WO2005078103A1 - Method for preparing high-purity agarose by using recombinant arylsulfatase - Google Patents

Abstract

Disclosed herein is a method for preparing high-purity agarose by using arylsufatase. The method comprises ligating arylsulfatase gene derived from Pseudoalteromonas carrageenovora into an expression vector, inserting the expression vector into a microorganism for transformation, culturing the transformed microorganism for mass production of the recombinant arylsulfatase and reacting the recombinant arylsufatase with agar to produce high-purity agarose. According to the present invention, it is possible to realize localization of agarose for biotechnological and medical use which as depended on importation so far and to avoid excessive use of organic solvents which is a main problem in the conventional production process of agarose by substituting an enzymatic production process. Thus, the amount of the used organic solvents can be reduced and waste treatment plant for the solvent Recovery process may be minimized, providing positive economic effect Such as import substitution effect and foreign export opportunity.

Description

METHOD FOR PREPARING HIGH-PURITY AGAROSE BY USING RECOMBINANT ARYLSULFATASE

Technical Field The present invention relates to a method for preparing high-purity agarose by using arylsulfatase and more particularly, a method for preparing high-purity agarose by ligating arylsulfatase gene derived from Pseudoal teromonas carrageenovora to an expression vector, inserting the expression vector in a microorganism for transformation, culturing the transformed microorganism to mass produce recombinant arylsulfatase and producing agarose using the recombinant arylsulfatase.

Background Art Agar is a kind of brown algae such as Gelidium and Gracilaria and has cell wall comprising 70% of agarose and 30% of agaropectin which are polysaccharides ( Carbohydro . Res . 16, ppl89-197, 1971). Agarose comprises agarobiose repeating unit containing modified 1,4 linked 3,6-anhydro- L-galactosyl residues and 1,3 linked D-galactosyl residues ( Carbohydra te polymers . 49, pp491-498, 2002) . On the other hand, the structure of agaropectin, though being similar to agarose, has sulfate group or carboxyl group in a side chain of the agarose saccharide { Carbohydro . Res . 17, pp234-236, 1971) . It has been disclosed a method for producing agarose for biotechnological and medical use by separating agarose and agaropectin from agar using an organic solvent. US Patent Application Serial No. 086,751, which is titled as "Agarose purification method using glycol" discloses a method for purifying agarose using glycol as an organic solvent. In Korea, there is disclosed a method for preparing agarose by treating agar extracted from Gelidium with chitosan, polyetholeneglycol (PEG), cetylpyridium (CPC) by Korea Food Research Institute (KOREAN J. FOOD SCI.TECHNOL, 30(1) , ppllO-114, 1999) . However, these methods using organic solvents requires excessive consumption of organic solvents and labor and additional cost for the recovery process of the organic solvents and the waste treatment plant For example, US Patent Application Serial No. 086,751 produces about 40 to 150 kg of waste solvents (PEG, DMSO, CPC, IPA, acetone, surfactants and the like) in preparing 300g of high-purity agarose by treatment of 1 kg of low-purity agarose . Meanwhile, in Korea, 700 ton of agar is annually produced (2001, Fishieries Yearbook) . Its 60 to 70% is exported and the rest is domestically consumed for food processing (MSC Co. Ltd., Korea, producing and selling edible agar) . Since the agar produced in Korea has excellent quality, it is exported to many major agarose producing companies such as FMC in USA. The producer price of the agar row material is about 1000 won per 1 kg and when formulated in agar powder or agar string, the selling price is about 24,000 about per 1 kg. However, the whole quantity of the exported agar is simply processed products and high-purity agarose for medical use requiring high quality entirely depends on importation due to imperfection of agarose producing technology. Though the price of the imported agarose varies according to usage, it is 1,000 to 2,000 times higher added value than the price of the raw material. Most of the researches of agar and agarose conducted in Korea include the component differences of agar according to seasons and regions [ Bull . Korean Fish . Soc . 18(1), 37- 43(1985)], conditions of pre-treatment for extraction of agar [ J. Korean Fish . Soc . 30(3), 423-427(1997), Korean J. Food SCI . Technol . 17(5), 340-344 (1985)] and ingredients of agar. There has not been reported a method for producing agarose and high-purity agarose from agar (criteria: sulfur content 0.2 % or less) and the researches are limited to preparation of agar for food and microorganism cultivation ( J. Korean Fish . Soc. 31(5), 673-676, 1998). Accordingly, in order to solve the problems in the process owing to excessive use of organic solvents, the present inventors introduce enzymatic production process, thereby minimizing the amount of the organic solvent, eliminating the recovery process of the organic solvent and miniaturizing the treatment plant. Thus, we have tried to develop a process for converting agaropectin to agarose by using an enzyme, instead of an organic solvent, to remove sulfate group of agaropectin.

Disclosure of Invention In order to solve the foregoing problems, the present inventors paid attention to a microorganism capable of decomposing carrageenan which is a substance known to have a molecular structure similar to that of agar. Thus, it is an object of the present invention to provide a method for preparing high-purity agarose by producing a recombinant arylsulfatase gene derived from Pseudoal teromonas carrageenovora , transforming a microorganism with the recombinant gene for mass expression, reacting the expressed arylsulfatase with agar, thereby solving the problems of environment pollution and high production cost caused by excessive use of organic solvents, which are involved in the prior art. To achieve the above object, according to the present invention, there is provided an arylsulfatase gene represented by the nucleotide sequence of SEQ NO. 3 comprising recognition sites of Bamti I and Hind ID? restriction enzymes and the gene of arylsulfatase by amplifying Pseudoal teromonas carrageenovora ATCC43555 chromosome and primers of SEQ NOs . 1 and 2 by PCR. Also, in accordance with the present invention, there is provided arylsulfatase represented by the amino acid sequence of SEQ NO. 4, which is translated from the above- described gene. In another aspect of the present invention, there is provided a recombinant expression vector comprising the above-described arylsulfatase gene. In a further aspect of the present invention, there is provided a microorganism transformed by the above-described recombinant expression vector. In a further aspect of the present invention, there is provided a method for producing arylsulfatase comprising culturing the above-described microorganism. In a further aspect of the present invention, there is provided a method for preparing high-purity agarose comprising reacting agar with the arylsulfatase produced by the above-described method. The agarose prepared by reacting agar with the recombinant arylsulfatase produced according to the present invention shows no difference from the agarose conventionally produced to be used in biotechnological and medical application in the aspect of performance and effect.

Brief Description of Drawings Further objects and advantages of the invention can be more fully understood from the following detailed description taken in conjunction with the accompanying drawings in which: Fig. 1 shows a gene map of pAST-Al, which is a recombinant expression vector; Fig. 2 shows arylsulfatase activity of the microorganisms which have been cultured on a solid plate medium, in which (A) is E. coli DH5I p pAST-Al, (B) is E. coli BL2KDE3) /pET21a, and (C) is E. coli BL21 (DE3) /pAST-Al; Fig. 3 shows a graph of strain multiplication (A) and arylsulfatase activity (B) in the E. coli BL21 (DE3) /pAST-Al strain (37^°C) according to the culture time, in which the activity is measured by adding 10 uM of IPTG at the exponential phase of the strain (the arrow represents the point at which IPTG is added) ; Fig. 4 shows a histogram of strain multiplication and arylsulfatase activity of the E. coli BL21 (DE3) /pAST-Al strain which is treated with IPTG at various concentrations after 2a hours and cultured for 8 hours; Fig. 5 shows a photograph of electrophoresis of the product treated with λ HindJR , a standard DNA, using agarose, in which A lane represents a normal agar gel, E lane represents agarose according to the present invention and Ag lane represents a normal agarose; and Fig. 6 is a photograph showing the result of electrophoresis (A) of the transformed strain lysate and the arylsulfatase active site shown by the zymogram reaction.

Best Mode for Carrying Out the Invention The present invention is directed to a method comprising the steps of: amplifying arylsulfatase structural gene by designing a novel primer to introduce a site of a novel restriction enzyme capable of recognizing the gene and reacting the designed primer with chromosomal DNA of Pseudoal teromonas carrageenovora as template DNA by PCR; preparing a recombinant expression vector by digesting the gene DNA amplified in the above step with a restriction enzyme and inserting the digested DNA into an expression vector which has been digested with the same restriction enzyme; introducing the recombinant expression vector prepared in the above step in to a microorganism (strain) for transformation and placing the transformed microorganism in a selected medium, followed by storage in a freezer; culturing the microorganism prepared in the above step under a condition to overexpress arylsulfatase; homogenizing the cultured cell in the culture medium of the microorganism strain, followed by centrifuging to give a crude enzyme solution, and adding ammonium sulfate to the crude enzyme solution, followed by centrifuging to prepare an enzyme solution; reacting the crude enzyme solution or the enzyme solution prepared in the above step with agar to produce high-purity agarose; separating agarose only by purifying and dehydrating the product of the above step and measuring gel strength and electrophoresis of the produced agarose; separating arylsulfatase by purifying the enzyme solution obtained by culturing the transformed microorganism; and analyzing optimal conditions for growth of the transformed microorganism, optimal conditions for expression of the recombinant arylsulfatase and optimal conditions for activity of the recombinant arylsulfatase. Here, the recombinant expression vector which is used according to the present invention is pAST-Al produced by inserting arylsulfatase gene derived from Pseudoal teromonas carrageenovora into pET21a vector. For amplification, the pAS-Al recombinant expression vector is transformed into E. coli DH5 I and E. coli DH5 Ip, followed by culturing. Furthermore, the amplified pAST-Al recombinant vector is preferably transformed into E . coli BL21 (DE3) so that the recombinant arylsulfatase produced by the microorganism transformed according to the present invention is active and stable upon reuse even after storage in a freezer. The finally transformed microorganism is called E. coli BL21 (DE3) /pAST-Al and deposited in Korean Agricultural Culture Collection (KACC) , National Institute of Agricultural Biotechnology in Korean Rural Development Administration on January 26, 2004 as Accession Number KACC 91091. Also, it is deposited in Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology on February 14, 2005 as Accession Number KCTC 10778BP for international patent application. Preferably, the E. coli BL21 (DE3) /pAST-Al according to the present invention is cultured in a medium having a IPTG concentration of 5 to 50 μM, since pET21a vector which is used in the present invention comprises a gene which may be over-expressed by IPTG Also, the arylsulfatase produced according to the present invention is reacted with 0.4 to 0.8% of agar at 35 to 50°C for 6 to 12 hours. Here, the concentration of arylsulfatase is preferably 0.5 to 4 Units per agar 1 g. This is because arylsulfatase cannot homogeneously react with agar at a high concentration of 0.8% or more due to high viscosity. When arylsulfatase is used at a concentration of 0.5 Unit relative to agar concentration, it is impossible to produce high-purity agarose while when arylsulfatase is used at a concentration of 4 Unit or more, the production efficiency is lowered as compared to the increase in production cost. Further, according to the present invention, the supernatant of the culture medium from the cultivation to express the recombinant arylsulfatase, strain homogenate or a mixture thereof, and the recombinant arylsulfatase purified and isolated from the foregoing liquids may be used in the reaction with agar in the method for preparing high purity agarose. General molecular biological experiment methods related to the gene manipulation follow the known methods (see: Sambrook, J. et al . (1989) Molecular Cloning. A laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory. Cold Spring Harbor. New York.; Ausubel, F. et al . (1995) Short Protocols in Molecular Biology. 3rd. John & Wiley Sons, Inc.). Now, the present invention is explained in further detail using the following examples. However, it should be understood that the present invention is not limited thereto.

Example 1. Preparation of Recombinant Expression Vector For amplification of the arylsulfatase structural gene and introduction of the new restriction enzyme recognition site, primer ( I ) and (II) having the following nucleotide sequences were designed and composed by Bioprogen Co. (Korea) . 5'-CGGGATCCCATGCAATTAGTATTATA-3' ( I ) 5 ' -CCAAGCTTTTAGCGTTTTAGTTCGTAAC-3 ' ( II ) 1 ≠ (100 pmoles) of primer ( I ) and (II), 50 ng of Pseudoal teromonas carrageenovora (ATCC 43555 chromosomal DNA as template DNA, 10 μi of 10X polymerase buffer and 10 μi of 10X dNTP mixture (2 mM of dGTP, dATP, dTTP and dCTP, produced by TAKARA, Japan) were mixed with 72 μi of sterile distilled water to make the total volume 100 μi . The reaction was heated at 95^°C for 5 minutes and cooled to 72^°C. The PCR (Polymerase Chain Reaction) was performed by repeating a cycle including 1 minute at 95^°C, 55 seconds at 55^°C and 2 minutes at 72^°C 25 times using 1 μi (5 Unit/ β) of Taq DNA polymerase (TAKARA, Japan) . The DNA fragment obtained from the PCR amplification was digested with BamHI and HindiII. The digested DNA fragment was ligated to plasmid pET21a (Novagen, Germany) which had been digested with BamHI and Hindi11 to prepare plasmid pAST-Al (6,416bp). The plasmid pAST-Al comprised sequentially T7-Lac fusion promoter, the arylsulfatase structural gene starting with ATG initiation codon and T7 transcription terminator. The chromosome map of plasmid pAST-Al of the recombinant expression vector is shown in Fig. 1.

Example 2. Transformation The plasmid pAST-Al prepared in Example 1 were transformed into E . coli DH5I p { recAl endAl gyrA96 thil hsdRl l supE44 relAl lacZ M15) by the calcium chloride method. For transformation into the strain, E. coli DH5I p was inoculated into 2 ml of LB medium (0.5% yeast extract, 1% Bacto-tryptone, 0.5% sodium chloride) and cultured at 37^°C for 12 hours while shaking. 100 μi of the culture fluid was then inoculated into 20 ιn£ of fresh LB medium and cultured at 30^°C overnight while shaking until the optical density of the medium at 600 nm was 0.5. Then, 10 ml of the culture fluid was centrifuged at 4 °C and at a speed of 5000 rpm for 10 minutes to remove the medium and 5 ml of 25 mM calcium chloride solution which had been previously cooled to 4 ^°C was added to the resulting cell precipitates, left at 4 °C for 10 minutes and centrifuged at 4 ^°C at a speed of 6000 rpm for 10 minutes to remove the calcium chloride solution. Again, 5 ml of calcium chloride solution was added thereto and the above- described procedures were repeated to remove the calcium chloride solution. To the residue, 1 ml of 25mM calcium chloride + 10% glycerol solution was added and kept at 4 ^°C for at least 30 minutes. The solution was then dispensed in 100 μi aliquots to Eppendorf tubes and kept at -70^°C, to be used as the strain for transformation. E. coli DH5 α was primarily transformed by adding the plasmid pAST-Al prepared in Example 1 to the Eppendorf tubes containing the strain. The transformed E. coli DH5I p pAST- Al was cultured by the liquid culture method for amplification. The amplified plasmid pAST-Al was secondarily transformed into E. coli BL21(DE3) which was the strain capable of stably mass-expressing arylsulfatase by the calcium chloride method.

Example 3. Screening of Transformed Strain In order to select the transformed strain, LBA medium

(LB medium containing ampicillin 50

or LBAIM medium

(LB medium containing ampicillin 50 μg/ml, 1 mM IPTG, 5-100 mM 4-methylumbelliferyl-sulfate) was used to screen the transformed strains. As can be seen from Fig. 2, only E. coli BL21 (DE3) /pAST-Al produced by transforming E. coli BL21(DE3) which was the E. coli strain capable of producing T7 RNA polymerase with pAST-Al showed arylsulfatase activity in the LBAIM medium. The E. coli BL21 (DE3) /pAST-Al was deposited in Korean Agricultural Culture Collection (KACC) , National Institute of Agricultural Biotechnology in Korean Rural Development Administration on January 26, 2004 as Accession Number KACC 91091.

Example 4. Conditions for Expression of Recombinant Arylsulfatase This experiment was performed to examine the activity level of arylsulfatase according to the concentration of IPTG (isopropyl-D-thio-galactopyranoside) , an inducing agent, as follows . Here, the growth of the strain was measured by absorption (OD₆oo) as compared to the culture fluid and the arylsulfatase activity was represented by Unit (unit/m ) , in which 1 Unit was an amount of enzyme releasing 1 μmole of p- nitrophenol (pNP) at 40 ^°C for 1 minute. The measurement of pNP was performed by measurement of absorption at 410 nm. The E. coli BL21 (DE3) /pAST-Al strain of Example 3 was inoculated into 5 ml of LBA medium (LB + ampicillin 30 μg/ml) for pre-culturing. The resulting strain culture fluid was inoculated into a test tube containing 10 ml of fresh LBA medium at a concentration of 1 to 5%. When the strain concentration was 0.5 to 1.0(OD6oo)/ that is, at the exponential phase of the strain, 10 μM of IPTG was added to the tube, the growth of the strain and arylsulfatase activity was examined at various times. As shown in Fig. 3, it was noted that the arylsulfatase activity in the E . coli transformant appeared as the strain grew and the activity was about 2 Unit per ml . Also, in order to examine the aspect of arylsulfatase expression of E. coli BL21 (DE3) /pAST-Al according to the concentration of the IPTG inducing agent, after culturing for 2 hours, IPTG at different concentrations was added to the medium, followed by culturing for further 8 hours. Then, growth of the strain and arylsulfatase activity in the supernatant of the culture fluid and the soluble fraction of the strain lysate were examined. The strain lysate was obtained by centrifuging the culture fluid after completion of the cultivation at 6,000rpm for 10 minutes, removing the supernatant and suspended the precipitates in 20 to 50mM of Tris-HCl (pH 8.5) buffer. The suspension was homogenized by using Ultrasonocator (Germany) and the soluble fraction of the homogenate was used as the lysate of the strain. As shown in Fig. 4, when IPTG was 10 μM, the strain lysate showed the highest arylsulfatase activity of 2.1 Unit. When the IPTG concentration between 50 to 5000 μM IPTG, both the supernatant of the culture fluid and the strain lysate showed arylsulfatase activity. At the low IPTG concentration, the supernatant of the culture fluid showed higher arylsulfatase activity than the strain lysate. Thus, in order to express the maximum enzyme activity, the strain would be cultured at an IPTG concentration of 5 to 50 μM, preferably 1 to 20 μM. In particular, when the active enzyme is intended to be produced in the supernatant of the culture fluid, the strain is preferably over-expressed at an IPTG concentration of 20 μM or more. In other hand, the cultivation is aimed at growth of E. coli and production of recombinant protein which is active in cells, it is performed at an IPTM concentration of 1 to 20 μM. Therefore, it was noted that there is secretory location of recombinant arylsulfatase according to the IPTG concentration .

Example 5. Production of High-purity Agarose Using Strain lysate The strain lysate of the E. coli BL21 (DE3) /pAST-Al was mixed with 0.6% agar (Junsei agar, Japan) solution in a mixing rate of 1 Unit to agar lg and reacted at 40 ^°C for 12 hours. The product was subjected to the freezing and thawing process and examined for gel strength (Compac-100, Sun Scientific Co., Japan), electrophoresis of gel using reference DNA (λ DNA-Hindlll marker) and sulfate removal of the product. Here, the measurements were performed on the basis of 1% gel and the results are shown in Table 1 and Fig. 5.

[Table l]

From the result, it is noted that it is possible to remove 75% of sulfur and the agarose produced according to he present invention has a gel strength lower than that of the conventional agarose (Invitron Co.) but has a electrophoresis result similar to that of the conventional agarose. Meanwhile, it is noted that the mobility of the conventional agarose on the electrophoresis was poor.

Example 6. Zymogram of Lysate of Transformed Strain The strain lysate of Example 4 was subjected to electrophoresis to separate a protein. The separated protein part on the gel was reacted with a specific substrate visible to the naked eyes to examine the activity of arylsulfatase in the strain lysate, thereby determining the molecular weight of arylsulfatase. The electrophoresis was performed by using 10% SDS PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel to separate the lysate of the strain. After separation, the gel was washed several times with SDS washing buffer (50mM Tris-HCl pH8.5) while stirring at 4 ^°C for 4 hours to remove the SDS component from the gel. The acrylamide gel with the SDS component removed was overlaid on 0.8% agarose gel containing 1 to 5mM of MUFS (4-methylumbelliferyl sulfate) , a synthetic substrate and subjected to zymogram analysis in a thermostat at 37 to 40^°Cfor 30 minutes. After completion of the zymogram reaction, the agarose gel was irradiated with UV at 360nm to examine the change of the synthetic substrate. As shown in Fig. 6, A shows the aspect of the protein separation in the strain lysate which has been stained with 10% SDS PAGE and B shows a photograph of the colored part of the agarose gel after the zymogram reaction which has been irradiated by UV rays, in which M lane represents standard molecular weight, E lane represents the strain lysate of the E . coli BL21 (DE) /pET21a and Al represents the electrophoresis of the strain lysate of the E. coli BL21 (DE) /pAST-Al . It was shown that only the lysate of the strain having the recombinant vector pAST-Al with arylsulfatase gene inserted had enzyme activity, which corresponds to the site of about 33.1kDa of the SDS PAGE gel, indicating the molecular weight of arylsulfatase.

Industrial Applicability According to the present invention, it is possible to realize localization of agarose for biotechnological and medical use which depends on importation, thereby reducing foreign currency waste and providing the obtaining of foreign currencies . Also, according to the present invention, it is possible to avoid excessive use of organic solvents which is a main problem in the conventional production process of agarose by substituting an enzymatic production process. Thus, the amount of the used organic solvents can be reduced and waste treatment plant for the solvent recovery process may be minimized.

Applicant's or agent's International app licanon No. file reference 2004P-0021

INDICATIONS RELATING TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL

A. The indications made below relate to the deposited microorganism or other biological material referred to in the description on page 33 , line B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet | | Name of depositary institution Korea Research Institute of Bioscience and Biotechnology Address of depositary insttrution (including postal code and country) Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology 52, Oun-dong, Yusong-gu, Taejon, 305-333, Korea

Date of deposit Accession Number February 14, 2005 KCTC 10778BP C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet | |

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States)

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable) The indications listed below will be submitted to the International Bureau later (specify the general nature of the indications e g , "Accession Sianber of Deposit")

For receiving Office use only For International Bureau use onlv | | This sheet was received with the international application I | This sheet was received by the Inte naπonal Bureau on:

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Form PCT/RO/134 (Juiyl998; reprint January 2004)

Claims

What Is Claimed Is:

1. An arylsulfatase gene represented by the nucleotide sequence of SEQ NO. 3 comprising recognition sites of Bamti I and Hind TR restriction enzymes and the gene of arylsulfatase, by amplifying Pseudoal teromonas carrageenovora ATCC43555 chromosome and primers of SEQ NOs . 1 ^• and 2 by PCR.

2. Arylsulfatase represented by the amino acid sequence of SEQ NO. 4, which is translated from the gene as defined in claim 1.

3. A recombinant expression vector comprising the arylsulfatase gene as defined in claim 1.

4. The recombinant expression vector according to claim 3, in which the vector is pAST-Al.

5. A microorganism transformed by the recombinant expression vector as defined in claim 4.

6. The microorganism according to claim 5, in which the microorganism is E. coli BL21 (DE3) /pAST-Al (Accession Number KCTC 10778BP) .

7. A method for producing arylsulfatase comprising culturing the microorganism as defined in claim 5.

8. The method according to claim 7, in which the culturing is performed at an IPTG concentration of 5 to 50 μM.

9. A method for preparing high-purity agarose comprising reacting agar with the arylsulfatase as defined in claim 2.

10. The method according to claim 9, in which the reaction is performed at a arylsulfatase concentration of 0.5 to 4 Units per agar lg at 35 to 50^°C for 6 to 12 hours.

11. The method according to claim 10, in which the agar concentration is 0.4 to 0.8%.