JPS62208283A - Recombinant vector and bacterium containing same - Google Patents

Abstract

PURPOSE:To produce the aimed cellulase under an aerobic condition efficiently, by preparing a recombinant vector containing a cellulase producing chromosome DNA of a bacterium belonging to the genus Ruminococcus, capable of producing cellulase. CONSTITUTION:Ruminococcus albus a bacterium belonging to the genus Ruminococcus is treated with lysozyme to dissolve a cell wall, mildly subjected to bacteriolysis with a surface active agent, treated with phenol and centrifuges to give a DNA fraction, from which DNA is obtained by ethanol precipitation. This chromosome DNA is cleaved with restricted endonuclease, DNA and a vector are scissored with the same restricted enzyme, then both are linked by the use of DNA ligase to give a recombinant vector. Then, the recombinant vector is transduced into Escherichia coli capable of being cultivated under an aerobic condition by transformation method to produce cellulase.

Description

[Detailed description of the invention] [Industrial application field] It is useful in the field of microbial utilization industry.

[Summary of the invention]

The present invention relates to chromosomal DNA that carries genetic information involved in cellulase production of cellulase-producing bacteria belonging to the Ruminococcus genus.
The present invention relates to a recombinant vector containing the fragment, and a cellulase-producing bacterium belonging to the genus Escherichia transformed with the recombinant vector.

[Conventional technology]

Biomass, a renewable resource, is attracting attention as a long-term solution to food and energy crises. Among these, cellulose-based resources are regarded as important, and there is active research into their conversion and use into energy compounds, raw materials for the chemical industry, food and feed, etc. For effective use of cellulose resources,
It is necessary to convert this into a stable and easily usable form by reducing its molecular weight, and this is why cellulase-producing bacteria are being screened from various microorganisms.

The rumen bacteria that have the ability to decompose cellulose live in the first blood cells of ruminants, including Ruminococcus flavifaciens, Ruminococcus alpus, and Bacteroides
Plevis, Bacteroides ruminocola, Bacteroides succinogenes, etc. have been isolated and fixed.

Ruminococcus alpus is a bacterium that has the strongest ability to decompose cellulose among these, and its culture conditions have been carefully studied. (edited by the Japanese Society of Agricultural Chemistry: Biomass,
Breakfast Bookstore [) 51-67 (+985)) [Problems to be solved by the invention] The composition of a completely synthetic medium for Ruminococcus alpus has already been established, but since this bacterium is an obligate anaerobic bacterium, Moreover, it is necessary to maintain the conditioned medium and culture in an anaerobic state, making it unsuitable for industrialization. Furthermore, anaerobic bacteria have disadvantages such as slower growth and lower bacterial cell concentration than aerobic bacteria. Therefore, in order to mass-produce cellulase possessed by this bacterium, it is necessary to increase its production capacity and establish an easier culture method.

[Means to solve the problem]

As a result of intensive research into improving cellulase production methods using cellulase-producing bacteria belonging to the genus Ruminococcus, the present inventors created a recombinant vector using a cellulase-producing gene obtained from a cellulase-producing bacteria belonging to the genus Ruminococcus. We have discovered that by introducing this into a microorganism that can be cultured under aerobic conditions and transforming it, it is possible to efficiently produce cellulase under aerobic conditions. completed.

That is, the present invention provides a recombinant vector containing a chromosomal DNA fragment carrying genetic information involved in cellulase production obtained by cleaving the chromosomal DNA of a cellulase-producing bacterium belonging to the genus Ruminococcus; The present invention provides a transformant obtained by introducing a vector into a microorganism belonging to the genus Escherichia that can be cultured under aerobic conditions.

Examples of the microorganism of the genus Ruminococcus used as the chromosomal DNA donor used in the present invention include Ruminococcus alps (a strain stored in the Faculty of Engineering, Nagoya University).

The method for preparing chromosomal DNA from Ruminococcus alpus is not particularly limited. For example, after lysing the cell wall with lysozyme, etc., gentle lysis using a surfactant, phenol treatment, and centrifugation obtains a DNA fraction.
DNA' is obtained by ethanol precipitation and further purified.

Alternatively, after lysis with a surfactant such as SDS, bacterial cell residue and DNA fraction are separated by centrifugation, and the DNA fraction is further obtained by cesium chloride equilibrium density gradient centrifugation.

The prepared chromosomal DNA is then cut for ligation with the vector. The donor chromosomal DNA is usually cleaved by a method using a restriction endonuclease, but is not particularly limited to this method; for example, a method in which cleavage is performed by physically applying a shearing force may be used. When using a restriction enzyme to cleave donor chromosomal DNA, any restriction endonuclease that does not have a cleavage site in the cellulase-producing gene can be used as long as reaction conditions that cause complete cleavage are used. Furthermore, any restriction endonuclease can be used, provided that reaction conditions that cause only partial cleavage are used. In this way, various restriction enzymes can be selected depending on the conditions used, but from the viewpoint of ease of ligation with the vector, it is preferable to use a restriction enzyme that has a unique cleavage site for the vector used. For example, the plasmid pBR3 as a vector
22, IEcoRI, Sal I
, Bam1l I.

Hindll, Pst I, etc. can be used.

On the other hand, as a vector, any vector can be used regardless of whether it is a plasmid or a phage, as long as it can be replicated in the microorganism of the genus Escherichia used as a host.

When using a plasmid as a vector, any plasmid can be used as long as it can be replicated in the host, but
Examples of plasmids that have a unique cleavage site with a specific restriction enzyme and have markers such as antibiotic resistance are preferred from the viewpoint of binding with donor chromosomal DNA fragments and ease of selection of transformed strains. pcR
l, pMI19. pBR313゜pBR322,p
BR324, pBR325, pAcYc177, pA
Examples include cYc184.

In order to insert and link the cut donor chromosomal DNA into a vector, it is generally used to cut the donor chromosomal DNA and the vector with the same restriction enzyme, and then use DNA ligase to join the two together. This is how it is done. However, the method is not limited to this method; any conventionally known method can be used.

Examples of DNA ligases include E. coli DNA ligase and T4 phage DNA ligase.

The recombinant vector, which is a conjugate of the donor chromosomal DNA fragment prepared in this manner and the vector, is then introduced into a host microorganism belonging to the genus Escherichia.

Microorganisms of the genus Escherichia used as hosts include:
Various strains of Escherichia coli can be used. For example, C600, χ1776, LE392.11810
Examples include 1°RPI, 3110r-m'', and JM103.

The method for introducing a recombinant vector into a microorganism of the genus Escherichia, which is a host microorganism, ie, the transformation method, is not particularly limited, but a method in which treatment is performed in the presence of calcium ions is common.

Target cellulase-producing gene from the transformed strain or donor chromosomal DNA containing the cellulase-producing gene
The method for selecting and isolating microorganisms into which the fragments have been introduced is not particularly limited, but the first selection is based on the expression of markers such as antibiotic resistance that the vector has, and the second selection is based on the cellulase activity of the host. A method that makes a selective selection is preferred.

There are no particular limitations on the method for producing and purifying cellulase using the rt-transformed strain thus finally selected.

The composition of the medium used for culturing includes appropriate carbon sources, nitrogen sources, organic nutrient sources, inorganic salts, etc. in order to allow the microorganisms of the genus Escherichia to grow well and to smoothly produce cellulase.

[For production]

A chromosomal DNA fragment encoding cellulase was selectively isolated from a cellulase-producing bacterium belonging to the genus Ruminococcus, which is an obligate anaerobic bacterium, and this was used as a recombinant vector to produce Escherichia coli, which can be cultivated under aerobic conditions. cellulase can be produced by introducing the cellulase into a cellulose by a transformation method.

〔Effect of the invention〕

By cloning cellulase-producing genes of anaerobic bacteria, cellulase of anaerobic bacteria can be efficiently produced under aerobic conditions.

〔Example〕

Next, examples of the present invention will be shown.

Example 1 (Preparation method of chromosomal DNA) When chromosomal DNA was prepared from Ruminococcus alpus, the complete synthetic medium 450.1 shown below was used and grown under complete anaerobic conditions.

R. albus complete synthetic medium (per 1 fi) saline solution, 75 proverbs K, I (PO
40.45g Na, Co,, 4
,0gL-cystine hydrochloric acid 0.2
5gNa, S・911.0
0.25*Fe50.・7H,OO, 01#c ZnSO4・71120 0
．． 01gMnSO4・4~61(200,0IHCoC
], ・6 Summer f, 0
0.01 fC resazurin sodium 1.0ml volatile fatty acid mixture b 3.1+sl pyridoxine hydrochloric acid 2. OmgP-aminobenzoic acid 0. IH biotin
0.05mg mono-arginine
0.02g ball mill cellulose
5.0ga: 0.6%KH,PO,,1,2
%NaCl, 1, 2% (NH4), So.

0.12%Mg5O, -78,0,0,12%CaCl
, ab=acetic acid 17-1, propionic acid 6ml. 4 ml of n-butyric acid.

1 ml of isobutyric acid. 1 ml of n-valeric acid, 1 ml of isovaleric acid, 1. After collecting OL-α-methylbutyric acid 1-1°, lysozyme solution (0,15M NaCl, 0,1M EDTA).

pH 8.0, 5 ml of lysozyme 5 g10+1) was reacted at 37°C for 60 minutes to form protoplasts, and then SDS solution (0.1 M Tris buffer pH 9,
Add 0,0,1% SO5, 0,1M NaCl),
Bacteriolysis was carried out at 60°C. This was treated with phenol and centrifuged to obtain a DNA solution, from which DNA was recovered as a filamentous precipitate with acceptable ethanol. Furthermore, it is treated with RNA degrading enzyme and proteolytic enzyme to reduce the refined fiDNA to about 2.5B.
Obtained.

Example 2 (Construction and Adjustment of Recombinant Plasmid) The chromosomal DNA obtained in Example 1 was completely degraded in Hind m at 37°C for 2 hours. Additionally, plasmid pBR3
After 22 was completely degraded with Hind III, the 5'-phosphate residue was excised with alkaline phosphatase.

Then, both were mixed and a ligation reaction was performed at 16° C. for 16 hours using DNA ligase derived from T4 phage.

Complete ligation was confirmed by agarose gel electrophoresis.

Example 3 (Competent cell preparation and transformation) Competent cell preparation and transformation were carried out using Molecular Clo
ning A Laboratory Manual
p249 (1982).

That is, Escherichia coli C600 (F-, thi
-1°thr-1,1auB6.1acY1. tor
A21.5upE44. λ-) in L medium until the early stage of logarithmic growth, the bacteria were collected, cooled with water, and added to a calcium chloride solution (pH
8,10mM hlis buffer.

The cells were suspended in 50mM CaC1□) to prepare competent cells.

L medium (per IQ) Peptone 10g Yeast
Extract 5gNaC,I
5R pH 7.2 Next, the recombinant plasmid prepared in Example 2 was added to this cell suspension and reacted for 3 minutes under water cooling.
A heat shock was applied at 0.degree. C. for 5 minutes to incorporate the plasmid into the cells. Add 5 times the amount of L-medium to this and
The mixture was left standing at ℃ for 1 hour to perform a transformation reaction.

This was plated on L agar medium containing ampicillin to form colonies of plasmid-containing strains, which were used as master plates. Next, this was replicated on L agar medium (containing ampicillin) and grown, and then agar containing 0.5% CMC1Lsg/resistant lysozyme was added. "The layer was reacted at 137°C for 4 hours.

A strain having the target CMCase and J gene forms a transparent halo around the colony on a CMC-containing agar plate by Congo red staining.

The obtained strain was transformed into Escherichia coli C600 (pRA
It was named l). This bacterial strain was deposited at the Microtech Institute as FERM P-8668.

Example 4 (Confirmation of plasmid pRA1) Escherichia coli C600 (PRAI), FERM P-8668
was cultured in M9CA medium until mid-logarithmic growth, then chloramphenicol was added and cultured overnight.

The presence of plasmid pHAI was confirmed by centrifuging Clear Lyse 1~ obtained by lysing the cultured bacterial cells in a cesium chloride density gradient.

M9CA medium Na2HP0 per IQ. 1 in 6g
1□PO43g NaCl 0.5g
N114CI IgM
gSO, (1M solution) 2m
l Glucose (2% solution) 10ml
CaCl, (1M solution) 0
．． 1ml Casamino 2pH
7,4 The restriction enzyme cleavage map of plasmid PRAI is shown in Figure 1. By analysis using restriction enzymes, plasmid pRA1 was
It was confirmed that it had an insert fragment of approximately 3.6 kb derived from Ruminococcus alpus.

Example 5 (Production of CMCase) After culturing Escherichia coli C600 (pRA1) in L medium, the cells were harvested and the cells were disrupted using ultrasound to obtain a crude enzyme solution. When a crude enzyme solution was applied to CMC as a substrate, CM
Case activity was confirmed.

The properties of C M Cass encoded in PRAI are shown in FIGS. 2, 3, and 4.

[Brief explanation of drawings]

FIG. 1 is a restriction enzyme cleavage map of plasmid ρItA1. The value in parentheses represents kb. FIG. 2 is a diagram showing the properties of CMCase in terms of the relationship between the viscosity reducing blade (vertical axis) and the ability to produce reducing sugars (horizontal axis). here. η5p=t/1o-1t: Flow time after reaction in Ostwald viscometer tO: Flow time of buffer solution Δη5p-1=ηSρ-1-η5pb-1ηspb:
Value of ηSρ in inactivated enzyme solution Figure 3 shows the optimum pH of CMCase investigated by measuring the reducing sugar produced. FIG. 4 is a diagram showing the optimum temperature of CMCase investigated by measuring the reducing sugar produced. Agent Patent Attorney 1) Parent Male Figure 3 Figure 4
Figure pH ('C) Figure 1 Gila vuII Figure 2 (μg/m text) Procedure Ne 11ff Original Book April 15, 1986

Claims (2)

[Claims] (1) A recombinant vector containing cellulase-producing chromosomal DNA of a cellulase-producing bacterium belonging to the genus Ruminococcus. (2) A cellulase-producing bacterium belonging to the genus Escherichia transformed with a recombinant vector containing cellulase-producing chromosomal DNA of a cellulase-producing bacterium belonging to the genus Ruminococcus.