Method for producing glycolic acid using immobilization technology

Abstract

This invention provides a method for producing glycolic acid using immobilization technology. [Solution] The method includes providing a modified strain containing a specific gene sequence, preparing a polyvinyl alcohol bacteria pellet containing the modified strain, and supplying a carbon source to the polyvinyl alcohol bacteria pellet and converting the carbon source to glycolic acid using the modified strain. The polyvinyl alcohol bacteria pellet and an auxiliary solution are mixed, and air is introduced while supplying the carbon source to the polyvinyl alcohol bacteria pellet.

Description

Technical Field

[0001] The present invention relates to a method for producing glycolic acid, and particularly to a method for producing glycolic acid by an immobilization technique.

Background Art

[0002] Glycolic acid (GA) is the smallest α-hydroxy acid and is also called hydroxyacetic acid or ethanol acid. Glycolic acid contains both a carboxyl group and a hydroxyl group and has the dual properties of a carboxylic acid and an alcohol. Therefore, glycolic acid is easy to decompose and absorb and has characteristics such as strong water solubility and high permeability. Furthermore, glycolic acid and its polymers are excellent in biodegradability and biocompatibility, can be decomposed in vivo, metabolized into water and carbon dioxide, and excreted outside the body. For this reason, the uses of glycolic acid are very wide. For example, glycolic acid and its polymers are used for the release of peptide and protein-based pharmaceuticals or can be used as pharmaceutical intermediates in the production of esters of menthol and quinine and the synthesis of other pharmaceuticals. Oligomers or derivatives of glycolic acid are used as food additives and can suppress the growth of harmful microorganisms by acidification.

[0003] In the prior art, glycolic acid is mainly produced by chemical synthesis methods (for example, there are methods such as the cyanation method of formaldehyde, the hydrolysis method of chloroacetic acid, and the oxonioation method of formaldehyde). However, these chemical synthesis methods have problems such as severe reaction conditions, strong toxicity of raw materials, high costs, difficulty in subsequent separation, and environmental pollution. Therefore, with the spread of the microbial enzyme catalyst method and the total biosynthesis method, it is gradually becoming the mainstream production method of glycolic acid instead of the chemical synthesis method.

Summary of the Invention

Problems to be Solved by the Invention

[0004] It is noteworthy that microbial enzyme-catalyzed methods require the use of toxic and expensive acetonitrile or hydroxyacetonitrile (glyconitrile) as raw materials, making them unsuitable for industrial production needs. While total biosynthesis holds promising potential in the glycolic acid production market, production volume limitations hinder large-scale application. Therefore, overcoming these shortcomings by improving the manufacturing process is a crucial challenge to address in this technological field. [Means for solving the problem]

[0005] To solve the above-mentioned technical problems, the present invention provides a method for producing glycolic acid by immobilization technology. The method for producing glycolic acid by immobilization technology includes providing a modified strain containing the gene sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; preparing a polyvinyl alcohol bacteria pellet containing the modified strain; mixing the polyvinyl alcohol bacteria pellet with an auxiliary solution; supplying a carbon source to the polyvinyl alcohol bacteria pellet; and converting the carbon source to glycolic acid using the modified strain, while introducing air while supplying the carbon source to the polyvinyl alcohol bacteria pellet.

[0006] In one embodiment of the present invention, the preparation of the polyvinyl alcohol bacteria pellets includes: preparing a 1% to 20% polyvinyl alcohol solution, adding the modified strain to the polyvinyl alcohol solution and mixing uniformly to obtain a polyvinyl alcohol bacteria solution; providing a saturated boric acid solution and a phosphoric acid solution; dropping the polyvinyl alcohol bacteria solution into the saturated boric acid solution to obtain a semi-finished polyvinyl alcohol bacteria pellet; and removing the semi-finished polyvinyl alcohol bacteria pellet, draining the water, and placing it in the phosphoric acid solution and leaving it for a predetermined time to obtain the polyvinyl alcohol bacteria pellets.

[0007] In one embodiment of the present invention, the preparation of the polyvinyl alcohol bacterial pellets further includes the preparation of 0.5% to 1.5% alginic acid.

[0008] In one embodiment of the present invention, the predetermined time is 30 minutes to 2 hours.

[0009] In one embodiment of the present invention, the concentration of the boric acid solution is 1% to 5%, and the concentration of the phosphoric acid solution is 0.1M to 1M.

[0010] In one embodiment of the present invention, the carbon source is ethylene glycol, and the air permeability is 0.3vvm to 1vvm.

[0011] In one embodiment of the present invention, the modified strain is a modified Escherichia coli, Gluconobacter sp., Rhodococcus, or oxidizing bacteria.

[0012] In one embodiment of the present invention, the auxiliary solution is composed of yeast extract, peptone, sorbitol, (NH4)2SO4, and MgSO4.7H2O.

[0013] In one embodiment of the present invention, the GC% of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 are all 50-60%.

[0014] In one embodiment of the present invention, the modified strain has the ability to produce alcohol dehydrogenase, aldehyde dehydrogenase, and bacterial hemoglobin.

[0015] One of the beneficial effects of the present invention is that the method for producing glycolic acid by immobilization technology provided in the present invention can improve the efficiency of glycolic acid production by oxidation of ethylene glycol by utilizing strain immobilization technology, through the technical means of "providing a modified strain containing the gene sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3" and "preparing a polyvinyl alcohol bacterial pellet containing the modified strain."

[0016] To further understand the features and technical content of the present invention, please refer to the following detailed description and drawings of the invention, however, the drawings provided are for reference and illustrative purposes only and are not intended to limit the invention. [Brief explanation of the drawing]

[0017] [Figure 1] This is a flowchart of the method for converting a carbon source to glycolic acid according to the present invention. [Figure 2] This is a schematic diagram of the plasmid according to the present invention. [Figure 3] This is a schematic diagram showing a comparison between SEQ ID NO:1 according to the present invention and an existing EGADH gene sequence. [Figure 4] Figures 4A and 4B are schematic diagrams showing a comparison between SEQ ID NO:2 according to the present invention and existing EGAlDH gene sequences. [Figure 5] This is a schematic diagram showing a comparison between SEQ ID NO:3 according to the present invention and an existing VHb gene sequence. [Figure 6] These are bar graphs and curve graphs showing the amount of glycolic acid converted according to the first embodiment of the present invention. [Figure 7] These are bar graphs and curve graphs showing the amount of glycolic acid converted according to the second embodiment of the present invention. [Figure 8] This is a flowchart showing the method for producing glycolic acid using immobilization technology. [Modes for carrying out the invention]

[0018] Hereinafter, embodiments of the "method for producing glycolic acid by immobilization technology" disclosed in the present invention will be described by way of specific examples. A person skilled in the art can understand the advantages and effects of the present invention from the disclosed content. The present invention can be implemented or applied through other different specific embodiments, and various detailed descriptions in this specification can also be modified and changed in various ways without departing from the idea of the present invention based on different viewpoints and uses. Also, it should be noted in advance that the drawings of the present invention are only schematically shown and not drawn based on actual dimensions. The following embodiments are used to explain the technical content of the present invention in more detail, but the disclosed content is not intended to limit the protection scope of the present invention.

[0019] In the specification, terms such as "first", "second", "third", etc. may be used to describe various elements or signals, but it should be understood that these elements or signals should not be limited by these terms. These terms are mainly used to distinguish one element from another element or one signal from another signal. Also, the term "or" in this specification should be understood to include any one or a combination of multiple of the related listed items according to the actual situation.

[0020] The present invention will be described with reference to FIGS. 1 and 2. The present invention provides a method for converting a carbon source into glycolic acid. This method includes step S10 of providing a plasmid containing the gene sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, step S20 of introducing the plasmid into a strain to obtain a modified strain, and step S30 of supplying a carbon source to the modified strain and converting the carbon source into glycolic acid by the modified strain. More specifically, before step S10, it may further include a step of synthesizing a DNA sequence. In the step of synthesizing the DNA sequence, a sequence having gene code discrimination compatible with the target strain is artificially synthesized so that the target strain can recognize and generate the corresponding substance. In the present invention, a sequence having gene code discrimination compatible with Gram-negative bacteria may be artificially synthesized. For example, the strain according to the present invention may be Escherichia coli, Gluconobacter sp, Rhodococcus, or an oxidizing bacterium. However, the above examples are merely realizable examples and do not limit the present invention.

[0021] In one embodiment of the present invention, SEQ ID NO:1 is a gene encoding alcohol dehydrogenase (EGADH), or a gene having a sequence identity of 80% or more with SEQ ID NO:1 and having EGADH activity; SEQ ID NO:2 is a gene encoding aldehyde dehydrogenase (EGAlDH), or a gene having a sequence identity of 80% or more with SEQ ID NO:2 and having EGAlDH activity; SEQ ID NO:3 is a gene encoding vitreoscilla hemoglobin (VHb), or a gene having a sequence identity of 80% or more with SEQ ID NO:3 and having VHb activity.

[0022]

[0023] In the process of converting genes into proteins in each species of organism, even if DNA can be transcribed into RNA due to the different effects of codons, the conversion of the gene to protein will not be successful without the corresponding tRNA. Therefore, in this invention, the coding recognition of artificially synthesized genes is adapted to the target bacterial strain by optimizing the coding sequences of EGADH, EGA1DH, and VHb. In other words, the gene sequences according to the present invention are optimized to match the tRNA corresponding to the target bacterial strain. Referring to Figures 3, 4A, 4B, and 5, these figures are schematic diagrams showing a comparison between SEQ ID NO:1 according to the present invention and an existing EGADH gene sequence, a schematic diagram showing a comparison between SEQ ID NO:2 according to the present invention and an existing EGA1DH gene sequence, and a schematic diagram showing a comparison between SEQ ID NO:3 according to the present invention and an existing VHb gene sequence, respectively.

[0024] Furthermore, one factor influencing codon optimization is the difference in ribosome residence times between sequences. To optimize ribosome residence time, codons with short residence times are aligned, meaning that ribosomes do not terminate on extremely long codons (related to translation rate). Such codons tend to have a low GC content (GC%).

[0025] In one embodiment of the present invention, the GC% of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 are all 50-60%. Specifically, the length of the original EGADH sequence and the optimized EGADH sequence is 1032 bp, the GC% of the original EGADH sequence is approximately 63.57%, and the GC% of the optimized EGADH sequence is approximately 56.2%. The length of the original EGADH sequence and the optimized EGADH sequence is 2316 bp, the GC% of the original EGADH sequence is approximately 64.77%, and the GC% of the optimized EGADH sequence is approximately 56.87%. The length of the original VHb sequence and the optimized VHb sequence is 441 bp, the GC% of the original VHb sequence is approximately 45.35%, and the GC% of the optimized VHb sequence is approximately 52.83%.

[0026] Since the bond between AT and GC is primarily a hydrogen bond, a higher bond energy leads to greater stability during replication and, consequently, a lower error rate. In other words, GC% is related to the stability of subsequent gene replication and the process of converting genes into proteins. As can be understood from the above, the specific GC% of the optimized gene sequence according to the present invention contributes to achieving both ribosome residence time and stability in transcription and translation.

[0027] In step S20, plasmids may be introduced into the bacterial strain by methods such as gene gun, electroporation, microinjection, or chemical transfection. Preferably, plasmids are introduced into the bacterial strain by electroporation or chemical transfection. Electroporation involves applying an electric current to the bacterial strain for a very short time (microseconds to milliseconds), placing the strain in a high-voltage, low-capacity environment. This creates a potential difference in the cell membrane, causing a change in the cell membrane structure. As a result, the cell membrane is compressed and thinned, creating countless tiny pores, allowing the plasmid to pass through the cell membrane and enter the cells of the bacterial strain. In one embodiment of the present invention, to obtain the maximum number of colonies, the electroporation treatment is preferably performed at a voltage of 0.5kV for 10msec, more preferably at a voltage of 1.5kV for 5msec, and even more preferably at a voltage of 1.0kV for 5msec. In another embodiment of the present invention, the electroporation method may be performed using a MicroPulser Electroporator instrument and in accordance with the BIORAD standard protocol (see section 5 high efficiency electroporation of E. coli).

[0028] Chemical transfection methods involve using a vector to deliver a plasmid and introducing a foreign gene into the cell by direct transmembrane or membrane fusion. For example, the vector may be a liposome used to encapsulate the foreign DNA, which can then fuse with the cell membrane after being taken up into the cell, thereby releasing the foreign DNA inside to enter the cell. Thus, in the present invention, the modified strain can produce at least alcohol dehydrogenase and aldehyde dehydrogenase, and a single strain can convert a carbon source to glycolic acid.

[0029] In step S30, a carbon source is supplied to the modified strain, and the modified strain converts the carbon source into glycolic acid. In the present invention, the carbon source may be ethylene glycol. Specifically, the ethylene glycol may be ethylene glycol produced by the conversion of carbon dioxide by modified cyanobacteria, ethylene glycol decomposed by PET recycling (by decomposition with ethylene glycol, discarded PET can be decomposed into dimethyl terephthalate and ethylene glycol), and ethylene glycol produced by any chemical process.

[0030] Furthermore, the modified strain according to the present invention can produce alcohol dehydrogenase and aldehyde dehydrogenase, thereby converting ethylene glycol to glycolaldehyde, and further converting glycolaldehyde to glycolic acid. The process of converting ethylene glycol to glycolic acid using the modified strain according to the present invention is shown in General Formula 1 below. [ka]

[0031] [First Embodiment] In the first embodiment of the present invention, Escherichia coli was modified as a strain, and the constructed plasmids pAAL-122 and pAALV-122 were introduced into the E. coli host E. coli DH5a by chemical transfection. Here, the difference between pAAL-122 and pAALV-122 is that pAAL-122 does not contain the VHb gene sequence. Subsequently, the strains were screened using an antibiotic (LB solid medium with 30 mg / mL kanamycin), and modified strains that yielded SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 upon successful homologous recombination were screened. The single strains selected on the solid medium were placed in 4 mL of LB + kanamycin medium and cultured with shaking at 170 rpm in a 37°C incubator. On a different day, the initial strain concentration OD was measured. 600The OD was adjusted to approximately 0.05, and the culture was expanded to 50 mL of medium. Samples were taken over time to determine the strain concentration OD. 600 The results were analyzed, and when the bacterial strain grew in the early logarithmic growth phase (24h), a conversion test was performed by adding 50g / L ethylene glycol, and the amount of glycolic acid converted was analyzed by HPLC. The results are shown in Figure 6.

[0032] [Second Example] In the second embodiment of the present invention, Gluconobacter sp. was modified as a strain, and the constructed plasmids pAAL-122 and pAALV-122 were introduced into the Gluconobacter by electroporation. Strains were then screened using a 5 mg / mL kanamycin YPS solid medium (yeast extract 5 g / L, peptone 3 g / L, sorbitol 60 g / L, (NH4)2SO4 5 g / L, MgSO4.7H2O 5 g / L). The single strains selected on the solid medium were placed in 4 mL of YPS + kanamycin medium and cultured with shaking at 170 rpm in a 28°C incubator. On a later date, the initial strain concentration OD was measured. 600 The OD was adjusted to approximately 0.05, and the culture was expanded to 50 mL of medium. Samples were taken over time to determine the strain concentration OD. 600 The solution was analyzed, and a conversion test was conducted by adding 50 g / L of ethylene glycol at 24 hours. The amount of glycolic acid converted was analyzed by HPLC, and the results are shown in Figure 7.

[0033] Notably, the modified strain according to the present invention has the ability to produce bacterial hemoglobin. As shown in Table 1 below, by introducing the hemoglobin-like protein gene (VHb), the expression levels of EGADH and EGAlDH in the strain can be increased, and consequently, the yield of ethylene glycol converted to glycolic acid is improved. In this specification, yield refers to the amount of product that can be converted per liter of culture medium per unit time. Conversion rate refers to the number of moles of conversion product divided by the number of moles of initial raw materials. Unless otherwise specified, % referred to in this specification refers to mole percentages.

[0034] [Table 1] Plasmid pAAL-122: It possesses EGADH and EGAlDH, but lacks VHb. Plasmid pAALV-122: Contains EGADH, EGAlDH, and VHb.

[0035] Furthermore, the present invention can also improve the efficiency of glycolic acid production by oxidation of ethylene glycol by using immobilization technology for modified strains. As shown in Figure 8, the present invention provides a method for producing glycolic acid by immobilization technology, comprising the steps of: providing a modified strain containing the gene sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 (step S100); preparing a polyvinyl alcohol (PVA) bacterial pellet containing the modified strain (step S200); and supplying a carbon source to the polyvinyl alcohol bacterial pellet and converting the carbon source to glycolic acid using the modified strain (step S300). The preparation of the polyvinyl alcohol bacterial pellet involves preparing a 1% to 20% polyvinyl alcohol solution, preferably a 10% to 15% polyvinyl alcohol solution.

[0036] The procedure for immobilizing the modified strain is as described in Examples 3 to 6. For Examples 3 to 6, strains into which Gluconobacter sp. pAALV-122 was introduced were used.

[0037] [Third Embodiment] In the third embodiment of the present invention, the procedure for immobilizing the modified strain involved weighing 2 g of polyvinyl alcohol and 16 g of water to prepare a 10% PVA solution. Next, the 10% PVA solution was heated to 80°C or higher and maintained for 30 minutes to ensure complete dissolution of the PVA. After the PVA was completely dissolved in the water, it was cooled to 37°C. Subsequently, 2 g of concentrated bacterial solution (bacterial cell concentration CDW 17.32 g / L) was added to the PVA solution to obtain a 10% PVA bacterial solution. At this time, the PVA solution was considered 100%, and the concentration of the added bacterial solution was 10%. The higher the bacterial strain content, the better the conversion efficiency. In the preferred embodiment, a bacterial solution with a concentration of 15% may be prepared. In addition, 120 mL of 4% saturated boric acid solution and 50 mL of 0.5 M phosphate solution (Na2HPO4, NaH2PO4) were prepared. Using a 10 mL syringe, slowly drop the 10% PVA bacterial solution into a 4% saturated boric acid solution, allow it to solidify and mold for 1 hour, then remove it, drain the water, and immerse it in a 0.5 M phosphate solution for 1 hour to form 10% PVA bacterial balls. Finally, immerse the bacterial balls directly in the phosphate solution and store at 4°C.

[0038] [Fourth embodiment] In the fourth embodiment of the present invention, the procedure for immobilizing the modified strain involved weighing 2 g of polyvinyl alcohol, 0.2 g of alginic acid (SA), and 15.8 g of water to prepare a 10% PVA-1% SA solution. Next, the 10% PVA-1% SA solution was heated to 80°C or higher and maintained for 30 minutes to ensure complete dissolution of the PVA-SA. After the PVA-SA was completely dissolved in water, it was cooled to 37°C. Subsequently, 2 g of concentrated bacterial solution (bacterial cell concentration CDW 17.32 g / L) was added to the 10% PVA-1% SA solution to obtain a 10% PVA-1% SA bacterial solution. In addition, 120 mL of a mixed solution of 4% saturated boric acid and 1% calcium chloride and 50 mL of a 0.5 M phosphate solution (Na2HPO4, NaH2PO4) were prepared. Using a 10 mL syringe, slowly drop the 10% PVA-1% SA bacterial solution into a mixed solution of 4% saturated boric acid and 1% calcium chloride, allow it to solidify and mold for 1 hour, then remove it, drain the water, and immerse it in a 0.5 M phosphate solution for 1 hour to form 10% PVA-1% SA bacterial balls. Finally, immerse the bacterial balls directly in the phosphate solution and store at 4°C.

[0039] [Fifth Example] In the fifth embodiment of the present invention, the procedure for immobilizing the modified strain involved weighing 0.3 g of alginic acid and 17.7 g of water to prepare a 1.5% SA solution. Next, the 1.5% SA solution was heated to over 80°C and maintained for 30 minutes to ensure that the SA was dissolved. After the SA was completely dissolved in the water, it was cooled to 37°C. Subsequently, 2 g of concentrated bacterial solution (bacterial cell concentration CDW 17.32 g / L) was added to the 1.5% SA solution to obtain a 1.5% SA bacterial solution. In addition, 120 mL of 2% calcium chloride was prepared. Using a 10 mL syringe, the 1.5% SA bacterial solution was slowly added dropwise to the 2% calcium chloride solution, allowed to solidify for 1 hour, washed the bacterial balls, and immersed in the aqueous solution for storage at 4°C.

[0040] [Sixth Example] In the sixth embodiment of the present invention, the procedure for immobilizing the modified strain involved weighing 2 g of polyvinyl alcohol and 16 g of water to prepare a 10% PVA solution. Next, the 10% PVA solution was heated to over 80°C and maintained for 30 minutes to ensure complete dissolution of the PVA. After the PVA was completely dissolved in the water, it was cooled to 37°C. Subsequently, 2 g of concentrated bacterial solution (bacterial cell concentration CDW 17.32 g / L) was added to the PVA solution to obtain a 10% PVA bacterial solution. The 10% PVA bacterial solution was poured into a mold, the mold was frozen at -20°C for 2 to 12 hours depending on the size of the mold, then removed and thawed by immersion in a water bath at room temperature for 30 to 60 minutes, and this was repeated 5 times to form a mass of bacteria. Finally, the mass of bacteria was thawed, cut into pieces, and stored at 4°C.

[0041] Modified strain-immobilized particles from Examples 3-6 were taken out at the same wet weight and tested at different hydraulic retention times (HRT). The feed consisted of a 50 g / L ethylene glycol solution + 5% YYS (yeast extract 5 g / L, peptone 3 g / L, sorbitol 60 g / L, (NH4)2SO4 5 g / L, MgSO4.7H2O 5 g / L), with a total reaction volume of 400 ml and an air flow rate of 0.5 vvm. vvm represents the ratio of air flow rate per minute to the actual liquid volume in the tank (air volume / culture volume / min). Hydraulic retention time indicates the average time the reacting liquid remains in the reactor. That is, volume / inlet flow rate is the hydraulic retention time. In this experiment, the reacting liquid is an ethylene glycol solution, and air refers to a gas containing oxygen.

[0042] [Table 2]

[0043] As shown in Table 2 above, comparing Examples 3 to 5, Example 4, which used a mixture of 10% PVA and 1% SA, yielded a higher yield than Examples 3 and 5, which used either PVA alone or SA alone. Here, yield indicates the amount of glycolic acid produced per unit time per unit volume. In other words, by using a mixture of PVA and SA, the efficiency of converting ethylene glycol to glycolic acid can be improved. It should be noted that differences in process operation also affect the efficiency of converting ethylene glycol to glycolic acid. Notably, comparing Examples 3 and 6, the bacterial mass produced by the freeze-thaw cycle did not need to be immersed in boric acid or phosphoric acid solution, resulting in relatively less damage to the bacterial cells. Therefore, even when using the same 10% PVA, the bacterial mass produced by the freeze-thaw cycle had the highest efficiency in converting ethylene glycol to glycolic acid.

[0044] [Beneficial effects from the examples] One of the beneficial effects of the present invention is that the method for producing glycolic acid by immobilization technology provided in the present invention can improve the efficiency of glycolic acid production by oxidation of ethylene glycol by utilizing strain immobilization technology, through the technical means of "providing a modified strain containing the gene sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3" and "preparing a polyvinyl alcohol bacterial pellet containing the modified strain."

[0045] Furthermore, by introducing the hemoglobin-like protein gene (VHb) into the modified strain according to the present invention, the expression levels of EGADH and EGAlDH in the strain can be increased, and consequently, the yield of ethylene glycol converted to glycolic acid is improved. Since the modified strain according to the present invention can produce hemoglobin-like protein, introducing 0.3vvm to 1vvm of air during the reaction process contributes to further improving the yield of ethylene glycol converted to glycolic acid.

[0046] Furthermore, this invention employs biomodification strategies to establish a glycolic acid production / purification process using bioconversion methods, with ethylene glycol produced by cyanobacteria through carbon dioxide conversion, PET recycling and decomposition, and chemical plant processes as raw materials. This allows for the acquisition of high-purity glycolic acid raw materials, and further improves the efficiency of glycolic acid production through the oxidation of ethylene glycol by utilizing strain immobilization technology.

[0047] The information disclosed herein represents only preferred embodiments of the present invention and does not limit the scope of the claims. Accordingly, all equivalent technical modifications made using the specification and drawings of the present invention are included within the scope of the claims. [Explanation of symbols]

[0048] S10~S30, S100~S300: Step

Claims

1. To provide a modified strain containing the gene sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, To prepare polyvinyl alcohol bacteria pellets containing the modified strain, The polyvinyl alcohol bacteria pellet and the auxiliary solution are mixed, a carbon source is supplied to the polyvinyl alcohol bacteria pellet, and the modified strain is used to convert the carbon source into glycolic acid. Includes, Air is introduced to the polyvinyl alcohol bacterial pellet while supplying the carbon source. A method for producing glycolic acid by an immobilization technique characterized by the above.

2. The preparation of the polyvinyl alcohol bacterial pellets is A 1% to 20% polyvinyl alcohol solution is prepared, and the modified strain is added to the polyvinyl alcohol solution and mixed uniformly to obtain a polyvinyl alcohol bacterial solution. Provide saturated boric acid solution and phosphoric acid solution, The polyvinyl alcohol bacterial solution is dropped into the saturated boric acid solution to obtain a semi-finished polyvinyl alcohol bacterial pellet. The polyvinyl alcohol bacterial pellet semi-finished product is removed, drained, and placed in the phosphoric acid solution and left for a predetermined time to obtain the polyvinyl alcohol bacterial pellet. including, A method for producing glycolic acid by the immobilization technique described in claim 1.

3. The preparation of the polyvinyl alcohol bacterial pellets further includes preparing 0.5% to 1.5% alginic acid. A method for producing glycolic acid by the immobilization technique described in claim 2.

4. The aforementioned predetermined time is 30 minutes to 2 hours. A method for producing glycolic acid by the immobilization technique described in claim 2.

5. The concentration of the boric acid solution is 1% to 5%, and the concentration of the phosphoric acid solution is 0.1 M to 1 M. A method for producing glycolic acid by the immobilization technique described in claim 2.

6. The carbon source is ethylene glycol, and the air permeability is 0.3 vvm to 1 vvm. A method for producing glycolic acid by the immobilization technique described in claim 1.

7. The aforementioned modified strains are modified Escherichia coli, Gluconobacter sp., Rhodococcus, or oxidizing bacteria. A method for producing glycolic acid by the immobilization technique described in claim 1.

8. The aforementioned auxiliary solution contains yeast extract, peptone, sorbitol, (NH ４ ) ２ SO ４ and MgSO ４ 7H ２ Composed of O, A method for producing glycolic acid by the immobilization technique described in claim 1.

9. The GC% for SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 are all between 50% and 60%. A method for producing glycolic acid by the immobilization technique described in claim 1.

10. The modified strain has the ability to produce alcohol dehydrogenase, aldehyde dehydrogenase, and bacterial hemoglobin. A method for producing glycolic acid by the immobilization technique described in claim 1.

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