KR20130119231A - Method for gamma-aminobutyric acid with glycerol as substrate by biotransformation process - Google Patents

Abstract

PURPOSE: A manufacturing method of gamma aminobutyric acid by biotransformation using glycerol as a substrate is provided to manufacture the gamma aminobutyric acid economically and eco-friendly since the glycerol produced as a by-product in biodiesel production is converted into high-priced gamma aminobutyric acid (GABA). CONSTITUTION: A method for manufacturing gamma aminobutyric acid (GABA) from glycerol using glutamate decarboxylase variant. Provided is a method of manufacturing the gamma aminobutyric acid (GABA) it is done by the substrate. The glutamate decarboxylase variant has an activity at a neutral pH. The glutamate decarboxylase variant has the sequence number of 1 to 3. The glutamate decarboxylase variant is Escherichia coli, Lactobacillus, or Bacillus.

Description

Method for producing gamma-aminobutyric acid by biotransformation process using glycerol as a substrate {method for gamma-aminobutyric acid with glycerol as substrate by biotransformation process}

The present invention relates to a method for preparing gamma-aminobutyl acid (GABA) using glycerol as a substrate by solving the limitation of the enzyme's active pH range.

Gamma-aminobutyl acid (GABA) is an inhibitory neurotransmitter of non-protein amino acids consisting of four carbons, which stabilizes neurons, relieves stress, improves memory, lowers blood pressure, reduces depression, prevents stroke and dementia It is known to have an effect on obesity, menopausal disorders, etc., and is a global food material that has been found to have an effect of inhibiting metastasis and proliferation of stroke, colon cancer and colon cancer cells. There is also increasing interest in biomass-derived materials in recent years, and GABA is an important monomer for obtaining polyamide 4, a useful plastic that can be produced from biomass.

Industrial methods for GABA production include synthesis, extraction and bioconversion. In the case of the synthesis method, the solvent used is mainly toxic, and in the case of the extraction method, a large amount of waste is generated, and the manufacturing cost of GABA is high. Bioconversion is a method of producing GABA by decarboxylation of L-glutamate by using an isolated enzyme or a whole cell containing the enzyme as a biocatalyst. The price of biologically produced L-glutamate is not relatively high compared to GABA, but is still not understood as a cheap raw material.

The enzyme used for the conversion to GABA is pyridoxal 5-phosphate (PLP) -dependent glutamate decarboxylase (GAD) (EC 4.1.1.15), which is derived from microorganisms such as lactic acid bacteria, Escherichia coli, and Bacillus. Used. Bacterial glutamate decarboxylase has a pH range of 3.5 to 6.0, an optimal pH of 3.8 to 4.6, and when the pH is below 3.5 or above 6.0, little reaction occurs. That is, it can be said that glutamate decarboxylase has no activity under normal culture conditions.

Glycerol, which is produced as a by-product from the production of biodiesel, is currently used as a raw material for cosmetics and does not generate much use, which is an obstacle to the overall development of the biodiesel industry. As a result, studies on using glycerol as a carbon source for microbial culture, such as Escherichia coli, are underway, and its success is expected to have a great influence on the commercialization of biodiesel.

KR 10-2011-0082052

Accordingly, the present inventors have developed a method of converting glycerol to GABA by overexpressing glutamate decarboxylase variants in E. coli so that glutamate can be converted to GABA at neutral pH.

The present invention relates to a method for preparing gamma-aminobutyl acid (GABA) using glycerol as a substrate, using a glutamate decarboxylase variant which is also active in neutral acidity.

The present invention enables the production of economical and environmentally friendly gamma-aminobutyl acid by converting glycerol produced as a by-product from biodiesel production into expensive GABA. In addition, it can improve the overall economic efficiency of the biodiesel industry, thereby improving the price competitiveness of biodiesel.

Figure 1 shows the growth curve of each strain in culture in glycerol culture.
Figure 2 relates to the results of culturing each strain in glycerol culture solution and analyzed the GABA contained in the culture solution with Gabase.

The present invention relates to a method for preparing gamma-aminobutyl acid (GABA) by a bioconversion process using glycerol as a substrate. More specifically, the present invention relates to the conversion of glycerol to glutamate and the preparation of GABA in glutamate by using glutamate decarboxylase variants which also show activity at neutral pH.

Glutamate dicarboxylase (GAD) in the present invention is a pyridoxal 5-phosphate (PLP) -dependent intracellular enzyme, which catalyzes the irreversible decarboxylation of L-glutamate to γ-aminobutyrate. GAD plays an important role in the vertebrate central nervous system by being involved in GABA production. GAD is considered to be unique among the enzymes involved in the synthesis of neurotransmitters, because both the substrate and the product of GAD are neurotransmitters and have opposite effects. In the nervous system, L-glutamate acts as an inhibitor of stimulant GABA, each of which is involved in glutamine producing synapses and GABA producing synapses.

Glutamate dicarboxylase of the present invention is not limited thereto, but may be derived from microorganisms such as E. coli, lactic acid bacteria, Bacillus. GABA can be produced through bioconversion using glutamate decarboxylase isolated and purified from microorganisms.

Glutamate decarboxylase isolated and purified from the microorganism is a host cell suitable for not only purifying the glutamate decarboxylase protein originally possessed by the microorganism, but also the glutamate decarboxylase gene possessed by the microorganism. Cloned and expressed in the host cells, and the purified glutamate decarboxylase protein from the host cell.

The glutamate decarboxylase variant of the present invention is not limited thereto as long as it has activity at neutral pH, but the glutamate 89 of glutamate decarboxylase β is glutamine, and the 15 amino acids at the carboxy terminus are removed. Variant (SEQ ID NO: 1), complex variant wherein glutamate no. 89 is glutamine, histidine no. 465 is substituted with alanine (SEQ ID NO: 2), complex variant no. 2 amino acid at the carboxy terminus is removed Number 3).

The present invention introduces the above variants into E. coli, and the E. coli is cultured in a glycerol culture and at the same time overexpressing the mutant glutamate decarboxylase so that GABA is produced.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example  1> In Glycerol GABA Production of

E. coli-derived glutamate decarboxylase (WT) and a variant (E89Q / ΔC) with modifications in glutamate No. 89 and carboxy terminus 15 amino acid sequences were cloned into pET-28b to transform E. coli BL21 (DE3). In addition, E. coli BL21 (DE3) having no plasmid was used as a control and cultured in a minimal medium of M9. In this case, 0.4% glycerol was used as the carbon source, and ammonium sulfate salt was used as the nitrogen source. After each E. coli was grown and reached the mid-log phase, protein overexpression was induced for 12 hours using 0.1 mM IPTG (isopropyl β-D-1-thiogalactopyranoside). In order to accurately measure the effect of protein overexpression, after overexpression was induced for 12 hours, the cells were recovered, suspended again in the same medium containing 0.1 mM IPTG, secondary cultured, and cell growth was observed (FIG. 1).

As a result, Escherichia coli overexpressing glutamate decarboxylase variant (E89Q / ΔC), which is active at neutral acidity, showed a significantly lower growth rate than E. coli overexpressing native glutamate decarboxylase (WT). This is because the intracellular glutamate concentration is lowered and negatively affects cell growth as the glycerol introduced into the cell is converted to glutamate and then converted to GABA by E89Q / ΔC having activity at neutral pH. This is further evident when the 50 mM addition of glutamate to the minimal medium shows similar growth rates for E. coli overexpressing the native form and E89Q / ΔC.

As a result of recovering and analyzing the medium after the second culture, E. coli overexpressing E89Q / ΔC produced GABA and added to the medium without adding glutamate (FIG. 2). The synthesis of GABA was plurally examined by TLC and gabase analysis.

<110> dongguk university <120> Method for gamma-aminobutyric acid with glycerol as substrate by          biotransformation process <130> P120387 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 471 <212> PRT <213> glutamate decarboxylase mutants <400> 1 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro   1 5 10 15 Arg Gly Ser His Met Asp Lys Lys Gln Val Thr Asp Leu Arg Ser Glu              20 25 30 Leu Leu Asp Ser Arg Phe Gly Ala Lys Ser Ile Ser Thr Ile Ala Glu          35 40 45 Ser Lys Arg Phe Pro Leu His Glu Met Arg Asp Asp Val Ala Phe Gln      50 55 60 Ile Ile Asn Asp Glu Leu Tyr Leu Asp Gly Asn Ala Arg Gln Asn Leu  65 70 75 80 Ala Thr Phe Cys Gln Thr Trp Asp Asp Glu Asn Val His Lys Leu Met                  85 90 95 Asp Leu Ser Ile Asn Lys Asn Trp Ile Asp Lys Glu Gln Tyr Pro Gln             100 105 110 Ser Ala Ala Ile Asp Leu Arg Cys Val Asn Met Val Ala Asp Leu Trp         115 120 125 His Ala Pro Ala Pro Lys Asn Gly Gln Ala Val Gly Thr Asn Thr Ile     130 135 140 Gly Ser Ser Glu Ala Cys Met Leu Gly Gly Met Ala Met Lys Trp Arg 145 150 155 160 Trp Arg Lys Arg Met Glu Ala Ala Gly Lys Pro Thr Asp Lys Pro Asn                 165 170 175 Leu Val Cys Gly Pro Val Gln Ile Cys Trp His Lys Phe Ala Arg Tyr             180 185 190 Trp Asp Val Glu Leu Arg Glu Ile Pro Met Arg Pro Gly Gln Leu Phe         195 200 205 Met Asp Pro Lys Arg Met Ile Glu Ala Cys Asp Glu Asn Thr Ile Gly     210 215 220 Val Val Pro Thr Phe Gly Val Thr Tyr Thr Gly Asn Tyr Glu Phe Pro 225 230 235 240 Gln Pro Leu His Asp Ala Leu Asp Lys Phe Gln Ala Asp Thr Gly Ile                 245 250 255 Asp Ile Asp Met His Ile Asp Ala Ala Ser Gly Gly Phe Leu Ala Pro             260 265 270 Phe Val Ala Pro Asp Ile Val Trp Asp Phe Arg Leu Pro Arg Val Lys         275 280 285 Ser Ile Ser Ala Ser Gly His Lys Phe Gly Leu Ala Pro Leu Gly Cys     290 295 300 Gly Trp Val Ile Trp Arg Asp Glu Glu Ala Leu Pro Gln Glu Leu Val 305 310 315 320 Phe Asn Val Asp Tyr Leu Gly Gly Gln Ile Gly Thr Phe Ala Ile Asn                 325 330 335 Phe Ser Arg Pro Ala Gly Gln Val Ile Ala Gln Tyr Tyr Glu Phe Leu             340 345 350 Arg Leu Gly Arg Glu Gly Tyr Thr Lys Val Gln Asn Ala Ser Tyr Gln         355 360 365 Val Ala Ala Tyr Leu Ala Asp Glu Ile Ala Lys Leu Gly Pro Tyr Glu     370 375 380 Phe Ile Cys Thr Gly Arg Pro Asp Glu Gly Ile Pro Ala Val Cys Phe 385 390 395 400 Lys Leu Lys Asp Gly Glu Asp Pro Gly Tyr Thr Leu Tyr Asp Leu Ser                 405 410 415 Glu Arg Leu Arg Leu Arg Gly Trp Gln Val Pro Ala Phe Thr Leu Gly             420 425 430 Gly Glu Ala Thr Asp Ile Val Val Met Arg Ile Met Cys Arg Arg Gly         435 440 445 Phe Glu Met Asp Phe Ala Glu Leu Leu Leu Glu Asp Tyr Lys Ala Ser     450 455 460 Leu Lys Tyr Leu Ser Asp His 465 470 <210> 2 <211> 486 <212> PRT <213> glutamate decarboxylase mutants <400> 2 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro   1 5 10 15 Arg Gly Ser His Met Asp Lys Lys Gln Val Thr Asp Leu Arg Ser Glu              20 25 30 Leu Leu Asp Ser Arg Phe Gly Ala Lys Ser Ile Ser Thr Ile Ala Glu          35 40 45 Ser Lys Arg Phe Pro Leu His Glu Met Arg Asp Asp Val Ala Phe Gln      50 55 60 Ile Ile Asn Asp Glu Leu Tyr Leu Asp Gly Asn Ala Arg Gln Asn Leu  65 70 75 80 Ala Thr Phe Cys Gln Thr Trp Asp Asp Glu Asn Val His Lys Leu Met                  85 90 95 Asp Leu Ser Ile Asn Lys Asn Trp Ile Asp Lys Glu Gln Tyr Pro Gln             100 105 110 Ser Ala Ala Ile Asp Leu Arg Cys Val Asn Met Val Ala Asp Leu Trp         115 120 125 His Ala Pro Ala Pro Lys Asn Gly Gln Ala Val Gly Thr Asn Thr Ile     130 135 140 Gly Ser Ser Glu Ala Cys Met Leu Gly Gly Met Ala Met Lys Trp Arg 145 150 155 160 Trp Arg Lys Arg Met Glu Ala Ala Gly Lys Pro Thr Asp Lys Pro Asn                 165 170 175 Leu Val Cys Gly Pro Val Gln Ile Cys Trp His Lys Phe Ala Arg Tyr             180 185 190 Trp Asp Val Glu Leu Arg Glu Ile Pro Met Arg Pro Gly Gln Leu Phe         195 200 205 Met Asp Pro Lys Arg Met Ile Glu Ala Cys Asp Glu Asn Thr Ile Gly     210 215 220 Val Val Pro Thr Phe Gly Val Thr Tyr Thr Gly Asn Tyr Glu Phe Pro 225 230 235 240 Gln Pro Leu His Asp Ala Leu Asp Lys Phe Gln Ala Asp Thr Gly Ile                 245 250 255 Asp Ile Asp Met His Ile Asp Ala Ala Ser Gly Gly Phe Leu Ala Pro             260 265 270 Phe Val Ala Pro Asp Ile Val Trp Asp Phe Arg Leu Pro Arg Val Lys         275 280 285 Ser Ile Ser Ala Ser Gly His Lys Phe Gly Leu Ala Pro Leu Gly Cys     290 295 300 Gly Trp Val Ile Trp Arg Asp Glu Glu Ala Leu Pro Gln Glu Leu Val 305 310 315 320 Phe Asn Val Asp Tyr Leu Gly Gly Gln Ile Gly Thr Phe Ala Ile Asn                 325 330 335 Phe Ser Arg Pro Ala Gly Gln Val Ile Ala Gln Tyr Tyr Glu Phe Leu             340 345 350 Arg Leu Gly Arg Glu Gly Tyr Thr Lys Val Gln Asn Ala Ser Tyr Gln         355 360 365 Val Ala Ala Tyr Leu Ala Asp Glu Ile Ala Lys Leu Gly Pro Tyr Glu     370 375 380 Phe Ile Cys Thr Gly Arg Pro Asp Glu Gly Ile Pro Ala Val Cys Phe 385 390 395 400 Lys Leu Lys Asp Gly Glu Asp Pro Gly Tyr Thr Leu Tyr Asp Leu Ser                 405 410 415 Glu Arg Leu Arg Leu Arg Gly Trp Gln Val Pro Ala Phe Thr Leu Gly             420 425 430 Gly Glu Ala Thr Asp Ile Val Val Met Arg Ile Met Cys Arg Arg Gly         435 440 445 Phe Glu Met Asp Phe Ala Glu Leu Leu Leu Glu Asp Tyr Lys Ala Ser     450 455 460 Leu Lys Tyr Leu Ser Asp His Pro Lys Leu Gln Gly Ile Ala Gln Gln 465 470 475 480 Asn Ser Phe Lys Ala Thr                 485 <210> 3 <211> 484 <212> PRT <213> glutamate decarboxylase mutants <400> 3 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro   1 5 10 15 Arg Gly Ser His Met Asp Lys Lys Gln Val Thr Asp Leu Arg Ser Glu              20 25 30 Leu Leu Asp Ser Arg Phe Gly Ala Lys Ser Ile Ser Thr Ile Ala Glu          35 40 45 Ser Lys Arg Phe Pro Leu His Glu Met Arg Asp Asp Val Ala Phe Gln      50 55 60 Ile Ile Asn Asp Glu Leu Tyr Leu Asp Gly Asn Ala Arg Gln Asn Leu  65 70 75 80 Ala Thr Phe Cys Gln Thr Trp Asp Asp Glu Asn Val His Lys Leu Met                  85 90 95 Asp Leu Ser Ile Asn Lys Asn Trp Ile Asp Lys Glu Gln Tyr Pro Gln             100 105 110 Ser Ala Ala Ile Asp Leu Arg Cys Val Asn Met Val Ala Asp Leu Trp         115 120 125 His Ala Pro Ala Pro Lys Asn Gly Gln Ala Val Gly Thr Asn Thr Ile     130 135 140 Gly Ser Ser Glu Ala Cys Met Leu Gly Gly Met Ala Met Lys Trp Arg 145 150 155 160 Trp Arg Lys Arg Met Glu Ala Ala Gly Lys Pro Thr Asp Lys Pro Asn                 165 170 175 Leu Val Cys Gly Pro Val Gln Ile Cys Trp His Lys Phe Ala Arg Tyr             180 185 190 Trp Asp Val Glu Leu Arg Glu Ile Pro Met Arg Pro Gly Gln Leu Phe         195 200 205 Met Asp Pro Lys Arg Met Ile Glu Ala Cys Asp Glu Asn Thr Ile Gly     210 215 220 Val Val Pro Thr Phe Gly Val Thr Tyr Thr Gly Asn Tyr Glu Phe Pro 225 230 235 240 Gln Pro Leu His Asp Ala Leu Asp Lys Phe Gln Ala Asp Thr Gly Ile                 245 250 255 Asp Ile Asp Met His Ile Asp Ala Ala Ser Gly Gly Phe Leu Ala Pro             260 265 270 Phe Val Ala Pro Asp Ile Val Trp Asp Phe Arg Leu Pro Arg Val Lys         275 280 285 Ser Ile Ser Ala Ser Gly His Lys Phe Gly Leu Ala Pro Leu Gly Cys     290 295 300 Gly Trp Val Ile Trp Arg Asp Glu Glu Ala Leu Pro Gln Glu Leu Val 305 310 315 320 Phe Asn Val Asp Tyr Leu Gly Gly Gln Ile Gly Thr Phe Ala Ile Asn                 325 330 335 Phe Ser Arg Pro Ala Gly Gln Val Ile Ala Gln Tyr Tyr Glu Phe Leu             340 345 350 Arg Leu Gly Arg Glu Gly Tyr Thr Lys Val Gln Asn Ala Ser Tyr Gln         355 360 365 Val Ala Ala Tyr Leu Ala Asp Glu Ile Ala Lys Leu Gly Pro Tyr Glu     370 375 380 Phe Ile Cys Thr Gly Arg Pro Asp Glu Gly Ile Pro Ala Val Cys Phe 385 390 395 400 Lys Leu Lys Asp Gly Glu Asp Pro Gly Tyr Thr Leu Tyr Asp Leu Ser                 405 410 415 Glu Arg Leu Arg Leu Arg Gly Trp Gln Val Pro Ala Phe Thr Leu Gly             420 425 430 Gly Glu Ala Thr Asp Ile Val Val Met Arg Ile Met Cys Arg Arg Gly         435 440 445 Phe Glu Met Asp Phe Ala Glu Leu Leu Leu Glu Asp Tyr Lys Ala Ser     450 455 460 Leu Lys Tyr Leu Ser Asp His Pro Lys Leu Gln Gly Ile Ala Gln Gln 465 470 475 480 Asn Ser Phe Lys                

Claims (4)

A method for producing gamma-aminobutyl acid (GABA) using glycerol as a substrate using glutamate dicarboxylase variants. The method of claim 1, wherein said glutamate decarboxylase variant has activity at neutral acidity. The method of claim 1 or 2, wherein the glutamate decarboxylase variant is SEQ ID NO: 1 to 3. A method for preparing gamma-aminobutyl acid (GABA). The method of claim 1, wherein the glutamate decarboxylase variant is Escherichia coli, lactic acid bacteria or Bacillus.

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