Novel asparaginases and uses thereof

a technology of asparaginase and polypeptide, which is applied in the field of newly identified asparaginase polypeptide variants and to polynucleotide sequences comprising genes, to achieve the effect of significantly increasing the expression of a variant asparaginase according to the invention and increasing the activity of the produced asparaginas

Inactive Publication Date: 2010-06-03
DSM IP ASSETS BV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0020]In another embodiment, the invention provides recombinant host cells wherein the expression of a variant asparaginase according to the invention is significantly increased or wherein the activity of the produced asparaginase is increased.

Problems solved by technology

Since acrylamide is considered as probably carcinogenic for animals and humans, this finding had resulted in world-wide concern.
Although no official limit is yet set for acrylamide that forms during cooking, the fact that a lot of products exceed this value, especially cereals, bread products and potato or corn based products, causes concern.
Although all L-asparaginases catalyze the same chemical conversion, this does not mean that they are suitable for the same applications.

Method used

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  • Novel asparaginases and uses thereof

Examples

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Effect test

example 1

Fermentation, Isolation and Purification of Asparaginases According to the Invention

[0347]Asparaginases of the invention were obtained by the construction of expression plasmids containing a DNA sequence encoding the asparaginase of the invention, transforming an Aspergillus niger strain with the plasmid and growing the Aspergillus niger strains as described in WO2004 / 030468.

[0348]After growing Aspergillus niger containing the proper expression plasmids cell free supernatants were prepared by centrifugation of the fermentation broth at 5000×g for 30 minutes at 4° C. If necessary the supernatants were filtered further over a Miracloth filter (Calbiochem cat #475855) and a GF / A Whatmann Glass microfiber filter (150 mm {acute over (Ø)}), respectively, to remove any solids. To remove any fungal material the supernatants could be adjusted to pH=5 with 4N KOH and sterile filtrated over a 2 μm (bottle-top) filter with suction. The supernatants were stored until use at 4° C. or frozen at −2...

example 2

Performance of Asparaginase According to the Invention

[0351]Specific Activity as a Function of pH

[0352]The specific activity of the asparaginase variants was determined at pH=4, pH=5, pH=6, pH=7, pH=8 at 37° C. in 50 mM phosphate / citrate buffer using cell-free supernatants. Specific enzyme activity, is herewith defined as the activity determined for an enzyme sample in units / mg of pure enzyme polypeptide. Specific asparaginase activity is therefore the activity determined for an asparaginase sample in units / mg of pure asparaginase polypeptide.

TABLE 1The specific activity of the variants relative to wild type (wt) A. nigerasparaginase (WO2004 / 030468) using asparagine as a substrateat the indicated pH values. For each pH the wild type specific activityis set to 100%. Activity was determined at 37° C.pH = 4pH = 5pH = 6pH = 7pH = 8(%)(%)(%)(%)(%)wt100100100100100ASN002141252263741033ASN001552464186971758

[0353]For determination of the specific activity the asparaginase, concentration in ...

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Abstract

The present invention relates to an asparaginase having the width of the pH activity profile which is at least 3.5. Furthermore the invention relates to newly identified asparaginase polypeptide according to any one of SEQ ID NO: 2 or SEQ ID NO: 4 and to variants thereof and to polynucleotide sequences that encode such novel asparaginase variants. Furthermore the invention relates to the use of these novel asparaginase variants in industrial processes.

Description

FIELD OF THE INVENTION[0001]The invention relates to newly identified asparaginase polypeptide variants and to polynucleotide sequences comprising genes that encode these novel asparaginases. The invention features the amino acid sequence of the full-length functional protein and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods of using these variant proteins in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins and cells, wherein a protein according to the invention is genetically modified to enhance or reduce its activity and / or level of expression. The invention also relates to methods of using these proteins in industrial processes.BACKGROUND OF THE INVENTION[0002]Recently, the occurrence of acrylamide in a number of heated food products was published (Tareke et al. Chem. Res. Toxicol. 13, 517-522 (2000)). Since acryla...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/82C07H21/04A23L1/03A21D8/04C12N15/63C12N1/15C12P21/06A23L1/27A23L5/40A23L19/00A23L29/00
CPCA23L1/0153C12Y305/01001C12N9/82A23L1/034A23L5/25A23L29/06A61P35/00
Inventor VAN DER LAAN, JAN METSKESTOR, MARK CRISTIAANLANGE DE, ILSEMOHRMANN, LISETTE
Owner DSM IP ASSETS BV
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