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Novel lipases and uses thereof

A polynucleotide and carrier technology, applied in the field of lipase and its application, can solve the problem of reducing the hardening rate of softened debris

Inactive Publication Date: 2015-06-17
DSM IP ASSETS BV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Complexation of monoglycerides with starch prevents complete recrystallization of the starch, which leads to softening of the initial crumb of the baked product and / or a reduction in the crumb firming rate during shelf life

Method used

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  • Novel lipases and uses thereof
  • Novel lipases and uses thereof
  • Novel lipases and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0209] Fermentation of Aspergillus niger

[0210] The lipolytic enzymes encoded by the nucleotide sequences SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 provided herein were constructed as follows: construction of expression plasmids containing DNA sequences, transformation of A. niger strains with such plasmids , and Aspergillus niger strains were cultured in the following manner.

[0211] Fresh spores of A. niger (10 6 -10 7 ) was inoculated in 20 ml CSL-medium (100 ml flask with a lid) and cultured at 34° C. and 170 rpm for 20-24 hours. After inoculating 5-10 ml CSL preculture in 100 ml CSM medium (500 ml capped flask), the strain was fermented at 34°C and 170 rpm for 3-5 days.

[0212] Cell-free supernatants were obtained by centrifugation (30 min, 5000 rpm) in 50 ml Greiner tubes. Pre-filter the supernatant on a GF / A Whatman Glass microfiber filter (150 mm AE) to remove larger particles, adjust to pH 5 with 4N KOH (if necessary), and filter over a 0.2 μm (bottle top)...

Embodiment 2

[0216] Purification of the lipolytic enzyme of the present invention

[0217] Step 1 - Preparation of ultrafiltrate

[0218] The culture supernatant obtained according to Example 1 was subjected to ultrafiltration to remove low-molecular contamination that may affect the enzyme activity assay and baking test. Ultrafiltration of 30 ml of the supernatant was carried out in a Millipore Labscale TFF system equipped with a filter membrane (with a cut-off of 10 kDa).

[0219] Depending on the color of the samples, wash them with 40 volumes of cold 100 mM phosphate buffer, pH 6.0 (containing 0.5 mM CaCl 2 ) wash 3-5 times. The final volume of the enzyme solution was 30ml, also called "ultrafiltrate".

[0220] The total protein content of the samples was determined using the Bradford method (The Protein Protocols Handbook, 2nd edition, Edited by J.M. Walker, Humana Press Inc, Totowa 2002, p15-21).

[0221] Step 2 - Determination of lipolytic enzyme concentration by A280 and HPSE...

Embodiment 3

[0229] activity measurement

[0230] The ultrafiltrate obtained in Example 2 can be used to perform the following enzyme activity measurements to establish the following lipolytic enzymes:

[0231] ·Lipase

[0232] · Phospholipase A1 or A2

[0233] · Lysophospholipase

[0234] ·Galactose esterase activity

[0235] specificity.

[0236]Lipase activity was measured spectrophotometrically by using the chromogenic substrate p-nitrophenyl palmitate (pNPP). In this assay, the chromogenic substrate p-nitrophenyl ester (pNPP) is dissolved in 2-propanol and suspended in phosphate in the presence of 0.1% gum arabic and 0.25% sodium deoxycholate Buffer pH 7.4. The lipase was incubated with this substrate solution at 37°C and the p-nitrophenyl (pNP) formation was measured at 405 nm for 2.6 minutes. This assay can also be performed at different pH values ​​to determine the pH dependence of lipase. It should be understood that different buffers may be required at different pH valu...

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Abstract

The present invention to newly identified polynucleotide sequences comprising genes that encode novel lipolytic enzymes LIP01-LIP03. The LIP01 enzyme may be isolated from Magnaporthe grisae, the LIP02 and LIP03 may be obtained by mutating the polynucleotide sequence encoding for LIP01. The invention features the full length coding sequence of the novel gene, which is suitable for expression in a suitable host cell as well as 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 proteins in industrial processes, for example in baking industry, vegetable oil degumming, production or modification of food emulsifiers and the production of glucose from wheat gluten.

Description

technical field [0001] The present invention relates to a newly identified polynucleotide sequence comprising a gene encoding a novel lipolytic enzyme. The enzyme can be isolated from Magnaporthe grisae. The invention describes the full-length coding sequence of the novel gene, as well as the amino acid sequence of the full-length functional protein and functional equivalents of the gene or amino acid sequence. The invention also relates to methods of using these enzymes in industrial processes such as the bakery industry. The invention also includes cells suitable for the production of these proteins transformed with the polynucleotides of the invention, and cells in which the lipases of the invention are typically genetically modified to enhance or decrease their activity and / or expression level. Background technique [0002] In order to improve the handling properties of the dough and / or the final properties of the baked product, there is a continuous effort to develop ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/37
CPCC07K14/37
Inventor 简·米特斯卡·拉恩·凡·德玛格特·伊丽莎白·弗兰克希斯·休恩瓦尔德-贝格曼斯
Owner DSM IP ASSETS BV
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