KR920008380B1 - Expression vector ptty2 and production of lipase - Google Patents

Abstract

(a) preparing lipase producing recombinant plasmid pJH92 with use of lipase coding DNA sequence-contg. gener fragment dirived from Pseudomonas fluorescens; (b) separating the DNA fragment contg. lipse gene from pJH92; (c) inserting the DNA fragment into tac promotor-contg. high expression plasmid pTTQ19 to obtain the plasmid pTTY2; (d) transforming the pTTY2 into E. coli and culturing; and (e) extracting and separating the lipase inculusion body from the E. coli culture, and refolding. E. coli BL21 contg. high expression plasmid pTTY2 is prepd. and heat resistant lipase can be produced with high yield by industrialmass prodn..

Description

Preparation of high yield plasmid pTTY2 and mass production method of heat resistant lipase using same

1 is a diagram showing the activity of lipase obtained in a new strain transformed by the present invention.

2 is a restriction map of recombinant plasmid pJH92 genetically engineered by the present invention.

3 shows a nucleotide sequence containing a lipase gene isolated by the present invention.

4 is a process chart for preparing a recombinant plasmid for high yield expression isolated by the present invention.

FIG. 5 is a photograph of Sodium dodesyl Sulfate Polyacrylamide Gel Electrophoresis for lipase produced from E. coli transformed with plasmid pTTY2 made using high yield expression plasmid vector pTTYQ19.

Figure 6 is a photograph of the E. coli transformed with the recombinant plasmid pTTY2 to induce lipase production and observed by electron microscopy.

The present invention relates to lipase production of microorganisms involved in making glycerol and fatty acids by hydrolysis of fats. That is, the gene encoding the lipase is isolated from Pseudomonas fluorescens SIK W1, which is reported to produce lipase with high thermal stability, to analyze genetic information, to prepare expression vectors, and in other host cells. The present invention relates to the production of recombinant plasmids for the purpose of regulation and production of gene expression and expression in E. coli and mass production.

Lipase (EC 3.1.1.3) is an enzyme that catalyzes the reaction of hydrolyzing fats with long chains to produce fatty acids and glycerol, mono- and diglycerides, obtained from natural plants and animals and from many microorganisms. In particular, oral lipase, pancreas and liver lipase have been studied for a long time. However, commercially available lipases derived from microorganisms that can be produced regardless of season or place and can be mass-produced within a short time due to the short growth period of cells are used as suitable catalysts.

Lipases have been used critically in dairy and food production, and are particularly important for enhancing the flavor of food. In the milk industry, lipases are used to provide a special flavor, and are also used in the analysis of serum fat and in the detergent industry. Recently, in order to increase the industrial value of these lipases, studies have been conducted in various aspects such as improvement of enzyme properties, synthesis of lipids using reverse reaction of hydrolysis reaction, conversion to high quality, and stabilization of lipase by immobilization.

However, despite their many applications, the industrial use of lipases is not yet active. The reason is that triglyceride, which is a substrate of lipase, is insoluble in aqueous solution, so it is necessary to improve the reaction process in order to maximize the lipase activity, improve the instability of extracellular secretion of lipase, and inexpensive lipase dissemination by mass production. This is a problem.

Lipases are obtained from natural plants and animals and from many microorganisms. It is widely distributed in fungi and bacteria, microorganisms producing lipase. Fungi include Rhizopus arrhizus, Rhizopus delemar, Aspergillus niger, and Muco javanicus at specific positions 1 and 3 of glycerides. It produces a lot of lipases, and bacteria have been studied for the production of enzymes in Staphylococcus aureus, Pseudomonas fluorescens, Pseudomonas fragi, and the like. In particular, Pseudemonas fluorescens, a low-temperature bacterium, has been reported to produce very heat resistant lipases. Driessen et al., Food Science Abstract, 6 (3), 130, 1973) have shown that lipase produced in Pseudomonas fluorescens 22F strain has a very high heat resistance (activity of 90 at 150 ° C). The time taken to decrease% was 4.8 minutes), and since this report, this phenomenon has been reported by Adams et al. (Adams et al. J, Dairy Sci., 64,1951,1981), Anderson et al. (Andersson et al. , J Dairy Sci. 62.361-367 1979), and have been the subject of intensive research for the purpose of identifying and exploiting the properties of these lipases.

The conventional chemical engineering method consumes a lot of energy because it requires high temperature (250 ° C.) and high pressure (55 atm) to hydrolyze fat insoluble in water to form glycerol and fatty acids. Recently, methods for producing fatty acids and mono- and di-glycerides using lipases or immobilized lipases liberated in organic solvents, and modification of fats have been attempted.

These enzyme engineering lipolysis and preparation method can be reacted at room temperature, easy to operate and process and high yield because there are no other side reactions, but lipase is not a protein that is always made in large quantities by cells, and induction of enzyme production for production Large-scale production is difficult because it requires the insoluble substrate needed for the process. In addition, since a small amount is produced and a complicated process is required for purification, the production price is high and its practicality is not suitable yet.

The present invention is to mass-produce the lipase gene of Pseudomonas fluorescens (Pseudomonas fluorescens) reported to be heat-resistant in E. coli using a high yield expression plasmid vector pTTY2. The lipase produced by Pseudomonas fluorescence, a low-temperature bacterium, has been reported by Anderson (Anderssom et al. J. Dairy Sci. 62,361,1979), which is a heat-resistant enzyme that takes about 50 minutes to reduce its activity by 90% at 100 ° C. have. They are also lipases produced from P. fluorescens SIK W1, which is sodium dodecyl sulfate. It has also been reported to be very resistant to chemical denaturants such as urea.

However, they were difficult to characterize because of the small amount of lipase produced, and the factors involved in thermal stability were difficult to study based on chemical composition and structural properties. In order to solve this problem, the present inventors attempted to produce a large amount of lipase by separating genes encoding lipase from P. fluorescens SKI W1 and expressing them in E. coli. As part of this, Pseudomonas fluorescens SKI W1 was donated by Anderson et al., And the lipase gene was isolated from the strain and cloned into Escherichia coli, and it was announced (Korea Industrial Microbiological Society Spring Conference, 1988). .

However, at this time, the amount of lipase produced in E. coli was very small, which made it difficult to use, and efforts were required to produce more efficient and large amounts of lipase. Therefore, in the present invention, when the gene cloned in E. coli is used as a gene source, the entire nucleotide sequence of the gene is determined, and the recombinant plasmid is transformed by linking to a strong expression vector based on this. The transformed strain was designed to have the ability to produce large amounts of lipase through expression induced by IPTG.

PTTQ19 (purchased from Amersham International Plc.UK), the expression vector used in the present invention, is a vector derived from pUC19 derived from IPTG having a strong promoter, the tac promoter and the rmB transcription termination sequence. In addition, since the vector contains the lac I ^q gene, the product produced in Escherichia coli by an external gene is designed to be produced in a precisely controlled state (MJR Stark, Gene, 51, 255, 1987).

On the other hand, when the production of protein by the expression of external genes in E. coli using a strong expression vector (expression) has been reported to form the inclusion body (inclusion body) is produced in most of them. The formation of such enclosures has several advantages, one of which is that the initial separation process is very simple. That is, a relatively high (about 50% or more) purity protein can be obtained from the inclusion body obtained by centrifugation at low speed through a simple operation of culturing E. coli and destroying the cell wall. This simple initial separation stage process provides a reduction in working capacity, saving time and equipment costs by reducing the purification stage, and maximizing purification efficiency.

Another advantage of inclusion body formation is that inclusion body formation increases the stability and accumulation of product during the production of proteins in Escherichia coli by foreign genes. This is in good agreement with the report that proteolytic enzymes present in E. coli do not attack inclusion bodies (Cheng et al. Gene, 14,121,1981). In this case, the separated inclusion body should be able to be dissolved in water and converted into a biologically active form.

The present invention is characterized in that a lipase is produced by an expression vector in Escherichia coli to form an inclusion body, which is then separated into an active lipase. Lipase production in Pseudomonas Pluorescens (P) also requires long generation times while good productivity at temperatures as low as 8 ° C. Thus, lipase production by transformed Escherichia coli with a short generation time at 37 ° C. has a great advantage.

Most of the genetic manipulations used in the present invention were carried out in accordance with the methods listed in the literature (Molecular cloning, A laboratory manual (Cold Spring Harbor Laboratory publication, T. Maniatis et al., 1982)). Specific embodiments of the present invention are as follows.

Example 1

A method of cloning a gene that encodes a lipase from P. fluorescens.

The present invention was carried out by the following method using E. coli strain JM83 and plasmid DNA pUC19.

Since pUC19 plasmid contains ampicillin resistance gene, DNA of P. fluorescens is isolated purely, cleaved with restriction enzyme and linked with pUC19 plasmid to make recombinant plasmid, transformed into E. coli, and then transformed into ampicillin and Genetically engineered microorganisms with ampicillin resistance and lipase activity can be selected from a lipid-coated solid medium.

First, E. coli strain JM83 / pUC19 was extracted in a liquid medium (LB medium) containing bactopeptone (10 g / l), bacto yeast extract (5 g / l), sodium chloride (10 g / l), and pH 7.5 in order to extract the plasmid in large quantities. After shaking incubation at a temperature of 37 ℃, harvested by centrifugation, lysozyme was added, dissolved in sodium dodecyl sulfonate and separated by CsCl-EtBr ultracentrifugation.

Separation of chromosomal DNA from P. fluorescens, a donor strain of the lipase gene, was performed using an improved Marmur method (J. Marmur, J. Mol Biol., 3, 208, 1961). First, P. fluorescens (P. fluorescens) was incubated for 5 hours in 100 ml of the liquid medium described above, harvested using a centrifuge (Beckman Model J2-21 M / E), and added to the 100 mg / ml lysozyme. Cells were destroyed by suspending small amounts of SDS solution. To this, 1 ml of 5 molar sodium chloride was added, suspended in a phenol / chloroform solution, followed by centrifugation to obtain a supernatant, and 2 times 95% ethanol was added to precipitate the resulting DNA. 10 mM Tris buffer solution containing 8.0 mM EDTA pH 8.0 Dissolved in. After treatment with Rnase (RNase), CaCl was dissolved to a final density of 1.55 g / ml, separated in a centrifuge (Beckman, Model TL-100), and used as a chromosome DNA sample. To prepare a recombinant plasmid, the plasmid pUC19 prepared above was treated with BamH I restriction enzyme, and then the isolated chromosomal DNA was mixed with that treated with Sau3A I restriction enzyme and linked using T4 DNA ligase. This linked recombinant plasmid was mixed with E. coli strain JM83, a strain for pretreatment with calcium chloride, and applied in a solid medium containing 0.5% tri-butyrin and ampicillin at a final concentration of 50 µg / ml at 37 ° C. Colonies that hydrolyze tri-butyrin were selected from ampicillin resistant strains after 24 h incubation.

In this way, eight strains (esterase) or lipase (lipase) showing a strain showing the activity was selected. These selected strains were incubated for 24 hours in ROM solid medium (1% olive oil and 0.001% rhodamine B), a screening medium that can distinguish esterase and lipase activity. It was adopted as a lipase producing bacteria that shows orange fluorescence (Fig. 1).

Example 2

Mapping Restriction Enzymes of DNA Encoding Lipases

Recombinant plasmid containing the lipase gene was isolated from the genetically engineered strain and transformed back into E. coli strain JM83 for lipase activity in solid LB medium contained in tri- butyrin, olive oil or ampicillin. Investigation confirmed 100% reproducibility. Plasmids were isolated from this identified strain, and the separated plasmids were named pJH9. The isolated recombinant plasmid pJH9 was digested with EcoR I and Hind III, and the inserted chromosomal DNA fragments were subjected to electroelution after electrophoresis. This was partially digested with Sau3 AI restriction enzyme and electrophoresed on agarose gel, followed by elution of 1-2 kb length DNA fragments. These DNA fragments were bound to pUC 19 carriers digested with BamH I and transformed to select E. coli strains with lipase activity. Examination of these plasmids revealed that they had a 2 kb inserted DNA fragment, from which the final plasmid pJH92 was obtained having a 1.65 kb chromosomal DNA fragment from which 350 base Pst I fragments were removed. Restriction maps were prepared by comparing plasmid pJH92 single or double cleavage with various restriction enzymes and comparing the electrophoretic patterns on agarose gels. 2 shows a restriction map of plasmid pJH92. Recombinant plasmid pJH92 is a new plasmid containing the gene of P. fluorescens lipase.

Example 3

Determination of the Sequence of Nucleotides of DNA Encoding Lipases

Plasmid pJH91 was digested with restriction enzymes Pst I, EcoR I, HindIII, Sal I, Ava II Sau3A I, HaeIII and the like to separate DNA fragments of several hundred bases in length. Each of these isolated DAN fragments was inserted into the polylinker site of the M13 bacterial phage DAN, and then the nucleotide sequence was measured according to Sanger dideoxy chain termination method. The result of the nucleotide sequence measured in this way is shown in FIG.

Example 4

Lipase Expression Plasmids Prepared

In order to mass-produce lipase in Escherichia coli, a recombinant plasmid for expression was prepared using the recombinant plasmid pJH92 obtained above as a gene source. First, the restriction enzymes EcoR I and Pst I were simultaneously treated to decompose the plasmid pJH92, and then a DNA fragment corresponding to 1.65 kilobase (kb) was isolated. The DNA fragment solution was extracted by removing impurities with phenol and chloroform, and then precipitated with ethanol to recover the DNA fragments. Meanwhile, pTTQ19, an expression vector, was treated with EcoR I and Pst I as restriction enzymes at the same time, and then cleaved for 2 hours at 37 ° C., extracted with phenol and chloroform to remove impurities and precipitated with ethanol. After purification was recovered. To this plasmid fragment, DNA fragments containing the lipase gene purified by the method described above were added and reacted in the presence of a DNA ligase of T4 bacterial phage for 3 hours at 16 ° C (recombinant plasmid in FIG. 4). The connected reaction solution was transformed into Escherichia coli strain JM103 (Escherichia coli JM103), Escherichia coli strain HB101 (E. coli 101), and Escherichia coli strain BL 21 (E. coli BL21), and then the selection medium. E. coli showing lipase activity was selected from (LB, ampicillin 100 μg / ml, tri-butyrin 5 mg / ml).

Transformation used a method known by Hanahan (DNA Cloning, Vol. 1, 109, 1985). In addition, to investigate whether the lipase gene was inserted into the desired position in the vector, lipase-activated ampicillin-resistant strains were cultured in LB medium at 37 ° C. for 8-10 hours, and the plasmid was isolated and purified according to a known method. Treatment with the restriction enzymes EcoR I and Pst I and the like confirmed the results after agarose gel electrophoresis. The recombinant plasmid prepared in this manner was named pTTY2.

Example 5

Expression of Lipase Gene in Escherichia Coli Using Expression Plasmid pTTY2

The resulting plasmid pTTY2 regulates the expression of the lipase gene by the tac promoter. In particular, the lac I ^q gene has a plasmid on the plasmid, thereby producing a repressor under normal culture conditions, thereby limiting the action of the tac promoter. Therefore, the following manipulations were carried out to mass-express lipase in Escherichia coli.

First, E. coli containing the recombinant plasmid pTTY2 was inoculated into LB liquid medium containing 100 µg / ml ampicillin, followed by aeration for 8-10 hours at 37 ° C, followed by subculture at 600 nm until the turbidity became 0.5. (isopropyl-thiogalactopyranoside) was added to a final concentration of 1.0 mM and further incubated for 2.5-3 hours.

The expression of lipase in Escherichia coli was examined by 1 ml of the culture medium and SDS-polyacrylamide electrophoresis (see FIG. 5). E. coli HB101 tested. The lipase production by JM103 in JM103 and BL21 is not reproducible and the stability of recombinant plasmid in E. coli is a problem. Therefore, in the present invention, E. coli BL21 was selected as the lipase mass producing bacterium through the operation of screening E. coli BL21. This strain was found to be the highest lipase producing bacterium. (E. coli BL21 containing pTTY2 plasmid was 1990 On November 14, 2014, it was deposited with KTCC 8498 P to the Genetic Engineering Center of the Korea Institute of Science and Technology and can be distributed by lawful procedure). HB101 also showed lower enzyme productivity than BL21. The amount of lipase expressed was examined by densitometer and the results corresponded to about 40% of the total E. coli protein content. In addition, the produced lipase was confirmed in the form of inclusion bodies in the E. coli cytoplasm through electron micrographs (see FIG. 6).

Example 6

Isolation of Inclusion Body and Restoration of Lipase Activity

Enzymes produced in inclusion body form are not active. Therefore, it is necessary to convert to enzymes having biological activity. Enzyme activity recovery of the lipase produced in the form of inclusion bodies in the present invention was carried out according to the known method of Marston F.A.O (DNA Cloning. Vol 3. IRL press. 1985).

The cultured E. coli was harvested by centrifugation, sonicated and centrifuged at 4000 × g for 10 minutes to remove cell debris. The cell aggregates thus obtained were lysed with 8M Urea and the solution was dialyzed in dilution buffer and the lipase activity was measured. Investigation of the lipase activity was performed as follows. The preparation of the substrate solution for the enzymatic reaction was according to the known Anderson method (Andersson: J. Dairy Sci. 62,361, 1979). The gum arabic was dissolved in 50 mM sodium phosphate buffer (pH 7.0) to a final concentration of 5%. 45 ml of this solution was added with 5 ml of olive oil as a substrate, followed by sonication for 10 minutes to form an emulsion. To this was added 30 ml H ₂ O, 30 ml CaCl ₂ (0.075 M), 20 ml NaCl (3 M), 20 ml sodium taurocholate (15 mg / ml). 2 ml of the substrate solution thus prepared was adjusted to a desired temperature and reacted for a predetermined time by adding a pre-prepared enzyme solution. After the enzyme reaction, 0.5 ml of 6N HCl was added to stop the reaction, and the amount of fatty acid produced was measured according to a known fatty acid quantitative method (Kwon et al: JAOCS, 63,89,1986). And the investigation of the thermal stability of the enzyme was carried out as follows. That is, 1.5 μl of an enzyme solution (2.8 mg / ml) was dispensed into an Eppendorf tube in 1.5 ml, and then heat-treated at a specific desired temperature. The heat treated enzyme solution was left at 4 ° C. for 10 minutes and then reacted at 34 ° C. for 15 minutes to determine the residual activity of the enzyme. As a result, when the lipase produced was heat-treated at 80 ° C. and 95 ° C. for 60 minutes, the lipase was 85% and 70%, respectively.

Claims (5)

Plasmid pTTY2, which produces large amounts of lipase and the lipase production is under the control of a tac promoter. The nucleotide sequence below, encoding a heat resistant lipase. (a) a recombinant plasmid pJH92 having lipase production characteristics was prepared using a gene fragment encoding a heat resistant lipase derived from P. fluorescens, and (b) contains a lipase in pJH92. The gene fragment was linked to a high expression plasmid pTTQ19 containing a tac promotor to prepare plasmid pTTY2, (c) transforming Escherichia coli with plasmid pTTY2, and (d) culturing the transformed Escherichia coli And expressing, extracting and separating the lipase inclusion body from the expressed E. coli, and then refolding the active lipase into the active lipase. The method according to claim 3, wherein when cloning the lipase gene in step (a), a strain showing esterase activity in a medium containing tri-butyrin is first selected, followed by olive oli and rhodamine ratios. (rhodamine B) Reselection in a medium containing the pigment to adopt a strain having only lipase activity. 4. The method according to claim 3, wherein the gene encoding the heat resistant lipase of P. fluorescens is the following nucleotide sequence.