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Asparaginase from basidiomycetes

Inactive Publication Date: 2013-08-15
NESTEC SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides an asparaginase enzyme with high activity and operational stability. Additionally, the invention aims to reduce the formation of acrylamide in food products by utilizing the asparaginase enzyme.

Problems solved by technology

Since carbohydrates are as ubiquitous as amino acids in food, there is a permanent risk of generating the cancerogenic and genotoxic acrylamide during the thermal treatment of food.
Genetic engineering of potato using an antisense asparagine synthase gene and tuber specific promoters have been reported to reduce, but not to eliminate asparagine from the potato tuber (Rommens 2007); a full elimination of asparagine is supposedly lethal for the plant.
However, no enzyme technology can be currently envisaged for the selective hydrolysis of a protein bound amino acid, such as asparagine, from a food protein, even less while maintaining the typical structural and sensory properties of the respective food material.

Method used

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  • Asparaginase from basidiomycetes
  • Asparaginase from basidiomycetes
  • Asparaginase from basidiomycetes

Examples

Experimental program
Comparison scheme
Effect test

example 1

Cultivation of Flammulina velutipes

[0119]All media and equipment were autoclaved prior to use and standard sterile techniques were applied throughout the procedure. Flammulina velutipes was maintained on standard agar plates (30.0 g L−1 glucose-monohydrate; 4.5 g L−1 asparagine-monohydrate; 1.5 g L−1 KH2PO4; 0.5 g L−1 MgSO4; 3.0 g L−1 yeast extract; 15.0 g L−1 agar agar; 1.0 mL L−1 trace metal solution containing 0.005 g L1 CuSO4.5H2O, 0.08 g L−1 FeCl3.6H2O, 0.09 g L−1 ZnSO4.7H2O, 0.03 g L−1 MnSO4H2O and 0.4 g L−1 EDTA. The pH of the medium was adjusted to pH 6 with 1 M NaOH prior to sterilisation. Precultures were prepared by homogenisation of a 10×10 mm agar plug with mycelium of Flammulina velutipes in 100 mL of sterile standard nutrition solution using an Ultra Turrax (Miccra D-9, Art, Müllheim, Germany). Submerged cultures were maintained at 24° C. and 150 rpm. After cultivation for 5 days, 50 ml preculture were transferred into 250 ml main culture medium consisting of minimal...

example 2

Enzyme Preparation from Flammulina velutipes

[0120]After 18 days of cultivation, the culture was filtrated and the extracellular enzyme-containing supernatant (200 mL) was reverse-foamed, the asparaginase and another protein being the only proteins left in the supernatant. The remaining liquid was concentrated using ultra-filtration (MWCO 10,000), and both proteins were separated via size exclusion chromatography at a Superose 6.

[0121]Most of the hydrolytic activity originally present was recovered indicating that this protocol yielded a useful enzyme concentrate through two steps only.

example 3

Hydrolysis of L-Asparagine Using Native Enzyme

[0122]100 μL of 10 mM asparagine in 0.1 M K2HPO4 / KH2PO4 buffer (pH 7.0) were preheated at 37° C. for 5 min. The reaction was started with the addition of 50 μL enzyme solution. After an incubation time of 20 min at 37° C. and 400 rpm in a thermoshaker, the assay was stopped by the addition of 20 μL TCA. A control experiment was carried out without substrate. The contents of aspartic acid were quantitatively measured with the HPLC after OPA-derivatisation, and the difference between sample and control was used to calculate then enzyme's activity.

[0123]The analytical evidence indicates a fast enzymatic hydrolysis of the substrate L-asparagine.

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Abstract

An asparaginase enzyme derived from the fungi Basidiomycete, in particular the Basidiomycete is Flammulina velutipes. A method for hydrolysing at least one of L-asparagine or L-glutamine. A method for reducing acrylamide formation in a substance comprising L-asparagine is also described.

Description

FIELD OF THE INVENTION[0001]The field of the present invention relates to an asparaginase enzyme obtainable from the fungi Basidiomycetes, esp. Basidiomycetes Flammulina velutipes. A method for the hydrolysis of L-asparagine and L-glutamine are also disclosed. A method for reducing the formation of acrylamide in a substance comprising L-asparagine is also disclosed.BACKGROUND OF THE INVENTION[0002]Applications of asparaginase enzymes in food technology originate from the finding that a thermal treatment of food converts asparagine in the presence of reducing carbohydrates partly to acrylamide. Since carbohydrates are as ubiquitous as amino acids in food, there is a permanent risk of generating the cancerogenic and genotoxic acrylamide during the thermal treatment of food. The thermal treatment is for example a baking, a roasting, a barbecuing or a deep-fat frying of the food. The onset of acrylamide formation during the thermal treatment of the food is observed at temperatures excee...

Claims

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Application Information

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IPC IPC(8): C12N9/82A23L1/015A23L5/20
CPCC12N9/82A23L1/0153A23K10/10A23L5/25A23V2002/00
Inventor BERENDS, PIETERRABE, SWENBERGER, RALF GUNTERLINKE, DIANAEISELE, NADINE
Owner NESTEC SA
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