KR20010007901A - β-Glucosidase obtained from oats and process for preparation of genistein or daidzein using it - Google Patents

Abstract

PURPOSE: The gene of beta-glucosidase derived from Avena sativa L. and a method for producing genistein or daidzein using the gene are provided, thereby genistein, which is useful for the prevention and treatment of osteoporosis and prostatic cancer, is effectively produced by using genetic engineering technology. CONSTITUTION: The method for producing genistein or daidzein comprises the steps of: performing PCR cDNA of Avena sativa L. with three primers consisting of a forward primer for alpha and beta-subunit, a reverse primer for alpha-subunit, and a reverse primer for beta-subunit for cloning alpha and beta-subunit of beta-glucosidase of Avena sativa L.; inserting the alpha and beta-subunit of beta-glucosidase of Avena sativa L. into the E. coli expression vector pET24a to prepare the recombinant expression vector pET24a-glu; transforming E. coli BL21 to produce E. coli BL21 transformant(pET24a-glu, KFCC11222); incubating E. coli BL21 transformant(pET24a-glu, KFCC11222); adding p-nitrophenyl-beta-D-glucopyranoside as a soybean extract component into the fermented solution of E. coli BL21 transformant(pET24a-glu, KFCC11222) and reacting them at 37deg.C for 15 minutes; adding 1M sodium carbonate to terminate the reaction; and analysing the production of genistein by HPLC.

Description

Β-glucosidase obtained from oats and process for preparation of genistein or daidzein using it}

The present invention relates to a beta-glucosidase, a recombinant microbial strain producing the enzyme or a method for producing genistein and dyedzein using the enzyme or recombinant strain. More specifically, the present invention provides a beta-glucosidase enzyme gene derived from oats, a recombinant vector comprising the gene, a recombinant strain transformed with the recombinant vector, and a recombinant beta- including culturing the transformed strain. A method for producing glucosidase, and a method for converting genistin or dydazine present in precursor form in soybean extract using recombinant beta-glucosidase or recombinant strain prepared by the method to genistein or dyedzein. .

In developed countries, natural replacement therapy has become one of the fields of prevention and treatment of diseases, and this tendency is changing the diet and culture of the general public, and the tendency to consume natural ingredients rather than chemical synthetic materials is becoming stronger. In women, postmenopausal osteoporosis is caused by a decrease in estrogens. Therefore, the administration of estrogens has many problems due to the side effects of increasing the incidence of cancers such as breast cancer and uterine cancer due to hormonal action. Therefore, it is emerging recently as a way to solve this is to periodically ingest the phytoestrogen (that is, the isoflavone component of soybean) as a natural food ingredient. However, the daily intake of isoflavones to maintain health is about 30 to 100mg, which is virtually impossible because you need to eat 1.2 to 2kg of beans every day. Therefore, it is required to develop derivatives with estrogen-like activity without side effects. Soybean is a crop that has been with humans for a long time, and its nutritional value is well known through many studies. The evidence suggests that many Asians have fewer incidences of adult disease, osteoporosis and prostate cancer than Westerners, and this result was attributed to isoflavone, one of the soybean ingredients. In particular, genistein, one of soy isoflavones, is used for the treatment of osteoporosis and anti-cancer, and many research institutes and organizations have demonstrated the effectiveness of preventing and treating genistein's osteoporosis and cancer. In particular, getting genistein has emerged as an important issue.

On the other hand, isoflavone compounds present in the legumes are present in a large amount in the form of precursors, that is, in the form of genistin and daidzin, which are linked to sugar molecules and β-1,4-glucoside bonds in soybean plants, It is present in about four times more than the content of genistein and daidzein, the active form of sugar. However, soybean fermented foods such as doenjang, cheonggukjang, soy sauce, and natto (Japanese style soybean paste) show more increased content of genistein than in soybean, which is produced by beta-glucosidase in fermenting bacteria. This is the result of the conversion. Therefore, Asian countries that have consumed soybean fermented foods as traditional foods, such as Japan and China, have reported lower incidences of breast cancer, colon cancer, and prostate cancer than Westerners. It is believed to be involved in the supply of isoflavonoids. When ingested soybeans, soybean Zenithine may be converted to Genistein by human intestinal flora, but the conversion rate is very small. Therefore, hydrolysis of sugar is an indispensable process in obtaining isoflavones of soybean.

To date, soybean isoflavones are obtained by chemical and enzymatic methods, and their yields are low, resulting in low yields, and not only high market value, but also the severity of environmental problems that can cause adverse effects on the natural ecosystem when chemical hydrolysis occurs. exist.

The present invention has been made to solve the above problems, the present inventors in spite of the presence of beta-glucosidase enzyme in various forms in organisms, including animals and plants, microorganisms, in particular soy isoflavones such as zenithine or die The gene was cloned from oat-derived beta-glucosidase, which can be used as a substrate, to convert genistin to genistein and produced from soybean extract, and from oat-derived enzyme beta-glucosidase. Examination of the conversion rate of Genistin to Genistein confirmed that the conversion is more than 95% and completed the present invention.

Accordingly, the present invention provides an oat-derived beta-glucosidase enzyme gene with excellent activity for use in in vitro enzymatic reactions for the production of genistein, which is effective for the prevention and treatment of osteoporosis and prostate cancer in relation to senile diseases. .

Another object of the present invention is to provide a recombinant vector comprising the gene.

Still another object of the present invention is to provide a recombinant strain transformed with the recombinant vector.

Still another object of the present invention is to provide a method for preparing a recombinant beta-glucosidase comprising culturing the transformed strain.

Still another object of the present invention is to provide a method for converting zenithine or dydazine present in a precursor form in soybean extract into genistein or dyedzein using the recombinant beta-glucosidase prepared by the above method.

The object of the present invention is to isolate beta-glucosidase from soybean, determine its nucleotide sequence and amino acid sequence, prepare a recombinant vector comprising the beta-glucosidase gene, and transform the recombinant vector into bacteria. A method for preparing a recombinant beta-glucosidase enzyme protein comprising converting and culturing the transformed bacteria, and also using the transformed bacterium or the beta-glucosidase protein, can be used to produce genistein or Achieved by preparing d-zane.

Hereinafter, the configuration of the present invention will be described.

Figure 1 shows a beta-glucosidase gene sequence derived from oats.

Figure 2 shows the amino acid sequence of the beta-glucosidase protein encoded by the beta-glucosidase gene from oats.

Figure 3 is a schematic diagram of the production of the recombinant expression vector pET24a-glu of the present invention for beta-glucosidase gene expression.

Figure 4 shows the result of confirming the production of Genistein and Dyzezein by reacting the recombinant strain E. coli BL21 transformant (pET24a-glu) (Accession Number: KFCC-11222) culture of the present invention with soybean extract.

The present invention comprises the steps of PCR screening the oat cDNA library using three primers to secure the beta-glucosidase gene; Culturing E. coli transformed by the recombinant vector into which the obtained beta-glucosidase gene is introduced to express the beta-glucosidase gene of oats; The transformed Escherichia coli culture is reacted with the soybean extract to produce genistein and dyedzein.

The present invention has the effect of securing economical efficiency by converting and converting genistin present in a relatively high content in soybean into genistein and dyedzein without any influence on the natural system by the bioenzymatic method as described above.

Hereinafter, the present invention will be described in detail through examples. However, the following examples are only for illustrating the present invention is not limited thereto. In addition, modifications, avoidances, or equivalents may be made by those skilled in the art within the technical spirit and scope of the present invention, but it will be clearly understood by those skilled in the art.

Example 1 Isolation of Beta-glucosidase Gene from Oat cDNA Library

In order to clone the α and β subunit genes of beta-glucosidase of oats registered in the Genebank, the following primers (I to III) were designed to screen oat cDNA libraries by PCR. Secured.

Forward primers for α and β-subunits:

5'-ATGGCTCTCCTTTGTTCAGCC CTGA-3 '(I)

Reverse primer for α-subunit:

5'-GGCATTAGTTCTTCATCACGC GGTGCCACCACC-3 '(Ⅱ)

Reverse primer for β-subunit:

5'-ACTTTTATTTTCCATACGGGTTTTG-3 '(Ⅲ)

PCR conditions were denatured DNA at 94 ℃ for 2 minutes, followed by 30 consecutive cycles of 1 minute at 94 ℃, 1 minute at 58 ℃, 2 minutes at 72 ℃ and then synthesized cDNA 10 minutes at 72 ℃ Secured.

Example 2 Expression of Oat Beta-Glucosidase Gene in Escherichia Coli

In order to clone only the gene corresponding to the mature protein of the oat gene obtained in Example 1 to Escherichia coli BL21, the EcoRI and XhoI sites may be included in the forward and reverse primers (IV and V) as shown below. After designing, synthesizing and PCR primers, the recombinant strain vector pET24a-glu was obtained by binding to EcoRI-XhoI site of E. coli expression vector pET24a, and transformed into E. coli BL21 according to the manual of the expression vector manufacturing company. E. coli BL21 transformant (pET24a-glu) was prepared (FIG. 3). The transformed strain E. coli BL21 transformant (pET24a-glu) was then cultured.

The inventors of the transformed strain E. coli BL21 transformant (pET24a-glu) on October 16, 2000, the accession number KFCC-11222 to the Korean spawn association.

Forward primer:

5'-AGCAAGAATAAGAATTCGCGCTTGAAAG-3 '(Ⅳ)

Reverse primer:

5'-CGTGGCATTCTCGAGTCATCACGCGG-3 '(Ⅴ)

At this time, PCR reaction conditions were the same as above.

Example 3 Preparation of Genistein and Dyzedine Using Oat Beta-Glucosidase from Soybean Extract

Synthetic substrate for beta-glucosidase 1 mM 1 mL of p-nitrophenyl-β-D-glucopyranoside (E. coli BL21 transformant (pET24a-glu) (Accession No .: KFCC- 11222) After mixing with 1mL of the culture and reacting at 37 ℃ for 15 minutes to stop the reaction with 2mL stop solution (1M sodium carbonate) and then measured the absorbance at 400nm to verify the enzyme activity and then directly reacted with soybean extract and genistein and Dyzezene production was confirmed by HPLC (FIG. 4). At this time, the developing solvent was 30% acetonitrile and 0.1% acetic acid, the column was used for Waters' symmetric C-18, 3.9X150mm, the flow rate was 1mL / min and the wavelength was measured at 260nm. Genistein's RT was 7.400 min.

As described above, according to the production method of the present invention can be produced by bioenzymatically converting a bioactive substance of genistein or dyedzein, which has a prophylactic or therapeutic effect in osteoporosis or cancer, and has a high content of genistein or dyedzein Fermented food can also be prepared, the present invention is a very useful invention in the food and pharmaceutical industry.

Claims (9)

CDNA clone comprising beta-glucosidase gene isolated from Oven (Avena sativa L.). The beta-glucosidase gene of claim 1, which has the following nucleotide sequence. The beta-glucosidase enzyme protein of claim 1, having the following amino acid sequence. The recombinant vector pET24a-glu comprising the beta-glucosidase gene according to claim 2. Strain E. coli BL21 transformant (pET24a-glu) transformed with the recombinant vector pET24a-glu according to claim 4 (Accession No .: KFCC-11222). A method for producing a recombinant beta-glucosidase protein comprising culturing the transformed strain E. coli BL21 transformant (pET24a-glu) (Accession No .: KFCC-11222) described in claim 5. A method for producing genistein or dydzein according to claim 5, wherein the transformed strain E. coli BL21 transformant (pET24a-glu) (Accession No .: KFCC-11222) culture is reacted with soybean extract. Recombinant beta-glucosidase protein prepared by claim 6. A method for producing genistein or dyedzein from soy extract using the beta-glucosidase enzyme protein of claim 3 or 8.