JPS6030B2 - Degradation of acrylamide monomer by bacteria - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to Nocardia belonging to the genus Nocardia. This invention relates to a method for decomposing and removing acrylamide monomers in acrylamide polymers using bacteria No. 10021.

Acrylamide polymers have the effect of rapidly solidifying the ground as a soil stabilizer, and are extremely useful for civil engineering works, but acrylamide monomers often remain in the polymers. Acrylamide polymer is almost non-toxic, but acrylamide monomer has LD5 in an oral toxicity test using mice. is 9'k9 of 252, which is highly toxic. Therefore, if the monomer remaining in the polymer used as a soil stabilizer dissolves in groundwater, there is a risk that those who drink it will be poisoned, mainly due to nerve effects. The present inventor isolated a large number of fungi from soil, well water, etc. into which acrylamide monomer has flowed, and searched for the ability of these bacteria to decompose and assimilate acrylamide monomer. We found that Bacteria No. 10021 of the genus Nocardia, which is particularly active among these bacteria, has the ability to decompose and assimilate the body. No. 10021 was grown in various culture media commonly used for culturing microorganisms, such as broth. It has been observed that when bacterial cells or enzymes extracted from them are brought into contact with acrylamide monomers, they are likely to decompose and disappear in an extremely short period of time.The present invention is based on these findings. This is a method for decomposing acrylamide monomers, which is characterized by decomposing acrylamide monomers remaining in an acrylamide polymer using Bacterium No. 10021 belonging to the genus Nocardia having decomposition ability or its enzyme.

No. used in the present invention. Bacterium No. 10021 has been deposited as Kouken Bacteria No. 3451, and its properties are as shown below.

1 Morphology {1} Cell shape and size (yeast/malt agar medium, 2?o, 7 hours) coccus or short-form (0.5-0
．． 8 x 1.0 to 1.5 A), aerial mycelia do not attach.

[2] Presence or absence of cell pleomorphism (yeast) - Malt agar medium, 270-slide culture shows zigzag division and growth in the early stages of long-shaped fungi (0.8 x 20 cells), and after 7 hours of culture It is re-cleaved into 4-2 cells in the form of a coccus or short camphorax.

The fracture surface is anchor-like. '3} Motility: None [
4} Presence or absence of spores: No spores or thorns observed.

Wind Gram staining: intestinal (dark staining).

{6} Anti-acidity: weakly positive by Ziehl-Nielsen method and Kinyon method.

'7 - Forehead Grain: Electron microscopically shows similar grains that are opaque to electron beams Growth status in each medium (1) Juicy plate culture (27o0, 24-4 hours) Growth is good, dry and irregular surroundings It forms small, flat, pale pink colonies (5 or more ribs), and no growth is seen invading the inside of the medium.

As the culture time progresses, the color of the colony becomes a slightly darker ibis red or salmon pink. (Color Harmony Manual color chart code required). Settlements can be easily scraped or destroyed with platinum ears. {2} Broth agar slant culture (27°C, 24-4 hours) Good growth, filamentous, dry and flat surface, pale pink.

'3' Broth liquid culture (270, 24-7 hours) The fungus forms a bacterial membrane or adheres to the upper tube wall and grows vigorously.

The culture solution is clear. A slight agitation will make the solution appear turbid, but the bacteria will settle out and the supernatant will become clear. [4- Beptone water culture (270, 24-7 hours) Same as broth liquid culture.

(5) High-rise yeast/malt agar puncture culture (27q0,
7 hours) It grows on the surface of the agar medium, with no growth observed inside or below the medium.

■ Synthetic culture medium culture (270, 9 hours) Glucose
Asparagine agar toji, Zuavek Dokus agar toji, glycerol/asparagine agar toji, calcium/
For malate agar Yasji, inorganic salt/starch agar, etc.
It shows only a slight growth, such as a milky white color.

'71 Meat juice/gelatin puncture culture (270, 7 hours)
It grows well on the surface, and grows in a funnel shape along the puncture site, but it hardly grows on the lower layer.

It doesn't liquefy either. {8} Litmus milk: No change.

m Physiological properties○} Nitrate reduction +{21
Denitrification reaction '3' MR test {4) Vp test {5} Production of indole (6' Production of hydrogen sulfide'7) Hydrolysis of starch Utilization of nitrogen sources Nitrate Ten ammonium salt Ten skin Pigment production (11) Urease Ten (12)
Oxidase (13) Catalase
+ (1 heart growth range: pH 6-9 Temperature 10-370 (15) Attitude towards oxygen: aerobic (16) ○-F test (Hu carp according to Lei$ou method): ○ (aerobic from glucose It becomes raw acid.

) (17) Generation of acid and gas from sugars: Yamada and Komagata method (journal.

of Generic and Aplytic Microbiology, Vol. 18, p. 399, 1972), and it grew well on the following sugars.

The generation of acid and gas is as follows. W Other properties '1' Assimilation of organic acids: Performed according to Higashimura's method (Journal of General Microbiology, Vol. 56, p. 265, 1969).

Assimilation was observed with sodium acetate, sodium citrate, sodium lactate, sodium malate, sodium propionate, sodium pyruvate, and sodium succinate, and no assimilation was observed with sodium benzoate and sodium oxalate. (2- Hemolytic leg Zein degradation {4'xanthine hydrolysis {5} Hypoxanthine hydrolysis {6} Tyrosine degradation + method of Gordon et al. (Journal of Bacteriology 6)
(Vol. 9, p. 147, 1955).

{7} Cell wall component The method of Becker et al. (Applied Microbiology 12, p. 421, 1964) shows that mesodiaminopimelic acid is enteric; 1970
) was positive for arabinose and galactose.

■ Types of lipid-component mycolic acids Mycolic acids were extracted using the method of Rushvaller et al. (Journal of Bacteriology, 105, p. 313, 1971), and fatty acids and aldehydes were identified by pyrolysis gas chromatography. , C, 4~
Only fatty acids of C and 8 were confirmed, and "C, s to C, 8 aldehydes were not observed," indicating that the mycolic acid that forms this bacterial cell lipid is nocardidomycolic acid.

{91 Antibiotic susceptibility (minimum inhibitory concentration) Penicillin G I unit/I Tetracycline
25hcg/Chloramphenicol 28hcg
'Upper streptomycin 5 marks hcg/'amphoteridine B >10 khcg/methanol 2
10% at Cape time, less than 10% at 7 hours 00 Toxicity to animals 3 week old mice were injected with 4.5 x 1/3' bacterial suspension.
No. 3 [intravenous injection or intraperitoneal injection, there was no decrease in body weight, and the body weight increased for 15 days at the same level as the control.

Autopsy findings revealed no abnormalities in any organs. Also the heart,
Reverse cultures from the lungs, liver, bran, kidneys, and brain were also negative.

When monkeys were orally administered 50 ml of bacterial suspension (2 x 1 per ml) cultured in yeast maltoki solution for 270 hours, no spitting was observed within 2 hours, and no diarrhea was observed. No abnormalities were found during observation for one month.

OU whole bacterial cell IR spectrum The first region (3000-2800 arc-1) by Arai et al. (Journal General Applied Microbiology vol. 9, p. 119, 1963) shows the B pattern, and the second region (1750 arc-1) ~i600 skin-1) shows the A pattern, the mth area (1500-1350cm-1) shows the A or B pattern, and the Nth area (1150-95cm-1) shows the A or B pattern.
0ka-1) showed eight patterns, and generally showed the patterns shown by non-pathogenic Streptomyces.

As mentioned above, No. Bacteria No. 10021 is an aerobic, Gram-positive, weakly acid-fast bacterium that does not produce endospores, and does not attach flagella or fimbriae.

According to Bergy's Manual of Determinative (Bacteriology, 197△ King), ``Gram-positive birch bacteria that do not produce endospores are classified as ``Lactobacilli'' in Chapter 16. They are all included in "Actinobacteria and their near green microorganisms" in Chapter 17. Among the Lactobacteriaceae, species of the genus Lactobacillus are anaerobic or suitably anaerobic, while species of the genus Listeria are pericardial and actively motile; The bacterial species is No. 1 because it does not contain diaminopimelic acid in its cell wall, and the bacterial species of the genus Karyophhanon have lateral flagella and are motile. It is clearly different from Bacterium No. 10021. Therefore, No. Bacterium No. 10021 should be included in the fungal genus in the 10,000-thousand "actinomycetes and their neargreen microorganisms." The cell wall composition of this group of microorganisms has been particularly well studied, and this serves as the standard for classification, but No.
Bacteria containing diaminopimelic acid, D-arabinose, and galactose in their cell walls like Bacteria No. 10021 (Bergie's classification, cell wall type W, 658)
p) Coryneform bacteria or four bacterial species of the order Actinobacteria: Mycobacterium, Nocardia, Thermomonospora, and Micropolyspora.

However, Micropolyspora and Thermomonospora grow on aerial mycelium, and are far away from these. Additionally, Mycobacterium generally has strong acid-fast properties, and is clearly the No. It is different from Bacterium No. 10021. Further NO
．． Bacteria No. 10021 also differs from coryneform bacteria in that it does not contain corinomicolic acid as a lipid constituent fatty acid.
Therefore, No. Bacteria No. 10021 can be considered to be a bacterium included in the genus Nocardia. However, regarding fungal genera related to the genus Nocardia, various mycological and biochemical properties are currently being studied at academic conferences, and some of them have been studied in the genus Rhodocotcus [Goodfellow et al., "The Biology of the World."

Nocardia, 39 pages, 1976 King and Goodfellow,
Proceedings of the International Symposium on Nocardia and Strebtomyces (Mordarski et al., Warsaw, 1978) ~ Or the genus Gordona (Higashimura, Journal of G of 9 General Microbiology, 6 beads) Basket, 15
There are reports that it is preferable to reclassify the classification as ``Page, 1971'', and a final conclusion has not yet been reached.
In the present invention, NO. 1002
Bacteria No. 1 and its mutant strain having the ability to decompose acrylamide monomer can be used.

Bacteria that degrade acrylamide include Gram-negative bacteria Pseudomonas aeruginosa and Pseudomonas aeruginosa (M. Kelly et al. Biochemical Journal Vol. 93, p. 566, 19
6), Mycobacterium smegmatis (Journal General Microbiology Vol. 46 “111
, 1967), but "production by bacteria of the genus Nocardia has not been reported."
Although the enzyme of Bacterium No. 1 has transferase activity like the amidase of P. aeruginosa, it has a broader range of substrate specificity, and the amitase of P. aeruginosa and Mycobacterium smegmatis is a transferase. , No. 1002
The amitase of Bacterium No. 1 is unique in that it is a constituent enzyme. No. The cells of Bacterium No. 10021 can be produced, for example, as follows.

Assimilable nitrogen sources include, for example, amino acids or their mixtures, peptides, proteins or their hydrolysis products (veptone, etc.), meat extracts, yeast extracts, soybean flour, corn impregnated liquid, fish meat extracts, and oil extracts. Bacteria are inoculated into a liquid culture medium (pH 6.0 to 8.0) containing a carbon source such as glucose, mannose, maltose, or other available carbohydrate derivatives, and the cells are kept stationary or aerated at 20 to 30°C. For example, in order to ensure that almost no acrylamide monomer remains within 2 hours at room temperature by the method of the present invention, 0.01 to 35% by weight of the monomer is preferably used. is 0.
1 to 5% by weight of bacterial cells or enzymes are used. The method for quantifying acrylamide in the following examples is as follows.

The acrylamide monomer was determined by gas chromatography and a total organic acid carbon analyzer.

That is, the decrease in the monomer was measured using a total organic carbon analyzer model 102A manufactured by Toshiba Beckman Corporation, and quantitatively determined using a gas chromatograph GC (using Chromosolve 101 as a carrier, column temperature 20,000, injection temperature 220 qo, N2 gas). 30
The peak observed at a retention time of about 5 minutes was measured using a quantitative curve under the following conditions: Example 1 Decomposition of Acrylamide Monomer by Growing Bacterial Cells 100 ppm of acrylamide monomer was added to a minimal culture medium, cultured in a shaking moat at 270℃, and the degree of decline and growth of acrylamide monomer was examined over time.

The results are shown in Table 1. From this result, NO. 100
Bacteria No. 21 was found to grow in the minimal culture medium while consuming acrylamide monomer as a nutrient source. Note that the above sample to which no bacteria were added was used as a control. Note to Table 1) The composition of the minimum culture medium is as follows.

FeC13・Mother LO O. 001
%MgC12・Anti-Japanese 2〇〇・
01%NaHS03 0.
01%K2HP04 0.2
%KH2P04 0.1%
CaC12・2 days 20 0.0
1% Example 2 Decomposition of acrylamide monomer by quiescent bacterial cells No. 270 in broth. Bacteria No. 10021 was cultured in a shaking moat, and the cells that reached mid-logarithmic phase were collected by centrifugation, resulting in a concentration of 0.9%.
After washing with physiological saline, it was subjected to a shaking reaction with an acrylamide monomer at 30 oo in a phosphate buffer solution of pH 7.0, and the amount of acrylamide monomer remaining in the reaction solution after centrifugation was examined over time.

The results are shown in Table 2. From this result, it was confirmed that the quiescent bacterial cells were able to decompose acrylamide 25 melon blood into two legs in a 1 hour reaction in a phosphate buffer. The numbers in the table represent the values for acrylamide. In the Examples shown in Table 2 and above, the enzyme may be extracted and used instead of the live bacteria.

In this case, the enzyme can be separated and purified as follows. No. Bacteria No. 10021 was cultured in broth at 270° C., and the cells in the logarithmic growth phase were collected by centrifugation and washed several times with physiological saline.
．． 09 phosphate buffer and sonicated for 20 minutes (
2 wind C'S) and then cooled and centrifuged (12,000 evenings, 2
0 minutes). The obtained supernatant solution can be used as a crude enzyme solution for decomposing acrylamide. Purification of this enzyme solution is carried out in accordance with a conventional method, using a combination of ammonium sulfate precipitation, DEAE cellulose, and a gel furnace such as Cephadex G-20.

The partially purified enzyme has a pH range of 7.4 to 9.0.
The optimum temperature was approximately 46 oo, but it lost its activity at 60 oo, and showed some activity even at 20 qo.

Claims (1)

[Claims] 1 Nocardia belonging to the genus Nocardia that has acrylamide decomposition ability. No. 10021 A method for decomposing acrylamide monomers, which comprises using bacteria or enzymes thereof to decompose acrylamide monomers remaining in an acrylamide polymer.