KR101844157B1 - Method of preparing d-lactic acid - Google Patents

Abstract

The present invention relates to a microorganism producing D-lactic acid from a disaccharide and a method for producing D-lactic acid using the microorganism. A commercial strain, which can produce D-lactic acid of high purity and high concentration by using wild-type Escherichia coil, is economical since a decomposition step of the carbon source into a monosaccharide can be simplified by using a disaccharide as the carbon source and is easy to scale up by improving the yield rate and productivity through rapid cell growth compared to glucose use, can be provided.

Description

METHOD OF PREPARING D-LACTIC ACID [0002]

The present invention relates to a method for producing D-lactic acid using microorganisms.

Lactic acid is widely used in various industries such as foods, medicines, cosmetics, etc. Recently, it has been utilized as a monomer of polylactic acid, and its demand has been greatly increased.

Lactic acid production methods can be divided into chemical synthesis and biological fermentation using carbohydrates as a substrate.

In addition to the problems of cost increase due to rising oil prices or environmental pollution, the conventional chemical synthesis method has the problem that an inactive D type or L type lactic acid in the form of a racemic mixture in which 50% of L-type lactic acid and 50% In this case, it is not possible to control the composition ratio of the mixture of D-type and L-type lactic acid, and when polyamic acid is produced from racemic mixture lactic acid as a raw material, the amorphous polymer having a low melting point is limited. There may be a problem that there are many.

In the case of biological fermentation using microorganisms, only D-type or L-type lactic acid can be selectively produced depending on the strain used. For example, microorganisms such as Lactobacillus sp., Bacillus sp., Rhizopus sp., Streptococcus sp., Enterococcus sp. L-lactic acid is mainly produced, and microorganisms of Leuconostoc sp. And Lactobacillus vulgaricus are known to mainly produce D-lactic acid.

Particularly, the D-lactic acid is characterized in that it is not metabolized in the body. Therefore, it can be used as an optically active herbicide by esterification and chlorination as well as being used for biosynthetic materials in the medical field. , It is confirmed that the effect is improved and the same effect is obtained even with a small dose, and the demand for the D type lactic acid is increasing. In addition, sc-polylactic acid (stereocomplex-PLA) has higher melting point and pyrolysis temperature than conventional polylactic acid and can be used as a high heat-resistant plastic raw material. This is because pure D-type poly lactic acid is added to pure L- Mixed and produced. As a result, since D-lactic acid is required as a monomer of D-type polylactic acid, the demand for D-type lactic acid is increasing.

Pure D-type lactic acid may be desirable for biological fermentation methods using optical specific substrate selectivity of microbial enzymes, but the general wild type D-lactic acid producing microorganisms present in nature are incomplete in terms of optical purity or productivity of lactic acid for industrial use There is a drawback. Representative D-lactic acid producing microorganisms include Lb. plantarum, Lb. pentosus, Lb. fermentum, and Lbdelbrueckii. However, it is generally inferior in terms of productivity and yield, and has the disadvantage of including L-lactic acid as an optical impurity.

Further, in the conventional fermentation industry, in order to facilitate steps such as acid, heat, saccharification enzyme treatment and the like in production of lactic acid, the carbon source had to be decomposed into a sugar monosaccharide which is easy to be applied with a microorganism.

Therefore, in order to economically produce D-lactic acid, research for producing high-purity L-lactic acid of high purity as a microorganism that can grow using disaccharides such as raw sugar, corn, malt, and the like instead of glucose is required.

PCT / JP2009 / 065957 (Mar. 25, 2010) Korean Patent No. 10-1037354 (2011.5.26)

It is an object of the present invention to provide a method for producing D-lactic acid from a disaccharide using microorganisms.

In order to solve the above problems, the present invention provides a method for producing D-lactic acid from a disaccharide by using microorganism Escherichia coli LC02 (Accession No .: KCTC13100BP, entrusted date: 2016.09.08.).

According to one embodiment, the LC02 may be incompatible with the biochemical properties of the E. coli RW-29 as a result of API 20E.

According to one embodiment, the lactic acid production method of the present invention comprises the steps of: inoculating the microorganism into a culture medium containing a yeast extract, a neutralizing agent and a disaccharide; Culturing under aerobic conditions, 40 ° C, pH 6.8; And secondary culturing at 40 [deg.] C, pH 6.8 under anaerobic conditions at the time of reaching OD ₆₀₀ 20.

According to one embodiment, maltose may be used as the disaccharide in the secondary culture.

According to another embodiment, sucrose may be used as a disaccharide in the secondary culture.

Other details of the embodiments of the present invention are included in the following detailed description.

According to the microorganism and the method for producing D-lactic acid using the microorganism according to the present invention, high purity and high concentration of D-lactic acid can be produced from disaccharides such as sucrose and maltose using a wild-type strain.

In addition, since a disaccharide is used as a carbon source in the production of lactic acid, it is possible to simplify the decomposition process of the carbon source into the monosaccharide, thereby making it possible to produce D-lactic acid economically.

Fig. 1 shows the results of the API 20E kit identification.
2 is a chiral column analysis graph showing the purity of D-lactic acid.
3 is a graph showing the results of production of D-lactic acid using disaccharides.
4 and 5 are graphs showing growth curves of microorganisms using disaccharides.
6 is a graph showing glucose availability of glucose, raw sugar or starch syrup on microorganisms.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention is capable of various modifications and various embodiments, and specific embodiments are illustrated in the drawings and described in detail in the description. It should be understood, however, that the present invention is not intended to be limited to any particular embodiment, but is intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

Hereinafter, a microorganism according to an embodiment of the present invention and a method for producing D-lactic acid from a disaccharide using the microorganism will be described in detail.

According to the present invention, it is possible to mass-produce D-lactic acid of high purity and high concentration from sugar components using E. coli.

The sugar component may include, for example, glucose, fructose, galactose and the like. In the present invention, the disaccharide may be used as a carbon source in the production of the D-lactic acid. Specifically, the disaccharide may include sucrose, maltose, lactose (lactose), and a combination thereof.

The present invention provides an LC02 microorganism which is a wild-type Escherichia coli producing D-lactic acid from a disaccharide (Accession No. KCTC13100BP, entrusted date: 2016.09.08).

LC02 microorganisms can be obtained from wastewater or soil through screening or identification steps,

Screening with 3M ™ Petrifilm ™ E. coli / Coliform Count Plate;

Screening of Gram-negative bacteria on EMB (Eosin methylene blue) agar plate;

Screening on Luria-Bertani (LB) plates;

Biochemical identification using API kit;

16s rDNA sequence identification step, and a combination thereof.

The 16s rRNA raw data is attached to SEQ ID NO: 1.

[SEQ ID NO: 1: 16s rRNA raw data]

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The LC02 microorganism has no resistance to antibiotics such as ampicillin and chloramphenicol, has no own plasmid, and can have a characteristic of being able to transform using an E. coli-derived vector.

In addition, the LC02 microorganism has 100% homology (identities 1412/1412, gap 0/1412) with the Escherichia coli strain RW-29 16S ribosomal RNA gene. However, the API 20E kit experiment showed biochemically opposite characteristics, (tryptophane deaminase) and INO (fermentation / oxidation, inositol) reagents. The results are shown in FIG.

According to one embodiment, the medium used in the culture may comprise yeast extract, neutralizing agent and disaccharide.

The disaccharide may be suitably replaced by those skilled in the art if it is a disaccharide component capable of producing lactic acid as a saccharide component. For example, raw sugar (sucrose), starch syrup (maltose) and the like can be used as disaccharides. For example, raw sugar (sucrose), starch syrup (maltose) or a combination thereof is used as a disaccharide Maltose can be used as the disaccharide in the secondary culture, and the types and combinations of the disaccharides used in the primary and secondary cultures can be appropriately selected by those skilled in the art. By using the disaccharide as a carbon source, it is possible to obtain an effect of rapidly growing the cells as compared with the case where glucose (glucose) is used as a carbon source.

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A neutralizing agent, for example, (NH ₄ OH), calcium hydroxide (Ca (OH) ₂₎ sodium hydroxide (NaOH), ammonium hydroxide may comprise, and combinations thereof, Ca ⁺⁺ ions to assist in the lactic acid production It may preferably contain calcium hydroxide (Ca (OH) ₂ ). However, in the case of using calcium carbonate (CaCO ₃ ) in the culture using Escherichia coli, the pH maintenance range is low and additional neutralizing agent can be added. Calcium hydroxide (Ca (OH) ₂ ) as well as calcium carbonate (CaCO ₃ ) hardly dissolves in water. Stirring can be maintained when the pump is used. In the case of calcium hydroxide (Ca (OH) ₂ ), it is necessary to add calcium hydroxide (Ca (OH) ₂ ) at a high concentration to minimize the increase of the culture liquid.

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Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Production Example 1: Screening for wild-type E. coli

Samples were taken from wastewater and soil for screening E. coli.

The collected samples were first screened using a 3M ™ Petrifilm ™ E. coli / Coliform Count Plate, and 1 ml of the undiluted sample was inoculated with a pipette. Lt; 0 > C incubator for 24 hours. Thereafter, the colonies of red colonies turned blue into the microorganisms grown in the plate, and blue colonies were isolated by a sample loop.

Then, the microorganisms selected in the first petrifilm were streaked on a BD EMB (eosin methylene blue) agar plate to secondary screen Gram-negative bacteria. On the EMB plate, a microorganism having metallic green was recognized as a gram-negative E. coli strain, and secondary selection was performed.

Secondary screened Escherichia coli anticipated microorganisms were streaked on LB (Luria-Bertani) plates for tertiary screening and O / N incubated at 37 ° C. Colonies of E. coli in pale yellow color were inoculated into 4 ml of liquid LB medium in a cap tube, incubated at 37 ° C for 6 hours, and analyzed by biochemical method using API 20E kit (bioMerieux, US). The API kit experiment was carried out according to the manufacturer's manual, and the results are shown in FIG. 1 at https://apiweb.biomerieux.com/. For the identification of E. coli strains by biochemical analysis, wild type Escherichia coli was finally identified by 16s rDNA sequence analysis. Bidirectional sequencing was performed using SOLIDENT, Inc., using universal primers 27F (AGAGTTTGATCCTGGAG) and 1492R (TACGGYTACCTTGTTACGACTT). A blast result using the NCBI database confirmed that the strain was 100% homologous to Escherichia coli ER1821R strain.

As shown in FIG. 1, the wild-type E. coli has 100% homology (identities 1412/1412, gap 0/1412) with Escherichia coli strain RW-29 16S ribosomal RNA gene. However, in the biochemical characteristics (API kit) Respectively. The new E. coli was named Escherichia coli LC02, and deposited with the BRC (Korea Access Association, National Institute of Bioscience and Biotechnology) (Accession No .: KCTC13100BP, deposit date: 2016.09.08).

Example 1 Production of Lactic Acid Using Disaccharide

The neutralizing agent, 6N Ca (OH) _2, was added to the modified M9 medium and cultured with 1 L fermentec FMT ST-M model / Korea. The trace metal may be suitably further added by those skilled in the art. The composition ratio of the modified M9 medium is shown in Table 1 below.

When the medium is diluted with the neutralizing agent added to the culture medium, the culture liquid is diluted to increase the set culture volume. When the culture liquid is not diluted, when the concentration exceeds 120 g / L, the whole culture liquid can be solidified due to Ca (LA) ₂ precipitation.

Modified M9 badge Na ₂ HPO ₄ .12H ₂ O 15.11 g / L KH ₂ PO ₄ 3g / L NH ₄ Cl 1 g / L NaCl 0.5 g / L Yeast extract 15g / L MgSO ₄ 1 ml / L saccharose 60 to 135 g / L

Specifically, the basic culture conditions were as follows: LC02 strain was inoculated 10% of the culture medium in the above medium, and then the mixture was inoculated at a total volume of 600 mL in a 1 L fermenter at 40 DEG C, and the pH was maintained at 6.8 . At this time, 6N Ca (OH) ₂ was used as a neutralizing agent. Culture was gradually proceeds into two by car, an aerobic atmosphere, cascade DO 40 maintained, 1 vvm, after the primary incubation with 40 ℃ and pH 6.8 conditions, at the time of reaching the measured value 20 at OD _600, an anaerobic atmosphere, 500 rpm, 40 ° C and pH 6.8, and the rpm condition can be adjusted to 300 to 700 rpm according to the culture scale. As the disaccharide contained in the Modified M9 medium, maltose or sucrose may be used.

As a result of the culture, chiral column analysis of lactic acid produced showed that 99.9% of lactic acid was D-type. The result is shown in FIG.

3 shows the results of culturing D-lactic acid with a 1 L scale fermenter using starch syrup (maltose) and raw sugar (sucrose) as industrial disaccharides.

As shown in Fig. 3, the concentration of D-lactic acid (D-lactic acid) was 67 g / L and the productivity was 2.2 g / L / h in the case of containing 60 g / L of maltose as a disaccharide , And acetic acid and succinic acid as other by-products.

In addition to maltose, a small amount of maltotriose and other saccharides are contained, resulting in a yield of 100% or more, and the production of D-lactic acid It is possible to improve the performance. In the case of raw sugar (sucrose), D-lactic acid of 43 g / L was produced using 135 g / L of initial raw sugar. In the case of raw sugar, the initial cell mass increase was fast, but the conversion to D-lactic acid in anaerobic condition was slower than the increase in cell mass. As the productivity increases after 30 hours of incubation, it can be expected that the metabolic pathway is activated after a certain period of time.

Experimental Example 1: Analysis of sugar availability of LC02

In order to measure the growth of LC02 using sucrose and maltose (maltose, glucose, syrup) as industrial and reagent grade disaccharides, 50 mL of modifided M9 medium, 10 to 12 g / L of carbon source and nitrogen source yeast extract After addition of 15 g / L of LC02, LC02 was inoculated and it was confirmed that disaccharides such as maltose and sucrose could be used through flask cultivation under aerobic conditions at 40 DEG C for 24 hours. Growth stopped at pH 5 due to the production of organic acids in the flask culture. The cell growth curve was measured by an OD ₆₀₀ value (UV spectrophotometer), and the reduction of sugar content was measured by HPLC and plotted in FIGS. 4 and 5. As the carbon source of Fig. 4, raw sugar or pure sucrose was used. As the carbon source of Fig. 5, disaccharide availability was measured by using maltose (starch syrup) or refined disaccharide, i.e. pure maltose I confirmed it again.

HPLC conditions for organic acid and sugar analysis are as follows.

Organic acid Analysis conditions:

- HPLC: ultimate 3000 LC system (Dionex, Thermo Scientific, US)

- RI detector: RI-101 (Shodex, Showa Denko, Japan)

- column: Aminex HPX-87h (BIORAD, US)

- mobile phase: 2mM H2SO4

- 0.6 ml / min, column oven 55, RID 35 ° C, 10ul injection

- The sample was diluted 1/2,

Sucrose analysis conditions:

- HPLC: ultimate 3000 LC system (Dionex, Thermo Scientific, US)

- RI detector: RI-101 (Shodex, Showa Denko, Japan)

- column: bondapak amino column (waters, US)

- mobile phase: ACN: water = 75: 25

- 0.9 ml / min, column oven 35, RID 35 ° C, 5ul injection

- The sample was diluted 1/2,

As can be seen from Fig. 4, LC02 rapidly grows using non-treated water crossover (raw sugar) and pure sucrose under aerobic conditions, and rapidly grows using starch syrup and pure maltose as shown in Fig. 5 .

In addition, in order to measure the sugar-using ability of glucose, raw sugar, and syrup of LC02, Ca (OH) _{2 was} neutralized with a total volume of 600 mL using a 1 L scale fermenter instead of the flask under the same conditions as the primary culture of Example 1 The results are shown in Fig. As a result of the same analysis as in the above flask culturing, it was confirmed that the disaccharide was used more than glucose in the growth of cells.

As described above, the present invention can promote the growth of microorganisms through primary culture using disaccharide and secondary culture using starch syrup or maltose (maltose), and can produce high concentration of D-lactic acid, The step of decomposing into monosaccharides can be simplified.

Therefore, according to the present invention, it is possible to provide Escherichia coli capable of producing 99.9% of D-lactic acid at a high concentration by using disaccharides, and a gene for blocking byproducts using a conventional genetic tool for E. coli It is possible to provide strains that are easy to manipulate and can provide optimization conditions for gene recombination and culture.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> LOTTE CHEMICAL CORPORATION          LOTTE FOOD CO., LTD <120> METHOD OF PREPARING D-LACTIC ACID <130> DPA-0818 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1423 <212> DNA <213> Escherichia coli <400> 1 ggctancatg caagtcgaac ggtaacagga agaagcttgc ttctttgctg acgagtggcg 60 gacgggtgag taatgtctgg gaaactgcct gatggagggg gataactact ggaaacggta 120 gctaataccg cataacgtcg caagaccaaa gagggggacc ttcgggcctc ttgccatcgg 180 atgtgcccag atgggattag ctagtaggtg gggtaacggc tcacctaggc gacgatccct 240 agctggtctg agaggatgac cagccacact ggaactgaga cacggtccag actcctacgg 300 gaggcagcag tggggaatat tgcacaatgg gcgcaagcct gatgcagcca tgccgcgtgt 360 atgaagaagg ccttcgggtt gtaaagtact ttcagcgggg aggaagggag taaagttaat 420 acctttgctc attgacgtta cccgcagaag aagcaccggc taactccgtg ccagcagccg 480 cggtaatacg gagggtgcaa gcgttaatcg gaattactgg gcgtaaagcg cacgcaggcg 540 gtttgttaag tcagatgtga aatccccggg ctcaacctgg gaactgcatc tgatactggc 600 aagcttgagt ctcgtagagg ggggtagaat tccaggtgta gcggtgaaat gcgtagagat 660 ctggaggaat accggtggcg aaggcggccc cctggacgaa gactgacgct caggtgcgaa 720 agcgtgggga gcaaacagga ttagataccc tggtagtcca cgccgtaaac gatgtcgact 780 tggaggttgt gcccttgagg cgtggcttcc ggagctaacg cgttaagtcg accgcctggg 840 gagtacggcc gcaaggttaa aactcaaatg aattgacggg ggcccgcaca agcggtggag 900 catgtggttt aattcgatgc aacgcgaaga accttacctg gtcttgacat ccacggaagt 960 tttcagagat gagaatgtgc cttcgggaac cgtgagacag gtgctgcatg gctgtcgtca 1020 gctcgtgttg tgaaatgttg ggttaagtcc cgcaacgagc gcaaccctta tcctttgttg 1080 ccagcggtcc ggccgggaac tcaaaggaga ctgccagtga taaactggag gaaggtgggg 1140 atgacgtcaa gtcatcatgg cccttacgac cagggctaca cacgtgctac aatggcgcat 1200 acaaagagaa gcgacctcgc gagagcaagc ggacctcata aagtgcgtcg tagtccggat 1260 tggagtctgc aactcgactc catgaagtcg gaatcgctag taatcgtgga tcagaatgcc 1320 acggtgaata cgttcccggg ccttgtacac accgcccgtc acaccatggg agtgggttgc 1380 aaaagaagta ggtagcttaa ccttcgggag ggcgctacca ctt 1423

Claims (6)

E. coli LC02 (accession number: KCTC13100BP, date of deposit: 2016.09.08.) As a method for producing D-lactic acid from a disaccharide using microorganisms,
Inoculating said microorganism into a culture medium containing yeast extract, neutralizing agent and disaccharide;
Culturing under aerobic conditions, 40 DEG C, pH 6.8; And
Culturing the mixture at 40 DEG C and pH 6.8 under anaerobic conditions at an OD ₆₀₀ value of 20; The method according to claim 1,
Wherein the disaccharide is sucrose, maltose or a combination thereof in the primary culture. The method according to claim 1,
Wherein the sucrose, maltose, or a combination thereof is used as the disaccharide in the secondary culture. delete delete delete

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