KR100343543B1 - Discovery of Bacillus stearothermophilus L1(KCTC 8954P) producing a novel lipase and development of its efficient production method using Escherichia coli BL21(DE3)/pSLE2(KCTC 8955P) - Google Patents

Abstract

The present invention is a bacterium Bacillus stearothermophilus L1 (KCTC 8954P), which is isolated from surrounding soils of hot spring water and has excellent heat resistance lipase activity, and is produced from these new strains. Purification method of L1 lipase using E. coli BL21 (DE3) / pSLE2 (KCTC 8955P) and CM-Sepharose column and DMEM-Sepharose column to mass-produce L1 lipase It is about.

Description

Production of L1 lipase Mass production of L1 lipase using Bacillus steareromophilus L1 (XCTC 8954P) and transformed E. coli ＢL21 (DE3) / WSLE2 (XCTCC 8955P) (Discovery of Bacillus stearothermophilus L1P) producing a novel lipase and development of its efficient production method using Escherichia coli BL21 (DE3) / pSLE2 (KCTC 8955P)}

The present invention is a bacterium Bacillus stearothermophilus L1 (KCTC 8954P), which is isolated from surrounding soils of hot spring water and has excellent heat resistance lipase activity, and is produced from these new strains. Purification method of L1 lipase using E. coli BL21 (DE3) / pSLE2 (KCTC 8955P) and CM-Sepharose column and DMEM-Sepharose column to mass-produce L1 lipase It is about.

Lipase is an enzyme that hydrolyzes long chain triglycerides to produce diglycerides, monoglycerides, glycerol and free fatty acids.

To date, many kinds of animals, plants, and microorganisms have been known to produce lipases, and studies on lipase biochemical properties and lipase genes have been actively conducted. Lipases are industrially important enzymes that catalyze the production of free fatty acids, transesterification of fats, and the synthesis of useful esters and peptides because of their unique properties such as substrate specificity, optical activity specificity and site specificity.

This important reaction is sometimes carried out effectively at high temperatures and under organic solvents. In particular, bee tallow and palm oil, which are mainly used in the production of free fatty acids in the food industry, are solid forms at normal enzymatic reaction temperatures, and therefore, heat-resistant lipases stable at high temperatures are required for effective enzymatic processing. In addition, the use of nontoxic Bacillus-derived enzymes is advantageous for safe use in the food industry.

However, the heat-resistant lipases developed to date have been developed only by lipases produced by Bacillus thermocatenulatus , and the development of effective heat-resistant lipases is very poor.

Accordingly, the present inventors can find useful strains for the production of free fatty acids, transesterification of fats, synthesis of esters and peptides, and search for strains producing stable heat-resistant lipases at high temperatures, and mass production of such lipases. By developing the present invention, the present invention has been completed.

Accordingly, the present invention provides a large amount of novel heat resistant lipases that are stable at high temperatures for the efficient enzymatic processing of bee tallow and palm oil, which are solid at room temperature, which are mainly used for the production of free fatty acids in the food industry. The purpose is.

1 is a schematic diagram for the production of recombinant plasmid pSLE2 for mass production of heat resistant L1 lipase.

2 is a photograph showing L1 lipase and pure purified lipase mass-produced 4 hours after IPTG treatment of transformed Escherichia coli BL21 (DE3) / pSLE2.

3A is a graph showing the thermal stability of L1 lipase; 3b is a graph showing the activity of L1 lipase with pH; 3C is a graph showing pH stability of L1 lipase.

4 is a graph comparing the thermal stability of L1 lipase and Candida cylindracea lipase.

The invention features a writing brush Russ (Bacillusstearothermophilus) and L1 is produced from which stable, L1 lipase at a high temperature as a novel strain of Bacillus stearate.

The present invention also features a method for purifying transformed E. coli BL21 (DE3) / pSLE2 and lipases produced therefrom for mass production of L1 lipases.

The present invention will be described in detail as follows.

In the present invention, after growing at 55 ℃ using the soil around the hot spring as a sample, after separating the Bacillus strain producing a lipase that can decompose olive oil, the strain is identified through the morphological and biochemical characteristics of the isolated strain It was.

The isolated new strain produced spores in the form of motility bacilli. In addition, the new strain had catalase activity, oxidized glucose, hydrolyzed starch, and failed to grow in 7% NaCl.

As mentioned above, the morphological and biochemical properties of the new strain were most similar to Bacillus acidocaldarius and Bacillus stearothermophilus . However, the lowest temperature for the growth of Bacillus Ecija even more lower than the growth minimum temperature of kalda less (Bacillus acidocaldarius), Bacillus Ecija FIG kalda less (Bacillus acidocaldarius) is on the other hand, well grow at acidic pH conditions, god of the invention Strains grew well at neutral pH conditions.

Therefore, the new strain of the present invention was identified as Bacillus stearothermophilus .

In addition, by cloning the gene encoding the lipase produced from the new strain and inferring the amino acid sequence (U78785) from the nucleotide sequence, it consists of a signal peptide consisting of 29 amino acids and a mature lipase consisting of 388 amino acids In addition, it was recognized as a new lipase different from the lipase produced by Bacillus thermocatenulatus .

Thus, the new strain producing the new lipase was named Bacillus stearothermophilus (hereinafter abbreviated as ' B. stearothermophilus ') L1, and deposited and deposited in the Biotechnology Research Institute Gene Bank on August 18, 1999. Was given the number KCTC 8954P.

In addition, as a result of examining the properties of the lipase produced by B. stearothermophilus L1 (hereinafter, weakly referred to as 'L1 lipase'), the L1 lipase showed optimal activity at 68 ° C. It can be very useful for the production of fatty acids, transesterification of fats, synthesis of esters and peptides. In addition, L1 lipase was stable in a wide range of pH 5-11, and the optimal pH was 9-10.

In particular, heat-resistant lipase that is stable at high temperatures is essential for the efficient enzymatic processing of solid fats, bee tallow and palm oil, which are mainly used for the production of free fatty acids in the food industry. In addition to having a heat resistance, such as non-toxic Bacillus enzymes widely used in the food industry, industrial utility is very large.

Therefore, the present inventors have studied for mass production of heat resistant L1 lipase suitable for such industrial applications.

That is, by producing a recombinant plasmid for mass production of L1 lipase, and transforming the recombinant plasmid into E. coli BL21 (DE3), transformed E. coli BL21 (DE3) / pSLE2 to the Biotechnology Research Institute Gene Bank It was deposited on the 18th of February and was granted KCTC 8955P.

As a result of the high expression experiment of L1 lipase using the transformed Escherichia coli BL21 (DE3) / pSLE2, the amount of intracellular L1 lipase increased to 132,000 U per liter of cell extract after 4 hours of culture. The production of L1 lipase is over 132 times greater than B. stearothermophilus L1.

In addition, the present invention has established an effective separation and purification method of lipase mass-produced from transformed Escherichia coli BL21 (DE3) / pSLE2. That is, L1 lipase was purely separated using a CM-Sepharose column and a DEAE-Sepharose column, and showed a recovery of about 62%.

Therefore, through mass production of the lipase of the mass expression system using the transformed E. coli strain, it is very usefully applied to the production of expensive free fatty acids from low-fat fat and the synthesis of various esters and peptides, thereby securing price competitiveness. have.

Hereinafter, Escherichia coli BL21 transformed with Bacillus stearothermophilus L1, a gene encoding L1 lipase, and a recombinant plasmid containing the gene, which produce a heat resistant lipase that hydrolyzes fat stably even at high temperatures. The purification method of (DE3) / pSLE2 and mass-produced L1 lipase will be described in detail through the following examples, but the present invention is not limited thereto.

Example 1 Cloning and Amino Acid Sequence Determination of L1 Lipase DNA

DNA of the new strain B. stearothermophilus L1 was completely cut with the restriction enzyme Hin dIII, cut with the same restriction enzyme and ligation with plasmid pUC19 treated with calf intestinal phosphatase, which was soluble in water. (competent) E. coli RR1 was transformed.

Transformed Escherichia coli RR1 was loaded with ampicillin in TCN plate (1% tryptone, 0.5% yeast extract, 0.5% NaCl, 1% tricaprylin (TCN), 20 mM NaCl, 1 mM CaCl) ₂ , 0.5% gum arabic and 1.5% agar were plated and incubated at 37 ° C. for 24 hours, and then TCN was decomposed to obtain a colony forming a transparent ring. As a result of culturing the colonies to isolate the plasmid, a 7.2 kb sized plasmid containing 4.5 kb of inserted DNA was identified and named pLIP1.

Retransformation of pLIP1 into E. coli RR1 revealed that 100% of the resulting colonies exhibited L1 lipase activity. pLIP1 was cut with restriction enzymes Hin dIII and Not I, and then linked with pBluescript SK cut with the same restriction enzyme and transformed into E. coli RR1 to obtain pLIP2 containing 2.7 kb of insert DNA.

The sequencing of the 2.7 kb insert DNA of pLIP2 was analyzed by Sanger dideoxy chain termination method using a sequence kit, M13 primer and reverse primer. As shown in SEQ ID NO: 3 and SEQ ID NO: 4 using the MacMolly program, an open reading frame (ORF) of L1 lipase consisting of 1,254 bps (417 amino acids) was found. ORF consists of a signal peptide consisting of 29 amino acids and a mature L1 lipase having a molecular weight of 43,125 consisting of 388 amino acids.

The L1 lipase was compared to the amino acid sequences in the GenBank using the Beauty program and found to be 23 amino acids different from the lipase (BTL2) produced by Bacillus thermocatenulatus .

Example 2 Preparation of Recombinant Plasmids for Mass Production of L1 Lipase

In order to produce a large amount of L1 lipase, a plasmid was prepared as shown in FIG. 1. B. stearothermophilus L1 DNA containing L1 lipase DNA was used as template DNA, and PCR was performed using the primers described in SEQ ID NOS: 1 and 2. Recombinant plasmid pSLE2 for mass production was prepared by cutting both ends of the DNA synthesized with restriction enzymes Nde I and Bam HI and then ligation with the mass production vector pET-22b (+) treated with the same restriction enzyme.

Example 3 Preparation of Transformed Escherichia Coli with Mass Production of L1 Lipase

Recombinant plasmid pSLE2 has a strong T7 promoter and readout signal, and thus, when Escherichia coli BL21 (DE3) having a T7 RNA polymerase gene is used as a host, it is possible to mass-produce a desired L1 lipase.

The activity of L1 lipase was measured by a pH stat method in which the amount of fatty acids produced by the hydrolysis of olive oil by L1 lipase at a constant pH was titrated with NaOH, and 1 unit (U) of the enzyme was 1 μmol per minute. It is defined as the amount of enzyme that produces fatty acids.

Transformed Escherichia coli BL21 (DE3) with recombinant plasmid pSLE2 was OD 600 nm at 0.6 in LB medium (1% tryptone, 0.5% yeast extract and 0.5% NaCl) containing ampicillin (100 μg / ml) After culturing until the IPTG (final concentration 1 mM) is added to the culture medium, the amount of intracellular lipase increases rapidly. After the addition of IPTG, the activity of the intracellular lipase was measured by culturing the bacteria, and after 4 hours, the amount of intracellular L1 lipase increased to 132,000 U per liter of the cell extract as shown in FIG. The main band appeared at the molecular weight 43,000 position in the phase.

Therefore, Escherichia coli BL21 (DE3) transformed with the recombinant plasmid pSLE2 was named Escherichia coli BL21 (DE3), which was deposited on August 18, 1999 to the Biotechnology Research Institute Gene Bank to receive accession number 8955P.

L1 lipase produced by the new strain B. stearothermophilus L1 was able to detect activity by decomposing TCN on the TCN plate to form a transparent ring, but the activity was not measured by the pH titration method. The amount of L1 lipase produced by transformed Escherichia coli BL21 (DE3) / pSLE2 is increased by 132 times or more.

Example 4: Separation and Purification Method of L1 Lipase

L1 lipase mass-produced from E. coli BL21 (DE3) / pSLE2 transformed in Example 3 was purified as follows.

That is, after transforming the transformed E. coli BL21 (DE3) / pSLE2 in 10 mM phosphate (pH 6.0) buffer by ultrasonic grinding method, the supernatant obtained by centrifugation is added to the CM-Sepharose column. After eluting the L1 lipase with potassium chloride salt, the buffer was changed to 10 mM Tris-HCl (pH 8.8) and added to a DEAE-Sepharose column, followed by eluting with potassium chloride salt to purify the lipase. The purely isolated L1 lipase has an activity of 1,700 U / mg for olive oil.

Purification of Heat-resistant L1 Lipase from Transformed Escherichia Coli step Total protein (mg) Full active (U) Specific activity (U / mg) Refined (pear) collection(%) Cell free extract 876 128,000 146 One 100 CM-Sepharose 122 89,800 736 5.04 70.1 DEAE-Sepharose 47 79,600 1,690 11.6 62.2

Example 5: Properties of L1 Lipase

The properties of the purely isolated L1 lipase in Example 4 were investigated as follows.

1) Characterization of L1 Lipase

The effect of enzyme temperature and pH on the purely isolated L1 lipase was investigated by pH titration method. As shown in Figure 3a, L1 lipase showed the optimum activity at 68 ℃, the enzyme activity was sharply reduced above 68 ℃. While this property exhibits optimal activity at 30 ° C, it is completely different from typical commercial Candida cylindracea lipase widely used in the food and pharmaceutical industries that do not exhibit activity above 55 ° C (FIG. 4). .

Therefore, L1 lipase is a typical heat resistant enzyme and has the characteristics of an enzyme suitable for various bioconversion reactions that perform the reaction at high temperatures. In order to measure the thermal stability of the lipase, it was allowed to stand at each temperature for 30 minutes, and then enzyme activity was measured at 50 ° C., which was stable up to 65 ° C. in the presence of calcium.

As a result of measuring the lipase activity while varying the pH, as shown in FIG. 3B, the optimum pH was 9 to 10, and after remaining at various pHs for 1 hour, the residual activity of the lipase was measured. It appeared to be stable in the wide range of -11.

A number of proteolytic enzyme inhibitors, diethyl p used as-nitro phenyl phosphate (diethyl p -nitrophenyl phosphate) (E600 ), phenylmethylsulfonyl fluoride (phenylmethylsulfonyl fluoride) (PMSF), ethylenediamine tetraacetic acid (ethylenediaminetetraacetic The effect of L1 lipase on acid (EDTA) and iodoacetamide was slightly inhibited by E600 and iodoacetamide, and little activity was inhibited by PMSF and EDTA. .

In addition, as a result of examining the effects of various metal ions, the activity was not inhibited by most of the ions except copper ions and mercury ions.

On the other hand, as a result of examining the effect of the surfactant, the activity was inhibited reversibly by SDS, little by the surfactant such as Triton X-100 and sodium deoxycholate (sodium deoxycholate).

2) Degradation of Triglycerides, Natural Fats and Natural Fats and Fats Using L1 Lipase

Relative Hydrolytic Capacity of L1 Lipase to Triglycerols, Natural Fats, and Natural Oils Type of fat Relative hydrolysis power (%) Natural Fats and Natural Oils Olive oil 100 Palm oil 152 Beef tallow 150 Coconut oil 139 Cotton seed oil 138 Soybean oil 119 Corn oil 109 Malt (wheat germ oil) 87 Peanut oil 75 Linseed oil 64 Triglycerol Triacetin 30 Tripropionin 548 Tributyrin 480 Tricaproin 210 Tricaprylin 170 Tricaprin 210 Trilaurin 360 Trimyristin 240 Tripalmitin 240 Tristearin 39 Triolein 100 Trilinolein 77 Trilinolenin 93

L1 lipase had a high hydrolytic ability to tripropionin, tributyrin and trilaurin as shown in Table 2, and decomposes solid fats such as palm oil and tallow well among natural fats. It was. This property is different from Bacillus thermocatenulatus lipase, which hydrolyzes tributyrin well, and the amino acid sequence of L1 lipase was similar to Bacillus thermocatenulatus lipase, It was confirmed that the specificity is another novel lipase.

As described above, the present invention relates to a mass production method of L1 lipase using B. stearothermophilus L1 and transformed Escherichia coli BL21 (DE3) / pSLE2. The L1 lipases obtained by the inventors of the mass production method exhibited optimum activity at 68 ° C. and high hydrolytic activity against Uji and palm oil, which are solid fats at room temperature. There is an effect that can be very useful for the synthesis of various esters and peptides.

Claims (6)

Bacillus stearothermophilus L1 (KCTC 8954P), characterized by producing L1 lipase. E. coli BL21 (DE3) / pSLE2 (KCTC 8955P) characterized in that the mass production of L1 lipase. Produced by Bacillus stearothermophilus L1 (KCTC 8954P) or Escherichia coli BL21 (DE3) / pSLE2 (KCTC 8955P), having stable heat resistance at high temperatures and comprising the amino acid sequence described in SEQ ID NO: 4 L1 lipase. A nucleotide sequence encoding L1 lipase set forth in SEQ ID NO: 3. Amino acid sequence for L1 lipase as set forth in SEQ ID NO: 4. The lipase of claim 3, wherein the L1 lipase is separated using a CM-Sepharose column and a DMEM-Sepharose column.