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Enzymatic treatment of oils

A glycerin and edible oil technology, applied in the direction of enzymes, transferases, hydrolytic enzymes, etc., can solve problems such as expensive, ineffective enzymes, and difficult industrial control of enzymatic reactions

Inactive Publication Date: 2007-01-17
AS DE DANSKE SUKKERFABRIKKER
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Acetyl CoA is quite expensive and not suitable for commercial applications to process edible oils
However, these enzymes do not work in the absence of acetyl CoA
Furthermore, enzymatic reactions dependent on fatty acid-CoA are difficult to industrially control

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0821] Example 1: Cloning, sequencing and heterologous expression of a transferase from Aeromonas salmonicida subsp. salmonicida

[0822] Strains used:

[0823] Aeromonas salmonicida subsp. salmonicida (ATCC 14174) was obtained from ATCC and grown overnight at 30°C in Luria-Bertani medium (LB). Cells were centrifuged and genomic DNA was isolated using the procedure for genomic DNA isolation purchased from Qiagen Ltd. Genomic DNA Buffer Set (Cat. No. 19060), Proteinase K (Cat. No. 19131) and RNase A (Cat. No. 19101) were purchased from Qiagen Ltd (Boundary court Gatwick Court, West Sussex, RH10 2AX).

[0824] The recombinant Aeromonas enzyme was produced with the host bacterial strain BL21(DE3)pLysS (Novagen). Competent cells of BL21(DE3)pLysS were used as hosts for the transformation of the expression vector pet12-AsalGCAT=pSM. Transformants containing appropriate plasmids were cultured at 37°C on LB agar medium containing 100 µg / ml ampicillin.

[0825] Construction of the...

Embodiment 2

[0839] Example 2: Cloning and expression of Aeromonas hydrophila transferase in Escherichia coli

[0840] Aeromonas hydrophila (ATCC #7965) was obtained from ATCC and grown overnight at 30C in Luria-Bertani medium (LB). Cells were centrifuged and genomic DNA was isolated using the procedure for genomic DNA isolation purchased from Qiagen Ltd. Genomic DNA buffer set (Cat. No. 19060), Proteinase K (Cat. No. 19131) and RNase A (Cat. No. 19101) were purchased from Qiagen Ltd (Boundary court Gatwick Court, West Sussex, RH10 2AX).

[0841] The recombinant Aeromonas enzyme was produced with the host bacterial strain BL21(DE3)pLysS (Novagen). Competent cells of BL21(DE3)pLysS were used as hosts for the transformation of the expression vector pet12a-A.h.GCAT=pSMa. Transformants containing appropriate plasmids were cultured at 37°C on LB agar medium containing 100 µg / ml ampicillin.

[0842] Construction of the expression vector pet12a-A.h.GCAT=pSMa:

[0843] For all DNA amplificatio...

Embodiment 3

[0856] Example 3: Expression of Aeromonas transferase in Bacillus subtilis 163

[0857] Plasmid construction

[0858]Aeromonas genes were heterologously expressed in B. subtilis using two different B. subtilis vectors (pUB110 and pBE5). The pUB110 vector contains the α-amylase promoter, while the pBE vector has the P32 promoter as a regulatory region for expression of the fused Aeromonas gene. In pUB110, the first amino acid of the mature GCAT gene of Aeromonas was fused in-frame to the last amino acid of the xylanase signal peptide sequence from Bacillus subtilis via the restriction site NheI, in the mature protein. 2 additional amino acids are generated ahead. pBE5 contains a cgtase signal sequence fusion at the NcoI site for secretion of the recombinant protein into the culture filtrate.

[0859] PCR reactions were performed to obtain Aeromonas genes fused in-frame to the signal sequences of the pUB110 and pBE5 vectors. The following primer pairs were used for PCR of th...

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PUM

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Abstract

The present invention relates to a method of reducing and / or removing diglyceride from an edible oil, comprising a) admixing an edible oil with an acyl acceptor substrate and a fatty-acid CoA independent diglyceride: glycerol acyltransferase, wherein the fatty-acid CoA independent diglyceride: glycerol acyltransferase is characterized as an enzyme which in an edible oil is capable of transferring an acyl group from a diglyceride to glycerol. Preferably, the diglyceride: glycerol acyltransferase comprises the amino acid sequence motif GDSX, wherein X is one or more of the following amino acid residues L, A, V, I, F, Y, H, Q, T, N, M or S. Furthermore the present invention relates to the use of a fatty-acid CoA independent diglyceride: glycerol acyltransferase characterized as an enzyme which in an edible oil is capable of transferring an acyl group from a diglyceride to glycerol, in the manufacture of an edible oil, for reducing and / or removing (preferably selectively reducing and / or removing) diglyceride from said edible oil, and to the use of said enzyme in the manufacture of a foodstuff comprising an edible oil for improving the crystallization properties of said foodstuff.

Description

[0001] References to related applications [0002] This application references the following related applications: U.S. Patent Application Serial No. 09 / 750,990, filed July 20, 1999; U.S. Patent Application Serial No. 10 / 409,391; U.S. Patent Application Serial No. 60 / 489,441, filed July 23, 2003 ; British Patent Application No. GB ​​0301117.8 filed on January 17, 2003; British Patent Application No. GB ​​0301118.6 filed on January 17, 2003; British Patent Application No. GB ​​0301119.4 filed on January 17, 2003; January 2003 British patent application number GB 0301120.2 filed on the 17th; British patent application number GB 0301121.0 filed on January 17, 2003; British patent application number GB 0301122.8 filed on January 17, 2003; British patent application number GB 0301122.8 filed on December 24, 2003 Patent application number GB 0330016.7; and International patent application number PCT / IB2004 / 000655 filed 15 January 2004. Each of these patent applications and each docum...

Claims

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Application Information

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IPC IPC(8): C12P7/64C12N9/10C12N9/14
CPCC12N9/20C12P7/6445C12N9/1029C12Y203/01073C12P7/6418C12Y301/01003C12P7/6458
Inventor 安娜·C·J·克里斯滕森保罗·瓦塞尔乔恩·D·米克尔森乔恩·B·索伊
Owner AS DE DANSKE SUKKERFABRIKKER
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