# Patent

## Title
Enhanced heterologous production of lipoxygenases

## Abstract

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.

## Background
REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application No. 61/817,077 of the same title filed Apr. 29, 2013, which is incorporated by reference in its entirety.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 28, 2014, is named 3060.002.US_SL.txt and is 29,528 bytes in size.


BACKGROUND
1. Field of the Invention
The invention is directed to systems, compositions and methods for the expression and purification of lipoxygenases, to amino acid and nucleic acid sequences of all or portions of lipoxygenases, to molecular constructs for the expression of lipoxygenases, and, in particular, to methods for the large scale production and use of lipoxygenases in food products.
2. Description of the Background
Lipoxygenases (LOXs; EC1.13.11_), also known as lipoxydases, are non-heme iron-containing dioxygenases distributed in plants and animals. LOXs catalyze hydroperoxidation of polyunsaturated fatty acids in the first step of fatty acid metabolite synthesis, to produce an unsaturated fatty acid hydroperoxide. A LOX definition according to enzyme classification is linoleate: oxygen oxidoreductase (for plant LOX) and arachidonate: oxygen oxidoreductase (for mammalian LOX). In plants, the most common LOX substrates linoleic acid and linolenic acids are converted into a variety of bioactive mediators involved in plant defense, senescence, seed germination, as well as plant growth and development (Grechkin A. Recent developments in biochemistry of the plant lipoxygenase pathway; Prog Lipid Res. 1998 November 37(5):317-52). Lipoxygenases with different specificities, subcellular location, and tissue-specific expression patterns have been identified as ubiquitously found across kingdoms from bacteria to mammals.
LOXs are of commercial value in various industries including but not limited to food-related applications in food processing including bread making (bleaching and improved texture), aroma and flavor enhancement as well as for production of perfumes, paint driers (lipoxygenases: potential starting biocatalysts for the synthesis of signaling compounds. Joo YC, Oh DK. 2012) and pitch control in softwood pulp (Microbial and enzymatic control of pitch in the pulp and paper industry, Ana Gutiérrez & José C. del Río & Angel T. Martínez, Appl Microbiol Biotechnol (2009) 82:1005-4018). Lipoxygenase is present in seeds (e.g. soybeans), grains and many other plant tissues. In the presence of oxygen, lipoxygenase oxidizes unsaturated fatty acids and produces lipid hydroperoxides, which improve dough structure through the oxidation of unsaturated fatty acids and subsequently react with specific chemical components of flour. As a consequence, dough stability and rising is increased, which either or together can increase the volume of the final product.
Regarding the processing of bread, lipoxygenase enzymes offer an advantage over current chemical additives. The flour ingredient industry had long been using chemical bleach, mostly benzoyl peroxide (BPO). Because of potential health concerns over BPO, some Euro countries and China banned the usage of BPO in flour. In the U.S., BPO is still widely used, but the demand keeps shirking although there is currently no safe alternative. Azodiformamide is another chemical alternative, but the dosage is limited to 40 ppm. At this trace dosage, the bleaching effect is quite restrained. In contrast, enzyme additives especially LOXs can replace chemicals to allow for the processing of flour, resulting in the bleaching of bread and its improved texture. In addition, lipid hydroperoxidases decolorize dough and oxidizes carotinoids, converting them into colorless compounds. This blanching of the dough results in lighter colored product, which is highly desired.
With regard to enzymes employed in the food industry, regulations frequently require enzymes to be recognized or proven as safe for use. In the case of lipoxygenases, considering that they are ubiquitously found in plants and consumed by humans and animals alike, plant lipoxygenases are considered safe for use, and therefore, of major value to the industry. Although soy extracts containing high levels of lipoxygenases have been used as an additive for bread manufacturing, soy produces an undesirable taste and smell and, accordingly, not often a useful option.
Because of plant LOX value, many attempts at high-level expression of recombinant plant derived LOX from soy, rice, potato and other sources by heterologous expression in microbial hosts including, but not limited to bacteria such as E. coli (BL21 strain), Bacilli, and in yeast has been attempted, though production was limited [3-8]. The best of these, although still a poor expression from E. coli, was observed at very cold temperatures [8]. Only one lipoxygenase was produced in Bacilli at high-levels ( ̃160 mg/L), but the lipoxygenase was from a bacterial enzyme, not a plant and consequently not approved for use in the human food industry [9, 10]. In addition, yields still could not achieve desired levels. Accordingly, a need exists for high level expression of plant lipoxygenases that is generally recognized as safe for use in foods, and easily produced in large quantities.
SUMMARY OF THE INVENTION
The present invention overcomes the problems and disadvantages associated with current strategies and designs, and provides new methods and compositions involving the heterologous expression, purification and use of lipoxygenases.
One embodiment of the invention is directed to the heterologous expression of lipoxygenases in microbes.
Another embodiment of the invention is directed to methods for the purification of lipoxygenases, preferably from heterologous expression systems according to the invention.
Another embodiment of the invention is directed to lipoxygenase polypeptide and nucleic acids sequences and molecular constructs of lipoxygenase coding sequences, preferably for the high level expression of lipoxygenase as compared to expression in wild-type host cells. Preferably wild-type host cells are cells that do not contain a protease deficiency and/or cells that do not contain one or more chaperones.
Another embodiment of the invention is directed to methods for the manufacture of bread products comprising adding lipoxygenases of the invention to a dough containing unsaturated fatty acids and/or carotinoids. Preferably the lipoxygenase reacts with components of the flour forming lipid hydroperoxides increasing the stability of the dough and enhancing the volume of the baked goods.
Another embodiment of the invention is directed to purified lipoxygenase enzyme made by the methods of the invention. When the purified enzyme is added to dough, another embodiment of the invention comprises products made with the purified enzyme added to dough such as, preferably, bread products. The manufacture of bread products of the invention preferably comprises adding lipoxygenases to a dough containing unsaturated fatty acids and/or carotinoids. Preferably the lipoxygenase reacts with components of the flour forming lipid hydroperoxides increasing the stability of the dough and enhancing the volume of the baked goods.
Other embodiments and advantages of the invention are set forth in part in the description, which follows, and in part, may be obvious from this description, or may be learned from the practice of the invention.



DESCRIPTION OF THE FIGURES
FIG. 1 Western analysis of SLP1 indicates varying profiles of degradation. M=marker. Different K12 cells with different genotypes are presented in sets of “U” (uninduced) and “I” (induced).
FIG. 2 Co-expression of the GroEL-GroES chaperone enhances SLP1 production. Four chaperones A-D were co-expressed in E. coli with SLP1. Left Panel: SLP1 is directed detected as a weak band in SDS-PAGE as a result of Co-expression with Chaperone B. Right Lane: Enhanced expression of SLP1 with co-expressed GroEL-GroES is confirmed by standard Western analysis.
FIG. 3 Single step purification of SLP1 from the 424 vector (native protein sequence, no polyhistidine tag). All lanes: SLP1 lacking a his tag was eluted from Zinc-NTA (IMAC) columns and run in SDS PAGE followed by protein staining: Lanes 1—crude lysate; Lane 2—column flow-through; Lane 3—column wash; Lanes 4-7—Zinc-NTA (IMAC) elution with SLP1 loaded in the presence of 0, 2, 5 and 10 mM imidazole, each eluted with 80 mM imidazole.
FIG. 4 E. coli cell lines used to verify experiments.
FIG. 5 SLP1 expression in E. coli; SDS PAGE protein gel of whole cell K12 E. coli lysate expressing SLP1.



DESCRIPTION OF THE INVENTION
Lipoxygenase enzymes (also referred to herein as LOX) are widely used in commercial processing of food products, the manufacture of perfumes and painting products, and in the processing of wood pulp. Although all lipoxygenase catalyze the same basic function, only plant lipoxygenases have been approved by the United States Food and Drug Administration for use in foods and food products. Despite their broad uses, lipoxygenase enzymes are only expressed at low levels and, consequently, commercial quantities are both expensive and difficult to produce.
Despite previous failures in achieving high level LOX expression, it has been surprisingly discovered that considerable enhancement of plant lipoxygenase expression can be achieved. At least part of this high-level of expression is attributed to the selection of sequences being expressed, expression of the sequences in a protease deficient host, and/or the co-expression with one or more chaperone plasmid sequences. Preferable, the increased expression achieved is at a higher level than expression in host cells that do not contain a protease deficiency and/or cells that do not contain one or more chaperone plasmids. Preferably the expression of the one or more proteases is eliminated, reduced to an undetectable level using conventional detection or reduced by at least 90%, all as compared to wild-type expression levels.
One embodiment of the invention comprises a system containing a bacterial cell host, preferably with a deficiency or one or more proteases, containing a coding sequence for lipoxygenase enzyme and preferably a chaperone system comprising one or more chaperone molecules. The system is preferably inducible and also preferably maintained from about 10° C. to about 37° C. for a period of time for maximal expression of enzyme product. The period of time is preferably from minutes to hours to days, and more preferably from about 1 to about 24 hours, more preferably from 2 to 12 hours and more preferably from about 2 to about 4 hours. The cells are preferably maintained at temperatures from about 15° C. to about 25° C. during this period.
The lipoxygenase enzyme may be derived from animal or bacterial cells, and is preferably derived from plant cells. Expression constructs may contain all or a portion of the lipoxygenase gene or coding region. Preferably constructs contain a portion of the coding region sufficient to create functional lipoxygenase activity. Preferably the constructs of the invention encode the sequences of SEQ ID NOs 1-3, or contain the nucleic acid sequences of SEQ ID NOs 4-6. Also preferably the sequence is a functional sequences that generates functional lipoxygenase activity.
Preferably the host cell is a microorganism that rapidly and economically proliferates in vitro such as, for example, one or more of the bacterial cell strains of K12 cells, E. coli cells, Bacillus cells, Lactococci or yeast cells. Also preferably, the host cells contain one or more protease deficiencies as compared to wild-type cells. For E. coli host cells, the deficiency is preferably of one or more of the proteases Lon, OMPT, and/or Lon/ClpP.
Preferably the host cells further contain one or more chaperone plasmid expression vectors. Chaperones function in assisting protein folding, benefiting the co-expressed molecules.
Expression of lipoxygenase in the systems of the invention typically involves inducing expression of the lipoxygenase sequence and also preferably the chaperone sequences before, during or after expression of the lipoxygenase, and preferably simultaneously or nearly simultaneously to allow for maximal expression of the enzyme.
Lipoxygenase produced according to methods of the invention can be further isolated and purified. Preferably, purification of lipoxygenase produced according to the methods of the invention involves contact the with immobilized-metal affinity chromatography media. The enzyme remains bound and can be washed with wash buffer and subsequently eluted with elution buffer.
Preferably the increased lipoxygenase expression of the invention is 5 fold greater as compared to expression in wild-type cells (e.g., cells that are not protease deficient and/or cells without one or more expression chaperones), more preferably 10 fold greater, more preferably 50 fold greater, more preferably 100 fold greater, more preferably 200 fold greater, more preferably 300 fold greater, more preferably 400 fold greater, and more preferably 500 fold greater or more.
Lipoxygenase made according to the invention is preferably useful in the manufacture of food products such as bread products (for either, or both bleaching and improving texture), the manufacture of paints thinners, perfumes, aroma and flavor enhancers, as signaling compounds, and for pitch control in softwood pulp in paper industry.
The following examples illustrate embodiments of the invention, but should not be viewed as limiting the scope of the invention.
EXAMPLES
Example 1
LOXs Employed for Protein Production
SLP1 (seed linoleate 13S-lipoxygenase-1 [Glycine max] NCBI Reference Sequence: NP_001236153.1, length 839 amino acids) and SLP3 seed linoleate 9S-lipoxygenase-3 [Glycine max] NCBI Reference Sequence: NP_001235383.1) were employed as LOXs for production in microbes. In addition, a shortened version of SLP1 (herein minilox) from amino acid Serine 278 containing an additional methionine before the Serine 278 were cloned and expressed in microbes.
Example 2
Synthesis of DNA Encoding Protein Sequences for SLP1, SLP3 and Minilox Optimized for Expression
Optimal gene codon usage in plants and bacteria differ. New DNA encoding sequences for SLP1, SLP3 and minilox were determined and synthetically generated according to instructions (Genscript USA Inc.). The sequence for minilox was identical as that of SLP1 with the exception of having an ATG encoding for an initiator methionine prior to nucleotide bases encoding for SLP1 Serine 278. Optimized sequences with desired cloning sites were created.
Example 3
Cloning of Soybean Lipoxygenase 1 (SLP1) and 3 (SLP3) and MiniLox
Initially, SLP1 and SLP3 were cloned into the pET 47b vector (Novogen) using SmaI-XhoI restriction sites, so that each contained the pET47b initiating methionine and a 6× histidine tag (SEQ ID NO 7). The SLP1 and minilox encoding DNA were then transferred to the DNA2.0 expression vector 424 purple, a low copy number plasmid without the histidine tags using NdeI-XhoI sites, so that expressed proteins would not contain the histidine tags. Similarly, the SLP1 encoding sequence was cloned into the 424-purple vector (herein 424 vector) with the exception of using NdeI-EcoRI cloning sites. The SLP1 encoding sequences were then transferred to the DNA2.0 purple-444 vector (herein 444 vector), a high copy number plasmid using restriction sites NdeI-XhoI. The vectors contained promoters for expression of the insert DNA with the pET47 containing a T7-promotor, and the DNA2.0 vectors contained a T5-promotor.
Example 4
Expression of SLP1, Minilox and SLP3
Vectors were transfected into cell lines. Initially, expression of SLP1 was performed with the 6 histidine (SEQ ID NO 7) tagged SLP1 vector in E. coli BL21 cells, an E. coli B cell line suitable for the expression of the pET47b vectors. Thereafter, all expression was performed in E. coli K12 strains. Expression was tested in LB media, with 50-100 μg/ml ampicillin, and induction of expression for all vectors was with 0.5-1 mM IPTG.
Example 5
Activity Assay
The activity assay utilized linoleic acid as a substrate and colorimetric detection of product. Detected values for the assay varied depending on the substrate preparation, age of substrate, and substrate batch, which may be subject to variation due to oxidation from the environment. As such, approximate expression levels of SLP1 in BL21 are presented as 1 unit/cell OD550 SLP1-LB culture and relative and approximate values for expression in other strains is relative to the BL21 expression. Cell OD550 is defined as a cell density at OD550.
Example 6
Improvement of SLP1 Production
SLP1 expressed with or without a histidine tag using the pET47b vector and BL21 cells was very poor when induced at room temperature. The standard level of activity, 1 unit/OD cells was established for induction at 15° C. with and overnight expression. Dramatically improved activity was observed using the purple-424 vector (herein 424 vector), in the K12 HMS174 cell line (4 units/Cell OD550). Unlike BL21 cells, activity was also observed when induced at 20° C.-25° C. with overnight expression. Slp1 activity could be further enhanced by growing cells at 15° C. for up to several days. In all E. coli strains tested, growth at 37 C of was found to generate little or no SLP1 activity, and protein degradation products were observed upon western analysis (FIG. 2).
An additional increase in activity was discovered using protease deficient E. coli K12 strains with the 424 vector. Lon, OMPT, or Lon/ClpP mutants all showed a further minimum two-fold increase in activity with ( ̃10 units/cell OD550). The specific E. coli cell lines with specific protease deficiencies also showed some similar characteristics of protein degradation (FIG. 3) yet some cell lines had less degradation than others, signifying that proteases play a role in the limited production of lipoxygenases.
An additional enhancement of activity was observed when using the 444 high plasmid copy vector in the K12 Pam155 (lon protease deficient) E. coli cell line and with chaperones. Chaperone plasmid sets consisting of five different plasmids from Takara Bio Inc. each designed to express a single or multiple molecular chaperone sets can enable optimal protein expression and folding and reduce protein misfolding. Each Takara plasmid carries an origin of replication (ORF) derived from pACYC and a chloramphenicol-resistance gene (Cmr) gene, which allows the use of E. coli expression systems containing ColE1-type plasmids that confer ampicillin resistance. The chaperone genes are situated downstream of the araB or Pzt-1 (tet) promoters and, as a result, expression of target proteins and chaperones can be individually induced if the target gene is placed under the control of different promoters (e.g., lac). These plasmids also contain the necessary regulator (araC or tetr) for each promoter. Takara Bio Inc. plasmids containing chaperones or sets thereof either tetracycline or arabinose inducible were coexpressed with SLP1. These include: groES-groEL, dnaK-dnaJ-grpE, groES-groEL-tig, or tig in plasmids (TakaraBo Inc.). Expression of SLP1 in the presence of groES-groEL alone or with tig (groES-groEL-tig) enhanced the amount of active enzyme produced roughly to 40-60 units/cell OD. Activity was optimal at 15° C. but also observed at or below 25° C. At 37° C., expression was more limited.
Expression of SLP1 in the Pam153 cell with concomitant GroESL chaperone expression, in LB, produces 68 micrograms of SLP1 per milliliter at a bacterial OD550 of 3, when grown in test tubes at 37° C. and induced at 20° C. overnight. However, SLP1 expression in an E. coli strain that is not a protease deficient strain and without chaperone expression can either not be detected at all with standard SDS PAGE analysis, or western analysis, or expresses less than 1 μg per milliliter LB under similar conditions at an OD550 of 3 (see FIG. 4). In general, expression of SLP1 in E. coli strains grown and induced under optimal conditions was undetectable or less than 1 microgram per milliliter when appropriate chaperones were absent and strains were not protease deficient. However when expressing SLP1 in E. coli K12 protease deficient strains with co-expression of an appropriate chaperone, 68 micrograms of SLP1 per milliliter at a bacterial OD550 of 3 was attained.
Example 7
Purification of SPL1
Purification of SLP1 with the 6×his tag (SEQ ID NO 7) was highly effective using standard Ni-NTA IMAC purification. In the 424 or 444 vectors lacking the 6×his tag (SEQ ID NO 7), where SLP1 was encoded by the native SLP1 sequence alone, IMAC was equally efficient though under modified conditions. Nickel and zinc were each tested with similar results and calcium or other divalent metals should do as well. Buffers for IMAC were either 50 mM phosphate or Tris-HCl at pH 7-9, with 400 mM NaCl and 10% glycerol. Cells were disrupted using B-PER (Peirce) or by a homogenizer, in the presence of PMSF as a protease inhibitor. Employing Zinc-NTA, it was discovered that loading the sample in buffer with 10 mM imidazole and elution in buffer with 80 mM imidazole was effective in purification of SLP1. Other column media that effectively binds SLP1 include MonoQ and DEAE, but not negatively charged resins.
Example 8
Novel Information Provides Improved SLP1 Expression
Preliminary studies indicate that relatively poor production of SLP1 is the result of rapid proteolysis accompanied by improper folding of the enzyme. The limited soluble SLP1 and lack of insoluble protein suggests that most of the protein produced was rapidly degraded. Degradation products of SLP1 are visible in different E. coli strains with different protease deficient genetic backgrounds (see FIG. 1). An increase in both active enzyme and total protein was observed when inducing at suboptimal growth temperatures, where proteases are less functional. A relative increase in production and activity of SLP1 when protein folding is enhanced by an over-expressed chaperone.
Example 9
High Level Expression of Lipoxygenase in the E. Coli, K12
Unless otherwise stated, all bacterial media employed in this example was Luria Broth (herein LB, consisting of 10 grams Tryptone, 5 grams Yeast Extract, and 10 grams NaCl, dissolved in 1 liter water, and sterilized for a minimum of 20 minutes in an autoclave). Soybean Lipoxygenase 1 (herein SLP1) was expressed from a plasmid transfected into E. Coli K12 cells. FIG. 5 represents an SDS-PAGE protein gel of whole cell soluble proteins extracted from the K12 cells employing the commercial B-PER Protein Extraction Reagent (Pierce, Cat#78243), following company protocols. The highest level of soluble SLP1 protein relative to total soluble protein in the cell extract was 30% or greater and approximated at 34% as estimated by the ImageJ (National Institute of Health public software) analysis software. These levels are consistent with high level production of the enzyme. M=marker, 1 Uninduced, 2 SLP1 induced with 0.5 mM IPTG 3&4 Induced with 0.5 mM IPTG and expressing a molecular chaperone.
CITED REFERENCES


    1. Permiakova, M. D. and V. A. Trufanov, Effect of soybean lipoxygenae on baking properties of wheat flour, Prikl Biokhim Mikrobiol, 2011. 47(3): p. 348-54.
    2. Permiakova, M. D., et al., [Role of lipoxygenase in the determination of wheat grain quality]. Prikl Biokhim Mikrobiol, 2010. 46(1): p. 96-102.
    3. Kanamoto, H., M. Takemura, and K. Ohyama, Cloning and expression of three lipoxygenase genes from liverwort, Marchantia polymorpha L., in Escherichia coli. Phytochemistry, 2012. 77: p. 70-8.
    4. Osipova, E. V., et al., Recombinant maize 9-lipoxygenase: expression, purification, and properties. Biochemistry Biokhimiia, 2010. 75(7): p. 861-5.
    5. Hwang, I. S, and B. K. Hwang, The pepper 9-lipoxygenase gene CaLOX1 functions in defense and cell death responses to microbial pathogens. Plant Physiol, 2010. 152(2): p. 948-67.
    6. Padilla, M. N., et al., Functional characterization of two 13-lipoxygenase genes from olive fruit in relation to the biosynthesis of volatile compounds of virgin olive oil. J Agric Food Chem, 2009. 57(19): p. 9097-107.
    7. Knust, B. and D. von Wettstein, Expression and secretion of pea-seed lipoxygenase isoenzymes in Saccharomyces cerevisiae. Appl Microbiol Biotechnol, 1992. 37(3): p. 342-51.
    8. Steczko, J., et al., Effect of ethanol and low-temperature culture on expression of soybean lipoxygenase L-1 in Escherichia coli. Protein Expr Purif, 1991. 2(2-3): p. 221-7.









Sequence Information


SLP1 polypeptide sequence 


(SEQ ID NO 1)


MFSAGHKIKGTVVLMPKNELEVNPDGSAVDNLNAFLGRSVSLQLISATK


 


ADAHGKGKVGKDTFLEGINTSLPTLGAGESAFNIHFEWDGSMGIPGAFY


 


IKNYMQVEFFLKSLTLEAISNQGTIRFVCNSWVYNTKLYKSVRIFFANH


 


TYVPSETPAPLVSYREEELKSLRGNGTGERKEYDRIYDYDVYNDLGNPD


 


KSEKLARPVLGGSSTFPYPRRGRTGRGPTVTDPNTEKQGEVFYVPRDEN


 


LGHLKSKDALEIGTKSLSQIVQPAFESAFDLKSTPIEFHSFQDVHDLYE


 


GGIKLPRDVISTIIPLPVIKELYRTDGQHILKFPQPHVVQVSQSAWMTD


 


EEFAREMIAGVNPCVIRGLEEFPPKSNLDPAIYGDQSSKITADSLDLDG


 


YTMDEALGSRRLFMLDYHDIFMPYVRQINQLNSAKTYATRTILFLREDG


 


TLKPVAIELSLPHSAGDLSAAVSQVVLPAKEGVESTIWLLAKAYVIVND


 


SCYHQLMSHWLNTHAAMEPFVIATHRHLSVLHPIYKLLTPHYRNNMNIN


 


ALARQSLINANGIIETTFLPSKYSVEMSSAVYKNWVFTDQALPADLIKR


 


GVAIKDPSTPHGVRLLIEDYPYAADGLEIWAAIKTWVQEYVPLYYARDD


 


DVKNDSELQHWWKEAVEKGHGDLKDKPWWPKLQTLEDLVEVCLIIIWIA


 


SALHAAVNFGQYPYGGLIMNRPTASRRLLPEKGTPEYEEMINNHEKAYL


 


RTITSKLPTLISLSVIEILSTHASDEVYLGQRDNPHWTSDSKALQAFQK


 


FGNKLKEIEEKLVRRNNDPSLQGNRLGPVQLPYTLLYPSSEEGLTFRGI


 


PNSISI


 


SLP3 polypeptide sequence 


(SEQ ID NO 2)


MLGGLLHRGHKIKGTVVLMRKNVLDVNSVTSVGGIIGQGLDLVGSTLDT


 


LTAFLGRSVSLQLISATKADANGKGKLGKATFLEGIITSLPTLGAGQSA


 


FKINFEWDDGSGIPGAFYIKNFMQTEFFLVSLTLEDIPNHGSIHFVCNS


 


WIYNAKLFKSDRIFFANQTYLPSETPAPLVKYREEELHNLRGDGTGERK


 


EWERIYDYDVYNDLGDPDKGENHARPVLGGNDTFPYPRRGRTGRKPTRK


 


DPNSESRSNDVYLPRDEAFGHLKSSDFLTYGLKSVSQNVLPLLQSAFDL


 


NFTPREFDSFDEVHGLYSGGIKLPTDIISKISPLPVLKEIFRTDGEQAL


 


KFPPPKVIQVSKSAWMTDEEFAREMLAGVNPNLIRCLKDFPPRSKLDSQ


 


VYGDHTSQITKEHLEPNLEGLTVDEAIQNKRLFLLDHHDPIMPYLRRIN


 


ATSTKAYATRTILFLKNDGTLRPLAIELSLPHPQGDQSGAFSQVFLPAD


 


EGVESSIWLLAKAYVVVNDSCYHQLVSHWLNTHAVVEPFIIATNRHLSV


 


VHPIYKLLHPHYRDTMNINGLARLSLVNDGGVIEQTFLWGRYSVEMSAV


 


VYKDWVFTDQALPADLIKRGMAIEDPSCPHGIRLVIEDYPYTVDGLEIW


 


DAIKTWVHEYVFLYYKSDDTLREDPELQACWKELVEVGHGDKKNEPWWP


 


KMQTREELVEACAIIIWTASALHAAVNFGQYPYGGLILNRPTLSRRFMP


 


EKGSAEYEELRKNPQKAYLKTITPKFQTLIDLSVIEILSRHASDEVYLG


 


ERDNPNWTSDTRALEAFKRFGNKLAQIENKLSERNNDEKLRNRCGPVQM


 


PYTLLLPSSKEGLTFRGIPNSISI


 


Minilox 1 polypeptide sequence 


(SEQ ID NO 3)


MSTPIEFHSFQDVHDLYEGGIKLPRDVISTIIPLPVIKELYRTDGQHI


 


LKFPQPHVVQVSQSAWMTDEEFAREMIAGVNPCVIRGLEEFPPKSNLD


 


PAIYGDQSSKITADSLDLDGYTMDEALGSRRLFMLDYHDIFMPYVRQI


 


NQLNSAKTYATRTILFLREDGTLKPVAIELSLPHSAGDLSAAVSQVVL


 


PAKEGVESTIWLLAKAYVIVNDSCYHQLMSHWLNTHAAMEPFVIATHR


 


HLSVLHPIYKLLTPHYRNNMNINALARQSLINANGIIETTFLPSKYSV


 


EMSSAVYKNWVFTDQALPADLIKRGVAIKDPSTPHGVRLLIEDYPYAA


 


DGLEIWAAIKTWVQEYVPLYYARDDDVKNDSELQHWWKEAVEKGHGDL


 


KDKPWWPKLQTLEDLVEVCLIIIWIASALHAAVNFGQYPYGGLIMNRP


 


TASRRLLPEKGTPEYEEMINNHEKAYLRTITSKLPTLISLSVIEILST


 


HASDEVYLGQRDNPHWTSDSKALQAFQKFGNKLKEIEEKLVRRNNDPS


 


LQGNRLGPVQLPYTLLYPSSEEGLTFRGIPNSISI


 


SLP1 DNA optimized encoding sequence (with


restriction sites 5′SmaI and 3′XhoI with stop


codon for cloning into pET47b with 6X histidine


tag


(SEQ ID NO 7)) (SEQ ID NO 4)


CCCGGGATGTTTAGTGCTGGTCACAAAATCAAAGGTACCGTGGTCCT


 


GATGCCGAAAAATGAACTGGAAGTCAACCCGGATGGTAGCGCCGTTG


 


ATAACCTGAATGCGTTCCTGGGTCGTAGCGTGTCTCTGCAGCTGATT


 


TCCGCCACCAAAGCAGACGCTCACGGCAAGGGTAAAGTTGGCAAAGA


 


TACGTTTCTGGAAGGTATTAATACCTCCCTGCCGACCCTGGGTGCCG


 


GTGAATCAGCTTTCAACATCCATTTCGAATGGGATGGTTCAATGGGC


 


ATTCCGGGCGCCTTCTACATCAAAAACTACATGCAGGTGGAATTTTT


 


CCTGAAAAGTCTGACCCTGGAAGCAATCTCCAATCAGGGTACGATTC


 


GTTTTGTCTGCAACTCGTGGGTGTATAATACCAAACTGTACAAAAGC


 


GTTCGCATCTTTTTCGCGAACCACACCTATGTTCCGAGCGAAACCCC


 


GGCACCGCTGGTTTCTTACCGTGAAGAAGAACTGAAAAGTCTGCGCG


 


GCAATGGTACCGGCGAACGTAAAGAATATGATCGCATTTATGACTAC


 


GATGTTTACAACGACCTGGGCAATCCGGATAAAAGCGAAAAACTGGC


 


CCGTCCGGTCCTGGGCGGTAGCTCTACCTTCCCGTATCCGCGTCGCG


 


GTCGTACCGGTCGTGGTCCGACCGTGACCGATCCGAACACCGAAAAA


 


CAGGGCGAAGTCTTTTATGTGCCGCGCGACGAAAATCTGGGCCATCT


 


GAAATCTAAAGATGCCCTGGAAATCGGTACCAAAAGTCTGTCCCAGA


 


TTGTGCAACCGGCGTTTGAAAGCGCCTTCGATCTGAAATCTACGCCG


 


ATTGAATTTCACTCCTTCCAGGACGTTCATGATCTGTATGAAGGCGG


 


TATCAAACTGCCGCGTGACGTCATTTCAACCATTATCCCGCTGCCGG


 


TGATCAAAGAACTGTACCGCACGGATGGTCAGCACATTCTGAAATTT


 


CCGCAACCGCATGTGGTTCAGGTTTCACAATCGGCGTGGATGACCGA


 


TGAAGAATTCGCGCGTGAAATGATCGCCGGCGTTAACCCGTGCGTCA


 


TTCGCGGTCTGGAAGAATTTCCGCCGAAAAGCAATCTGGACCCGGCA


 


ATCTATGGCGATCAGAGTTCCAAAATTACCGCTGACTCTCTGGACCT


 


GGATGGCTACACGATGGATGAAGCCCTGGGTAGTCGTCGCCTGTTTA


 


TGCTGGACTATCACGATATCTTCATGCCGTACGTGCGTCAGATTAAC


 


CAACTGAATTCTGCAAAAACCTATGCTACCCGTACGATCCTGTTTCT


 


GCGCGAAGACGGCACGCTGAAACCGGTTGCAATTGAACTGAGCCTGC


 


CGCATTCTGCTGGTGATCTGAGTGCCGCGGTGTCCCAGGTTGTGCTG


 


CCGGCAAAAGAAGGCGTTGAAAGTACCATCTGGCTGCTGGCGAAAGC


 


CTATGTTATTGTCAACGATTCATGTTACCATCAACTGATGTCGCACT


 


GGCTGAATACCCATGCAGCTATGGAACCGTTTGTTATCGCAACGCAT


 


CGCCACCTGTCTGTCCTGCACCCGATTTATAAACTGCTGACCCCGCA


 


TTACCGTAACAATATGAACATCAATGCACTGGCTCGCCAGAGTCTGA


 


TTAACGCGAATGGTATTATCGAAACCACGTTCCTGCCGTCAAAATAT


 


TCGGTGGAAATGTCATCGGCCGTTTACAAAAACTGGGTCTTTACCGA


 


CCAGGCACTGCCGGCTGATCTGATCAAACGTGGCGTCGCGATTAAAG


 


ATCCGAGCACCCCGCATGGTGTGCGTCTGCTGATTGAAGACTATCCG


 


TACGCGGCCGATGGCCTGGAAATCTGGGCAGCTATTAAAACCTGGGT


 


GCAGGAATATGTTCCGCTGTATTACGCACGCGATGACGATGTGAAAA


 


ATGACTCCGAACTGCAACACTGGTGGAAAGAAGCTGTTGAAAAAGGT


 


CATGGCGACCTGAAAGATAAACCGTGGTGGCCGAAACTGCAGACCCT


 


GGAAGATCTGGTGGAAGTTTGTCTGATTATCATTTGGATTGCCAGCG


 


CACTGCATGCCGCGGTGAACTTTGGTCAATATCCGTACGGCGGTCTG


 


ATTATGAATCGTCCGACCGCAAGCCGTCGCCTGCTGCCGGAAAAAGG


 


CACGCCGGAATACGAAGAAATGATCAACAACCATGAAAAAGCGTACC


 


TGCGCACCATCACGAGCAAACTGCCGACCCTGATTAGCCTGTCTGTT


 


ATCGAAATTCTGTCAACGCACGCGTCGGATGAAGTCTATCTGGGTCA


 


GCGTGACAACCCGCATTGGACCAGTGATTCCAAAGCGCTGCAGGCCT


 


TCCAAAAATTCGGCAACAAACTGAAAGAAATCGAAGAAAAACTGGTC


 


CGTCGCAACAATGATCCGAGCCTGCAGGGTAACCGTCTGGGTCCGGT


 


GCAACTGCCGTATACCCTGCTGTATCCGTCCAGTGAAGAAGGTCTGA


 


CGTTTCGTGGTATTCCGAACTCCATTTCCATCTGACTCGAG


 


SLP3 DNA optimized encoding sequence (with


restriction sites 5′NdeI and 3′EcoRI and 3′stop


codon for cloning into the pJex purple 424


vector from DNA2.0 Inc.


(SEQ ID NO 5)


CATATGCTGGGCGGCCTGCTGCACCGTGGTCATAAAATCAAGGGCA


 


CCGTGGTCCTGATGCGTAAGAACGTCCTGGATGTGAATAGCGTGAC


 


CTCGGTCGGCGGTATTATCGGCCAGGGTCTGGACCTGGTGGGTAGC


 


ACGCTGGATACCCTGACGGCCTTTCTGGGCCGCTCAGTGTCGCTGC


 


AACTGATCAGCGCAACCAAAGCAGATGCTAACGGCAAAGGCAAGCT


 


GGGCAAGGCGACGTTCCTGGAAGGCATTATCACCTCCCTGCCGACG


 


CTGGGTGCAGGCCAGTCAGCCTTTAAAATTAATTTCGAATGGGATG


 


ACGGCTCTGGTATTCCGGGCGCCTTCTACATCAAGAACTTCATGCA


 


GACCGAATTTTTCCTGGTCAGCCTGACGCTGGAAGATATCCCGAAT


 


CATGGCTCGATTCACTTTGTGTGCAACAGCTGGATCTACAATGCGA


 


AACTGTTCAAGTCCGATCGCATTTTCTTTGCCAATCAGACCTATCT


 


GCCGTCAGAAACGCCGGCACCGCTGGTTAAATACCGTGAAGAAGAA


 


CTGCACAACCTGCGTGGTGACGGTACCGGTGAACGTAAAGAATGGG


 


AACGCATCTACGATTACGACGTTTACAACGATCTGGGTGATCCGGA


 


CAAAGGCGAAAACCATGCGCGTCCGGTCCTGGGCGGTAATGACACC


 


TTTCCGTACCCGCGTCGCGGTCGTACCGGTCGTAAACCGACGCGTA


 


AGGATCCGAACAGCGAATCTCGCAGTAATGATGTGTATCTGCCGCG


 


TGACGAAGCCTTTGGTCACCTGAAAAGCTCTGATTTCCTGACGTAC


 


GGCCTGAAGTCCGTTTCACAGAACGTCCTGCCGCTGCTGCAAAGCG


 


CATTTGATCTGAATTTCACCCCGCGCGAATTTGATTCGTTCGACGA


 


AGTTCATGGTCTGTATAGCGGCGGTATTAAGCTGCCGACCGACATT


 


ATCTCTAAAATCAGTCCGCTGCCGGTGCTGAAGGAAATTTTTCGCA


 


CGGATGGCGAACAGGCTCTGAAGTTCCCGCCGCCGAAAGTCATCCA


 


AGTGTCGAAAAGCGCGTGGATGACCGATGAAGAATTTGCACGTGAA


 


ATGCTGGCTGGTGTTAACCCGAATCTGATTCGCTGTCTGAAGGATT


 


TCCCGCCGCGTTCCAAACTGGATTCACAGGTGTATGGTGACCACAC


 


CAGTCAAATCACGAAAGAACATCTGGAACCGAACCTGGAAGGCCTG


 


ACCGTTGATGAAGCTATTCAGAATAAACGTCTGTTTCTGCTGGATC


 


ATCACGACCCGATCATGCCGTATCTGCGTCGCATTAATGCGACCTC


 


GACGAAAGCGTACGCCACCCGCACGATCCTGTTCCTGAAGAACGAT


 


GGTACCCTGCGTCCGCTGGCCATTGAACTGAGCCTGCCGCATCCGC


 


AGGGTGACCAATCGGGTGCGTTTAGCCAGGTTTTCCTGCCGGCCGA


 


TGAAGGCGTCGAAAGTTCCATCTGGCTGCTGGCAAAAGCTTATGTG


 


GTTGTCAACGATTCTTGCTACCATCAGCTGGTGTCTCACTGGCTGA


 


ATACCCATGCAGTGGTTGAACCGTTTATTATCGCTACGAACCGCCA


 


CCTGTCTGTCGTGCATCCGATCTATAAACTGCTGCATCCGCACTAC


 


CGCGACACCATGAACATTAATGGTCTGGCGCGTCTGAGTCTGGTCA


 


ACGATGGCGGTGTGATTGAACAGACGTTTCTGTGGGGCCGTTATTC


 


TGTTGAAATGAGTGCCGTTGTCTACAAAGATTGGGTCTTCACCGAC


 


CAAGCACTGCCGGCAGACCTGATCAAGCGTGGTATGGCAATTGAAG


 


ATCCGTCCTGTCCGCACGGCATCCGTCTGGTGATTGAAGATTATCC


 


GTACACCGTTGACGGTCTGGAAATCTGGGATGCAATTAAAACGTGG


 


GTGCATGAATACGTTTTTCTGTACTACAAGTCTGATGACACCCTGC


 


GCGAAGACCCGGAACTGCAGGCGTGCTGGAAAGAACTGGTGGAAGT


 


TGGTCACGGCGATAAAAAGAACGAACCGTGGTGGCCGAAAATGCAA


 


ACCCGTGAAGAACTGGTTGAAGCGTGTGCCATTATCATTTGGACGG


 


CAAGCGCTCTGCATGCGGCCGTGAACTTTGGCCAGTATCCGTACGG


 


CGGTCTGATTCTGAATCGCCCGACCCTGTCTCGTCGCTTCATGCCG


 


GAAAAAGGCAGTGCTGAATATGAAGAACTGCGTAAAAATCCGCAGA


 


AGGCGTACCTGAAAACCATCACGCCGAAATTTCAAACCCTGATTGA


 


CCTGAGCGTGATCGAAATTCTGTCCCGCCATGCGTCAGATGAAGTT


 


TATCTGGGTGAACGTGACAACCCGAATTGGACCTCCGATACGCGTG


 


CACTGGAAGCTTTTAAGCGCTTCGGCAACAAACTGGCCCAGATCGA


 


AAACAAGCTGTCAGAACGTAACAACGATGAAAAGCTGCGTAATCGC


 


TGCGGCCCGGTGCAAATGCCGTATACCCTGCTGCTGCCGTCCTCAA


 


AAGAAGGTCTGACGTTCCGTGGTATCCCGAATAGCATTAGCATCTA


 


AGAATTC


 


Minilox optimized encoding sequence (with 5′NdeI


and 3′XhoI restriction sites and 3′stop


codon for cloning into pJexpress purple 424 vector


from DNA2.0 Inc.)


(SEQ ID NO 6)


CATATGTCTACGCCGATTGAATTTCACTCCTTCCAGGACGTTCAT


 


GATCTGTATGAAGGCGGTATCAAACTGCCGCGTGACGTCATTTCA


 


ACCATTATCCCGCTGCCGGTGATCAAAGAACTGTACCGCACGGAT


 


GGTCAGCACATTCTGAAATTTCCGCAACCGCATGTGGTTCAGGTT


 


TCACAATCGGCGTGGATGACCGATGAAGAATTCGCGCGTGAAATG


 


ATCGCCGGCGTTAACCCGTGCGTCATTCGCGGTCTGGAAGAATTT


 


CCGCCGAAAAGCAATCTGGACCCGGCAATCTATGGCGATCAGAGT


 


TCCAAAATTACCGCTGACTCTCTGGACCTGGATGGCTACACGATG


 


GATGAAGCCCTGGGTAGTCGTCGCCTGTTTATGCTGGACTATCAC


 


GATATCTTCATGCCGTACGTGCGTCAGATTAACCAACTGAATTCT


 


GCAAAAACCTATGCTACCCGTACGATCCTGTTTCTGCGCGAAGAC


 


GGCACGCTGAAACCGGTTGCAATTGAACTGAGCCTGCCGCATTCT


 


GCTGGTGATCTGAGTGCCGCGGTGTCCCAGGTTGTGCTGCCGGCA


 


AAAGAAGGCGTTGAAAGTACCATCTGGCTGCTGGCGAAAGCCTAT


 


GTTATTGTCAACGATTCATGTTACCATCAACTGATGTCGCACTGG


 


CTGAATACCCATGCAGCTATGGAACCGTTTGTTATCGCAACGCAT


 


CGCCACCTGTCTGTCCTGCACCCGATTTATAAACTGCTGACCCCG


 


CATTACCGTAACAATATGAACATCAATGCACTGGCTCGCCAGAGT


 


CTGATTAACGCGAATGGTATTATCGAAACCACGTTCCTGCCGTCA


 


AAATATTCGGTGGAAATGTCATCGGCCGTTTACAAAAACTGGGTC


 


TTTACCGACCAGGCACTGCCGGCTGATCTGATCAAACGTGGCGTCG


 


CGATTAAAGATCCGAGCACCCCGCATGGTGTGCGTCTGCTGATTGA


 


AGACTATCCGTACGCGGCCGATGGCCTGGAAATCTGGGCAGCTATT


 


AAAACCTGGGTGCAGGAATATGTTCCGCTGTATTACGCACGCGATG


 


ACGATGTGAAAAATGACTCCGAACTGCAACACTGGTGGAAAGAAGC


 


TGTTGAAAAAGGTCATGGCGACCTGAAAGATAAACCGTGGTGGCCG


 


AAACTGCAGACCCTGGAAGATCTGGTGGAAGTTTGTCTGATTATCA


 


TTTGGATTGCCAGCGCACTGCATGCCGCGGTGAACTTTGGTCAATA


 


TCCGTACGGCGGTCTGATTATGAATCGTCCGACCGCAAGCCGTCGC


 


CTGCTGCCGGAAAAAGGCACGCCGGAATACGAAGAAATGATCAACA


 


ACCATGAAAAAGCGTACCTGCGCACCATCACGAGCAAACTGCCGAC


 


CCTGATTAGCCTGTCTGTTATCGAAATTCTGTCAACGCACGCGTCG


 


GATGAAGTCTATCTGGGTCAGCGTGACAACCCGCATTGGACCAGTG


 


ATTCCAAAGCGCTGCAGGCCTTCCAAAAATTCGGCAACAAACTGAA


 


AGAAATCGAAGAAAAACTGGTCCGTCGCAACAATGATCCGAGCCTGC


 


AGGGTAACCGTCTGGGTCCGGTGCAACTGCCGTATACCCTGCTGTAT


 


CCGTCCAGTGAAGAAGGTCTGACGTTTCGTGGTATTCCGAACTCCAT


 


TTCCATCTGACTCGAG






Other embodiments and uses of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. All references cited herein, including all publications, U.S. and foreign patents and patent applications, are specifically and entirely incorporated by reference. The term comprising, where ever used, is intended to include the terms consisting and consisting essentially of. Furthermore, the terms comprising, including, and containing are not intended to be limiting. It is intended that the specification and examples be considered exemplary only with the true scope and spirit of the invention indicated by the following claims.

## Claims

1. A method of producing lipoxygenase enzyme comprising:
providing a nucleic acid expression construct within a host microorganism that is generally recognized as safe for the production of food enzymes, wherein the nucleic acid construct encodes a plant lipoxygenase enzyme;
providing one or more chaperone plasmids within the host microorganism; and
inducing expression of the plant a lipoxygenase enzyme encoded by the nucleic acid expression construct, wherein the amount of soluble plant lipoxygenase enzyme expressed is 30% or greater relative to the total soluble protein expressed by the host microorganism.

2. The method of claim 1, further comprising purifying the expressed plant lipoxygenase enzyme and collecting purified plant lipoxygenase enzyme.

3. The method of claim 2, wherein purification comprises contacting the expressed plant lipoxygenase enzyme to immobilized-metal affinity chromatography media.

4. The method of claim 1, wherein the nucleic acid expression construct encodes the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

5. The method of claim 1, wherein the nucleic acid expression construct encodes the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

6. The method of claim 1, wherein the host microorganism is a bacterial cell containing one or more protease deficiencies.

7. The method of claim 6, wherein the bacterial cell is a strain of Lactoccocus, K12 cells, Bacillus cells, E. coli cells or yeast cells.

8. The method of claim 1, wherein the one or more chaperone plasmids are simultaneously co-expressed with the plant lipoxygenase enzyme.

9. The method of claim 1, wherein inducing comprises maintaining the host microorganism at from 10-37° C. for a period of time.

10. The method of claim 9, wherein inducing comprises maintaining the host microorganism at from 25-35° C. for a period of time.

11. The method of claim 9, wherein inducing comprises maintaining the host microorganism at from 10-25° C. for a period of time.

12. The method of claim 9, wherein inducing comprises maintaining the host 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 the expressed plant lipoxygenase enzyme does not contain a histidine tag.

15. The method of claim 1, wherein the amount of soluble plant lipoxygenase enzyme expressed is 5 fold greater or more than the amount of soluble plant lipoxygenase enzyme expressed from the host microorganism that are not protease deficient or expressed from the host microorganism without an expression chaperone.

16. The method of claim 1, wherein the amount of soluble plant lipoxygenase enzyme expressed is 10 fold greater or more than the amount of soluble plant lipoxygenase enzyme expressed from the host microorganism that are not protease deficient or expressed from the host microorganism without an expression chaperone.

17. The method of claim 1, wherein the amount of soluble plant lipoxygenase enzyme expressed is 100 fold greater or more than the amount of soluble plant lipoxygenase enzyme expressed from the host microorganism that are not protease deficient or expressed from the host microorganism without an expression chaperone.

18. A method of producing lipoxygenase enzyme comprising:
providing a nucleic acid expression construct within a host microorganism, wherein the nucleic acid expression construct encodes a plant 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 a plant lipoxygenase enzyme;
inducing expression of the plant lipoxygenase enzyme encoded by the nucleic acid expression construct;
purifying the expressed plant lipoxygenase enzyme by contacting the expressed plant lipoxygenase enzyme to immobilized-metal affinity chromatography media, wherein the amount of soluble plant lipoxygenase enzyme expressed relative to the total soluble protein expressed in the host microorganism is 30% or greater; and
collecting purified plant lipoxygenase enzyme.

19. The method of claim 18, wherein the expressed plant lipoxygenase enzyme does not contain a histidine tag.

20. A method of producing a plant lipoxygenase enzyme comprising:
providing a nucleic acid expression construct within a host microorganism, wherein the construct encodes the plant 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 plant lipoxygenase enzyme;
inducing expression of the plant lipoxygenase enzyme, which produces 30% or greater amount of soluble plant lipoxygenase enzyme relative to total soluble protein in the host microorganism; and
collecting the plant lipoxygenase enzyme.

21. The method of claim 20, wherein collecting the plant lipoxygenase enzyme comprises contacting the plant lipoxygenase enzyme to immobilized-metal affinity chromatography media.

22. The method of claim 20, wherein the amount of plant lipoxygenase enzyme collected is 5 fold greater or more than the amount of plant lipoxygenase enzyme collected from the host microorganism that are not protease deficient or from the host microorganism without an expression chaperone.

23. The method of claim 20, wherein the amount of plant lipoxygenase enzyme collected is 10 fold greater or more than the amount of plant lipoxygenase enzyme collected from the host microorganism that are not protease deficient or from the host microorganism without an expression chaperone.

24. The method of claim 20, wherein the amount of plant lipoxygenase enzyme collected is 100 fold greater or more than the amount of plant lipoxygenase enzyme collected from the host microorganism that are not protease deficient or from the host microorganism without an expression chaperone.