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Exo-specific amylase polypeptides, nucleic acids encoding those polypeptides and uses thereof

a technology of amylase and polypeptide, which is applied in the field of amylase polypeptides and nucleic acids encoding the polypeptides, can solve the problems of increasing bread firmness, affecting the activity of the enzyme, and affecting the stability of the enzyme, so as to improve the activity, the activity is higher, and the stability of the ph stability

Inactive Publication Date: 2007-01-25
BERG CASPER TUNE +9
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The PS4 variant polypeptides effectively retard bread staling by reducing amylopectin crystallization, maintaining crumb softness and preventing the formation of a leathery crust, while being more thermostable and pH-stable than their parent enzymes.

Problems solved by technology

Improved amylases can ameliorate problems inherent in certain processes, such as baking.
Crystallisation of amylopectin takes place in starch granules days after baking, which leads to increased firmness of bread and causes bread staling.
When bread stales, bread loses crumb softness and crumb moisture.
However, due to steam, the temperature in the crumb is only about 100° C. at the end of the baking process.
Endoamylase activity can negatively affect the quality of the final bread product by producing a sticky or gummy crumb due to the accumulation of branched dextrins.

Method used

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  • Exo-specific amylase polypeptides, nucleic acids encoding those polypeptides and uses thereof
  • Exo-specific amylase polypeptides, nucleic acids encoding those polypeptides and uses thereof
  • Exo-specific amylase polypeptides, nucleic acids encoding those polypeptides and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

Cloning of PS4

[0428]Pseudomonas saccharophila is grown overnight on LB media and chromosomal DNA is isolated by standard methods (Sambrook J, 1989). A 2190 bp fragment containing 5 the PS4 open reading frame (Zhou et al., 1989) is amplified from P. saccharophila chromosomal DNA by PCR using the primers P1 and P2 (see Table 3). The resulting fragment is used as a template in a nested PCR with primers P3 and P4, amplifying the openreading frame of PS4 without its signal sequence and introducing a NcoI site at the 5′ end of the gene and a BamHI site at the 3′ end. Together with the NcoI site a codon for a N-terminal Methionine is introduced, allowing for intracellular expression of PS4. The 1605 bp fragment is cloned into pCRBLUNT TOPO (Invitrogen) and the integrity of the construct analysed by sequencing. The E.coli Bacillus shuttle vector pDP66K (Penninga et al., 1996) is modified to allow for expression of the PS4 under control of the P32 promoter and the ctgase signal sequence. Th...

example 2

Site Directed Mutagenesis of PS4

[0430] Mutations are introduced into the mta gene by 2 methods. Either by a 2 step PCR based method, or by a Quick Exchange method (QE). For convenience the mta gene is split up in 3 parts; a PvuI-FspI fragment, a FspI-PstI fragment and a PstI-AspI fragment, further on referred to as fragment 1, 2 and 3 respectively.

[0431] In the 2 step PCR based method, mutations are introduced using Pfu DNA polymerase (Stratagene). A first PCR is carried out with a mutagenesis primer (Table 4) for the coding strand plus a primer downstream on the lower strand (either 2R or 3R Table 3).

[0432] The reaction product is used as a primer in a second PCR together with a primer upstream on the coding strand. The product of the last reaction is cloned into pCRBLUNT topo (Invitrogen) and after sequencing the fragment is exchanged with the corresponding fragment in pCSmta.

[0433] Using the Quick Exchange method (Stratagene), mutations are introduced using two complementary ...

example 3

Multi SDM

The PS4 variants were generated using a QuickChange® Multi Site Directed Mutagenesis Kit (Stratagene) according to the manufactures protocol with some modifications as described.

Step 1: Mutant Strand Synthesis Reaction (PCR)

[0437] Inoculate 3 ml. LB (22 g / l Lennox L Broth Base, Sigma)+antibiotics (0,05 μg / ml kanamycin, Sigma) in a 10 ml Falcon tube [0438] Incubate o / n 37° C., ca. 200 rpm. [0439] Spin down the cells by centrifugation (5000 rpm / 5 min) [0440] Poor off the medium [0441] Prepare ds-DNA template using QIAGEN Plasmid Mini Purification Protocol

[0442] 1. The mutant strand synthesis reaction for thermal cycling was prepared as follow:

PCR Mix:2.5μl10X QuickChange ® Multi reaction buffer0.75μlQuickSolutionXμlPrimers⁢ ⁢(primer⁢ ⁢length⁢ ⁢28⁢–35⁢ ⁢bp->10⁢ ⁢pmolprimer⁢ ⁢length⁢ ⁢24⁢–27⁢ ⁢bp->7⁢ ⁢pmolprimer⁢ ⁢length⁢ ⁢20⁢–23⁢ ⁢bp->5⁢ ⁢pmol)1∥ldNTP mixXμlds-DNA template (200 ng)1μlQuickChange ® Multi enzyme blend (2.5 U / μl)(PfuTurbo ® DNA polymerase)XμldH2O ...

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Abstract

This invention relates to amylase polypeptides, and nucleic acids encoding the polpypeptides and uses thereof. The amylases of the present invention have been engineered to have more beneficial qualities. Specifically, the amylases of the current invention show an altered exospecifity.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application claims benefit of and priority to U.S. Ser. No. 60 / 485,616, entitled “Exo-specific Amylase Polypeptides, Nucleic Acids Encoding Those Polypeptides and Uses Thereof” (Docket No. GC807P), filed Jul. 7, 2003, by Berg, et al, and U.S. Ser. No. 60 / 485,413, entitled, “Thermostable Amylase Polypeptides, Nucleic Acids Encoding Those Polypeptides and Uses Thereof (Docket No. GC806P), filed Jul. 7, 2003. These applications are related to U.S. Ser. No. 60 / 485,539, entitled “Polypeptides”, filed Jul. 7, 2003 (Docket number P016939USO).FIELD OF THE INVENTION [0002] This invention relates to amylase polypeptides, and nucleic acids encoding the polpypeptides and uses thereof. The amylases of the present invention have been engineered to have more beneficial qualities. Specifically, the amylases of the current invention show an altered exospecifity. BACKGROUND OF THE INVENTION [0003] Improved amylases can ameliorate problems inh...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12P21/06C07H21/04C12N9/32C12N15/74C12N1/21C12N9/28
CPCC12N9/2417
Inventor BERG, CASPER TUNEBOJSEN, KIRSTENDERKX, PATRICK M.F.FIORESI, CAROLGERRITSE, GIJSBERTKRAGH, KARSTEN MATTHIASLIU, WEISHAW, ANDREWTHOUDAHL, CHARLOTTE REFDAHLKELLETT-SMITH, ANJA HEMMINGSEN
Owner BERG CASPER TUNE
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