KR100665798B1 - - A novel gene of -glucosidase - Google Patents

Abstract

The present invention relates to a gene encoding beta-glucosidase, a key enzyme useful for the preparation of validoxylamine A, an intermediate for the preparation of the anti-diabetic bogglibose, recombinant plasmid and recombinant plasmid into which the gene is inserted. It relates to a transformant transformed into.

Beta-glucosidase, flavobacterium, diabetes mellitus, bolibos, balidoxylamine A

Description

A novel gene of β-glucosidase

1 shows a recombinant plasmid cloned with beta-glucosidase,

Figure 2a shows the results of high performance liquid chromatography analysis when the beta-glucosidase is not expressed ( E. coli DH5α / pHCE).

Figure 2b shows the results of high performance liquid chromatography analysis of beta-glucosidase ( E. coli DH5α / pHbgl).

The present invention relates to a gene encoding beta-glucosidase, a key enzyme useful for the preparation of validoxylamine A, an intermediate for the preparation of the anti-diabetic bogglibose, a recombinant plasmid and a recombinant plasmid into which the gene is inserted. It relates to a transformant transformed into.

Beta-glucosidase is an enzyme that catalyzes the hydrolysis of various beta-glucosides and oligosaccharides and has been reported to be present in a variety of plants, fungi, bacteria and the like (Zeljko, et al., Appl. Environ. Microbiol. 70 (7): 3941-394 (2004)), beta-glucosidase, which is active against various sugars, is known (Yaw-keun, et al., Enz. Microbiol. Tenchnol, 24, 144-150 (1990)). ).

On the other hand, it has been reported that beta-glucosidase derived from Flavobacterium sacchafilum has a function of hydrolyzing Validamycin A to generate validoxylamine A (Takeuchi et al., Chem. Pharm. Bull, 36 (9), 3540-3548 (1988)).

However, in addition to the above reports, there are no studies on the enzyme beta-glucosidase acting on validamycin A. Moreover, the process of separating enzyme beta-glucosidase from Flavobacterium sacchafilum is very complicated, and there is a problem that the separated enzyme is not stable, and because it is an enzyme in cells, it is difficult to secure a large amount of industrial use. This is an impossible problem.

Accordingly, the present inventors completed the present invention by separating the gene encoding beta-glucosidase from Flavobacterium sacchafilum and identifying the sequence to solve the problems of the prior art.

Accordingly, it is an object of the present invention to provide a gene encoding beta-glucosidase of Flavobacterium sacchafilum.

Still another object of the present invention is to provide a recombinant plasmid into which the gene is inserted and a transformant transformed with the recombinant plasmid.

In order to achieve the above object, the present invention provides a gene encoding beta-glucosidase consisting of the amino acid sequence of SEQ ID NO: 1.

The present invention also provides a gene encoding beta-glucosidase having the nucleotide sequence of SEQ ID NO: 2.

Hereinafter, the present invention will be described in more detail.

After preparing a gene library from Flavobacterium saccharophyllum cells, genes exhibiting beta-glucosidase activity may be selected from the prepared Flabobacterium sacchafilum gene library.

For example, to obtain a gene library of Flavobacterium sacchafilum, Flavobacterium cells are disrupted to isolate genomic DNA. Using the isolated genomic DNA as a template, PCR is performed using Primer 1 (SEQ ID NO: 3) and Primer 2 (SEQ ID NO: 4). Purify the gene fragments by performing gel electrophoresis on the PCR products, cloning them into an appropriate vector, for example pHCE (Takara, Korea), and transforming Escherichia coli to prepare transformants. Rofilum's gene library can be prepared.

In order to select candidate groups for transformants having activity against beta-glucosidase from the gene library of Flavobacterium sacchafilum prepared above, paranitrophenyl-glu in the action of beta-glucosidase The action of decomposing copyranoside (pNP-glucopyranoside) into paranitrophenol (pNP) and glucose can be utilized (Romaniec et al., Enzyme and Microbioal Technology , 15 (5), 1993, 393-). 400). Therefore, after reacting each culture of the gene library of Flavobacterium sacchafilum prepared above with a paranitrophenyl-glucopyranoside substrate solution, the absorbance of each reactant was measured and measured. Reactants free of nitrophenol (405 nm, yellow) are selected as transformant candidates.

As described above, the transformant candidate group selected as described above may be reacted with a solution containing validamycin A to determine the production amount of validoxalixamine A, thereby measuring activity against beta-glucosidase (see FIGS. 2A to 2B). ).

As a result of the activity measurement, a transformant exhibiting beta-glucosidase activity in the gene library of Flavobacterium sacchafilum can be recovered, and the recovered transformant can be recovered using a known method such as alkali. Recombinant plasmids can be isolated using the recovery method (J. Sambrook et al., Molecular cloning, Vol. 1, 1.25-1.28). According to the above method, a recombinant plasmid having a gene fragment of about 3.5 kb inserted therein was recovered, which is called pHbgl (see FIG. 1).

The nucleotide sequence of the gene encoding beta-glucosidase (SEQ ID NO: 2) and the sequencing of the recombinant plasmid pHbgl obtained above as a template using an appropriate primer, for example, SP6 primer (Takara, Korea) The amino acid sequence to be translated (SEQ ID NO: 1) can be identified.

In addition, E. coli DH5α / pHbgl is named E. coli transformants that exhibit activity against beta-glucosides transformed with the recombinant plasmid pHbgl recovered above. The transformant E. coli DH5α / pHbgl was deposited on February 17, 2005 to the Korea Agromicrobial Conservation Center (Accession Number: KACC 95030P).

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrating the present invention, and the present invention is not limited to these examples.

Example 1.Isolation of Genomic DNA from Flavobacterium sacchafilum and Construction of Gene Library

Flavobacterium sacchafilum (IFO 13984) cells were added to a lysozyme solution (5 ml), crushed cells, and then mixed with acid phenol (5 ml), followed by centrifugation ( 10,000 rpm, 10 minutes) to separate genomic DNA. Using the extracted DNA (5 µg) as a template, PCR was performed using Primer 1 (SEQ ID NO: 3) and Primer 2 (SEQ ID NO: 4). The sequence of the primer 1 and primer 2 was designed by deriving a common part by performing homology search of the base sequence of the known beta-glucosidase. PCR (polymerase chain reaction) was repeated 30 times using AmpliTaq polymerase (PerkinElmer Biosystems, USA) with reaction conditions of 1 minute at 94 ° C, 1 minute at 52 ° C, and 2 minutes at 72 ° C. Agarose gel (0.8%) electrophoresis was performed on the amplified PCR product, and stained for 20 minutes in an ethidium bromide solution (0.5 ㎍ / ml, 100 ml), followed by 100bp standard DNA (Bionia, Korea). ), The presence of the amplified gene fragment was confirmed.

Phenol (200 μl) and chloroform (200 μl) were added to the recovered gene fragments to remove impurities, and ethanol (2.5 ml) was added to purify the purified protein fragments. The purified gene fragment was digested with restriction enzymes BamH I and Nde I (Roche, USA), and then ligated with a pHCE vector (Takara, Korea) digested with the same restriction enzyme to prepare a recombinant plasmid. Transformants were obtained by transforming E. coli DH5α (Life Technologies, USA) with the prepared recombinant plasmid. The resulting transformants were incubated overnight in LB liquid medium containing 100 μg / ml of ampicillin to prepare a gene library of Flavobacterium sacchafilum. Colonies of the prepared gene libraries were inoculated in 96 deep well plates and cultured.

Example 2 Determination of Beta-Glucosidase Activity of the Gene Library of Flavobacterium Saccharophilus

Each culture solution of the Flavobacterium sacchafilum gene library obtained in Example 1 was reacted with a 0.1% paranitrophenyl-glucopyranoside substrate solution and the absorbance of the reactants was measured. As a result of the measurement, paranitrophenol (pNP) was freed from each of the reactants, and a reactant having an increased absorbance at 405 nm was selected as a transformant candidate group.

After adding validamycin A to a concentration of 1% in tris-hydrochloric acid buffer (pH 7.5), the transformant cultures selected above were added and reacted at 30 ° C. for 2 hours. After completion of the reaction, the cells were removed by centrifugation, the supernatant was taken, and the amount of ballidoxylamine A in the supernatant was measured using high performance liquid chromatography. High-performance liquid chromatography was performed using a MicroBondapak (Waters Co.) C18 column, and the separation solution was used as eluent 2 mM ammonium phosphate buffer (pH 8.0). Elution of ballidoxyl A was confirmed at a wavelength of 210 nm of the chromatography, and the activity of converting all of ballidamycin A in the reaction solution to balidoxylamine A was shown (see FIGS. 2A to 2B). As a result of the chromatographic analysis, the sample which confirmed the elution of the ballidoxyl A was selected as a transformant having activity by the beta-glucosidase gene.

Example 3. Transformant E. coli  Gene sequence identification of DH5α / pHbgl

Solution 1 (50 mM glucose, 25 mM Tris-hydrochloric acid, 10 mM EDTA) was added to the transformant selected in Example 2 to attenuate the cell membrane of the transformant, and then solution 2 (0.2 N NaOH, 1% SDS). ) To destroy the cell membrane, denature the proteins and chromosomes of the cells, add solution 3 (5 M potassium acetate, acetic acid) to agglomerate other components except the recombinant plasmid, and then centrifuge to recombine the plasmid. After separating the layers, ethanol was added to precipitate only the recombinant plasmid to recover the recombinant plasmid into which the 3.5 kb gene fragment was inserted, which was named pHbgl (see FIG. 1).

Using the plasmid pHbgl as a template and sequencing using an SP6 primer (Takara, Korea) that specifically binds to a commercially available plasmid pHCE vector, a sequencing sequence (SEQ ID NO: 2) and an amino acid sequence translated therefrom (sequence) Number 1) was obtained. Similarity search was performed on these base sequences and amino acid sequences, and found to be novel genes.

Therefore, E. coli DH5α / pHbgl transformed with the recombinant plasmid pHbgl recovered above was named E. coli DH5α / pHbgl. 95030P).

Gene sequences encoding beta-glucosidase provided by the present invention can be used to mass-produce beta-glucosidase, a key enzyme useful for the preparation of ballidoxylamine A.

Attach sequence list electronic file  

Claims (7)

A gene encoding beta-glucosidase consisting of the amino acid sequence of SEQ ID NO: 1. The gene according to claim 1, which encodes beta-glucosidase having the nucleotide sequence of SEQ ID NO: 2. Recombinant plasmid comprising the gene of claim 1. The recombinant plasmid of claim 3, wherein the recombinant plasmid has a genetic map of FIG. 1. E. coli cell line transformed with the recombinant plasmid of claim 3. The E. coli cell line according to claim 5, wherein the cell line is E. coli DH5α / pHbgl (Accession No .: KACC 95030P). A method of producing validoxamineamine A from validamycin A by an enzyme protein expressed from the gene of claim 1.

Images