KR19980069057A - Sorbitol dehydrogenase and its genes of Gluconobacter suboxydans - Google Patents

Abstract

The present invention relates to sorbitol dehydrogenase and genes encoding the same in the plasma membrane of Gluconobacter suboxydans (KCTC 2111) strain. The sorbitol dehydrogenase isolated and purified from Gluconobacter suboxydans consists of three subunits, the first subunit of 75 kDa molecular weight dehydrogenase subunit containing pyrroloquinoline quinone (PQQ) as a coenzyme, Membrane-bound sorbitol dehydrogenase consisting of a second subunit of cytochrome c of 50 kDa molecular weight and a third subunit of 29 kDa molecular weight which plays an important role in the stability of sorbitol dehydrogenase. And the gene of sorbitol dehydrogenase comprises a first subunit with 2265 bp and a second subunit with 1437 bp, which are linked in a 5.7 kb sized DNA fragment cleaved with restriction enzyme Pst I.

Description

Sorbitol dehydrogenase and its genes of Gluconobacter suboxydans

The present invention relates to sorbitol dehydrogenase and genes thereof of Gluconobacter suboxydans .

Sorbitol dehydrogenase is known as an enzyme involved in the preparation of sorbose, which acts as a catalyst for the conversion of sorbitol to sorbose, and is used as a precursor of vitamin C and glolic acid. The process for preparing sorbose from sorbitol is carried out by acetic acid bacteria such as Glunobacter suboxydans, Acetobacter xylinum , and 96-99% conversion within 24 hours at normal temperatures (Liebster, J. et al., Chem. List , 50, 395 (1956).

Sorbitol dehydrogenase found in Gluconobacter is classified into NAD-dependent, NADP-dependent, and NAD (P) -independent sorbitol dehydrogenase depending on the specificity of the coenzyme, among which is directly involved in industrial sorbose conversion. Is known to be a NAD (P) -independent enzyme (Cummins, JT et al., J. Biol. Chem ., 224, 323; 226, 301 (1957)).

The present inventors have already purified and analyzed the membrane-bound sorbitol dehydrogenase that is bound to the plasma membrane of Gluconobacter suboxydans (KCTC 2111), a representative acetic acid bacterium involved in the conversion of sorbitol to sorbose. And FAD-required sorbitol dehydrogenase (Shinagawa, E. et al., Agric. Biol. Chem. , 46, 135 (1982)) and coenzyme requirements from the Glunobacter suboxydans IFO 3254 strain with known enzyme properties . This other new sorbitol dehydrogenase was discovered, which contains pyrroloquinoline quinone (PQQ) as a coenzyme and consists of three subunits (Choi et al., FEMS Microbiol. Lett ., 125, 45 (1995). )).

In other words, the cells of Glucobacter suboxidans (KCTC 2111) obtained by culturing in SYP medium (Solbitol 5%, Bakto-Peptone 1%, Yeast extract 0.5%) were suspended and crushed in 10 mM acetate buffer (pH 5.0). The supernatant was ultracentrifuged to recover the plasma membrane fraction. Plasma membrane fractions solubilized by addition of 1.5% n-octylglucoside (Beringermannheim Co., Ltd.) were obtained by CM-TSK 650 (S) (Merck), DEAE-TSK 650 (S) (Merck), and Mono-S (Pharmacia). ) And NAD (P) -independent sorbitol dehydrogenase were purified using a series of columns such as Superose 12 (Pharmacia). The purified enzyme is active against a wide range of substrates such as 100%, 68%, and 70% conversion of various polyols such as sorbitol, mannitol, and ribitol, respectively. In particular, this enzyme requires a pyrroloquinoline quinone as a coenzyme, such as a 9-fold increase in activity when pyrroloquinoline quinone is added, and the fluorescence spectrum shows that the purified enzyme includes pyrroloquinoline quinone as coenzyme. Absorption spectra showed that this enzyme had cytochrome c . Purified enzymes were electrophoresed (Reisfeld, RA, et al., Nature, 195, 281 (1962)) under acidic conditions at pH 4.3 to form single bands, 75, 50, and 12.5% on SDS-PAGE. It was found to consist of three subunits having a molecular weight of 14 kDa, which were named first subunit, second subunit and third subunit in order of high molecular weight, respectively (Choi et al. , FEMS Microbiol. Lett , 125, 45 (1995).

More detailed experiments, however, have found that another subunit of 29 kDa molecular weight plays an important role in the stability of sorbitol dehydrogenase. In other words, while investigating various pH conditions in order to improve the protein resolution in the purification using the DEAE-TSK column, the peak having a sorbitol dehydrogenase activity at pH 6.0 rapidly maintains a stable peak and enzyme activity Was found to separate into two peaks. It was found that the peak of which the enzyme activity was stably retained had a further subunit of 29 kDa molecular weight, in contrast to the peak which rapidly lost enzymatic activity, and the subunit was renamed as the third subunit. That is, it is uncertain whether or not the third subunit of 14 kDa molecular weight is a true subunit in view of the relative amount with other subunits.

Hereinafter, the characteristics of sorbitol dehydrogenase were further investigated using purified enzymes having a third subunit of 29 kDa molecular weight.

Sorbitol dehydrogenase was determined to have a Michaelis-Menten constant of K _m = 120 mM and V _max = 3.9 x 10 ^-5 M / min when sorbitol was used as a substrate. In addition, dichlorophenol indophenol (DCIP) or ferricyanide acted effectively as an electron acceptor of sorbitol dehydrogenase, and its activity when phenazine methosulfate (PMS) was added as an electron mediator Increased. Purified enzymes showed a significant increase in activity by calcium or magnesium ions, while copper ions showed severe inhibition.

However, sorbitol dehydrogenase is present in combination with the plasma membrane, and the recovery rate is only about 0.05%. Therefore, there is a problem in that industrial use of the enzyme obtained by such extraction and purification is difficult.

Therefore, the present inventors are studying a method for mass production of sorbitol dehydrogenase, an enzyme required for the production of sorbose, which is used as an essential precursor of vitamin C and gloonic acid, and especially the sorbitol dehydrogenase present in the industrially important plasma membrane. The purified enzyme was isolated and purified from the Glunobacter suboxydans strain, and the purified enzymes were the first subunit and the cytochrome c phosphorus, a 75 kDa dehydrogenase subunit containing pyrroloquinoline quinone (PQQ) as a coenzyme. It was found that it was a membrane-bound sorbitol dehydrogenase having a second subunit of 50 kDa molecular weight and a third subunit of 29 kDa molecular weight, which played an important role in the stability of sorbitol dehydrogenase. The amino terminal amino acid sequence of each subunit was determined. . The DNA sequences of the first subunit and the second subunit can be determined by cloning the gene of sorbitol dehydrogenase and decoding the DNA sequence.

It is an object of the present invention to provide sorbitol dehydrogenase and genes thereof present in the plasma membrane of Gluconobacter suboxydans .

Figure 1 shows the DEAE-TSK column chromatography using acetic acid buffer solution of pH 5 and pH 6.

Figure 2 shows the SDS-PAGE results of peak I and peak II separated by DEAE-TSK column chromatography.

Figure 3 shows a restriction map of lambda GEM 5-1 containing 1.53 kb DNA fragment (#SDH 2-1) comprising the amino terminus of the sorbitol dehydrogenase first subunit of Gluconobacter suboxydans.

Figure 4 shows the positions of S1, S2 and S3 DNA fragments prepared by cleavage with various restriction enzymes in a 5.7 kb DNA fragment digested with restriction enzyme Pst I with lambda GEM 5-1.

Figure 5 shows the nucleotide sequence in a 5.7 kb DNA fragment digested with restriction enzyme Pst I.

In accordance with this object, in the present invention, a membrane of Gluconobacter suboxydans comprising a first subunit of 75 kDa, a second subunit of 50 kDa and a third subunit of 29 kDa Provided is a sorbitol dehydrogenase gene contained in a 5.7 kb sized DNA fragment cleaved by a bound sorbitol dehydrogenase and a restriction enzyme Pst I of Gluconobacter suboxydans (KCTC 2111).

Sorbitol dehydrogenase is isolated and purified from a plasma membrane of Gluconobacter suboxydans (KCTC 2111) using a series of columns. Several biochemical properties of the purified enzymes are determined, and each subunit is purified, and the amino terminal sequence of each subunit is determined using an amino acid sequence analyzer (Applied Biosystems, 477A).

Primers 1 and 2 are synthesized using codons based on the amino terminal amino acid sequences of the determined first subunit. Genomic DNA extracted from Gluconobacter suboxydans was ligated to plasmid pBluescript SK (Staratagene) and subjected to 30 cycles according to SSP-PCR method using primers 1 and 2 to isolate a 1.53kb size # SDH2-1 fragment. do. This fragment contains the amino terminal portion of the first subunit. This # SDH2-1 fragment is linked to the plasmid pBluescript SK, transformed into E. coli, and then transformed and cultured and the plasmid is extracted by alkali method.

To prepare a genetic library of sorbitol dehydrogenase, first, the genomic DNA extracted from Gluconobacter suboxydans was treated with a restriction enzyme, cloned into lambda GEM-11 (Promega), and then labeled with # SDH2-1. Lambda GEM 5-1 is isolated from the library. The lambda GEM 5-1 is then digested with restriction enzymes to obtain a DNA fragment of about 5.7 kb and analyzed for DNA sequence. This fragment was digested with restriction enzymes Kpn I and Pst I, restriction enzymes Not I and Sac II, and restriction enzymes Pst I and Sac I to prepare S1 DNA fragments, S2 DNA fragments and S3 DNA fragments, respectively. Each DNA fragment of S1, S2 and S3 obtained can be deciphered with DNA sequence using ExoIII-Mungbean (Stratagene).

Analyzing the decoded nucleotide sequence, the sorbitol dehydrogenase of Gluconobacter suboxydans was composed of 2265 bp, the first subunit having a molecular weight of 79,139 Da, and 1437 bp, and had a molecular weight of 51,368 Da. It can be seen that the second subunit is linked in a 5.7 kb sized DNA fragment cleaved with restriction enzyme Pst I.

Hereinafter, the present invention will be described in more detail based on the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

EXAMPLE

(Step 1) Separation and function of the third subunit

During the purification of sorbitol dehydrogenase from Gluconobacter suboxydans (KCTC 2111) strain, the stability of protein separation and enzyme activity according to the pH change of DEAE-TSK, an anion exchange column, was investigated.

Samples partially purified through a CM-TSK column were subjected to DEAE-TSK column chromatography using 10 mM acetic acid buffer at pH 5 or pH 6, and the enzyme activity of each fraction showing activity was measured.

As shown in FIG. 1, the elution at pH 6 (b) was much higher than that at pH 5 (a), and the peak I and peak II showing enzymatic activity were purely separated.

The SDS-PAGE results of the fractions separated by DEAE-TSK column chromatography eluting with pH 6 in which both peaks were completely separated are shown in FIG. 2. Figure 2a shows the fractions eluted at the first active peak, peak I, where column M represents the standard molecular weight protein, as shown in columns 1-4, the first and second subunits 75 kDa and 50 Although the kDa band was observed, the band of 29 kDa, which is the third subunit, did not appear, and in this case, the enzyme activity was reduced by about 10 times after 1 hour after elution. Figure 2b shows the fractions eluted at the second active peak, peak II, where column M represents the standard molecular weight protein, as shown in columns 5-8, in addition to the two subunits there is a third subunit 29 kDa band In the case of the peak II was confirmed that the enzyme activity is maintained as it is. As a result, a protein having a molecular weight of 29 kDa, the third subunit, is likely to be involved in the electron transfer system which is closely related to the action mechanism of sorbitol dehydrogenase and contributes to stabilizing the activity of sorbitol dehydrogenase.

(Step 2) Determining the amino terminal amino acid sequence of the first subunit and the third subunit

Sorbitol dehydrogenase with Pyrroloquinoline quinone (PQQ) of Gluconobacter suboxydans (KCTC 2111) as a coenzyme was isolated and purified (Choi, et al., FEMS Microbiol. Lett ., 125, 45 (1995)), Each subunit of the purified enzyme was purified and then the amino terminal sequence of the first subunit of SEQ ID NO: 1 and the third subunit of SEQ ID NO: 2 was determined using an amino acid sequence analyzer (Applied Biosystems, 477A).

[SEQ ID NO 1]

Glu-Asp-Thr-Gly-Thr-Ala-Ile-Thr-Asn-Ala-Asp-Gln-His-Pro-Gly

[SEQ ID NO 2]

Ala-Gly-Thr-Pro-Leu-Lys-Ile-Gly-Val-Thr-Phe-Gln

(Step 3) Primer construction and isolation of 1.53 kb DNA fragment

Primer 1 (5'-CCGGAATTC GAA (G) GAT (C) ACI GGI ACI GC-3 ') and Primer 2 (5'-ATT) based on the amino terminal amino acid sequence of the determined first subunit C, A) ACI AAT (C) GCI GAT (C) CAA (G) CAT (C) CC-3 ').

Genomic DNA extracted from Gluconobacter suboxydans was partially cut with restriction enzyme Bam HI according to Takeda Shimizu, J. Ferm. Bioeng. , 72, 1 (1991). Subsequently, the plasmid pBluescript SK (Stratagene) was digested with Nae I and Bam HI, and the genomic DNA fragmented at the Bam HI position was ligated.

SSP-PCR method using the primers 1 and 2, and T7 primer (White, B., SSP-PCR and genome walking, in Method in Mol. Biol. PCR protocols, Humana Press, 15, 339 (1993)) After 30 times, according to 1.53 kb fragment # SDH2-1 was isolated.

The isolated # SDH2-1 fragment was linked to plasmid pBluescript SK and transformed with E. coli DH5α, and then cultured in LB medium to which 100 μg / mL of ampicillin was added. Subsequently, the plasmid was extracted by alkali method (Sambrook, J. et al., Molecular Cloning , CSH Press, p.1.25 (1989)) and reacted positively when reacted with Primer 1 and Primer 2. This # SDH2-1 The fragment contained a portion of the amino terminus of the first subunit but did not have a carboxy terminus portion.

(Step 4) Isolation of 15 kb DNA fragment using 1.53 kb DNA fragment

# SDH2-1 was labeled with DIG (Boehringer Mannheim) (Ausubel, F. et al., Current Protocols in Mol. Biol., Wiley Sons Inc., p.2.4.1 (1987)).

To prepare a gene library of sorbitol dehydrogenase, genomic DNA was extracted from Gluconobacter suboxydans (KCTC 2111) in the same manner as in (step 3) and cut into 15-23 kb size partially digested with restriction enzyme Sau 3A. DNA was cloned into the Bam HI position of lambda GEM-11 (Promega).

The labeled # SDH2-1 was used to isolate 15 kb of lambda GEM 5-1 from the library.

3 shows a restriction map of lambda GEM 5-1.

(Step 5) Sequence translation of Pst I cleavage site in 15 kb DNA fragment

Lambda GEM 5-1 was digested with restriction enzyme Pst I to obtain a DNA fragment of about 5.7 kb containing # SDH2-1.

To facilitate sequencing, 5.7 kb DNA fragments were digested with restriction enzymes Kpn I and Pst I, S1 DNA fragments, restriction enzymes Not I and Sac II, respectively, S2 DNA fragments and restriction enzymes Pst I and Sac I. S3 DNA fragments were prepared.

FIG. 4 shows S1, S2 and S3 DNAs prepared by cleavage of various restriction enzymes in a DNA fragment of about 5.7 kb containing # SDH2-1 obtained by cleaving lambda GEM 5-1 with restriction enzyme Pst I. It shows the location of the fragment.

The base sequences of the respective S1, S2 and S3 DNAs were read using ExoIII-Mungbean (Stratagene).

Figure 5 shows the nucleotide sequence in the 5.7 kb DNA fragment cut with restriction enzyme Pst I.

As shown here, the shine-dalgano sequence 'AGGA' is located at 651-654 bp and the signal sequence of the first subunit is located at 665-766 bp, which encodes the first subunit from which the signal peptide was truncated. The sequence was between 767 and 2926 bp. It encodes 754 amino acids, and it can be seen that the amino terminal sequence coincides with the 15 amino acid sequence found by the amino terminal sequencing of the first subunit. There was then a Shine-Dalgano sequence (AGGA) at 2950-2953 bp, and the second subunit was located at 2964-4400 bp, which encodes 478 amino acids, followed by stop codons followed by several inversion repeats. . In addition, the molecular weight of the active protein inferred from the above sequences was 79 kDa for the first subunit and 51 kDa for the second subunit, which is similar to the molecular weights of 75 kDa and 50 kDa, which are the result of the electrophoresis of the enzyme. there was.

In addition, it can be seen that the sequences appearing in the first and second subunits of the amino and carboxy terminal sites of the dehydrogenases including pyrroloquinoline quinone (PQQ) found in bacteria as coenzymes were present in the first subunit. The specific consensus sequence present in is shown in SEQ ID NO: 3, and the specific consensus sequence present in the carboxy terminal region is shown in SEQ ID NO: 4 below. This is another proof that the first subunit contains pyrroloquinoline quinone as coenzyme (where Xaa is any amino acid).

[SEQ ID NO 3]

[Asp / Glu / Asn] -Trp-Xaa-Xaa-Xaa-Gly- [Arg / Lys] -Xaa-Xaa-Xaa-Xaa-Xaa-Xaa- [Phe / Tyr / Trp] -Ser-Xaa-Xaa- Xaa-Xaa- [Leu / Ile / Val / Met] -Xaa-Xaa-Xaa-Asn-Xaa-Xaa-Xaa-Leu- [Arg / Lys]

[SEQ ID NO 4]

Trp-Xaa-Xaa-Xaa-Xaa-Tyr-Asp-Xaa-Xaa-Xaa- [Asp / Asn]-[Leu / Ile / Val / Met / Phe / Tyr]-[Leu / Ile / Val / Met / Phe / Tyr]-[Leu / Ile / Val / Met / Phe / Tyr]-[Leu / Ile / Val / Met / Phe / Tyr] -Xaa-Xaa-Gly-Xaa-Xaa- [Ser / Thr / Ala]- Pro

In addition, it was confirmed that one or three heme binding sequences of SEQ ID NO: 5 exist in the first subunit and the second subunit (where Xaa is any amino acid, and Yaa is any amino acid other than Xaa). to be).

[SEQ ID NO: 5]

Cys-Xaa-Yaa-Cys-His

As a result of analyzing the DNA sequence identity, the DNA sequence of the first subunit showed high similarity to several dehydrogenases including pyrroloquinoline quinone as coenzyme, especially Acetobacter polyoxogenes and aceto . Alcohol dehydrogenase from Acetobacter aceti , 77% (Tamaki, T. et al., BBA ., 1088, 292 (1991)) and 70% (Inoue, T. et al., J. Bacteriol, respectively). , 171, 3115 (1989)). The DNA sequence of the second subunit showed the highest identity with the sequence of cytochrome c -553 (Takeda and Shimizu, J. Ferm. Bioeng. , 72, 1 (1991)) of Gluconobacter suboxydans IFO 12528 . , DNA base identity was 83%, amino acid sequence identity was 88%.

As such, it was confirmed that the first subunit and the second subunit are located in one operon. On the other hand, a third subunit encoding 29 kDa has not yet been found.

In the above, the gene of sorbitol dehydrogenase of Gluconobacter suboxydans (KCTC 2111) includes a first subunit encoded by 2265 bp, a second subunit encoded by 1437 bp, and these subunits are restriction enzymes. It can be seen that it is linked in a 5.7 kb DNA fragment that is cleaved with Pst I. These sequences are registered with GenBank on May 4, 1995 as U26263.

Claims (11)

A membrane bound sorbitol dehydrogenase of Gluconobacter suboxydans , comprising a first subunit of 75 kDa, a second subunit of 50 kDa, and a third subunit of 29 kDa. The method of claim 1, Sorbitol dehydrogenase comprising pyrroloquinoline quinone (PQQ) as a coenzyme in a first subunit. The method of claim 1, Sorbitol dehydrogenase, characterized in that the first subunit comprises the amino terminal amino acid sequence of SEQ ID NO: 1. [SEQ ID NO 1] Glu-Asp-Thr-Gly-Thr-Ala-Ile-Thr-Asn-Ala-Asp-Gln-His-Pro-Gly The method of claim 1, Sorbitol dehydrogenase, characterized in that the third subunit comprises the amino terminal amino acid sequence of SEQ ID NO: 2. [SEQ ID NO 2] Ala-Gly-Thr-Pro-Leu-Lys-Ile-Gly-Val-Thr-Phe-Gln The method of claim 2, Sorbitol dehydrogenase (where Xaa is any amino acid) comprising the amino acid sequence of SEQ ID NO: 3 at the amino terminal portion of sorbitol dehydrogenase comprising pyrroloquinoline quinone (PQQ) as a coenzyme in the first subunit ). [SEQ ID NO 3] Xaa-Leu- [Arg / Lys] The method of claim 2, Sorbitol dehydrogenase (where Xaa is any amino acid) comprising the amino acid sequence of SEQ ID NO: 4 at the carboxy terminus portion of sorbitol dehydrogenase comprising pyrroloquinoline quinone (PQQ) as a coenzyme in the first subunit ). [SEQ ID NO 4] Trp-Xaa-Xaa-Xaa-Xaa-Tyr-Asp-Xaa-Xaa-Xaa- [Asp / Asn]-[Leu / Ile / Val / Met / Phe / Tyr]-[Leu / Ile / Val / Met / Phe / Tyr]-[Leu / Ile / Val / Met / Phe / Tyr]-[Leu / Ile / Val / Met / Phe / Tyr] -Xaa-Xaa-Gly-Xaa-Xaa- [Ser / Thr / Ala]- Pro The method of claim 1, Sorbitol dehydrogenase, wherein the first subunit and the second subunit comprise a heme binding sequence of SEQ ID NO: 5, wherein Xaa is any amino acid and Yaa is any amino acid other than Xaa . [SEQ ID NO: 5] Cys-Xaa-Yaa-Cys-His A sorbitol dehydrogenase gene contained in a 5.7 kb sized DNA fragment cleaved by the restriction enzyme Pst I of Gluconobacter suboxydans (KCTC 2111). The method of claim 8, A sorbitol dehydrogenase gene, wherein the sorbitol dehydrogenase gene comprises a first subunit of 2265 bp and a second subunit of 1437 bp. The method of claim 9, Sorbitol dehydrogenase gene, characterized in that the first subunit comprises the base sequence of SEQ ID NO: 6. [SEQ ID NO: 6] The method of claim 9, Sorbitol dehydrogenase gene, characterized in that the second subunit comprises the following SEQ ID NO: 7. [SEQ ID NO: 7]