JP2967202B2 - Method for obtaining solvent-resistant microorganisms - Google Patents

Description

Description: FIELD OF THE INVENTION The present invention relates to a method for obtaining a solvent-resistant microorganism, and more particularly to a method for producing or decomposing a water-insoluble substance by a microorganism, which is resistant to a specific organic solvent. The present invention relates to a method for obtaining a microorganism.

[Prior Art and Problems to be Solved by the Invention] When a useful substance is produced using a microorganism, the microorganism is usually cultured in an aqueous solution. When an organic solvent is added to the culture, growth may be inhibited or even killed due to the toxicity of the solvent. Such a phenomenon is more remarkable as the partition coefficient of the solvent is smaller.

However, in producing or decomposing a water-insoluble substance by a microorganism, in order to uniformly add a compound that is hardly soluble in water to a culture solution, the compound may be dissolved in an organic solvent and then added, or the compound in the culture solution may be added. Extraction using an organic solution is conventionally known. In these cases, it is necessary to limit the amount of the solvent or to use a low-polarity solvent, such as hexane or octane, which does not always have a sufficient solvency, in order to avoid toxicity of the solvent to the microorganisms.

Some microorganisms, such as a small number of bacteria belonging to the genus Pseudomonas, are resistant to relatively toxic solvents such as toene and xylene, but these bacteria are not necessarily microorganisms used for substance production or decomposition. Not useful.

The present inventors have conducted intensive studies to establish a method for imparting a process-friendly resistance to an organic solvent to a microorganism useful as a means for producing and decomposing a substance, and as a result, have reached the present invention.

[Means for Solving the Problems] The present invention provides a distribution coefficient P Organic solvent S Grows in
But log P_{+ M}≧ log P Meets the condition of −0.6,
P Smaller distribution coefficient P_{+ M}Organic solvent S_{+ M}During ~
Microorganisms that do not grow in_{+ M}Organic solvent
Medium S_{+ M}Microorganisms that are in contact with
A method for obtaining a solvent-tolerant organism, characterized by selecting Organic solvent S Grows in, but P
Smaller distribution coefficient P_{+ (M + 1)}Organic solvent S
_{+ (M + 1)}Microorganisms that do not grow in
To obtain microorganisms resistant to the solvent.
A method for obtaining a solvent-resistant microorganism, characterized in that:_{+ M}≧ log P -0.6 condition
And P Smaller distribution coefficient P_{+ M}Organic solvents
S_{+ M}Microorganisms that are resistant to the solvent
(B) Select the microorganism obtained in (a) above using log P
_{+ (M + 1)}≧ 1og P_{+ M}Meets the condition of −0.6,
P_{+ M}Smaller distribution coefficient P_{+ (M + 1)}Organic solvent
Medium S_{+ (M + 1)}Resistant to the solvent by contact with
Distribution coefficient P Organic solvent S Grows in, but P
Smaller distribution coefficient P_{+ (M + k)}(However, k is 1
Is an integer greater than or equal to. ) Organic solvent S_{+ (M + k)}Inside
Is treated with a microorganism that does not grow by the following method,
Solvent characterized by obtaining microorganisms resistant to
Method for obtaining resistant microorganisms, (a) Log P_{+ M}≧ log P -0.6 condition
And P Smaller distribution coefficient P_{+ M}Organic solvents
S_{+ M}Microorganisms that are resistant to the solvent
(B) using the microorganism obtained in (b) above in an organic solvent S
_{+ (M + 1)}Contact with the solvent
(C) Then, the obtained microorganism is converted into an organic solvent S
_{+ (M + 2)}Contact with the solvent
Select organisms, organic solvent S_{+ (M + 2)}Of
Instead of S_{+ (M + 3)},…,
S_{+ (M + k-1)}, S_{+ (M + k)}Use organic solvent
And the same operation is repeated, and the organic solvent S
_{+ (M + 3)},…, S_{+ (M + k-1)}, S
_{+ (M + k)}Select microorganisms that are resistant to

(Here, the organic solvent S _{+ (m + 1)} ,
S _{+ (m + 2)} , ..., S _{+ (m + k-1)} , S
_{+ (M + k)} is P _{+ (m + 1)} > P _{+ (m + 2)}
>...> P _{+ (m + k-1)} > P _{+ (m + k)} , Distribution coefficient P _{+ (m + 1)} ,
P _{+ (m + 2)} , ..., P _{+ (m + k-1)} , P
_{+ (M + k))} .

The microorganism that can be used in the present invention is not particularly limited, but preferably has at least resistance to an organic solvent having a large partition coefficient. Specifically, Esherich
ia), Pseudomonas, Achromobacter, Agrobacterium (A)
grobacterium), Alcaligenes
Genus, Achromonas, Acinetobacter, Erwinia, Enterobacter, Brevibacterium, Citrobacte
r) genus, genus Flavobacterium, genus Paracoccus, Proteus
Genus, Klebsiella, Chromobacterium, Rhodococcu
s) genus, Microbacterium genus,
Nocardia, Bacillus
And microorganisms belonging to the genus Corynebacterium, the genus Saccharomyces, and the like, and include Escherichia coli ( E. coli ).
ATCC 33588, Pseudomonas putida (P. Putida) ATC
C 17453, Achromobacter delica turras ( A. delica)
tulus) IAM 1433, Agrobacterium tumefaciens (A. tumefaciens) IFO 3058, Alcaligenes faecalis (A. faecalis) JCM 1474, Akuromonasu hydrophila (A. hydrophila) JCM 1027, Acinetobacter calcoaceticus (A. calcoaceticus ) IFO 125
52, Bacillus cereus ( B. cereus ) IFO 3131, Bacillus subtilis ( B. subtilis ) AHU 1219, Brevibacterium paraffinoliticum ( B. paraffinolitic)
um) ATCC 21195, Brevibacterium Heruborumu (B. helvolum) ATCC 21342, Brevibacterium lactofermentum (B. lactofermentum) ATCC 2142
0, Brevibacterium ammoniagenes ( B. ammoni
agenes ) ATCC 13746, Brevibacterium ammoniagenes IFO 12072, Brevibacterium roseum ( B.
roseum ) ATCC 13825, Brevibacterium flavum ( B. flavum ) ATCC 13826, Chromobacterium cocolamatum ( C. chocolatum ) IFO 3758, Corynebacterium glutamicum ( C. glutamicum ) JCM 1318, Corynebacterium flaveticiens ( C. flavescens ) IMA
1642, Corynebacterium hydrocarboxy dance (C. Hydrocarbooxydans) ATCC 21767, Corynebacterium Hakyurisu (C. Herculis) ATCC 13868, Erwinia Habikora (E. Herbicola) ATCC 21434, Citrobacter freundii (C. Freundii) ATCC 675
0, Microbacterium ammonia filam ( M. am
moniaphilum) ATCC 15354, Flavobacterium Suabeorensu (F. suaveolens) IFO 3752, Flavobacterium Riteshiensu (F. litescens) IFO 3084, Enterobacter Kuroase (E. cloacae) IFO 3320, Klebsiella pneumoniae (K. pneumoniae ) IFO 3318,
Rhodococcus erythropolis ( R. erythropolis ) IF
O 12320, Rhodococcus equi, R. equi IFO 3730,
Nocardia sp . ( N. sp .) ATCC 21145, Paracoccus denitrificans ( P. denitrificans ) IFO
13301, Proteus mirabilis ( P. mirabilis ) IFO
3849, Saccharomyces uvarum ( S. uvarum ) ATCC
26602 and the like are suitable.

 Resistant to target organic solvents using the above microorganisms
To obtain a microorganism, first, the partition coefficient P Organic solvents
S Grows in the log P_{+ M}≧ log P −0.
6 Smaller distribution coefficient P_{+ M}of
Organic solvent S_{+ M}Microorganisms that do not grow in
Number P_{+ M}Organic solvent S_{+ M}Contact. here
And organic solvent S where microorganisms can grow Is the distribution coefficient
P Log value of (log P ) Is selected in consideration of
Minimum log P for living organisms Value (limit log
P ) The above organic solvents should be selected.

The partition coefficient (P) is expressed as the ratio of the activity of a substance at equilibrium in two immiscible solvent systems, and when the concentration of the substance is small, the concentration can be regarded as the activity. When determining which organic solvents can be used for biological reactions,
The polarity or hydrophobicity of the solvent is used as an index. When log P is used as an index of the polarity of the solvent, a good correlation with the biological reaction can be obtained. = Moderate reactivity in solvents of 2-4, log
It is known that non-polar solvents with P> 4 have high reactivity (C. Laane, S. Boeren, and K. Vos; Trends Biotec
hnol., 3, 251, 1985 and C. Laane, S. Boeren, K. Vos, and
C. Veeger; Biotechnology and Bioengineering, 30 , 81, 19
87).

The present inventors have elucidated a method for specifying an organic solvent that can be used as described above using the log P value as an index even in the growth of microorganisms in the presence of an organic solvent.

The log P value can be determined by the following method.

(2) Method of calculating by calculation The log P value is expressed as the sum of the hydrophobicity of each component constituting the solute molecule, and the substituent X is introduced into the reference substance (H) to obtain a hydrophobic substituent constant π in the compound. _X is represented by the following equation.

π _X = log Px−log P _H log P is expressed as the sum of π _{X of the} substituent constituting the substance and the logarithmic (log P _H ) value of the distribution coefficient of the reference substance.

Here, the log P or π value is desirably determined experimentally, but the hydrophobic fragment constant (eg, hydrophobicity such as cyclization, branching, multiple bonds, and substituents) affecting the partition (eg, hydrophobicity such as cyclization, branching, multiple bonds, and substituents) hydrophobic fragmental const
anty: f), and log P can be calculated from log P = Σf (Nys, G, G., Rekker, RF, Chim. Themp., 5, 52)
1,1973; RFRekker, The Hydrophobic Fragmental Const
ant, Elsevier, New York, 1977), and Table 1 shows log P values calculated by this calculation method. Where p
-For xylene, lo obtained by the following method (2)
gP value.

(2) Method determined by experiment (Flask shaking method) Determined by determining the solute concentration ratio between an organic solvent (for example, n-octanol) and water. Take a predetermined volume each of the organic solvent and water which have been saturated in the partner phase, and put them in a Meyer. Usually, the drug is dissolved in one of the phases in advance, and the concentration of the two phases after equilibration is quantified by an appropriate method (usually a colorimetric method) to determine the partition coefficient P. At this time, measurement is performed by changing the drug concentration, and a value in a region where the value of P does not change depending on the concentration is adopted (Terada
Hiro, Chemistry, Supplement No. 122, p73, 1979).

 Calculate the log P value of various organic solvents by the above method
After that, specific microorganisms are grown in an organic solvent with a predetermined log P value.
Perform a growth test and determine the lowest log P value at which growth is possible (limit log
 P value) can be determined. Here, for the present invention
Hexane, hepta
, Octane, isooctane, nonane, decane, dodeca
Aliphatic hydrocarbons such as cyclopentane and cyclohexane
Alicyclic hydrocarbons such as xane and cyclooctane,
Zen, toluene, p-xylene, m-xylene, o-xylene
, Styrene, ethylbenzene, propylbenzene,
Aromatic hydrocarbons such as lorobenzene and o-dichlorobenzene
Primates, 1-heptanol, 1-octanol, 1-decano
Alcohols, such as n-hexyl ether, diphe
Ethers such as nyl ether and methoxytoluene
There is. Further, a mixture of these can also be used.
S_{+ M}J kinds (j is an integer of 2 or more) of solvents S¹
_{+ M}, S^Two _{+ M},…, S^j _{+ M}When using
Distribution coefficient P¹ _{+ M}, P^Two _{+ M},…, P^j _{+ M}Is P ≧ P¹
_{+ M}, P ≧ P^Two _{+ M},…, P ≧ P^j _{+ M}AndAndS to be¹ _{+ M}, S^Two _{+ M},…, S^j _{+ M}Determined
I just need. J is usually 2 to 10. Also, S¹ _{+ M}, S
^Two _{+ M},…, S^j _{+ M}Is not particularly limited. This
Shows log P values for major of these organic solvents
It is as follows.

 As described above, the distribution coefficient P Organic solvent S Growing in
But P Smaller distribution coefficient P_{+ M}Organic solvent S
_{+ M}From the microorganisms that do not grow in the solvent S_{+ M}Inside
To obtain a viable microorganism, the microorganism must be
_{+ M}Contact.

Specifically, a microorganism is inoculated on an agar medium, and an organic solvent S
_{+ M} is added to the surface for culturing, or a strain that grows is obtained by inoculating and culturing a mutated microorganism in a liquid medium to which an organic solvent has been added.

In addition, when the microorganisms are brought into contact with the organic solvent S _{+ m} , it is desirable to carry out a mutation treatment in advance.

The method of the mutation treatment is not limited, and a method using a mutation agent such as N-methyl-N-nitroso-N'-nitroguanidine (NTG), ethyl methanesulfonic acid (EMS);
A method using radiation or the like as a mutagen; transposon mutagenesis, site-directed mutagenesis
And other genetic engineering mutation processing methods. When performing a mutation treatment using a mutagen, the mutagen is added to a concentration of 10 to 500 μg / ml in an appropriate buffer, and then the microorganism is added to the solution, and the pH is 5 to 8, and the temperature is 15 to 42. ° C
1 minute to 10 hours.

 The organic solvent S_{+ M}Is P_{+ M}≧ log P −0.6
It is necessary to satisfy the conditions. That is, S Distributor
Choose an organic solvent that is relatively close to the number. This condition
Is not satisfied, the organic solvent S is efficiently used._{+ M}Resistant to
Can not obtain the microorganisms.

 Although solvent-resistant microorganisms can be obtained as described above,
Distribution coefficient P Organic solvent S Partitioning from microorganisms growing in
Coefficient P_{+ (M + k)}(However, k is an integer of 1 or more.
You. ) Organic solvent S_{+ (M + k)}Microorganisms growing in
The above operation is repeated a predetermined number of times as described below.
It is necessary to return.

 The magnitude of the partition coefficient of the organic solvent used in the above operation
Is P > P_{+ M}> P_{+ (M + 1)}>…> P
_{+ (M + k)}It is. Also, log P_{+ M}≧ log P −
0.6,It is.

In this way, by repeatedly selecting the solvent-resistant bacteria stepwise, it is possible to obtain a microorganism capable of growing in the presence of a small partition coefficient, in other words, a highly toxic organic solvent.

When culturing the solvent-resistant microorganism obtained by the present invention, an appropriate medium containing nutrients necessary for the growth of the microorganism is selected in consideration of the purpose of use of the microorganism (cell production, fermentation production, etc.). The concentration and the method of addition of the organic solvent to be added to the medium should be appropriately selected.

EXAMPLES Next, the present invention will be described in more detail with reference to examples.

Reference Example 1 Peptone Escherichia coli JA 300 (ATCC 33588)
10 g /, then pour the 24 hours with shaking cultured bacteria in a liquid medium containing yeast extract 5 g / in to about 10 ⁸ cells / ml with physiological saline,
0.1 ml of this pour solution is added to an agar plate (diameter 8 cm) (peptone 10
g /, 5 g / yeast extract), and then 2.5 ml of propylbenzene was overlaid and cultured at 37 ° C for 20 hours.

As a result, colonies were formed, and it was found that the cells had resistance to propylbenzene.

Reference Example 2 When the culture was performed in the same manner as in Reference Example 1 except that p-xylene was used instead of propylbenzene, no colony was formed.

Reference Example 3 In Reference Example 1, Escherichia coli JA 300 (ATCC 3
3588) in place of Chaudmonas putida (ATCC 17453)
And using p-xylene instead of propylbenzene in the same manner as in Reference Example 1. As a result, colonies were formed, and it was found that p-xylene was resistant.

Reference Example 4 The procedure of Reference Example 3 was repeated, except that toluene was used instead of p-xylene. as a result,
No colonies formed.

Example 1 Escherichia coli JA300 (ATCC 33588) resistant to propylbenzene (log P value = 3.8) was shake-cultured in a liquid medium containing 10 g of peptone / 5 g / yeast extract for 12 hours, and then washed with physiological saline. Dilute to 10 ⁸ cells / ml
0.1 ml was inoculated on an agar plate medium having a diameter of 8 cm (containing 10 g of peptone and 5 g of yeast extract /), and then 2.5 ml of cyclohexane was stacked and cultured at 37 ° C for 20 hours. A strain having cyclohexane resistance was obtained from the formed colonies.

Next, after treating the cyclohexane-resistant strain in the same manner as described above, the strain was inoculated on an agar plate medium having the same composition as above, and further, 50% / 50% of cyclohexane and para-xylene.
2.5 ml of (v / v) solution was added to the surface of the agar and cultured at 37 ° C. for 20 hours. A strain having (cyclohexane + paraxylene) resistance was obtained from the formed colonies.

The obtained (cyclohexane + paraxylene) -resistant strain was contacted with 20 μg / ml NTG at 37 ° C. for 30 minutes in an NTG treatment (50 mM Tris / maleate buffer (pH 6.0)) for 30 minutes. To wash out NTG.)
Then, the cells were inoculated on an agar medium having the same composition as described above, and 2.5 ml of para-xylene (log P value = 3.1) was stacked on the agar medium.
The cells were cultured for 0 hours. Escherichia coli having paraxylene resistance was obtained by hooking the resulting colonies and culturing them in another medium.

Example 2 Laxylene (log P value = 3.1) resistant Pseudomonas
NTG treatment was performed on Putida (ATCC 17453) in the same manner as in Example 1 except that the mutation treatment temperature was 30 ° C. in the method described in Example 1. Next, inoculate the agar plate medium (containing 10 g of peptone and 5 g of yeast extract),
Further, 2.5 ml of toluene was stacked and cultured at 30 ° C. for 20 hours. Hook fungi were formed from the formed colonies and cultured in another medium to obtain a toluene-resistant strain.

[Effect of the Invention] By applying the method of the present invention to all microorganisms including useful microorganisms used as a means for producing or decomposing substances, a strain having resistance to a desired organic solvent can be obtained. In particular, microorganisms having resistance to solvents such as benzene, toluene, and xylene can be easily obtained.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12R 1:19) (C12N 1/20 C12R 1:40) (C12N 1/20 C12R 1:19)

Claims (5)

(57) [Claims] 1. The distribution coefficient P Organic solvent S Grow in
But log P_{+ M}≧ log P Meets the condition of −0.6
Then P Smaller distribution coefficient P_{+ M}Organic solvent S
_{+ M}Microorganisms that do not grow in_{+ M}
Organic solvent S_{+ M}Resistant to the solvent by contact with
Of solvent-resistant microorganisms characterized by selecting
Acquisition method. 2. The distribution coefficient Organic solvent S Grows in
Is P Smaller distribution coefficient P_{+ (M + 1)}Organic solvent
Medium P_{+ (M + 1)}Microorganisms that do not grow in
Method to obtain microorganisms resistant to the solvent.
A method for obtaining a solvent-resistant microorganism. (A) Log P_{+ M}≧ log P -0.6 condition
And P Smaller distribution coefficient P_{+ M}Organic solvents
S_{+ M}Microorganisms that are resistant to the solvent
(B) Select the microorganism obtained in (a) above using log P
_{+ (M + 1)}≧ 1og P_{+ M}Meets the condition of −0.6,
P_{+ M}Smaller distribution coefficient P_{+ (M + 1)}Organic solvent
Medium S_{+ (M + 1)}Resistant to the solvent by contact with
Microorganisms to be selected. 3. The distribution coefficient P Organic solvent S Grow in
But P Smaller distribution coefficient P_{+ (M + k)}(However
And k is an integer of 1 or more. ) Organic solvent S
_{+ (M + k)}Microorganisms that do not grow in
To obtain microorganisms resistant to the solvent.
And a method for obtaining a solvent-resistant microorganism. (A) Log P_{+ M}≧ log P -0.6 condition
And P Smaller distribution coefficient P_{+ M}Organic solvents
S_{+ M}Microorganisms that are resistant to the solvent
(B) using the microorganism obtained in (a) above in an organic solvent S
_{+ (M + 1)}Contact with the solvent
(C) Then, the obtained microorganism is converted into an organic solvent S
_{+ (M + 2)}Contact with the solvent
Select organisms, organic solvent S_{+ (M + 2)}Of
Instead of S_{+ (M + 3)},…,
S_{+ (M + k-1)}, S_{+ (M + k)}Use organic solvent
And the same operation is repeated, and the organic solvent S
_{+ (M + 3)},…, S_{+ (M + k-1)}, S
_{+ (M + k)}Select microorganisms that are resistant to (Where the organic solvent S_{+ (M + 1)},
S_{+ (M + 2)},…, S_{+ (M + k-1)}, S
_{+ (M + k)}Is P_{+ (M + 1)}> P_{+ (M + 2)}
>…> P_{+ (M + k-1)}> P_{+ (M + k)}In
AndDistribution coefficient P that satisfies_{+ (M + 1)},
P_{+ (M + 2)},…, P_{+ (M + k-1)}, P
_{+ (M + k)}Corresponding to each) (4) the microorganism is Pseudomonas putida ATCC 175;
43 and the distribution coefficient P Organic solvent S Is para-xylene
And the distribution coefficient P_{+ M}Organic solvent S_{+ M}Is Toen
The method of claim 1, wherein 5. The microorganism is Escherichia coli ATCC 33588.
Yes, distribution coefficient P Organic solvent S Is propylbenzene
And the distribution coefficient P_{+ M}Organic solvent S_{+ M}Is cyclo
Hexane, partition coefficient P_{+ (M + 1)}Organic solvents
S_{+ (M + 1)}Is cyclohexane / para-xylene (50
% / 50%) and the distribution coefficient P_{+ (M + k)}Organic solvent
Medium S_{+ (M + k)}Is para-xylene.
the method of.