sequences

[0196] Efforts were made to create AmyL and AmyS chimeras, combining preferred features of AmyL-type enzymes (AmyL and its variants) and AmyS-type enzymes (AmyS and its variants) into a single enzyme. Ideally, the resulting chimeric enzymes will have the best properties of each enzyme, such as thermostability similar to AmyL-type enzymes, combined with the high specific activity of AmyS-type enzymes towards starch substrates at high temperatures. These enzymes would be useful for starch liquefaction, such as ethanol production, since their catalytic activity would ideally result in a rapid initial viscosity reduction rate and low final viscosity.

[0197]A series of chimeric molecules (SEQ ID NO: 4-17) were constructed from AmyL and AmyS. The chimera comprises an N-terminal portion from AmyL (SEQ ID NO: 1) and a C-terminal portion from AmyS (SEQ ID NO: 2). The N-terminal portion of the chimera comprise...

Embodiment 2

[0203] Thermostability screening of first-generation chimeric α-amylases derived from AmyL and AmyS enzymes

[0204] The first generation of chimeras consisted of single crossover mutants in which the N-terminal part of the chimeric amylase was derived from AmyL and the C-terminal part from AmyS as described above. The chimeric amylases screened for thermostability were 186, 187, 200, 202, 228, 249, 254 and 259. Chimeric amylases were assayed at 95°C throughout the time course. Samples were removed at the indicated time points and activity was measured. For each chimera, the percent activity relative to the enzyme was maintained at 95 °C, pH 5.6 containing 2.6 mM CaCl 2 Plotted against minutes in 50 mM NaCl in 50 mM malate buffer. Assay conditions were as described in the Methods section above. For each enzyme, residual activity was calculated as the percentage of activity relative to that of the enzyme not incubated at 95 °C. The control enzyme was AmyS control (SEQ ID N...

Embodiment 3

[0207] Thermostability screening of second-generation chimeric α-amylases derived from AmyL and AmyS enzymes: addition of stabilizing structures

[0208] The stability data in Example 2 show that among the tested chimeric α-amylases, the chimera containing only the first 186 amino acid residues of the AmyL sequence was highly thermostable, while the other chimeras containing more AmyL sequences Alpha-amylases that are not thermostable include the 187 chimera, which differs from the 186 chimera only at position 187. The data suggest that the replacement of the Asp residue with a Ser residue at position 187 is directly related to the lack of thermostability of the 187 chimera, and possibly to the thermostability of each chimeric amylase containing more than 186 amino acid residues from AmyL Sexual lack is relevant.

[0209] Quite unexpectedly, other studies are known to report that the mutation S187D in AmyL-derived amylases reduces the thermostability of the amylases. For exa...

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Abstract

Chimeric alpha amylases having the characteristics of high thermostability and good performance in starch degradation, especially high-temperature liquefaction processes, are provided. The alpha-amylases are chimeras of AmyL and AmyS enzymes, and are useful in starch degradation processes. Methods of making the chimeric enzymes, and methods of using the chimeric alpha-amylases for liquefaction, cleaning starch residue from a surface, and treating woven material to remove coatings. Kits for practicing the methods are provided. Polynucleotides encoding the chimeric amylases, vectors, and expression hosts also are provided.

Description

[0001] cross reference [0002] This application claims priority to US Patent Application Serial No. 61 / 126,066, filed April 30, 2008, the entire contents of which are incorporated herein by reference. technical field [0003] The present application mainly relates to alpha-amylases for use in industrial processes, such as for liquefaction, baking and cleaning applications. More specifically, the present application relates to chimeric alpha-amylases having improved activity in high temperature applications and / or providing improved starch degradation properties. Background technique [0004] For many industrial processes that use starch, it is desirable to have amylolytic enzymes that can function at high temperatures to rapidly break down the starch, thereby reducing viscosity. Examples of such enzymes are known in the art. For example, the alpha-amylases from Bacillus licheniformis or Bacillus stearothermophilus, AmyL and AmyS, respectively, facilitate the liquefaction ...

Claims

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Application Information

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IPC IPC(8): C12N15/62C07K14/00C12N5/00C12N15/00A23L29/00A23L29/20
CPCY02E50/17C12N9/2417C12P19/14A23L29/06C12N9/96Y02E50/10C11D3/38618
Inventor S·D·鲍尔A·肖
Owner DANISCO US INC
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