Enzymes for starch processing

a technology of enzymes and starch, applied in the field of polypeptides, can solve the problems of energy-consuming conventional starch conversion process, achieve the effects of increasing the efficacy of various starch degrading processes, and reducing the cost of production

Inactive Publication Date: 2006-07-06
NOVOZYMES AS +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0009] The present inventor have surprisingly discovered that by adding a carbohydrate-binding module (CBM) to certain alpha-amylases the activity and specificity can be altered thereby increasing the efficacy o

Problems solved by technology

It will be apparent from the above discussion that the conventional starch conversion process is very

Method used

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  • Enzymes for starch processing

Examples

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example

Construction of the Nucleic Acid Sequence V019, Encoding Rhizomucor pusillus Alpha Amylases and Athelia rolfsii Glucoamylase CBM

[0218] Vector pLA1 was digested with the appropriate restriction endonuclease to cut out the region encoding A. niger alpha-amylase catalytic domain. The Rhizomucor pusillus alpha-amylase gene was amplified by PCR using the primers P001 (SEQ ID NO: 104) and P002 (SEQ ID NO: 105), the amplified fragment is shown as SEQ ID NO: 19.

PCR reaction system:Conditions:38.9 micro LH2O198° C. 10 sec  5 micro L10 × reaction buffer268° C. 90 sec  1 micro LKlen Taq LA (CLONTECH)1-230 cycles  4 micro L10 mM dNTPs368° C.10 min 0.3 micro L × 2100 pmole / micro L Primers 0.5 micro LTemplate DNA

[0219] DNA fragments were recovered from agarose gel by the Qiagen gel extraction Kit. The resulting purified fragments were mixed with the vector digest. The mixed solution was introduced into Saccharomyces cerevisiae to construct the expression plasmid pLAV019 by in vivo recombinatio...

example 2

Construction of the Nucleic Acid Sequence V022, Encoding Meripilus giganteus Alpha Amylase and Athelia rolfsii Glucoamylase CBM

[0220] The Meripilus giganteus alpha-amylase gene was amplified by PCR using the primers P003 (SEQ ID NO: 106) and P004 (SEQ ID NO: 107).

[0221] DNA fragments were recovered from agarose gel by the Qiagen gel extraction Kit. The resulting purified fragments and the appropriate restriction endonuclease digested vector pLA1 to cut out the region encoding A. niger alpha-amylase catalytic domain were mixed. The mixed solution was introduced into Saccharomyces cerevisiae to construct the expression plasmid pLAV022 by in vivo recombination.

example 3

Expression of Amylases With CBM in Aspergillus oryzae

[0222] The constructs comprising the alpha amylase genes with CBM described in examples 1 and 2 were used to construct expression vectors, pAspV019 and pAspV022, respectively. The two plasmids, pAspV019 and pAspV022, consist of an expression cassette based on the Asperigillus niger neutral amylas II promoter fused to the Aspergillus nidulans triose phosphate isomerase non translated leader sequence (Pna2 / tpi) and the Aspergillus niger amyloglycosidase terminator (Tamg). Also present on the plasmid was the Aspergillus selective marker amdS from Aspergillus nidulans enabling growth on acetamide as sole nitrogen source. The expression plasmids pAspV019 and pAspV022 were transformed into Aspergillus as described in Lassen et al. (2001), Applied and Environmental Micorbiology, 67, 4701-4707. Transformants expressing V019 and V022, were isolated, purified and cultivated in shake flasks. The culture broths from fermentations of Aspergil...

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Abstract

The present invention relates to polypeptides comprising a carbohydrate-binding module amino acid sequence and,an alpha-amylase amino acid sequence as well as to the application of such polypeptides.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit under 35 U.S.C. 119 of U.S. provisional application Nos. 60 / 638,614 and 60 / 650,612 filed Dec. 22, 2004 and Feb. 7, 2005, respectively, the contents of which are fully incorporated herein by reference.CROSS-REFERENCE TO SEQUENCE LISTING AND DEPOSITED MICROORGANISMS [0002] The present application contains information in the form of a sequence listing, which is appended to the application and also submitted on a data carrier accompanying this application. In addition, the present application refers to deposited microorganisms. The contents of the data carrier and the deposited microorganisms are fully incorporated herein by reference. FIELD OF THE INVENTION [0003] The present invention relates to polypeptides comprising a carbohydrate-binding module (“CBM”) and an alpha-amylase catalytic domain. Furthermore, the invention relates to wild type alpha-amylases polypeptides comprising useful alpha-amylase ca...

Claims

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Application Information

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IPC IPC(8): C07H21/04C12P21/06C12P7/06C12N9/32C12N15/74C12N1/18
CPCC07K2319/00C12N9/242C12N9/2428C12P7/06C12Y302/01003C12P19/14Y02E50/17C12Y302/01001C12P19/02Y02E50/10
Inventor FUKUYAMA, SHIROMATSUI, TOMOKOSOONG, CHEE LEONGALLAIN, ERICNIELSEN, ANDERS VIKSOUDAGAWA, HIROAKILIU, YEDUAN, JUNXINWU, WENPINGANDERSEN, LENE NONBOE
Owner NOVOZYMES AS
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