KR101634858B1 - Recombinant microorganisms metabolizing 3,6-anhydro-L-galactose and use thereof - Google Patents

Abstract

The present invention relates to a recombinant microorganism metabolizing 3,6-anhydro-L-galactose and a use thereof. More particularly, the present invention relates to a recombinant microorganism expressing 3,6-AHG, Can be manufactured.

Description

Recombinant microorganisms metabolizing 3,6-anhydro-L-galactose and its use {Recombinant microorganisms metabolizing 3,6-anhydro-L-galactose and use thereof}

The present invention relates to a recombinant microorganism metabolizing non-fermental sugar, 3,6-anhydro-L-galactose, and its use.

Efforts are underway to develop energy resources that replace fossil fuels worldwide due to the energy security crisis resulting from the depletion of petroleum resources. As part of these efforts, research is underway to replace fossil raw materials with biomass to convert to a sustainable carbon economy, and replace existing chemical processes with eco-friendly bioprocesses to develop biofuels and biochemical materials. This is a new industrial group that can change the paradigm of front-end industry and has the advantage of reducing greenhouse gas and waste generation.

Biomass to produce biofuels has been transformed from first-generation carbohydrate biomass, such as corn and sugar cane, to second-generation biomass derived from woody biomass. Recently, third-generation biomass derived from seaweeds is in the spotlight.

Among seaweed biomass, red algae such as mugwort are known to have a higher carbohydrate content than green algae and brown algae. Agarose, a major polysaccharide constituting red algae, is a polymer of 3,6-anhydro-L-galactose and D-galactose . Among them, D-galactose is a fermentable monosaccharide that can be easily used by microorganisms, and many studies have been carried out to produce bioethanol by fermenting D-galactose produced by hydrolysis of red algae biomass using a chemical or enzymatic treatment method as a microorganism It has been progressed. Recently, a microorganism that degrades agarose Saccharophagus ( Saccharophagus) degradans) 2-40, pseudo Alteromonas Atlantica (Pseudoalteromonas atlantica ) An enzyme that converts 3,6-anhydro-L-galactose at T6c has been identified (PCT / KR2012 / 000607). In addition, as the genome sequence of Vibrio sp. EJY3, a microorganism capable of metabolizing 3,6-anhydro-L-galactose, known as the non-inactivated rare saccharide, was identified, The function of 6-anhydro-L-galactose metabolism-related genes and genes has also been revealed.

The present inventors have reported studies on the production method of 3,6-anhydro-L-galactose and its functionality (Yun EJ , et al . Process Biochem . (2011) 46 (1): 88-93. Yun EJ , et al . Appl . Microbiol . Biotechnol. (2013) 97 (7) 2961-70). For example, the reducing end of 3,6-anhydro-L-galactose has a property of being easily hydrated, thereby exhibiting a moisturizing function. In addition, whitening and antioxidant functionalities have been identified and have been shown to have a preventive effect on colon cancer (Yun EJ , et al . Appl . Microbiol. Biotechnol . (2013) 97 (7) 2961-70).

However, 3,6-anhydro-L-galactose is generally known to be a non-bioactive monosaccharide that is not available to microorganisms. Even though about 60% of red algae biomass is composed of carbohydrates, production yield of biofuel is low do.

Korean Patent Publication No. 2012-0085364 PCT / KR2012 / 000607

 Michel G et al. Appl. Microbiol. Biotechnol. (2006) 71 (1): 23-33  Roh H, et al. J. Bacteriol. (2012) 194 (10): 2773-2774

It is an object of the present invention to provide a recombinant vector of an enzyme group involved in the metabolic pathway of 3,6-anhydro-L-galactose, a recombinant microorganism transformed with the recombinant vector, and a method of producing ethanol from the recombinant microorganism .

In order to accomplish the above object, the present invention provides a gene encoding 3,6-anhydro-L-galactose dehydrogenase (3,6-anhydro-L-galactose dehydrogenase) A gene encoding 3,6-anhydrogalactonic acid cycloisomerase (3,6-anhydrogalactonic acid cycloisomerase); A gene encoding 2-keto-3-deoxy-galactonic acid kinase; And a gene encoding 2-keto-3-deoxy-phosphogalactonic acid aldolase. The present invention also provides a recombinant vector for ethanol production comprising the gene encoding 2-keto-3-deoxy-phosphogalactonic acid aldolase.

The present invention also relates to a gene encoding 3,6-anhydro-L-galactose dehydrogenase (3,6-anhydro-L-galactose dehydrogenase); A gene encoding 3,6-anhydrogalactonic acid cycloisomerase (3,6-anhydrogalactonic acid cycloisomerase); A gene encoding 2-keto-3-deoxy-galactonic acid kinase; And a recombinant microorganism for producing ethanol transformed with a gene encoding 2-keto-3-deoxy-phosphogalactonic acid aldolase (2-keto-3-deoxy-phosphogalactonic acid aldolase).

The present invention also provides a method for producing ethanol comprising fermenting the recombinant microorganism according to the present invention as a carbon source using at least one selected from the group consisting of galactose and 3,6-anhydro-L-galactose.

The present invention also relates to a method for producing pyruvic acid by reacting a culture broth or strain extract of a recombinant microorganism according to the present invention with at least one substrate selected from the group consisting of galactose and 3,6-anhydro-L-galactose; And fermenting the pyruvic acid with alcohol.

The present invention provides a method for producing ethanol from a recombinant microorganism expressing an enzyme group involved in the metabolic pathway of 3,6-anhydro-L-galactose.

Therefore, it can be provided as a core technology for increasing production yield when producing high value-added materials using red algae biomass.

Brief Description of the Drawings Fig. 1 is a graph showing the results of the enzymatic reaction products of the enzymes isolated and purified from recombinant microorganisms expressing 3,6-anhydro-L-galactose dehydrogenase of the present invention or 3,6-anhydrogalactonic acid cyclo- GC-TOF MS, wherein A is a peak of 3,6-anhydro-L-galactose used as a substrate, B is a reaction product of 3,6-anhydro-L-galactose dehydrogenase Anhydrogalactonic acid, and C represents the peak of 2-keto-3-deoxy-galactonic acid, which is the reaction product of 3,6-anhydrogalactonic acid cyclase nitrification enzyme.
2 is a graph showing the results of an enzyme reaction of 3,6-anhydro-L-galactose dehydrogenase isolated and purified from a recombinant microorganism expressing 3,6-anhydro-L-galactose dehydrogenase of the present invention A is the result of 2-dimensional Heteronuclear single quantum coherence spectroscopy (HSQC) NMR analysis, and B is the result of 2-dimensional Heteronuclear Multibond Bond Correlation (HMBC) NMR analysis.
FIG. 3 is a graph showing the activity of the 3,6-anhydrogalactonic acid cyclase nitrase isolated and purified from the recombinant microorganism expressing the 3,6-anhydrogalactonic acid cyclase nitrase of the present invention by GC-TOF As a result of MS analysis and two-dimensional NMR analysis, A was a mass spectral analysis of GC-TOF MS of 2-keto-3-deoxy-gluconic acid standard material, and B was 3,6- anhydrogalactonic acid cyclic nitration As a result of the one-dimensional hydrogen NMR analysis of the enzyme reaction product of the enzyme, the result of the 2-dimensional HMBC NMR analysis of the enzyme reaction product of 3,6-anhydrogalactonic acid cyclase nitrification enzyme C showed that D was 3,6-anhydrogalactone 2-dimensional HMBC NMR analysis of the enzyme reaction product of the acid cyclase nitrase.
Fig. 4 is a graph showing the results obtained by measuring the activity of the 3,6-anhydro-L-galactose dehydrogenase of the present invention using 3,6-anhydro-L-galactose as the carbon source and the 3,6-anhydrogalactonic acid cyclo- Or concurrently expressing recombinant microorganisms.
Fig. 5 is a graph showing the results of the measurement of the agarose hydrolyzate (c) containing 3,6-anhydro-L-galactose (a), galactose (b), or 3,6-anhydro-L- galactose and galactose as a carbon source Anhydro-L-galactose dehydrogenase and 3,6-anhydrogalactonic acid cyclase nitrase of the present invention, respectively, or at the same time, under aerobic conditions of the recombinant microorganism.
Fig. 6 shows a quantitative analysis curve of ethanol using GC-FID.
FIG. 7 is a graph showing the results of the growth experiments of the recombinant microorganism expressing the 3,6-anhydro-L-galactose dehydrogenase and the 3,6-anhydrogalactonic acid cyclase nitrase of the present invention, respectively a), the substrate consumption measurement result (b), and the quantitative analysis result of ethanol produced by fermentation (c).
FIG. 8 is a graph showing the activity of the 3,6-anhydro-L-galactose dehydrogenase of the present invention, 3,6-anhydrogalactonic acid cyclase nitricase, 2-keto- 2-keto-3-deoxy-phosphogalactonic acid aldolase at the same time, according to the carbon source of the recombinant microorganism.
9 is a graph showing the activity of the 3,6-anhydro-L-galactose dehydrogenase of the present invention, 3,6-anhydrogalactonic acid cyclo-isomerizing enzyme, 2-keto- 2-keto-3-deoxy-phosphogalactonic acid aldolase in the fermentation conditions of the recombinant microorganism.
Fig. 10 is a graph showing the activity of the 3,6-anhydro-L-galactose dehydrogenase of the present invention, 3,6-anhydrogalactonic acid cyclase nitricase, 2-keto-3-deoxy- 2-keto-3-deoxy-phosphogalactonic acid aldolase at the same time as the recombinant microorganism.

Hereinafter, the structure of the present invention will be described in detail.

The present invention relates to a gene encoding 3,6-anhydro-L-galactose dehydrogenase (3,6-anhydro-L-galactose dehydrogenase) A gene encoding 3,6-anhydrogalactonic acid cycloisomerase (3,6-anhydrogalactonic acid cycloisomerase); A gene encoding 2-keto-3-deoxy-galactonic acid kinase; And a gene encoding 2-keto-3-deoxy-phosphogalactonic acid aldolase. The present invention also relates to a recombinant vector for producing ethanol, which comprises a gene coding for 2-keto-3-deoxy-phosphogalactonic acid aldolase.

The recombinant vector for ethanol production of the present invention is characterized in that ethanol can be produced as a final product using galactose and / or 3,6-anhydro-L-galactose (hereinafter referred to as '3,6-AHG') as a substrate . More specifically, the recombinant vector comprises a gene encoding 3,6-anhydro-L-galactose dehydrogenase which metabolizes 3,6-AHG, a gene encoding 3,6-anhydro-L-galactose dehydrogenase Anhydrogalactonic acid cyclic nitrification enzyme which is isomerized and converted to 2-keto-3-deoxy-galactonic acid by ring-opening the ring structure of 3,6-anhydrogalactonic acid produced by 3-anhydrogalactonic acid Keto-3-deoxy-galactonic acid phosphorylase which phosphorylates the 2-keto-3-deoxy-galactonic acid with 2-keto-3-deoxy-phosphogalactonic acid, -Keto-3-deoxy-phosphogalactonic acid aldolase which degrades keto-3-deoxy-phosphogalactonic acid with pyruvic acid. The pyruvic acid is used as a starting material of alcohol fermentation and fermented to finally produce ethanol.

The 3,6-anhydro-L-galactose dehydrogenase is an enzyme that converts 3,6-AHG to 3,6-anhydrogalactonic acid. Vibrio sp. EJY3, Saccharophagus degradans) 2-40, or pseudo Alteromonas Atlantica (Pseudoalteromonas atlantica ) T6c, and may be represented by, for example, the nucleotide sequence of SEQ ID NO: 1 from Vibrio sp. EJY3 and the amino acid sequence of SEQ ID NO: 2.

The 3,6-anhydrogalactonic acid cyclase nitricase is an enzyme which is isomerized by ring-opening the 3,6-anhydrogalactonic acid ring to convert it to 2-keto-3-deoxy-galactonic acid , Vibrio sp. EJY3, Saccharophagus ( Saccharophagus) degradans) 2-40, or pseudo Alteromonas Atlantica (Pseudoalteromonas which can be derived from such atlantica) T6c, more specifically, Saccharomyces par Guus de Gras Tansu (Saccharophagus degradans) of 2-40-derived SEQ ID NO: 3 of the sequence (amino acid sequence: SEQ ID NO: 4), Pseudomonas Alteromonas Atlantica (Pseudoalteromonas nucleotide sequence of the 5 (amino acid sequence:: atlantica) T6c derived from the SEQ ID NO sequences of 7 (amino acid sequence:: SEQ ID NO: 6) , Vibrio genus (Vibrio sp) EJY3 Origin SEQ ID NO. SEQ ID NO: 8). ≪ / RTI >

The present inventors obtained genes for clustering with 3,6-anhydro-L-galactose dehydrogenase, one of the AHG metabolizing enzymes, by using genetic engineering techniques, and performed enzymatic reactions. As a result, Anhydrogalactonic acid cyclic nitrile synthase which produces linear 2-keto-3-deoxy-galactonic acid without changing stoichiometry from anhydrogalactonic acid (hereinafter referred to as " AHGA & For the first time.

Accordingly, the present invention provides a method for producing 2-keto-3-anhydrogalactonic acid which comprises 3,6-anhydrogalactonic acid cycloisomerase represented by any one of the amino acid sequences of SEQ ID NOS: 4, -3-deoxy-galactonic acid. ≪ / RTI >

The present invention also relates to a 3,6-anhydrogalactonic acid cycloisomerase represented by any one of the amino acid sequences of SEQ ID NOS: 4, 6 or 8, the 3,6- A method for producing 2-keto-3-deoxy-galactonic acid by reacting a microorganism producing an anhydrogalactonic acid cyclo-isomerizing enzyme or a culture product of the microorganism with 3,6-anhydrogalactonic acid .

The 2-keto-3-deoxy-galactonic acid kinase is an enzyme which phosphorylates 2-keto-3-deoxy-galactonic acid with 2-keto-3-deoxy-phosphogalactonic acid, Vibrio sp. EJY3, Saccharophagus ( Saccharophagus) degradans) 2-40, or pseudo Alteromonas Atlantica (Pseudoalteromonas atlantica ) T6c, and the like, and may be represented by the nucleotide sequence of SEQ ID NO: 9 and the amino acid sequence of SEQ ID NO: 10.

The 2-keto-3-deoxy-phosphogalactonic acid aldolase is an enzyme which decomposes 2-keto-3-deoxy-phosphogalactonic acid into pyruvic acid. Vibrio sp. EJY3, Saccharophagus ( Saccharophagus) degradans) 2-40, or pseudo Alteromonas Atlantica (Pseudoalteromonas atlantica ) T6c, and the like, and may be represented by the nucleotide sequence of SEQ ID NO: 11 and the amino acid sequence of SEQ ID NO: 12.

The above-mentioned genes include polynucleotides having a physicochemical activity of an enzyme and encoding a protein in which one or more amino acids are deleted, substituted, inserted and / or added. For example, polynucleotides that contain any one of the nucleotides described in SEQ ID NO: 1, 3, 5, 7, 9 or 11 and that hybridize under stringent conditions to a polynucleotide encoding a protein having the physicochemical properties of the enzyme do. "Polynucleotide hybridizing under stringent conditions" refers to a polynucleotide that hybridizes under ECL direct nucleic acid labeling and detection system (Amersham Pharmacia Biotech), for example, under conditions described in the manual (washing at 42 ° C with primary wash buffer containing 0.5 x SSC) Refers to a polynucleotide that hybridizes to one or more probe DNAs comprising a sequence of at least 20, preferably at least 30 contiguous residues (e. G., 40, 60, or 100 contiguous residues) optionally selected from the enzyme protein. Polynucleotides of the invention include isolated polynucleotides. &Quot; Isolated nucleotide " refers to a polynucleotide that exists in a different form than naturally occurring polynucleotides. For example, vectors and polynucleotides incorporated into the genome of another organism are included in the isolated polynucleotide. In addition, the isolated polynucleotide includes a cDNA, a PCR product, or a polynucleotide obtained as a restriction fragment. In addition, the polynucleotide used as part of the polynucleotide encoding the fusion protein is also included in the " isolated polynucleotide ".

Polynucleotides encoding the above-described enzymes of the present invention can be isolated, for example, by the following method: [0158] Each PCR primer based on the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, And PCR is performed using a chromosomal DNA or cDNA library derived from an enzyme-producing strain as a template to obtain the DNA of the present invention.

The polynucleotide of the present invention can be obtained by introducing a restriction enzyme fragment of a chromosomal DNA derived from an enzyme-producing strain into a fibrin or plasmid using a DNA fragment obtained as a probe, by colony hybridization or plaque hybridization, A library obtained by transforming Escherichia coli cells with gDNA vector, or (b) screening against a cDNA library.

Alternatively, the polynucleotide of the present invention can be used to analyze the nucleotide sequence of a DNA fragment obtained by PCR; Design of PCR primers based on sequence analyzed to elongate strands outside known DNA sequences; And digestion of the chromosomal DNA of the enzyme-producing strain with appropriate restriction enzymes and reverse PCR by self-cyclizing reaction using the DNA as a template ( Genetics , 120, 621-623 (1988)). The polynucleotides of the present invention can also be obtained by the RACE method (Rapid Amplification of cDNA End, 'PCR Jikken Manual (Manual for PCR experiments)', 25-33, HBJ Publishing Bureau).

In addition to the genomic DNA and cDNA cloned by the above method, the polynucleotide of the present invention includes synthesized DNAs.

The term "recombinant vector" as used herein refers to a gene construct containing an essential regulatory element operatively linked to the expression of a gene insert, which is capable of expressing a desired protein in a suitable host cell. Such vectors include, but are not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector or a viral vector. Suitable expression vectors include signal sequence or leader sequences for membrane targeting or secretion in addition to expression control elements such as promoter, operator, initiation codon, termination codon, polyadenylation signal and enhancer, and may be prepared in various ways depending on the purpose. The promoter of the vector may be constitutive or inducible. Further, the expression vector includes a selection marker for selecting a host cell containing the vector, and includes a replication origin in the case of a replicable expression vector.

The recombinant vector of the present invention can be preferably prepared by inserting the above-mentioned enzyme-encoding nucleic acid separately or together into a vector for expression of a common E. coli strain. The Escherichia coli strain expression vector may be any commonly used Escherichia coli vector for expression.

The present invention also relates to a gene encoding 3,6-anhydro-L-galactose dehydrogenase (3,6-anhydro-L-galactose dehydrogenase); A gene encoding 3,6-anhydrogalactonic acid cycloisomerase (3,6-anhydrogalactonic acid cycloisomerase); A gene encoding 2-keto-3-deoxy-galactonic acid kinase; And a recombinant microorganism for producing ethanol transformed with a gene encoding 2-keto-3-deoxy-phosphogalactonic acid aldolase.

Such transformation includes any method of introducing the nucleic acid into an organism, cell, tissue or organ, and can be carried out by selecting a suitable standard technique depending on the host cell as is known in the art. Such methods include electroporation, protoplast fusion, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, agitation with silicon carbide fibers, Agrobacterium mediated transformation, PEG, dextran sulfate, Pectamine, and the like.

In addition, since the expression amount of the protein and the expression are different depending on the host cell, the host cell most suitable for the purpose may be selected and used.

Examples of host cells include Escherichia coli), Bacillus subtilis (Bacillus subtilis), Streptomyces (Streptomyces), Pseudomonas (Pseudomonas), Proteus Mira Billy's (Proteus but are not limited to, prokaryotes such as mirabilis or Staphylococcus . In addition, fungi (e.g., Aspergillus ), yeast (e.g., Pichia pastoris), Mai access to the Yasaka Auxerre Vichy kids (Saccharomyces cerevisiae), investigating car break in Rome Seth (Schizosaccharomyces), Castello La Neuro Chrysler Corporation (Neurospora crassa ) may be used, but the present invention is not limited thereto. The transformant can be easily prepared by introducing a recombinant vector containing the genes into an arbitrary host cell.

More specifically, a recombinant vector comprising a gene encoding 3,6-anhydro-L-galactose dehydrogenase; A recombinant vector comprising a gene encoding a 3,6-anhydrogalactonic acid cyclo-isomerase; A recombinant vector comprising a gene encoding 2-keto-3-deoxy-galactonic acid phosphorylase; And a gene encoding 2-keto-3-deoxy-phosphogalactonic acid aldolase.

Or a recombinant vector comprising a gene encoding a 3,6-anhydro-L-galactose dehydrogenase and a gene encoding a 3,6-anhydrogalactonic acid cycloheteridase; A recombinant vector comprising a gene encoding 2-keto-3-deoxy-galactonic acid phosphorylase; And a gene encoding 2-keto-3-deoxy-phosphogalactonic acid aldolase.

Or a gene encoding 3,6-anhydro-L-galactose dehydrogenase, a gene encoding 3,6-anhydrogalactonic acid cyclo-isomerizing enzyme, a 2-keto-3-deoxy-galactonic A gene encoding an acid phosphorylase and a gene encoding a 2-keto-3-deoxy-phosphogalactonic acid aldolase.

The recombinant microorganism according to the present invention may be a fermentation strain.

The present invention also relates to a process for producing ethanol comprising fermenting the recombinant microorganism according to the present invention with a carbon source using at least one selected from the group consisting of galactose and 3,6-anhydro-L-galactose.

The recombinant microorganism according to the present invention can produce pyruvic acid, which is a starting material of alcohol fermentation, and can produce ethanol from fermentation conditions using galactose and / or 3,6-AHG as a carbon source from enzymes capable of metabolizing 3,6-AHG Can be generated.

Arabinose may be used as an inducer in the fermentation.

The present invention also relates to a method for producing pyruvic acid by reacting a culture broth or strain extract of a recombinant microorganism according to the present invention with at least one substrate selected from the group consisting of galactose and 3,6-anhydro-L-galactose; And fermenting the pyruvic acid with alcohol.

The recombinant microorganism according to the present invention can express an enzyme group capable of metabolizing 3,6-AHG, and the culture solution or strain extract of the recombinant microorganism contains such an enzyme group, and galactose and / or 3,6-AHG Is provided as a substrate to produce pyruvic acid. The pyruvic acid is a starting material for alcohol fermentation and can produce ethanol under fermentation conditions.

Hereinafter, the present invention will be described in detail with reference to examples. The following examples illustrate the invention and are not intended to limit the scope of the invention.

Example 1 Preparation of 3,6-anhydro-L-galactose dehydrogenase recombinant microorganism

A 3,6-anhydro-L-galactose dehydrogenase derived from Vibrio sp. EJY3 was cloned. For this purpose, the following primer sets were prepared based on the nucleotide sequence information on the 3,6-anhydro-L-galactose dehydrogenase-coding gene (ORF Names: VEJY3_09240).

Forward primer 1: 5'-gaaggagatataaggatgaaacgttaccaaatgtacgttg-3 '(SEQ ID NO: 13)

Reverse primer 2: 5'-atgatggtgatggtggtcgaaattcacatagaatgtcttc-3 '(SEQ ID NO: 14)

Each of the enzyme genes amplified by polymerase chain reaction was cloned into pBAD (Invitrogen, Product no. V440-01 vector and modified pET21a (hereinafter referred to as pJL) vector to which 6 histidine residues were added at the amino terminus, . E. coli was expressed by transforming the (E. coli) BL21 (DB3) the PCR conditions used are as follows: 1) initial denaturation conditions (initial denaturation):. 97 ℃ , 30 seconds, 2) annealing conditions : 35 cycles: 97 ° C, 10 seconds - 57 ° C, 1 minute - 72 ° C, 2 minutes, 3) final extension: 72 ° C, 5 minutes.

The 3,6-anhydro-L-galactose dehydrogenase cloned into the pBAD vector was induced to express for 18 hours at 16 ° C and 200 rpm using 0.2% (w / v) arabinose as an inducer. The enzyme over-expressed in E. coli BL21 was purified using a His-trap column.

200 μg of the purified 3,6-anhydro-L-galactose dehydrogenase was incubated in 20 mM Tris-HCl (pH 7.4) together with 10 mM 3,6-AHG and 1.5 mM NADP cofactor at 30 ° C. for 1 hour The reaction products were analyzed by GC-TOF MS (gas chromatography-time of flight mass spectrometry), and the chemical structure was determined by two-dimensional NMR (nuclear magnetic resonance) analysis.

For the GC-TOF MS analysis, the enzyme reaction product dried was subjected to derivatization reaction. Methoxyamine hydrochloride (40 mg / mL) was dissolved in pyridine (Sigma-Aldrich, St. Louis, Mo.), and 5 μl of the solution was reacted at 30 ° C. for 90 minutes. Then 45㎕ of N - methyl - N - trimethylsilyl trifluoroacetate Loa theta polyimide into the (N -methyl- N -trimethylsilyltrifluoroacetamide, Fluka, Buchs, Switzerland) was reacted with 37 ℃ 30 minutes. The derivatized samples were analyzed using an Agilent 7890 A GC (Agilent Technologies, Wilmington, DE) coupled to a Pegasus HT TOF MS (LECO, St. Joseph, MI). RTX-5Sil MS column (30 mx 0.25 mm, 0.25-mu m film thickness; Restek, Bellefonte, PA) was used and 1 μl sample was injected in a splitless mode. The oven temperature was initially given a retention time of 1 minute at 50 占 폚, and thereafter the temperature was raised to 330 占 폚 at a rate of 20 占 폚 / min and a retention time of 5 minutes was given. The ion source temperature was 250 占 폚 and the transfer line temperature was 280 占 폚. The scan range of the mass spectra was 85-500 m / z.

The reaction product was purified using a Sephadex G-10 column. That is, 2 mg of each of the purified reaction products was characterized by ¹³ C NMR, ¹ H- ¹³ C HSQC NMR and ¹ H- ¹³ C HMBC NMR analysis using a Bruker Avance II 900 MHz NMR spectrometer, internal standard (internal standard) is 3- (trimethylsilyl) -propionic -2,2,3,3, -d ₄ acid (3- (trimethylsilyl) -propionic-2,2,3,3 -d 4 acid ).

1A is a 3,6-AHG used as a substrate, and FIG. 1B shows that 3,6-anhydrogalactonic acid is produced as a reaction product by an enzymatic reaction with 3,6-anhydro-L-galactose dehydrogenase will be.

FIG. 2 shows the result of NMR analysis of the reaction product of 3,6-anhydro-L-galactose dehydrogenase. As a result, the correlation spots between the first carbon and the first hydrogen did not appear and the aldehyde groups of 3,6- -A-anhydro-L-galactose dehydrogenase (FIG. 2A), and the correlation between the 3-carbon and the 6-hydrogen is shown. As a result, the 3,6- Since the spot of carbon number 1 appeared near 180 PPM, the aldehyde group of 3,6-AHG was oxidized to a carboxyl group (FIG. 2B).

Therefore, the enzyme reaction product of 3,6-anhydro-L-galactose dehydrogenase is 3,6-anhydrogalactonic acid.

Example 2: Preparation of 3,6-anhydrogalactonic acid cyclase nitrifying enzyme recombinant microorganism

Saccharophagus ( Saccharophagus) degradans) 2-40, pseudo Alteromonas Atlantica (Pseudoalteromonas atlantica) was cloned T6c, in Vibrio (Vibrio) 3,6- anhydro-D-galactose dehydrogenase -L- EJY3 of origin. To this end, Saccharophagus degradans) 2-40, pseudo Alteromonas Atlantica (Pseudoalteromonas based on information for each CP000282, 1152nt and CP000388, 1137nt, and CP003241, 1089nt): atlantica) T6c , Vibrio genus (Vibrio) EJY3 ACI gene sequence (European Molecular Biology Laboratory obtained from the genome sequence (EMBL) nucleotide sequence database identification number The following primer sets were prepared.

1) Sde

Forward primer 1: 5'- gaaggagatataaggatgaaaattcataacatgaaaaattttatcaa-3 '(47mer: SEQ ID NO: 15)

Reverse primer 2: 5'-atgatggtgatggtgtcattcagcaaaatacactgtcttc -3 '(40 mer: SEQ ID NO: 16)

2) Patl

Forward primer 1: 5'-gaaggagatataaggatgatgagtgtcattaccaaactagaca-3 '(43mer: SEQ ID NO: 17)

Reverse primer 2: 5'-atgatggtgatggtgagaatgtttaactaaatagggaagaag-3 '(42mer: SEQ ID NO: 18)

3) Vejy3

Forward primer 1: 5'- gaaggagatataaggatgaaaacaacaatcaaagacatcaaaa-3 '(43mer: SEQ ID NO: 19)

Reverse primer 2: 5'- atgatggtgatggtgcacttcgtactgagcaattttgt-3 '(38mer: SEQ ID NO: 20)

Each gene was amplified by PCR using the corresponding genomic DNA as a template. The N-terminal part of the amplified sde ACI, patl ACI, and vejy3 ACI DNA all had a Ligation Independent Cloning (LIC) method to the modified pET 21a ( pJL ) vector containing the gene sequence encoding 6 histidine residues for purification And then transformed into Escherichia coli E. coli BL21 (DE3) for expression. The polymerase chain reaction conditions used were as follows. 1) Initial denaturation: 97 ° C for 30 seconds, 2) annealing conditions: 35 cycles: 97 ° C, 10 seconds - 57 ° C, 1 minute - 72 ° C, 2 minutes, 3) final extension ): 72 占 폚, 5 minutes.

Escherichia coli transformed into E. coli BL21 (DE3) was inoculated with recombinant plasmids containing sdeACI, patlACI, and vejy3ACI genes, respectively, in Luria-bertani medium containing 50 mg / L of ampicillin antibiotics and OD ₆₀₀ = And cultured with shaking until the ratio became 0.5 to 1.0. After that, expression was induced at 16 ° C for 24 hours using isopropyl-β-D-thiogalactopranoside (IPTG) at a concentration of 0.5 mM / L. The culture broth was centrifuged at 4000 rpm for 15 minutes and suspended in 20 mM Tris-HCl, pH 8.0 buffer to make a crude extract from the cells using an ultrasonic crusher. The crude extract was centrifuged at 15,000 rpm for 40 to 60 minutes at 4 ° C to separate crude enzyme solution and precipitate. The crude enzyme solution was filtered through a 0.45 μm filter paper, and proteins were purified stepwise by affinity chromatography, ion exchange chromatography, and gel filtration chromatography.

10% SDS-PAGE analysis results in an enzyme of approximately 42 kDa size (not shown).

25 μg of the purified 3,6-anhydrogalactonic acid cyclase nitrating enzyme (vejy3ACI) was reacted with the reaction product obtained in Example 1 at 30 ° C for 1 hour. The reaction product was analyzed by GC-TOF MS, the chemical structure was identified by two-dimensional NMR analysis, and purification was carried out using Sephadex G-10 column. The analysis and purification were carried out in the same manner as in Example 1 above. As a standard substance, 2-keto-3-deoxy-gluconic acid (Sigma, Product No. 12271) was used.

As shown in Fig. 1C, the reaction product was confirmed to be 2-keto-3-deoxy-galactonic acid.

3 shows the results of NMR analysis of the reaction product of 3,6-anhydrogalactonic acid cyclase nitricase (vejy3ACI). As a result of NMR analysis, 2-keto-3-deoxy-gluconic acid ) Mass spectrometry of the reference material and the mass spectrum of the enzyme reaction product of the 3,6-anhydrogalactonic acid cyclase nitricase of Figure 1C are identical, it is concluded that the enzyme of 3,6-anhydrogalactonic acid cyclase nitrase The reaction product was found to be a substance having a molecular weight and a two-dimensional structure such as 2-keto-3-deoxy-gluconic acid (Fig. 3A). In addition, a peak of the hemi-ketal structure of the carboxyl group of No. 1 carbon and No. 2 carbon was shown (FIG. 3B), and no correlation spot between No. 1 carbon and No. 6 hydrogen appeared The carboxyl group of the first carbon is still present (FIG. 3C). In addition, no correlation spot between the 3-carbon and the 6-carbon was observed, indicating that the 3,6-anhydro bond was opened, and the correlation spot between the 2-carbon and the 6-hydrogen appeared (FIG. 3D ), As well as the carboxylic acid ( ¹³ C 180 PPM) signal present in 3,6-anhydrogalactonic acid remained intact, so that the carboxylic acid of carbon number 1 remained intact and the 3,6-anhydrogalactonic acid cyclic The enzymatic reaction product of nitrification enzyme was found to be 2-keto-3-deoxy-galactonic acid.

Example 3: Preparation of recombinant microorganisms of 3,6-anhydro-L-galactose dehydrogenase and 3,6-anhydrogalactonic acid cyclo-isomerase

In order to test the growth of recombinant E. coli, 3,6-anhydro-L-galactose dehydrogenase derived from Vibrio sp. EJY3 and 3,6-anhydrogalactonic acid cyclic isomerase were each treated with two enzymes The coding gene was cloned into a pBAD vector at the same time to produce E. coli . K12 MG1655. The primer information used at this time is as follows:

1. Enzyme Each Cloned  Occation

1.1. 3,6-anhydro-L-galactose dehydrogenase coding gene (ORF Names: VEJY3_09240)

Forward primer 1: 5'-gaaggagatataaggatgaaacgttaccaaatgtacgttg-3 '(SEQ ID NO: 13)

Reverse primer 2: 5'-atgatggtgatggtggtcgaaattcacatagaatgtcttc-3 '(SEQ ID NO: 14)

1.2. 3,6-anhydrogalactonic acid cyclase nitricase coding gene (ORF Names: VEJY3_09370)

Forward primer 1: 5'-gcgctcgagatgaaaacaacaatcaaagacatcaaaac-3 '(Tm: 61.9, Xho I) (SEQ ID NO: 21)

Reverse primer 2: 5'-gcgtacgtacacttcgtactgagcaattttgtc-3 '(Tm: 61.8, Snab I) (SEQ ID NO: 22)

2. Enzyme at the same time Cloned  Case: 3,6- Anhydro -L-galactose Dihydrogenase  Coding gene ( ORF Names : VEJY3 _09240) and 3,6- Anhydrogalactonic acid Cyclo Isomerizing enzyme coding gene ( ORF Names : VEJY3 _09370)

2.1. 3,6-anhydro-L-galactose dehydrogenase coding gene (VEJY3_09240)

Forward primer 1: 5'-gcgctcgagatgaaacgttaccaaatgtacgttg-3 '( Xho I) (SEQ ID NO: 23)

Reverse primer 2: 5'-gcgtctagattagtcgaaattcacatagaatgtct-3 '( Xba I) (SEQ ID NO: 24)

2.2. 3,6-anhydrogalactonic acid cyclo-nitriding enzyme coding gene (VEJY3_09370)

Forward primer 1: 5'-gcgtctagaatgaaaacaacaatcaaagacatcaaaac-3 '( Xba I) (SEQ ID NO: 21)

Reverse primer 2: 5'-gcgtacgtacacttcgtactgagcaattttgtc-3 '( Snab I) (SEQ ID NO: 22)

The 3,6-anhydro-L-galactose dehydrogenase coding gene (ORF Names: VEJY3_09240) and the 3,6-anhydrogalactonic acid cyclase nitrase coding gene (ORF Names: VEJY3_09370) were simultaneously cloned into pBAD If so Xba I restriction enzyme spot and Xba I restriction enzyme spot ligation reaction upon was to be connected to each other are connected VEJY3_09240 and VEJY3_09370 Xho I and Snab I distal position restriction place of VEJY3_09370 primer 1 of VEJY3_09240 primer 2 is associated with the pBAD vector Respectively.

pBAD vector to obtain E. coli . Each of the enzymes transformed into K12 MG1655 was expressed using 0.01% (w / v) of Arabidopsis. E. coli containing each enzyme coding gene K12 MG1655 strain was cultured in modified M9 medium. The production method of M9 medium is as follows. To make 5-fold concentrated M9 salt solution, 2.5 g of NaCl, 5 g of NH ₄ Cl and 250 mM of Tris-HCl buffer (pH 7.4) were dissolved in 1 L of water and sterilized. 2mL of a 5-fold concentrated M9 salt solution of 200mL 1M MgSO _4, 0.1mL of _{1M CaCl 2, 20% (w} / v) 3,6-AHG with 20mL 5% (w / v) YNB (yeast in 20mL of nitrogen base) was added and sterilized water was added so that the total volume became 1L. Recombinant E. coli The culture conditions of K12 MG1655 were 30 ° C and 200 rpm.

As a result of culturing the recombinant microorganism using 1% (w / v) 3,6-AHG as a carbon source and 0.01% (w / v) arabinose as an inducer, In the absence of the gene-free empty vector, recombinant E. coli , K12 MG1655 did not grow at all and recombinant E. coli expressing the respective enzyme 3,6-anhydro-L-galactose dehydrogenase and 3,6-anhydrogalactonic acid cyclase nitrite was slightly grown, Recombinant E. coli expressing 6-anhydro-L-galactose dehydrogenase and 3,6-anhydrogalactonic acid cyclo-isomerase simultaneously exhibited the highest growth.

The recombinant microorganism was cultured under aerobic conditions in M9 medium at 30 DEG C for 96 hours, and the absorbance at 600 nm was measured. At this time, 20 mL of 20% (w / v) 3,6-AHG, 3,6-AHG and agarose hydrolyzate mainly containing galactose were used as a carbon source. At this time, agarose hydrolyzate mainly containing 3,6-AHG, 3,6-AHG and galactose was used as a carbon source.

As a result, it was confirmed that when the recombinant Escherichia coli was cultured in the AHG carbon source, only the 3,6-anhydro-L-galactose dehydrogenase gene and the 3,6-anhydrogalactonic acid cycloheteritinase gene were grown under the recombinant conditions (Fig. 5A). In addition, in the galactose condition, similar growth curves were obtained under the conditions of an empty vector and two enzyme genes (Fig. 5B). In the condition of the agarose hydrolyzate carbon source mainly containing AHG and galactose, (Fig. 5 (c)), and thereafter, two genes were added.

In addition, the recombinant microorganism was subjected to ethanol fermentation experiment under microaerobic conditions. The ethanol produced by the fermentation of the recombinant microorganism was subjected to GC-FID analysis according to ethanol standard concentration to give a calibration curve. After centrifuging the culture (16,000 rpm, 4 ° C, 5 minutes), the supernatant was analyzed and the analysis conditions were as follows. Inlet temperature was 250 ℃, split ratio 20: 1, pressure 11.567 psi, total flow 24 mL / min, septum purge flow 3 mL / min, and 1 ㎕ of sample was injected. The oven flow was 1 mL / min. The oven temperature condition was first maintained at 40 DEG C for 3.5 minutes, then heated to 150 DEG C at a rate of 50 DEG C / min, and then allowed to stand for 1 minute. Thereafter, the temperature was raised to 180 ° C at a rate of 20 ° C / min, followed by a retention time of 2 minutes, and a total analysis time was 10.2 minutes. The temperature of the FID was 300 占 폚, hydrogen gas flow 40 mL / min, air flow 350 mL / min, and helium gas flow 15 mL / min. As a result of the calibration curve of FIG. 6, the equation of y = 245.18x + 12.24 (y = peak area, x = ethanol concentration, g / L) was obtained.

The cell density in the micro-expiratory condition was similar for both the empty vector and the two enzyme genes (Fig. 7a). As a result of measuring the substrate consumption, the consumption of galactose was similar, but the consumption of galactose From the time afterwards, it was found that AHG was consumed in the presence of two enzyme genes (Fig. 7B). Ethanol production was higher than that of the empty vector condition with two enzyme genes. At 42 hr, the ethanol concentration was 24% higher than the empty vector condition with 2 enzyme genes.

Example 4 Preparation of Recombinant Microorganisms of 3,6-AHG Metabolism Related Enzymes

An enzymes involved in 3,6-AHG metabolism, namely, 3,6-anhydro-L-galactose dehydrogenase, 3,6-anhydrogalactonic acid cycloheteridase, 2-keto- Galactonic acid phosphorylase and 2-keto-3-deoxy-phosphogalactonic acid aldolase were introduced into E. coli KO11 FL strain for fermentation. The primer information used at this time is as follows:

1) 3,6-anhydro-L-galactose dehydrogenase coding gene (VEJY3_09240)

Forward primer 1: 5'-gcgctcgagatgaaacgttaccaaatgtacgttg-3 '( Xho I) (SEQ ID NO: 23)

Reverse primer 2: 5'-gcgtctagattagtcgaaattcacatagaatgtct-3 '( Xba I) (SEQ ID NO: 24)

2) 2-keto-3-deoxy-galactonic acid phosphorylase coding gene + 2-keto-3-deoxy-phosphogalactonic acid aldolase coding gene + 3,6-anhydrogalactonic acid cyclic Nitric oxide coding genes (VEJY3_09380 + VEJY3_09375 + VEJY3_09370, respectively)

Forward primer 1: 5'-gcgtctagaatgagtttggaaataaaacaagatacg-3 '( Xba I) (SEQ ID NO: 21)

Reverse primer 2: 5'-gcgtacgta cacttcgtactgagcaattttgtc-3 '( Snab I) (SEQ ID NO: 22)

(ORF Names: VEJY3_09240) and the 2-keto-3-deoxy-galactonic acid phosphorylase coding gene + 2-keto-3-deoxy- When cloning of the phagalactonic acid aldolase coding gene + 3,6-anhydrogalactonic acid cyclase nitrase coding gene (VEJY3_09380 + VEJY3_09375 + VEJY3_09370, respectively) into pBAD simultaneously, the Xba I restriction site of VEJY3_09240 primer 2 VEJY3_09380 + VEJY3_09375 + VEJY3_09370 Xba I restriction of primer 1 enzyme spot Lai were to be connected to each other at the time of ligation reaction associated VEJY3_09240 and VEJY3_09380 + VEJY3_09375 + VEJY3_09370 end position of the Xho I and Snab I restriction enzyme spot was to be connected with the pBAD vector. Enzymes expression and culture of recombinant E. coli KO11 FL strains were performed in the same manner as in E. coli K12 MG1655 of Examples 1 and 2 above. At this time, 1% galactose or 1% galactose + 1% 3,6-AHG was used as the carbon source of the culture medium.

The recombinant microorganism was cultured to conduct a growth experiment. The medium was prepared by using the modified M9 medium described above. The medium was prepared by adding 1% (w / v) of 3,6-AHG without carbon source to the carbon source (control group) (w / v) arabinose alone, and 0.01% (w / v) arabinose in 1% (w / v) 3,6-AHG.

As shown in Fig. 8, an increase in cell density was observed only in the case of adding 1% (w / v) of 3,6-AHG and 0.01% (w / v) of arabinose.

The recombinant microorganism was fermented under the conditions of galactose and 3,6-AHG mixed sugar, which are agarose degradation products. At this time, fermentation was carried out under the condition of 1% (w / v) galactose and mixed sugar (1% (w / v) galactose + 1% (w / v) 3,6-AHG) % (w / v) of arabinose was used as the inducer.

As shown in FIG. 9, the density of cells was higher than that of the case where only 1% (w / v) of galactose was added under mixed sugar conditions.

The recombinant microorganism was subjected to an alcohol fermentation experiment in the same manner as in Example 3 under the condition of micro-expiration.

As shown in FIG. 10, 4.11 g / L of ethanol was produced during fermentation of mixed sugar and 1.93 g / L of ethanol was produced during fermentation of galactose.

<110> KOREAN UNIVERSITY RESEARCH AND BUSINESS FOUNDATION <120> Recombinant microorganisms metabolizing 3,6-anhydro-L-galactose          and use thereof <130> P14U13C0549 <150> 2013-0051134 <151> 2013-05-07 <160> 24 <170> Kopatentin 2.0 <210> 1 <211> 1443 <212> DNA <213> Vibrio sp. EJY3 <400> 1 atgaaacgtt accaaatgta cgttgacggc cagtggattg acgctgagaa cggcaaagtt 60 gatcaggtta ttaacccgtc aaccgaagaa gtgcttgctg agattcagga tggtgaccaa 120 gatgatgctg agcgcgtttt aagtgtggct aaacgtgcac aatctgactg gaaacgagtg 180 cccgcgcgtc aacgtgctga actgttgaga aagtttgctc aagaaatccg taataaccgt 240 gagcatcttg cagagttact cgtgagcgaa caaggcaaat tataccgagt tgcgttgggg 300 gaagtcgatg tagctgcatc ttttatcgaa tacgcctgtg actgggctcg tcagatggat 360 ggcgatattg ttcaatctga taatgtgaac gaacatatct ggattcaaaa aattcctcgt 420 ggtgtcgtgg tcgcgatcac tgcatggaat ttcccatttg cattagcagg tcgcaagata 480 ggaccagcac tggttgcggg taacactatt gttgttaaac caacctctga aactccgcta 540 gcaacgctag agttaggcta tattgctgaa aaagtaggta ttcctgcagg tgtactcaat 600 atagttaccg gtggtggagc aagcttaggt ggcgctttaa ctagtcaccg ttatacaaat 660 atggtcacta tgacaggttc aacacccgtt ggtcagcaga taatcaaagc atctgcgaat 720 aacatggctc acgttcaact agagctcggt ggtaaagcac cgttcatcgt aatggaagat 780 gctgatctgg agcaagctgc tgccgctgca ctacattcac gcttcgacaa ctgtggtcag 840 gtatgtacat gtaacgaacg tatgtatgtg cactctagtg tctacgatga attcatggcg 900 atctttatgg agaaagtcca aaatatcaaa gtgggtaatc ctatggatcc agaatctgat 960 atgggtccta aagtaaacaa acgagagctt gatcatatgg aagcattagt cgcgcaggca 1020 ttgaaagaag gcgcgcaact tttgcatggt ggcaagcgcc ttacggaggg tgagtttgga 1080 aagggcttct ggtttgaacc cacaatctta ggtaatgttc aacaatcaat gacgattgtt 1140 catgaagagg catttggtcc aattcttcct gttataaaat tcgacacttt tgaagaagtc 1200 attgattacg caaatgatag tgagtatggc ttggcaacta tgatttgtac gcgaaatatg 1260 aagtatgtac atcgcttaac tcacgagctt gaatgtggtg agatttatgt gaaccgtggt 1320 catggagaac agcatcaagg tttccataat ggatataaac tgagcggaac tggtggtgaa 1380 gatggaaaat atggcttcga acaatattta gagaagaaga cattctatgt gaatttcgac 1440 taa 1443 <210> 2 <211> 480 <212> PRT <213> Vibrio sp. EJY3 <400> 2 Met Lys Arg Tyr Gln Met Tyr Val Asp Gly Gln Trp Ile Asp Ala Glu   1 5 10 15 Asn Gly Lys Val Asp Gln Val Ile Asn Pro Ser Thr Glu Glu Val Leu              20 25 30 Ala Glu Ile Gln Asp Gly Asp Gln Asp Asp Ala Glu Arg Val Leu Ser          35 40 45 Val Ala Lys Arg Ala Gln Ser Asp Trp Lys Arg Val Val Ala Arg Gln      50 55 60 Arg Ala Glu Leu Leu Arg Lys Phe Ala Gln Glu Ile Arg Asn Asn Arg  65 70 75 80 Glu His Leu Ala Glu Leu Leu Val Ser Glu Gln Gly Lys Leu Tyr Arg                  85 90 95 Val Ala Leu Gly Glu Val Asp Val Ala Ala Ser Phe Ile Glu Tyr Ala             100 105 110 Cys Asp Trp Ala Arg Gln Met Asp Gly Asp Ile Val Gln Ser Asp Asn         115 120 125 Val Asn Glu His Ile Trp Ile Gln Lys Ile Pro Arg Gly Val Val Val     130 135 140 Ala Ile Thr Ala Trp Asn Phe Pro Phe Ala Leu Ala Gly Arg Lys Ile 145 150 155 160 Gly Pro Ala Leu Val Ala Gly Asn Thr Ile Val Val Lys Pro Thr Ser                 165 170 175 Glu Thr Pro Leu Ala Thr Leu Glu Leu Gly Tyr Ile Ala Glu Lys Val             180 185 190 Gly Ile Pro Ala Gly Val Leu Asn Ile Val Thr Gly Gly Gly Ala Ser         195 200 205 Leu Gly Gly Ala Leu Thr Ser His Arg Tyr Thr Asn Met Val Thr Met     210 215 220 Thr Gly Ser Thr Pro Val Gly Gln Gln Ile Ile Lys Ala Ser Ala Asn 225 230 235 240 Asn Met Ala His Val Gln Leu Glu Leu Gly Gly Lys Ala Pro Phe Ile                 245 250 255 Val Met Glu Asp Ala Asp Leu Glu Gln Ala Ala Ala Ala Leu His             260 265 270 Ser Arg Phe Asp Asn Cys Gly Gln Val Cys Thr Cys Asn Glu Arg Met         275 280 285 Tyr Val His Ser Ser Val Tyr Asp Glu Phe Met Ala Ile Phe Met Glu     290 295 300 Lys Val Gln Asn Ile Lys Val Gly Asn Pro Met Asp Pro Glu Ser Asp 305 310 315 320 Met Gly Pro Lys Val Asn Lys Arg Glu Leu Asp His Met Glu Ala Leu                 325 330 335 Val Ala Gln Ala Leu Lys Glu Gly Ala Gln Leu Leu His Gly Gly Lys             340 345 350 Arg Leu Thr Glu Gly Glu Phe Gly Lys Gly Phe Trp Phe Glu Pro Thr         355 360 365 Ile Leu Gly Asn Val Glu Gln Ser Met Thr Ile Val His Glu Glu Ala     370 375 380 Phe Gly Pro Ile Leu Pro Val Ile Lys Phe Asp Thr Phe Glu Glu Val 385 390 395 400 Ile Asp Tyr Ala Asn Asp Ser Glu Tyr Gly Leu Ala Thr Met Ile Cys                 405 410 415 Thr Arg Asn Met Lys Tyr Val His Arg Leu Thr His Glu Leu Glu Cys             420 425 430 Gly Ile Tyr Val Asn Arg Gly His Gly Glu Gln His Gln Gly Phe         435 440 445 His Asn Gly Tyr Lys Leu Ser Gly Thr Gly Gly Asp Gly Lys Tyr     450 455 460 Gly Phe Glu Gln Tyr Leu Glu Lys Lys Thr Phe Tyr Val Asn Phe Asp 465 470 475 480 <210> 3 <211> 1152 <212> DNA <213> Saccharophagus degradans 2-40 <400> 3 atgagtaaat gcctgatgga aagtatagtg gaattagagt tgcctcaagg taacccaatt 60 aaaataaaat ccgtagcagt cgagcattac aaggttccgc tagctgaagt gttgtctgat 120 gcaaagcacg gggatcatac ctattttgag ctaatagtta gccgtatcac ttgccaaaat 180 ggtgtagaag gtgtgggtta tacctatacc ggtgggtccg gcggttcggc aatctattcg 240 ctattagtcg atgaaattaa gcctatgctt gttgggcggg acgccaccca aattctagcc 300 atatgggaag aaatttattg gcgcttacat tatgttgggc gcggtggttt agttagcttt 360 gctcagtcag cggttgatat tgcattgtgg gatattcgct gcaagttgtt ggggcaaccc 420 ctgtggaaag tggcgggcgg tttaagcaat aaaacacgct gctatgccgg cggtatagat 480 ttaaattttt cgcaagaaaa actattaagc aatatacaag gttatttaga cgcgggcttt 540 aatgctgtaa aaattaaagt tggcaaagat aatattaaag aagatattgc gcgtgtacgc 600 gcagtgcgag agttaattgg caaagatacc acatttatgg tggatgccaa ctactccatg 660 accaaagaaa aagccattcg ttttgctaac gccatagaag accaaaatat tacttggttt 720 gaagagccaa cattgccaga cgattaccaa ggctatgccg atatcgctca agcaatatca 780 atacccctag ctatgggtga aaacctacac actattcacg aatttaccta tgccgtgcaa 840 caagccaagc ttggtttttt gcagcccgat gcttctaata ttggtggtat tactggttgg 900 ttgaacgttg caagtttagc aaacgcacac aacttaccgg tgtgcagtca cggcatgcaa 960 gagttgcacg tttcacttat gtcgtctcag cccaatgcgg gttatttaga agttcactcc 1020 tttcctatcg accaatacac aacacaaccg ctagcaatgg aaaacggtta cgcactagca 1080 ccagatatag aaggcacggg tgttgtgttt gtcgatgaat tattacgtgg ccatttggct 1140 aaaaaatcct aa 1152 <210> 4 <211> 383 <212> PRT <213> Saccharophagus degradans 2-40 <400> 4 Met Ser Lys Cys Leu Met Glu Ser Ile Val Glu Leu Glu Leu Pro Gln   1 5 10 15 Gly Asn Pro Ile Lys Ile Lys Ser Val Ala Val Glu His Tyr Lys Val              20 25 30 Pro Leu Ala Glu Val Leu Ser Asp Ala Lys His Gly Asp His Thr Tyr          35 40 45 Phe Glu Leu Ile Val Ser Arg Ile Thr Cys Gln Asn Gly Val Glu Gly      50 55 60 Val Gly Tyr Thr Tyr Thr Gly Gly Ser Gly Gly Ser Ala Ile Tyr Ser  65 70 75 80 Leu Leu Val Asp Glu Ile Lys Pro Met Leu Val Gly Arg Asp Ala Thr                  85 90 95 Gln Ile Leu Ala Ile Trp Glu Glu Ile Tyr Trp Arg Leu His Tyr Val             100 105 110 Gly Arg Gly Gly Leu Val Ser Phe Ala Gln Ser Ala Val Asp Ile Ala         115 120 125 Leu Trp Asp Ile Arg Cys Lys Leu Leu Gly Gln Pro Leu Trp Lys Val     130 135 140 Ala Gly Gly Leu Ser Asn Lys Thr Arg Cys Tyr Ala Gly Gly Ile Asp 145 150 155 160 Leu Asn Phe Ser Gln Glu Lys Leu Leu Ser Asn Ile Gln Gly Tyr Leu                 165 170 175 Asp Ala Gly Phe Asn Ala Val Lys Ile Lys Val Gly Lys Asp Asn Ile             180 185 190 Lys Glu Asp Ile Ala Arg Val Ala Val Arg Glu Leu Ile Gly Lys         195 200 205 Asp Thr Thr Phe Met Val Asp Ala Asn Tyr Ser Met Thr Lys Glu Lys     210 215 220 Ala Ile Arg Phe Ala Asn Ala Ile Glu Asp Gln Asn Ile Thr Trp Phe 225 230 235 240 Glu Glu Pro Thr Leu Pro Asp Asp Tyr Gln Gly Tyr Ala Asp Ile Ala                 245 250 255 Gln Ala Ile Ser Ile Pro Leu Ala Met Gly Glu Asn Leu His Thr Ile             260 265 270 His Glu Phe Thr Tyr Ala Val Gln Gln Ala Lys Leu Gly Phe Leu Gln         275 280 285 Pro Asp Ala Ser Asn Ile Gly Gly Ile Thr Gly Trp Leu Asn Val Ala     290 295 300 Ser Leu Ala Asn Ala His Asn Leu Pro Val Cys Ser His Gly Met Gln 305 310 315 320 Glu Leu His Val Ser Leu Met Ser Ser Gln Pro Asn Ala Gly Tyr Leu                 325 330 335 Glu Val His Ser Phe Pro Ile Asp Gln Tyr Thr Thr Gln Pro Leu Ala             340 345 350 Met Glu Asn Gly Tyr Ala Leu Ala Pro Asp Ile Glu Gly Thr Gly Val         355 360 365 Val Phe Val Asp Glu Leu Leu Arg Gly His Leu Ala Lys Lys Ser     370 375 380 <210> 5 <211> 1137 <212> DNA <213> Pseudoalteromonas atlantica T6c <400> 5 atgagtgtca ttaccaaact agacacacct gccatgaaca gttcacaaat tcagtcagtc 60 aatgttgagt tattcaacgt gccccttgac gaagtgttga acgatgctaa gcacggcgat 120 catacccact ttgagctgat cctatgcacc attacttgta ccgatggtac tcaaggcgtg 180 ggttatacct acacaggtgg taaaggtgga cgcgctatat actcactgct taatgacgaa 240 ctaaagcctt tcttgatggg taaagatgct agctgcatca atcatttatg ggaagaaatg 300 caatatcact tgcactatgt tggtcgtggc ggtttagtca gtttcgccat atccgcagtc 360 gatatcgccc tgtgggacat tcattgcaag gttcttaatc aacccttgtg gaaagtagct 420 gggggctgca gtgaccgcgt aaactgttat gcaggaggca ttgaccttaa tttttccact 480 gaaaaattgc tcggcaatat ccaaggctac ttagactgcg gctttgaagc ggtcaaaata 540 aaagtgggca aagaagatta tcgtgaagac gtggcccgcg tggcggctgt gcgcaacctg 600 attggtcctg atgcgatatt tatggtggat gcaaattatt cacttacagt caataaagcc 660 attaagtttg ctcaggcgat agagcagtat gatatcacct ggtttgaaga accgaccatt 720 cctgatgatt ttgccggttt tgctcacatt gccagcaaaa tcaatattcc gttggccatg 780 ggcgaaaacc tgcacactat ttacgaattc aaccaagcga taagccaagc caaacttggg 840 ttcttacaac ctgatgcatc gaatattggc ggtatcactg gttggctaac ggttgcccag 900 atgggctacg cgaacaactt acctatttgc agtcatggca tgcacgaatt acatgtatct 960 cttatggcat ctcagccaaa tgcgggttac ttggaagtac actcgtttcc cattgaccga 1020 tataccactc accctctgaa acttgaaaat ggcaaagccg ttgcgcccag tacacccggt 1080 gtaggcgtcg agttcaagac agagttactt cttccctatt tagttaaaca ttcttag 1137 <210> 6 <211> 378 <212> PRT <213> Pseudoalteromonas atlantica T6c <400> 6 Met Ser Val Ile Thr Lys Leu Asp Thr Pro Ala Met Asn Ser Ser Gln   1 5 10 15 Ile Gln Ser Val Asn Val Glu Leu Phe Asn Val Pro Leu Asp Glu Val              20 25 30 Leu Asn Asp Ala Lys His Gly Asp His Thr His Phe Glu Leu Ile Leu          35 40 45 Cys Thr Ile Thr Cys Thr Asp Gly Thr Gln Gly Val Gly Tyr Thr Tyr      50 55 60 Thr Gly Gly Lys Gly Gly Arg Ala Ile Tyr Ser Leu Leu Asn Asp Glu  65 70 75 80 Leu Lys Pro Phe Leu Met Gly Lys Asp Ala Ser Cys Ile Asn His Leu                  85 90 95 Trp Glu Glu Met Gln Tyr His Leu His Tyr Val Gly Arg Gly Gly Leu             100 105 110 Val Ser Phe Ala Ile Ser Ala Val Asp Ile Ala Leu Trp Asp Ile His         115 120 125 Cys Lys Val Leu Asn Gln Pro Leu Trp Lys Val Ala Gly Gly Cys Ser     130 135 140 Asp Arg Val Asn Cys Tyr Ala Gly Gly Ile Asp Leu Asn Phe Ser Thr 145 150 155 160 Glu Lys Leu Leu Gly Asn Ile Gln Gly Tyr Leu Asp Cys Gly Phe Glu                 165 170 175 Ala Val Lys Ile Lys Val Gly Lys Glu Asp Tyr Arg Glu Asp Val Ala             180 185 190 Arg Val Ala Ala Val Arg Asn Leu Ile Gly Pro Asp Ala Ile Phe Met         195 200 205 Val Asp Ala Asn Tyr Ser Leu Thr Val Asn Lys Ala Ile Lys Phe Ala     210 215 220 Gln Ala Ile Glu Gln Tyr Asp Ile Thr Trp Phe Glu Glu Pro Thr Ile 225 230 235 240 Pro Asp Phe Ala Gly Phe Ala His Ile Ala Ser Lys Ile Asn Ile                 245 250 255 Pro Leu Ala Met Gly Glu Asn Leu His Thr Ile Tyr Glu Phe Asn Gln             260 265 270 Ala Ile Ser Gln Ala Lys Leu Gly Phe Leu Gln Pro Asp Ala Ser Asn         275 280 285 Ile Gly Gly Ile Thr Gly Trp Leu Thr Val Ala Gln Met Gly Tyr Ala     290 295 300 Asn Asn Leu Pro Ile Cys Ser His Gly Met His Glu Leu His Val Ser 305 310 315 320 Leu Met Ala Ser Gln Pro Asn Ala Gly Tyr Leu Glu Val His Ser Phe                 325 330 335 Pro Ile Asp Arg Tyr Thr Thr His Pro Leu Lys Leu Glu Asn Gly Lys             340 345 350 Ala Val Ala Pro Ser Thr Pro Gly Val Gly Val Glu Phe Lys Thr Glu         355 360 365 Leu Leu Leu Pro Tyr Leu Val Lys His Ser     370 375 <210> 7 <211> 1089 <212> DNA <213> Vibrio sp. EJY3 <400> 7 atgaaaacaa caatcaaaga catcaaaacg agactgttta agattccgtt aaaggaaatt 60 ttatctgatg caaaacatgg tgatcatgac cactttgagc tgatcactac aacggtcacg 120 ttagaagatg gttcgcaggg aaccggctat acttatactg gtggcaaagg cggttactcg 180 atcaaagcga tgctagagta tgatattcag cctgcgctaa tcggcaaaga cgcgacgcaa 240 attgaagaga tctatgactt tatggagtgg catattcact atgtcggtcg tggcggtatc 300 tctacatttg cgatgtctgc ggtagacatt gcgctttggg atctaaaagg taaacgagaa 360 ggcttgccgt tatggaaaat ggctggtgga aaaaacaata cctgtaaagc gtactgtggt 420 ggcattgacc ttcagtttcc acttgagaaa ttgctcaaca atatttgtgg ttatttagaa 480 agtggcttca atgccgttaa gatcaagatt ggtcgcgaaa atatgcaaga agatattgac 540 cgcattaagg cggttcgcga gctgattggg ccagatatca cctttatgat cgatgccaac 600 tattcgttga cagtagaaca agcgatcaaa ctgtcaaaag cggtagagca atatgacatc 660 acgtggtttg aagagccaac attgccagat gactacaaag gttttgctga gattgctgac 720 aatacagcga ttccgttggc catgggggaa aaccttcaca ccattcatga gtttggttat 780 gcaatggacc aagcaaagct tggctactgc caaccagatg cctcaaactg tggtggcatt 840 accggttggt tgaaagcggc ggacttgatt acagaacata atatcccagt gtgtactcac 900 ggtatgcaag agctacacgt aagtcttgtt tcagcgtttg atacaggttg gctagaggtg 960 cacagcttcc cgattgatga atacaccaag cgtcctttgg ttgtagaaaa cttccgcgct 1020 gtggcgtcca atgagccggg tatcggggtc gagttcgatt gggacaaaat tgctcagtac 1080 gaagtgtaa 1089 <210> 8 <211> 362 <212> PRT <213> Vibrio sp. EJY3 <400> 8 Met Lys Thr Thr Ile Lys Asp Ile Lys Thr Arg Leu Phe Lys Ile Pro   1 5 10 15 Leu Lys Glu Ile Leu Ser Asp Ala Lys His Gly Asp His Asp His Phe              20 25 30 Glu Leu Ile Thr Thr Thr Val Thr Leu Glu Asp Gly Ser Gln Gly Thr          35 40 45 Gly Tyr Thr Tyr Thr Gly Gly Lys Gly Gly Tyr Ser Ile Lys Ala Met      50 55 60 Leu Glu Tyr Asp Ile Gln Pro Ala Leu Ile Gly Lys Asp Ala Thr Gln  65 70 75 80 Ile Glu Glu Ile Tyr Asp Phe Met Glu Trp His Ile His Tyr Val Gly                  85 90 95 Arg Gly Gly Ile Ser Thr Phe Ala Met Ser Ala Val Asp Ile Ala Leu             100 105 110 Trp Asp Leu Lys Gly Lys Arg Glu Gly Leu Pro Leu Trp Lys Met Ala         115 120 125 Gly Gly Lys Asn Asn Thr Cys Lys Ala Tyr Cys Gly Gly Ile Asp Leu     130 135 140 Gln Phe Pro Leu Glu Lys Leu Leu Asn Asn Ile Cys Gly Tyr Leu Glu 145 150 155 160 Ser Gly Phe Asn Ala Val Lys Ile Lys Ile Gly Arg Glu Asn Met Gln                 165 170 175 Glu Asp Ile Asp Arg Ile Lys Ala Val Arg Glu Leu Ile Gly Pro Asp             180 185 190 Ile Thr Phe Met Ile Asp Ala Asn Tyr Ser Leu Thr Val Glu Gln Ala         195 200 205 Ile Lys Leu Ser Lys Ala Val Glu Gln Tyr Asp Ile Thr Trp Phe Glu     210 215 220 Glu Pro Thr Leu Pro Asp Asp Tyr Lys Gly Phe Ala Glu Ile Ala Asp 225 230 235 240 Asn Thr Ala Ile Pro Leu Ala Met Gly Glu Asn Leu His Thr Ile His                 245 250 255 Glu Phe Gly Tyr Ala Met Asp Gln Ala Lys Leu Gly Tyr Cys Gln Pro             260 265 270 Asp Ala Ser Asn Cys Gly Gly Ile Thr Gly Trp Leu Lys Ala Ala Asp         275 280 285 Leu Ile Thr Glu His Asn Ile Pro Val Cys Thr His Gly Met Gln Glu     290 295 300 Leu His Val Ser Leu Val Ser Ala Phe Asp Thr Gly Trp Leu Glu Val 305 310 315 320 His Ser Phe Pro Ile Asp Glu Tyr Thr Lys Arg Pro Leu Val Val Glu                 325 330 335 Asn Phe Arg Ala Val Ala Ser Asn Glu Pro Gly Ile Gly Val Glu Phe             340 345 350 Asp Trp Asp Lys Ile Ala Gln Tyr Glu Val         355 360 <210> 9 <211> 966 <212> DNA <213> Vibrio sp. EJY3 <400> 9 atgagtttgg aaataaaaca agatacggag tctagctata gcgatattct tagctttggt 60 gagccaatgt ttgagtttag ccaagttgga caagcaggtt caggccagcc tgatttcttg 120 agtggttttg gtggtgatgc ttcgaacttt gctatcgcag cagcaagaca aggcgcatca 180 gttggaatgt tgacacaact tggcgacgat gaattcggta agcgttttgt tgagctgtgg 240 gaacagcagg gtgttagcag ttcagctgtg tgtatactac caaataaagc aacgggcgtt 300 tattttatta cgcacgatga tgagggacac catttttctt tcttgcgtaa gaactctgcg 360 gccagtttaa tgacaccgca agacttacca tcagatgcga ttgccaatgc taagcttctt 420 catatcactg ctattactca ggcgattagt gattcaagtt gtgactcagt gtttgcagca 480 attgaaacag cgaaagcgca cggcactcaa gtgtcctatg acaccaactt gcgcttaaag 540 ctatggtcac tgcaacgcgc tcgcgccatc attaatgaaa ccgcgtcact agtcgatgtc 600 tgcttcccta gtattgacga agcacgcttg gtgactggcc ttgaacatgc tgacgatatc 660 atcgattttt acctaaaagc aggcgcgaaa gttgtcgtac ttaaacaggg tggtgacggt 720 gcgacagtgg ctaatgagca tattaggcat ttcatccttc cgcataaagt gacacctgtt 780 gatgcgaccg ctgctggtga ttcatttgca ggctcattct gtacgcatta tgtcaacgga 840 gagtctttag agcagtgtct tgcgtatgca aatgccaccg cgtctatcac gattactggt 900 tttggtgcag ttgccccttt accgacattt gagcaagtgc ttgagaaaat caacgaatct 960 aaatag 966 <210> 10 <211> 321 <212> PRT <213> Vibrio sp. EJY3 <400> 10 Met Ser Leu Glu Ile Lys Gln Asp Thr Glu Ser Ser Tyr Ser Asp Ile   1 5 10 15 Leu Ser Phe Gly Glu Pro Met Phe Glu Phe Ser Gln Val Gly Gln Ala              20 25 30 Gly Ser Gly Gln Pro Asp Phe Leu Ser Gly Phe Gly Gly Asp Ala Ser          35 40 45 Asn Phe Ala Ile Ala Ala Ala Arg Gln Ala Ser Val Gly Met Leu      50 55 60 Thr Gln Leu Gly Asp Asp Glu Phe Gly Lys Arg Phe Val Glu Leu Trp  65 70 75 80 Glu Gln Gln Gly Val Ser Ser Ser Ala Val Cys Ile Leu Pro Asn Lys                  85 90 95 Ala Thr Gly Val Tyr Phe Ile Thr His Asp Asp Glu Gly His His Phe             100 105 110 Ser Phe Leu Arg Lys Asn Ser Ala Ala Ser Leu Met Thr Pro Gln Asp         115 120 125 Leu Pro Ser Asp Ala Ile Ala Asn Ala Lys Leu Leu His Ile Thr Ala     130 135 140 Ile Thr Gln Ala Ile Ser Asp Ser Ser Cys Asp Ser Val Phe Ala Ala 145 150 155 160 Ile Glu Thr Ala Lys Ala His Gly Thr Gln Val Ser Tyr Asp Thr Asn                 165 170 175 Leu Arg Leu Lys Leu Trp Ser Leu Gln Arg Ala Arg Ala Ile Ile Asn             180 185 190 Glu Thr Ala Ser Leu Val Asp Val Cys Phe Pro Ser Ile Asp Glu Ala         195 200 205 Arg Leu Val Thr Gly Leu Glu His Ala Asp Asp Ile Ile Asp Phe Tyr     210 215 220 Leu Lys Ala Gly Ala Lys Val Val Leu Lys Gln Gly Gly Asp Gly 225 230 235 240 Ala Thr Val Ala Asn Glu His Ile Arg His Phe Ile Leu Pro His Lys                 245 250 255 Val Thr Pro Val Asp Ala Thr Ala Ala Gly Asp Ser Phe Ala Gly Ser             260 265 270 Phe Cys Thr His Tyr Val Asn Gly Glu Ser Leu Glu Gln Cys Leu Ala         275 280 285 Tyr Ala Asn Ala Thr Ala Ser Ile Thr Ile Thr Gly Phe Gly Ala Val     290 295 300 Ala Pro Leu Pro Thr Phe Glu Gln Val Leu Glu Lys Ile Asn Glu Ser 305 310 315 320 Lys     <210> 11 <211> 621 <212> DNA <213> Vibrio sp. EJY3 <400> 11 atggatctca atcaacgttt agctaagctc aaagttgtgc ctgtgattgc tgtagataat 60 gcgcaagata ttttgccttt aggtaaggcg ctggtagaga atggtttacc agtcgcagaa 120 attacctttc gctctgacgc ggcgactgaa gccattcgtt tacttcgtac tacttatcca 180 gacatcttga ttggtgcggg tacggtattg aacgaagctc aagtaattga ggcaaaagag 240 gcgggtgctg actttattgt ttctccaggc ttgaacccaa tcacagtaaa agcatgtcaa 300 aaacataaaa taaccatcgt ccctggtgta aacagcccat cgttggttga gcaagctctt 360 gagcttggtg ttgatactgt taaatttttc ccagcagaag cgtcgggcgg tctagcgatg 420 ttgaagtcgt tgcttggccc ttatcaacaa atcaaagtga tgcctacagg tggtatcaat 480 caaaacaaca ttcatgatta tctggctctt cctgctgtac ttgcttgtgg tggtacgtgg 540 atggtggata aatcactggt acataaaggt gcttgggatg aaattggccg attggtcaga 600 gaaattgtcg ccgcagtata g 621 <210> 12 <211> 206 <212> PRT <213> Vibrio sp. EJY3 <400> 12 Met Asp Leu Asn Gln Arg Leu Ala Lys Leu Lys Val Val Pro Val Ile   1 5 10 15 Ala Val Asp Asn Ala Gln Asp Ile Leu Pro Leu Gly Lys Ala Leu Val              20 25 30 Glu Asn Gly Leu Pro Val Ala Glu Ile Thr Phe Arg Ser Asp Ala Ala          35 40 45 Thr Glu Ala Ile Arg Leu Leu Arg Thr Thr Tyr Pro Asp Ile Leu Ile      50 55 60 Gly Ala Gly Thr Val Leu Asn Glu Ala Gln Val Ile Glu Ala Lys Glu  65 70 75 80 Ala Gly Ala Asp Phe Ile Val Ser Pro Gly Leu Asn Pro Ile Thr Val                  85 90 95 Lys Ala Cys Gln Lys His Lys Ile Thr Ile Val Pro Gly Val Asn Ser             100 105 110 Pro Ser Leu Val Glu Gln Ala Leu Glu Leu Gly Val Asp Thr Val Lys         115 120 125 Phe Phe Pro Ala Glu Ala Ser Gly Gly Leu Ala Met Leu Lys Ser Leu     130 135 140 Leu Gly Pro Tyr Gln Gln Ile Lys Val Met Pro Thr Gly Gly Ile Asn 145 150 155 160 Gln Asn Asn Ile His Asp Tyr Leu Ala Leu Pro Ala Val Leu Ala Cys                 165 170 175 Gly Gly Thr Trp Met Val Asp Lys Ser Leu Val His Lys Gly Ala Trp             180 185 190 Asp Glu Ile Gly Arg Leu Val Arg Glu Ile Val Ala Ala Val         195 200 205 <210> 13 <211> 40 <212> DNA <213> Artificial Sequence <220> Forward primer 1 for 3,6-anhydro-L-galactose dehydrogenase <400> 13 gaaggagata taaggatgaa acgttaccaa atgtacgttg 40 <210> 14 <211> 40 <212> DNA <213> Artificial Sequence <220> Reverse primer 2 for 3,6-anhydro-L-galactose dehydrogenase <400> 14 atgatggtga tggtggtcga aattcacata gaatgtcttc 40 <210> 15 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Forward primer 1 for SdeACI <400> 15 gaaggagata taaggatgaa aattcataac atgaaaaatt ttatcaa 47 <210> 16 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer 2 for SdeACI <400> 16 atgatggtga tggtgtcatt cagcaaaata cactgtcttc 40 <210> 17 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Forward primer 1 for Pat1ACI <400> 17 gaaggagata taaggatgat gagtgtcatt accaaactag aca 43 <210> 18 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer 2 for Pat1ACI <400> 18 atgatggtga tggtgagaat gtttaactaa atagggaaga ag 42 <210> 19 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Forward primer 1 for Vejy3ACI <400> 19 gaaggagata taaggatgaa aacaacaatc aaagacatca aaa 43 <210> 20 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer 2 for Vejy3ACI <400> 20 atgatggtga tggtgcactt cgtactgagc aattttgt 38 <210> 21 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Forward primer 1 for Vejy3ACI <400> 21 gcgctcgaga tgaaaacaac aatcaaagac atcaaaac 38 <210> 22 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer 2 for Vejy3ACI <400> 22 gcgtacgtac acttcgtact gagcaatttt gtc 33 <210> 23 <211> 34 <212> DNA <213> Artificial Sequence <220> Forward primer 1 for 3,6-anhydro-L-galactose dehydrogenase <400> 23 gcgctcgaga tgaaacgtta ccaaatgtac gttg 34 <210> 24 <211> 35 <212> DNA <213> Artificial Sequence <220> Reverse primer 2 for 3,6-anhydro-L-galactose dehydrogenase <400> 24 gcgtctagat tagtcgaaat tcacatagaa tgtct 35

Claims (5)

Keto-3-deoxy-galactonic acid containing 3,6-anhydrogalactonic acid cycloisomerase represented by the amino acid sequence of SEQ ID NO: 8 Composition.
The method according to claim 1,
The 3,6-anhydrogalactonic acid cyclic nitrification enzyme is characterized in that it is isomerized while ring-opening the 3,6-anhydrogalactonic acid ring to convert it to 2-keto-3-deoxy-galactonic acid 2-keto-3-deoxy-galactonic acid.
The method according to claim 1,
3,6-anhydrogalactonic acid cyclase nitricase is encoded from a gene represented by the nucleotide sequence of SEQ ID NO: 7. 2. A composition for producing 2-keto-3-deoxy-galactonic acid according to claim 1,
The method according to claim 1,
The 3,6-anhydrogalactonic acid cyclase nitrification enzyme is a 2-keto-3-anhydrogalactonic acid cyclic isomerase obtained from the culture product of the transformant transformed with the recombinant vector into which the gene represented by the nucleotide sequence of SEQ ID NO: -Deoxy-galactonic acid.
3,6-anhydrogalactonic acid cycloisomerase represented by the amino acid sequence of SEQ ID NO: 8, the 3,6-anhydrogalactonic acid cyclic isomerase Or a culture product of the microorganism is reacted with 3,6-anhydrogalactonic acid to produce 2-keto-3-deoxy-galactonic acid.

Images