JPH03198778A - New plasmid, transformant containing the same plasmid and production of thermostable lipase using the same transformant - Google Patents

Abstract

PURPOSE:To efficiently produce a thermostable lipase by synthesizing plasmid pHTLip1 having a wide host vector integrated with DNA fragment containing a thermostable lipase gene derived from Pseudomonas. CONSTITUTION:Plasmid pHTLip1 integrated with DNA fragment containing a thermostable lipase gene derived from Pseudomonas and capable of manifesting in a bacterium of the genus Pseudomonas is synthesized. A cell of bacterium belonging to the genus Pseudomonas is transformed by using the plasmid pHTLip1 and the transformant is cultured in a nutritive medium to form thermostable lipase in the culture solution. Then the thermostable lipase is collected from the culture solution and purified. Productivity of the thermostable lipase by the bacteria of gene recombination by this procedure is about 20 times as much as that of original strain.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention provides a recombinant DNA containing a thermostable lipase gene,
The present invention relates to a Pseudomonas bacterium containing the same and a method for producing heat-stable lipase using the same. Lipase is known as an enzyme that hydrolyzes lipids, but in recent years, ester synthesis reactions that utilize the reverse reaction by lipase have been attracting attention.

Therefore, the thermostable lipase produced by the present invention is
This enzyme is expected to have industrial uses such as processing and modifying fats and oils.

[Prior art]

Conventionally, industrial methods such as processing and modifying fats and oils have mostly been carried out using chemical methods, which require harsh reaction conditions such as high temperature, high pressure conditions, or alkali treatment. Recently, biochemical methods using lipase have been attempted in order to save energy consumption and prevent pollution caused by industrial wastewater.

[Problems with conventional technology]

For industrial use of lipases, it is more preferable to use heat-stable lipases, but the ones known so far as heat-stable lipases are, for example,
Thermostable lipase of eudomonas mephitica [, ], Ferment, Techno 1. ．． 5
Volume 1, 895-903 (1,973), Special Publication 1977-2
No. 5553] and Pseudomonas cepacia (Pseu
Thermostable lipase (domonas cepacia)
(Japanese Patent Publication No. 57-59753), etc., and all of these had low productivity and were problematic for industrial use as they were.

[Means for solving problems]

In order to solve the problem mentioned above, the present inventors created a strain with high production of heat-stable lipase using genetic recombination technology.
We thought that it would be possible to supply heat-stable lipase to the market at a low cost by cultivating the obtained strain, producing a significant amount of heat-stable lipase in the culture solution, and recovering it, and conducted extensive studies. The present inventors isolated a DNA fragment containing a heat-stable lipase gene from the chromosome of a thermostable lipase-producing bacterium, Pseudomonas spp., obtained a plasmid containing the DNA fragment, and used this to infect Pseudomonas spp. By transforming and culturing the transformant, they were able to produce a large amount of heat-stable lipase outside the bacterial cells, and succeeded in harvesting it.

The source of the chromosomal DNA containing the thermostable lipase gene of the present invention is supplied from a Pseudomonas bacterium capable of producing thermostable lipase. For example, Kogata et al.
Pseudomonas mefitica varietus lipolytei (No. 53) isolated by Pseudomonas mefitica varietus
s mephitica var, 1ipolyti
A strain named ca) is mentioned. The mycological properties of this strain are described in Japanese Patent Publication No. 50-25553, and this strain has been published in FERM P-5
20) has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology.

Preparation of Transformants Chromosomal DNA and transformants according to the present invention can be prepared generally by the following steps.

(1) Preparation method of chromosomal DNA Pseudomonas mephitica varietus (ca.
var.

1ipolytica) FERM P-520 as LB
Medium (tryptone = 4-1.0%, yeast extract 0.5%, sodium chloride 1.0
%) at 30° C. overnight aerobically. After collecting the bacterial cells by centrifugation, the method of Saito and Miura [Biochim
.

Biophys, Acta, Vol. 72, 619-629.
(1,963)) using known methods such as chromosomal DNA
A is extracted and purified to obtain DNA.

(2) Method for inserting a DNA fragment into a plasmid vector and transforming the plasmid vector. Preferably, the plasmid vector is one that can be replicated within the host, has a known restriction enzyme cleavage site, and has a selection marker such as drug resistance. For example R3F1010, R1b6
Examples include broad host range plasmid vectors such as 79 and R1162. More specifically, ρFL210, etc., in which a kanamycin resistance gene, a chloramphenicol resistance gene, etc. are introduced into R3FIOIO, can be used.

The vector DNA thus obtained is cut with a restriction enzyme such as Hindlrl, mixed with a chromosomal DNA fragment cut with the same restriction enzyme and purified, and reacted by a known method using ligase or the like to obtain a recombinant plasmid.

The recombinant plasmid is then used to infect Pseudomonas bacteria, such as Pseudomonas mephiti.
Monas mephj, tica) is transformed.

For transformation, methods such as the calcium chloride method, the rubidium chloride method, and the electroporation method are used.

For selective isolation of transformants capable of producing thermostable lipase, it is convenient to use an agar medium containing a triglyceride such as tributyrin or triolein emulsified with polyvinyl alcohol and an antibiotic at a predetermined concentration.

That is, by isolating strains that form a large rear zone on the agar medium from among the transformed bacteria, bacteria containing the lipase gene can be selectively isolated. Plasmid DNA containing a heat-stable lipase gene is obtained from this bacterium using a known method such as an alkaline method or a boiling method.

Furthermore, thermostable lipase is produced using Pseudomonas bacteria obtained by introducing the plasmid DNA of the present invention thus obtained. For example, Pseudomonas mephitica varietus lipotei (Pseudomonas mephitj, ca.
var, Hpol, ytj, ca) FERN P-5
It can be obtained by culturing No. 20 using a known medium, and separating and purifying the thermostable lipase from the culture supernatant. The enzymatic properties of the obtained lipase are described in Japanese Patent Publication No. 50-25553, and the main properties are as follows.

1. Action: Has lipolytic properties and decomposes beef tallow, coconut oil, etc. to a decomposition rate of 92'A at a reaction temperature of 70°C.

2. Substrate specificity: Decomposes various glycerides, esters, lecithin, etc.

3. Stable pH range: pH 3~], ], , 5 (3
7°C) 4, optimal pl+ range: p86.5-7.05,
Suitable temperature for action = 70°C 6. Heat resistance: In the form of an aqueous solution, it does not lose its activity even when kept at 60°C for 14 hours, and becomes 75% in 20 minutes at 73°C.

7. Molecular weight Estimated molecular weight by bimolecular sieve is 160,000
It is.

8. Effect of bile acids: Even when bile acids are added to the enzyme solution at pH 7, there is no change in activity.

Lipase activity measurement method Substrate solution 5IIIQ made by mixing and emulsifying 2% polyvinyl alcohol (PVA) solution 2251 and olive oil 75 mΩ and 0.2 M pinequilpine buffer (pH 7,0) 4 m
Place Q in a flat bottom test tube and pre-humidify at 37°C for 5 minutes. Add sample solution 1n+9 to this, shake well, and immediately raise the temperature to 37°C.
, leave for 30 minutes. After 30 minutes, add 10 ml of acetone/ethanol mixture (1:1) and mix well. To this, add 10 mL of 0.05N sodium hydroxide solution and 10+J of acetone/ethanol mixture (1:1), and further add 2 drops of phenolphthalein test solution, and stir with a Starsea while blowing nitrogen gas onto the liquid surface. Tsutsu, 0.
Titrate to pH 10,00 with 05N hydrochloric acid. For the blank value, use purified water instead of the sample solution and perform the same operation. For enzyme activity, one unit is the amount of enzyme that produces 1 micromole of fatty acid per minute.

The present invention will be specifically explained below with reference to Examples.

8- Arrow 1 (1) Preparation of chromosomal DNA Pseudomonas mephitica varietus (Pseudomonas mephitica)
var.

1ipolytica) FERM P-520 as LB
Culture with aerobic shaking overnight at 30°C in a medium (1.0% tryptone, 0.5% yeast extract, 1% to 1.0% lithium chloride), and after collecting the bacteria, use the method of Kirei and Miura ( Biochim,
Biophys, Acta, vol. 72, 61.9-
629 (1963)), chromosomal DNA was extracted and purified to obtain 4.6 mg of chromosomal DNA.

(2) Insertion of chromosomal DNA fragment into plasmid vector Preparation of chromosomal DNA fragment involves inserting 8.0μ of the chromosomal DNA into
The restriction enzyme Hjndm was added to g, and the reaction was carried out at 37°C for 15 minutes to partially cleave. On the other hand, as a plasmid vector, broad host range plasmid vector R3
A plasmid pFL210 was prepared in advance and constructed by introducing a kanamycin resistance gene derived from pUC-4, a chloramphenicol resistance gene derived from pBR328, and a multi-cloning site derived from pUc18 into FIOIO, and this pFL210 was limited to 2.5 μg. Enzyme Hind
m was added, reacted at 37° C. for 2 hours to completely cleave it, and used after dephosphorylation with alkaline phosphatase. The prepared chromosomal DNA fragment and plasmid vector were mixed, DNA ligase was added, and the mixture was reacted overnight at 16°C to perform a ligation reaction.

(3) Transformation using plasmids When selecting a lipase gene, it is convenient if the host is a lipase gene-deficient strain. Therefore, Pseudomonas mephytica varietus lipolyteis
onas mephitica var, lipol
ytica) FERM P-520 was treated with nitrosoguanidine and treated with Antibiotic Medium 3 containing 0.1% triolein emulsified with polyvinyl alcohol
Mutant strain KW-1, which does not form a halo, was selected using
was separated. KW-1 strain was transformed with the ligated DNA by electroporation, and 200 μg/I
As a result of selection on LB medium containing IIQ chloramphenicol and 0.1% triolein, 17 transformants that formed a halo were obtained.

(4) Analysis of lipase gene Plasmids were extracted and isolated from the 17 halo-forming strains, cut with the restriction enzyme Hindm, and the inserted DNA fragments were analyzed by agarose electrophoresis. All 17 strains contained approximately 8 Kb Hindm. m fragments were commonly identified. The 8Kb Hindm fragment was transferred to plasmid vector PFL2.
], recombinant DN linked to the Hindll site of 0
KW-1 by the Elek 1-loboration method using A
When the strain was transformed, a halo-forming strain was obtained, so it was concluded that the fragment-ase gene was present on the 118 Kb)lindm fragment. A plasmid vector containing the 8 Kb Hindm fragment was named pHTLipl, and its restriction enzyme map is shown in FIG. Each DNA fragment obtained by cleaving pHTLipl with various restriction enzymes was subcloned into plasmid vector pFL21Q, and the KW-1 strain was transformed, and the presence or absence of halo formation in triolein medium was examined in the same manner as above. The results are shown in Figure 2. In addition, the 3Kb EcoRI-XhoI fragment was
21. KW by plasmid subcloned into O.
-1- strain transformant also showed lipase producing ability.

(5) Production of lipase Pseudomonas mephiteica varietus lipolytei (P
seudomonas mephitjca var,
lipolytica) FERM P-520 was transformed. This strain was mixed with 2.0% soybean oil and 0.5% peptone.
(manufactured by Difco), meat extract 0.3% (manufactured by Difco), KH2PO40.1%, MgSO4.7H200
, 02%, Fe50. ' 78200.001% and 1 drop of Adekanol in a liquid medium at 30°C.
The cells were cultured with shaking for 1 day and centrifuged to obtain a culture supernatant. In addition,
Lipase activity was determined using a mixed emulsified solution of 2% polyvinyl alcohol solution and olive oil as a substrate, and pH 7.0.3.
The reaction was carried out for 30 minutes at 7°C, and the amount of free fatty acids was determined by titration. For enzyme activity, one unit was defined as the amount of enzyme that produced 1 micromole of fatty acid per minute.

The lipase activity of the culture supernatant was approximately 400 U/++bQ, and the lipase productivity of the recombinant tree was higher than that of Pseudomonas.
Pseudomonas mephitica 1 2 Pseudomonas mephitica
var, lipolytica) FERM P-5
The lipase productivity was about 20 times higher than that of No. 20.

〔Effect of the invention〕

The present invention involves isolating a DNA fragment containing a heat-stable lipase gene of a Pseudomonas bacterium, obtaining a plasmid containing the DNA fragment, transforming a Pseudomonas bacterium using this, and culturing the transformant. This is a method in which heat-stable lipase is mass-produced outside the bacterial body and then collected. The productivity of heat-stable lipase by the genetically modified bacterium obtained by the present invention is 20 times that of the original strain.

[Brief explanation of drawings]

Figure 1 shows the field of Pseudomonas mephitica varietus.
ticavar, 1ipolytica) FEBM
A recombinant DNA in which the DNA encoding P-520 lipase was integrated into the plasmid vector pFL210.
This is an example of a restriction enzyme map prepared by cutting A (pHTLipl) with various restriction enzymes and based on the molecular weight measured using agarose gel electrophoresis. Figure 2 shows the DNA fragments obtained by cutting the lipase gene with various restriction enzymes and subcloning them into pFL210 to generate Pseudomonas mephiteica varietus.
Pseudomonasmephiti
cava var, 1ipolytica) KW-1
The transformed strain was examined for halo formation in triolein medium.

Claims (1)

[Scope of Claims] 1) DNA having the restriction enzyme map shown in FIG. 1 and containing a heat-stable lipase gene derived from a Pseudomonas bacterium
Plasmid pHTLip1, which can be expressed in Pseudomonas bacteria, is a fragment incorporated into a wide host range vector. 2) Pseudomonas transformant into which plasmid pHTLip1 was introduced. 3) Heat resistance characterized by culturing the Pseudomonas genus transformant into which the plasmid pHTLip1 has been introduced, producing heat-stable lipase in the culture solution, and then collecting the heat-stable lipase from the culture solution. Method for producing lipase.