ENHANCED HETEROLOGOUS PRODUCTION OF LIPOXYGENASES
The invention is directed to the enhanced expression and purification of lipoxygenase enzymes. These enzymes are of wide-spread industrial importance, often produced in heterologous microbial systems. Preferably, the lipoxygenase produced by the methods of the invention is a plant-derived enzyme and expressed at high-levels in a microbial system that includes a protease-deficient host and one or more chaperone expression plasmids. The invention is also directed to amino acid and nucleic acid fragments of the lipoxygenase enzyme including fragments in expression constructs encoding all or a portion of one or more lipoxygenase genes. The invention is also directed to methods of manufacturing bread and other food and also non-food products with lipoxygenase manufactured by the methods of the invention.
1 . A method of producing lipoxygenase enzyme comprising:
providing a nucleic acid expression construct within a host microorganism, wherein the construct encodes a plant-derived lipoxygenase enzyme;
providing one or more chaperone plasmids within the host microorganism; and
inducing expression of a lipoxygenase polypeptide encoded by the construct.
2 . The method of claim 1 , further comprising purifying the expressed lipoxygenase polypeptide and collecting isolated lipoxygenase polypeptide.
3 . The method of claim 1 , wherein the nucleic acid expression construct encodes all or a functional portion of the sequence of SEQ ID NO 1, SEQ ID NO 2, or SEQ ID NO 3.
4 . The method of claim 1 , wherein the nucleic acid expression construct contains all or a functional portion of the sequence of SEQ ID NO 4, SEQ ID NO 5, or SEQ ID NO 6.
5 . The method of claim 1 , wherein the host microorganism is a bacterial cell containing one or more protease deficiencies.
6 . The method of claim 5 , wherein the bacterial cell is a strain of K12 cells, E. coli cells, Bacillus cells, Lactoccocus or yeast cells.
7 . The method of claim 5 , wherein the bacterial cell is an organism generally recognized as safe for the production of food enzymes.
8 . The method of claim 1 , wherein the one or more chaperone plasmids are simultaneously co-expressed with the lipoxygenase polypeptide.
9 . The method of claim 1 , wherein inducing comprises maintaining the heterologous microorganism at from 10-37° C. for a period of time.
10 . The method of claim 9 , wherein inducing comprises maintaining the heterologous microorganism at from 25-35° C. for a period of time.
11 . The method of claim 9 , wherein inducing comprises maintaining the heterologous microorganism at from 10-25° C. for a period of time.
12 . The method of claim 9 , wherein inducing comprises maintaining the heterologous microorganism at from 20-25° C. for a period of time.
13 . The method of claim 9 , wherein the period of time is from 1 hour to 2 days.
14 . The method of claim 1 , wherein purification comprises contacting the expressed lipoxygenase to immobilized-metal affinity chromatography media.
15 . The method of claim 1 , wherein the expressed lipoxygenase polypeptide does not contain a histidine tag.
16 . The method of claim 1 , wherein the collected lipoxygenase polypeptide comprises a plant lipoxygenase.
17 . The method of claim 1 , wherein the expressed lipoxygenase polypeptide does not contain a histidine tag.
18 . The method of claim 1 , wherein soluble lipoxygenase polypeptide relative to total soluble protein in the cell extract is 30% or greater.
19 . A composition comprising lipoxygenase polypeptide made by the method of claim 1 .
20 . A method for the manufacture of a bread product, comprising adding the composition of claim 19 to a dough.
21 . The method of claim 20 , wherein the dough contains unsaturated fatty acids, carotinoids or both unsaturated fatty acids and carotinoids.
22 . A bread product made by the method of claim 21 .
23 . A method of producing lipoxygenase enzyme comprising:
providing a nucleic acid expression construct within a host microorganism, wherein the construct encodes a plant-derived lipoxygenase enzyme and the host microorganism is generally recognized as safe for the production of food enzymes;
providing one or more chaperone plasmids within the host microorganism wherein the one or more chaperone plasmids are simultaneously co-expressed with the lipoxygenase polypeptide;
inducing expression of a lipoxygenase polypeptide encoded by the construct;
purifying the expressed lipoxygenase polypeptide by contacting the expressed lipoxygenase to immobilized-metal affinity chromatography media, wherein soluble lipoxygenase polypeptide relative to total soluble protein in the cell extract is 30% or greater; and
collecting isolated lipoxygenase polypeptide.
24 . An artificial and functional lipoxygenase enzyme produced by the method of claim 23 .