KR100251523B1 - Gene encoding the heat-resistant glutamate racease derived from the high-temperature microbial bacillus strain, a symbiotic strain of the high-temperature absolute symbiotic Symbiobacterium, and a method for producing a heat-resistant glutamate racease using the same - Google Patents

Abstract

PURPOSE: Provided is a gene coding thermostable glutamateracemase(GluRA) originated from a thermophile Bacillus sp. strain, a symbiotic strain of thermophilic absolute symbiont, a Symbiobacterium. And a preparation method of thermostable glutamateracemase is also provided. The glutamateracemase has excellent activity and enzymatic stability at high temperature, and is thus useful as biological catalyst in preparation of D-glutamic acid. CONSTITUTION: A gene of glutamateracemase(GluRA) originated from a thermophile Bacillus sp. strain has base sequence represented by the sequence 1. The method for preparation of thermostable glutamateracemase is composed of the following steps of: (a) culturing microbes transformed with the expression vector pGSK23 to express GluRA; (b) heating stem cell eluate obtained from the expressed GluRA; and (c) separation GluRA by non ion exchange chromatography and nonhydrophilic chromatography.

Description

Gene encoding a heat-resistant glutamate racease derived from a high-temperature microbial bacillus strain, a symbiotic strain of the high-temperature absolute symbiotic Symbiobacterium, and a method for producing a heat-resistant glutamate racease using the same

The present invention is the thermophilic microorganism Bacillus (Bacillus sp.) La resistant strains of the glutamate-derived using the genetic code to him and prophylaxis azepin relates to a method for producing a heat-resistant Sema azepin la glutamate.

More specifically, the gene encoding the glutamate racemase (EC 5.1.1.3; hereinafter abbreviated as 'GluRA') and its base derived from the Bacillus strain, a symbiotic strain of the pyrogenic absolute symbiotic microorganism Symbiobacterium. The present invention relates to a sequence, an expression vector comprising the gene and an E. coli transformant, and a method for producing GluRA using the same, wherein the heat-resistant GluRA prepared according to the present invention has excellent activity and high enzyme stability even at high temperature. It can be usefully used as a biocatalyst in bioconversion process for preparation.

D-glutamic acid is widely used as a raw material for pharmaceuticals, bioactive substances, antibiotics and synthetic sweeteners, and as a substrate material for the synthesis of high value-added pharmaceutical products containing other D-amino acids. Since D-glutamic acid is complex and expensive, the demand is expected to increase rapidly if economical manufacturing methods can be developed and supplied at low prices.

Expensive D-glutamic acid preparation can be largely divided into chemical preparation and enzymatic preparation. The production of amino acids by the chemical preparation method has a difficulty in going through a complicated optical division process since the product is obtained in a racemic mixture of D, L-amino acids. On the other hand, D-glutamic acid synthesis using a biocatalyst has attracted attention as a low pollution clean technology because it can directly prepare optically pure D-glutamic acid and can reduce by-products that cause environmental pollution due to no side reactions.

The enzymatic method is a method for preparing D-glutamic acid directly from L-glutamic acid using GluRA as a biocatalyst, and using D-amino acid aminotransferase (EC 2.6.1.21) as a biocatalyst. And a method for producing D-glutamic acid from -ketoglutarate.

GluRA used in the enzymatic preparation is a lactic acid bacterium (N.Nakajima et al., Agric. Biol. Chem., 52, 3099-3104 (1988)), in ester Rica (G. Balico Venetianer and P., J. Bacteriol. , 175, 6571-6577 (1993)), genus Bacillus (L. Liu et al., Biochem. , 121, 1155-1161 (1997)) and genus Staphylococcus (MJ Pucci et al., J. Bacteriol. , 177, 336-342 (1995)) strains, and the like.

GluRA is an enzyme that directly prepares D-glutamic acid, one of the major cell wall components of bacteria, from L-glutamic acid and has high substrate specificity for L-glutamic acid. In the enzymatic synthesis of D-glutamic acid using GluRA, when an appropriate amount of GluRA is added to the reaction solution to which L-glutamic acid is added, L-glutamic acid is converted to D-glutamic acid to reach an equilibrium condition. After completion of the enzyme conversion reaction, glutamate decarboxylase (EC 5.1.1.3) can be added to the reaction solution to decompose the remaining L-glutamic acid remaining in the reaction solution to prepare optically pure D-glutamic acid.

In the bioconversion process using an enzyme as a biocatalyst, the stability of the biocatalyst is the most important factor in determining the productivity of the enzyme conversion synthesis reaction. The stability of biocatalyst is very important factor in productivity improvement in enzymatic process for producing high value-added medicinal amino acid D-glutamic acid and various kinds of D-amino acid using biocatalyst conversion reaction of heat-resistant GluRA. In general, thermophilic microorganisms that live in hot growing environments have heat-resistant enzymes that are stable to heat under the influence of high temperature extremes in the habitat. In addition to heat-stable enzymes, these heat-resistant enzymes have been reported to have stability to organic solvents, stability to pH and chemical modifiers. Therefore, the heat-resistant GluRA derived from the thermophilic microorganism has the stability of the above-mentioned biocatalysts, and thus can be usefully used as a biocatalyst in the development of D-glutamic acid and various kinds of D-amino acids.

Therefore, the present inventors searched for a new high temperature microorganism that produces heat-resistant GluRA from about 1,300 types of high temperature microorganisms separated from high temperature microbial habitats such as soil, wastewater, manure, geyser, etc. in Korea, and it is possible to grow at 65 ° C. A new high temperature microbial bacillus SK-1 strain (KCTC 0306BP), which produces heat resistant GluRA having very high thermal stability, has been isolated and filed (Korean Patent Application No. 97-15427).

The present inventors conducted research to develop a genetic engineering method for mass-producing heat-resistant GluRA derived from high-temperature microorganisms . As a result, we cloned the heat-resistant GluRA gene from the SK-1 strain of the high-temperature microorganism Bacillus and expressed an expression vector including the same. The present invention was completed by transforming an expression vector into E. coli and preparing a heat-resistant GluRA therefrom to confirm its activity.

The present invention is the thermophilic microorganism Bacillus (Bacillus sp.) Genes that code for the strain-derived heat-resistant GluRA and its base sequence, conversion expression vector and the E. coli material containing the gene and to provide a method for preparing GluRA using the same There is a purpose.

1 shows the construction of a recombinant plasmid vector pGSK 23 comprising a gene encoding a heat resistant glutamate racease,

Figure 2 shows the effect of temperature in the production of D- glutamic acid using the heat-resistant glutamate racease prepared according to the present invention.

In order to achieve the above object, the present invention provides a gene encoding the heat-resistant GluRA produced from the strain of the genus Bacillus strain and its nucleotide sequence.

In another aspect, the present invention provides an expression vector comprising the gene and E. coli transformants thereof.

In another aspect, the present invention provides a method for producing GluRA by transforming a microorganism with an expression vector containing a heat-resistant GluRA gene, culturing the transformant to obtain a crude cell eluate, and heat-treating the same, followed by chromatography. do.

The present invention also provides an amino acid sequence of the heat resistant GluRA from the Bacillus sp.

In another aspect, the present invention provides a thermophilic microorganism Bacillus sp. SK-1 applications that use the heat-resistant strain derived from biological GluRA catalytic conversion of D- glutamic acid The reaction for obtaining a D- glutamic acid as substrate the L- glutamic acid.

Hereinafter, the present invention will be described in detail.

The invention of suitable size by cutting the thermophilic microorganism strain of Bacillus in order to obtain a gene that code the heat GluRA produced from sp, the thermophilic part the total chromosome was separated from the cells obtained by culturing a Bacillus strain with the restriction enzyme DNA Isolate fragments.

Specifically, the present invention is the high temperatureBacillusgenus Thermophilic Isolated from Korean Soil as StrainsBacillusGenus SK-1 strain (KCTC 0306BP) is used.

A clone containing a gene encoding heat-resistant GluRA was isolated by transforming a D-glutamic acid-required mutant Escherichia coli strain with a DNA fragment of the Bacillus sp . Strain prepared above, which did not die even in a medium not containing D-glutamic acid. do.

Specifically, Escherichia coli WM335 is used as the D-glutamic acid requirement mutant Escherichia coli strain. Escherichia coli WM335 is a D-glutamic acid-required mutant strain that loses the function of synthesizing D-glutamic acid constituting the cell wall and cannot grow in medium without D-glutamic acid (Lugtenberg, EJJ et al., J. Bacteriol. 114, 499-506, (1973).

Clonal DNA containing the gene encoding the heat-resistant GluRA is analyzed using restriction enzymes to isolate the GluRA gene.

Specifically, the clone had a 6.2 kb sized recombinant plasmid containing a gene encoding a heat resistant GluRA, and an unnecessary portion of the inserted DNA of the plasmid was cut with a restriction enzyme to finally separate a 792 bp GluRA gene DNA fragment.

The plasmid containing the heat-resistant GluRA gene was named pGSK 23, and the plasmid was transformed into Escherichia coli and the transformant ( E. coli XL1-Blue / pGSK23) was placed on October 10, 1997. Deposited to the Engineering Research Institute Gene Bank (Accession Number: KCTC 0393BP)

In addition, using the plasmid vector pGSK 23 including the heat resistant GluRA gene, the base sequence of the heat resistant GluRA gene is determined by Sanger et al. (Sanger, F. et al., Science 214, 1205-1210 (1981)). Specifically, the heat resistant GluRA gene isolated from the thermophilic Bacillus SK-1 strain (KCTC 0306BP) includes the nucleotide sequence of SEQ ID NO: 1.

In addition, the present invention determines the amino acid sequence of the heat-resistant GluRA using the base sequence. Specifically, the heat resistant GluRA derived from the high temperature Bacillus strain SK-1 comprises the amino acid sequence of SEQ ID NO: 2 consisting of 264 amino acids.

In another aspect, the present invention provides a method for producing GluRA by transforming a microorganism with an expression vector containing a heat-resistant GluRA gene, culturing the transformant to obtain a crude cell eluate, and performing heat treatment followed by chromatography.

Specifically, the heat-resistant GluRA produced from the thermophilic Bacillus strain is inoculated with the E. coli transformant (KCTC 0393BP) transformed by the expression vector pGSK23 in LB medium and cultured for 24 hours, and then, the cells were harvested and the cells were harvested by an ultrasonic mill. Crushed to obtain crude enzyme solution.

The heat treatment of the crude enzyme solution effectively removes E. coli-derived proteins and enzymes. The preferred temperature for heat treatment is 55 ° C.

The enzymatic liquid partially purified by the heat treatment is purified by ion exchange chromatography, hydrophobic chromatography, and the like, to obtain purely purified heat resistant GluRA.

In order to investigate the activity of the heat-resistant GluRA derived from the thermophilic Bacillus sp . Strain prepared above, the heat-resistant GluRA was added to the reaction solution containing L-glutamic acid, and the production amount of D-glutamic acid was measured by a high-performance liquid phase analyzer.

In addition, in order to investigate the effect of temperature on the activity of heat-resistant GluRA, the production reaction of D-glutamic acid was measured at different temperatures.

As a result, the heat-resistant GluRA of the present invention showed high D-glutamic acid productivity and excellent activity even at high temperatures.

Hereinafter, the present invention will be described in more detail based on the following examples. However, these Examples are only for illustrating the present invention, the present invention is not limited to these.

<Example 1> Bacillus Gene cloning coding for heat resistant GluRA from the genus SK-1 (KCTC 0306BP)

A thermophilic microorganism Bacillus sp. SK-1 strain isolated from soil (KCTC 0306BP) to 100 ㎖ 1.5% polypeptone (polypeptone), 0.2% yeast extract (yeast extract), 0.2% meat extract (meat extract), 0.2% glycerol (glycerol), 0.2% K ₂ HPO ₄ , 0.2% KH ₂ PO ₄ , 0.026% NH ₄ Cl inoculated in MY liquid medium and incubated for 8 hours at 55 ℃ centrifuged at 5000rpm for 20 minutes to recover the cells It was. Total chromosomes were isolated from the recovered cells, and total chromosome separation was performed by Saito et al. (Saito, H. and Miura, K. Biochem. Biophys. Acta 72, 619-629 (1963)). Specifically, the recovered cells were washed with SE (0.15 M NaCl, 0.1 M EDTA (pH 0.8)) and suspended in 10 ml of ST (0.1 M NaCl, 0.01 M EDTA, 0.1 M Tris-HCl (pH 9.0)). To the suspension was added 5-10 mg of lysozyme, 2.5 ml of TS (0.14 M NaCl, 0.001 M EDTA, 0.02 M Tris-HCl, pH5 and 5% SDS) and 12 mg of protease at 37 ° C. After standing for 3 hours, 12.5 ml of phenol was added thereto, and the mixture was left at room temperature for 15 minutes and centrifuged. After the supernatant was taken, two-fold volume of ethanol was added to separate chromosomal DNA with a glass rod. The chromosomal DNA was washed with a solution of TE (0.01 M Tris-HCl, 0.1 mM EDTA (pH 7.5)): ethanol (v / v) 3: 7, 2: 8, 1: 9, respectively, followed by SSC ( 0.1 M NaCl, 0.15 M sodium citrate). RNase A was added to the solution, and the resultant was left at 37 ° C. After the same amount of phenol was added, centrifugation was carried out, and the supernatant was carefully collected, 2 times of ethanol was added thereto, and total chromosome DNA was separated with a glass rod. This was dissolved in TE and dialyzed overnight in 100-fold TE to obtain 2 ml of crude chromosomal DNA.

The crude chromosomal DNA was partially hydrolyzed with restriction enzyme Sau3AI, and DNA fragments of about 3 to 10 kb in size were separated by sucrose density gradient by centrifugation. The isolated DNA fragment was hydrolyzed with restriction enzyme BamHI, 5'-terminal phosphate was removed, mixed with vector pBR 322 (Pharmacia, Sweden) and reacted with T4 DNA ligase at 16 ° C. for 16 hours to generate a recombinant plasmid. It was made. Recombinant plasmids were used for transformation of E. coli WM335 according to the electrophoration method.

Escherichia coli WM335 is a D-glutamic acid-required mutant strain that loses the function of synthesizing D-glutamic acid, which constitutes the microbial cell wall, and cannot grow in medium without D-glutamic acid (Lugtenberg, EJJ et al., J. Bacteriol). 14, 499-506 (1973)).

Therefore, when the transformed recombinant E. coli is cultured in an LB plate medium containing ampicillin, only recombinant E. coli, which has a function of synthesizing D-glutamic acid, possesses the GluRA gene derived from SK-1 of thermophilic Bacillus, D- Growth is also possible in a medium containing no glutamic acid. Through this screening process, one recombinant strain was isolated that could be grown in a medium without heat-resistant GluRA activity and without addition of D-glutamic acid.

Example 2 Determination of DNA Sequence Encoding Heat Resistant GluRA

The recombinant plasmid pGSK23 having a size of 6.2 kb was extracted and purified from the recombinant E. coli having the selected heat resistant GluRA activity, and a restriction map was prepared to determine the portion containing the recombinant heat resistant GluRA.

Unnecessary portions of the inserted DNA of pGSK23 were cut with restriction enzyme Hind III to remove approximately 1.1 kb of Hind III DNA fragment and self-ligation to obtain subclone pGSK 231. Using this plasmid as template DNA, the nucleotide sequence was determined according to Sanger's method (Sanger, F. et al., Science 214, 1205-1210 (1981)) and from this the amino acid sequence of the heat resistant GluRA was determined. Reference). A gene of 792 bp size encoding the heat resistant GluRA was identified from the determined nucleotide sequence.

Example 3 Production of Heat-Resistant GluRA from Recombinant Escherichia Coli

The plasmid vector pGSK23 containing the recombinant heat resistant GluRA gene was transformed into E. coli XL1-Blue strains and then 24 hours in LB-Amphicillin medium containing 1.0% tryptone, 0.5% yeast extract, 0.5% sodium chloride and 0.01% ampicillin. After culturing during the production of recombinant heat resistant GluRA, cells were recovered from the culture medium and crushed to prepare a coenzyme solution containing recombinant heat resistant GluRA. The crude enzyme solution prepared by the above method was left at 60 ° C. for 30 minutes to selectively denature recombinant E. coli-derived proteins and enzymes, and then centrifuged at 12000 rpm for 30 minutes to remove them from the crude enzyme solution. The crude enzyme solution partially purified by heat treatment was completely purified by recombinant heat-resistant GluRA by ion exchange chromatography and non-hydrophilic chromatography. Recombinant GluRA activity of the prepared coenzyme solution was reacted with 0.2 ml of 0.1 Tris buffer solution (pH 8.5) at 10 ° C. for 10 hours with D-glutamic acid and 0.01 ml of coenzyme solution. Then, the amount of D-glutamic acid produced by the GluRA reaction was analyzed by a high-performance liquid phase analyzer (HPLC) equipped with a fluorescence detector.

Example 4 Production of D-Glutamic Acid Using Heat-Resistant GluRA

The recombinant heat resistant GluRA obtained by the method of Example 3 was used as a biocatalyst source for the production of D-glutamic acid. The reaction solution for preparing D-glutamic acid by the biocatalyst conversion method was prepared by adding 10 mM L-glutamic acid and 50 mM Tris buffer (pH 8.5) to a concentration of 10 mg / ml GluRA purified as an enzyme source. As a result of reacting the reaction solution in a 55 ° C. water bath, D-glutamic acid was produced at a productivity of 3.5 g / l / h.

Example 5 Investigation of the Effect of Temperature on the Preparation of D-Glutamic Acid Using Heat-Resistant GluRA

Exhibited a maximum at 70 ℃ As with addition of heat to the reaction mixture containing the GluRA L- glutamic acid in the above Example 4 as a biological catalyst, and varying the reaction temperature as shown in Figure 2, a result of the manufacturing D- glutamic acid, 70 High D-glutamic acid production activity was shown even at high temperature above ℃.

Therefore, since the heat-resistant GluRA derived from the Bacillus SK-1 strain produced from the recombinant E. coli of the present invention has high thermal stability, it was confirmed that it can be usefully used as a biocatalyst for producing D-glutamic acid even at high temperatures.

As described above, the gene encoding GluRA included in the thermophilic Bacillus sp . Strain of the present invention is a large amount of heat-resistant GluRA having an activity of catalyzing isomer formation by converting L-glutamic acid to D-glutamic acid by genetic engineering method. It can be used to produce and ultimately used to prepare industrially useful D-glutamic acid, the production method is one example and the heat-resistant GluRA produced by the method of the present invention has a high thermal stability, so simple heat treatment It can be easily purified through, and its excellent activity even at high temperatures can be usefully used as a biocatalyst for enzymatic synthesis of D-glutamic acid.

(Sequence list)

SEQ ID NO: 1

Sequence length: 792

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: gene

(Sequence list)

SEQ ID NO: 2

Sequence length: 264

Type of sequence: amino acid

Number of chains: 1 chain

Form: Straight

Type of sequence: protein

Claims (11)

A heat resistant glutamate racease (GluRA) gene, characterized in that it has the nucleotide sequence set forth in SEQ ID NO: 1. The method of claim 1, wherein the gene is thermophilicBacillusgenus Glutamate racease gene, characterized in that derived from the strain. The method of claim 2, wherein the high temperatureBacillusgenus StrainsBacillusGlutamate racease gene, characterized in that the genus SK-1 (KCTC 0306BP). An expression vector comprising the gene of claim 1. The method of claim 4, wherein the expression vector is pGSK 23. Escherichia coli transformant transformed by the expression vector pGSK 23 of claim 5 XL1-Blue / pGSK23 (Accession Number: KCTC 0393BP). A method for obtaining heat-resistant GluRA by culturing the microorganism transformed with the expression vector of claim 4, expressing GluRA, obtaining a crude cell eluate therefrom, and performing heat treatment, followed by ion exchange chromatography and non-hydrophilic chromatography. The method of claim 7, wherein the heat treatment is carried out at 55 ℃. 8. The production method according to claim 7, wherein the transformed Escherichia coli transformant XL1-Blue / pGSK23 is used. A heat resistant glutamate racease having an amino acid sequence of SEQ ID NO. A method for preparing D-glutamic acid using L-glutamic acid as a substrate using the glutamate racemate of claim 10 as a biocatalyst.

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