JPH068365B2 - Degradation of isoprene rubber by microorganisms - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for decomposing isoprene-based rubber by a microorganism.

Isoprene rubber has a wide range of uses as a rubber product, but if rubber can be strongly decomposed, waste rubber products such as waste tires are decomposed and reused as raw materials instead of simply treating them as waste. can do. Such products can be used again in the rubber product manufacturing process (mastication process, etc.) in the state of oligomers, and can also be effectively used in various fields as raw materials for the production of pharmaceuticals such as vitamins and quinones, and fragrances. be able to.

[Conventional technology]

Conventionally, the present inventors have reported a method for decomposing natural rubber by a microorganism of the genus Nocardia or Rhodococcus, and a method for producing an isoprene oligomer, but these microorganisms have weak degrading activity and are associated with growing cells. The rubber-degrading activity was detected only in all cases.

[Problems to be solved by the invention]

Therefore, the present inventors, as a result of obtaining a microorganism having a stronger rubber-degrading activity from the natural world, among strains having a decomposing ability for isoprene-based rubber, a strain recognized as belonging to the genus Xanthomonas has a strong isoprene-based rubber. It was found that the strain was decomposed into, and this strain was isolated. Next, a method of strongly decomposing the isoprene-based rubber by culturing the present strain and contacting the obtained cultured bacterial cells, culture filtrate, or a treated product thereof with the isoprene-based rubber is developed.

[Means for solving problems]

Examples of representative microorganisms used in the present invention include the above-mentioned Xanthomonas spp., And the mycological properties of this strain are as shown below.

[Mycological properties]

a) Morphology Cell morphology Cell size (μm) 0.6 × 3-5 Motility + flagella Polar flagella 1-2 spores (heat resistance) -Gram stainability-b) Growth state in each medium Meat juice agar yellow , Glossy glucose yeast extract yellow, mucilage production and malt extract medium broth gelatin stab culture gelatin liquefaction weak c) Physiological properties (1) Reduction of nitrate (2) Denitrification- (3) Starch hydrolysis + (4) Use of NH ₄ and NO ₃ + (5) Pigment formation Brown (water-soluble) (6) Urease- (7) Oxidase + (8) Catalase + (9) ph4 growth − (10) 40 ° C growth − (11) 10 ° C growth − (12) Attitude toward oxygen Absolute aerobic (13) OF test Oxidative (glucose) (14) Sugar to acid (-) Glycerol, lactose, sucrose, mannitol, sorbitol, trehalose, inositol, fructose, mannose, xylose, arabinose (+) galactose, starch, maltose (15) Cellulase- (16) 5% NaC growth- (17 ) Auxotrophy Glutamic acid, promoted by methionine (18) Growth inhibition by 1 × 10 ^-6 M / crystal violet (19) Growth inhibition by 0.1% SDS (20) 0.02% Inhibition by triphenyltetrazolium chloride (21) Growth with aspartic acid as the only N and C source
± (22) Growth as a sole carbon source (+): threonine, ornithine, cellobiose, xylose, arabinose, trehalose, β-hydroxybutyric acid, p-hydrosibenzoic acid (−): fucose, fructose, rhamnose, lactose, inositol, Ethanol, tryptamine, butanediol, acetic acid, citric acid, lactic acid Based on the above-mentioned mycological properties, Verge's Manual of Systematic Tertiology Vol. 1, 1984 (Bergey's Man
ual of Systematic Bacteriology Vol 1,1984), it was confirmed that it belongs to the genus Xanthomonas,
This strain was named Xanthomonas NR-35Y strain. This strain has been deposited as Microorganism Research Institute No. 9640.

This strain grows in a medium containing synthetic or natural isoprene-based rubber as a main carbon source. As a general growth medium, for example, a synthetic medium containing the following inorganic salts is used.

Table _{-1 (NH 4) 2 SO 4} ( or _{KNO 3) 1.0g KH 2 PO 4} 0.2g ( or _{0.8g) K 2 HPO 4 1.6g MgSO} 4 · 7H 2 O 0.2g NaC 0.1g CaC 2 · 2H 2 O 0.02g FeSO ₄ 0.01g Na ₂ MeO ₄・ 2H ₂ O 0.5mg Na ₂ WO ₄・ 2H ₂ O 0.5mg MnSO ₄ 0.5mg Distilled water 1 pH 7.5 (or 7.0) Usually 50mg ~ 500 mg / 100 m of natural rubber or synthetic isoprene rubber latex is added, and if necessary, 10 to 100 mg of an organic nutrient source such as yeast extract is added, and then a degrading microorganism is inoculated. 3 at 30 ° C
Incubate for ~ 15 days. The isoprene-based rubber decomposing activity can be found in the cultured cells (bacteria), the culture filtrate, or a treated product of these. From this, it is confirmed that this strain produces an isoprene-based rubber-degrading enzyme outside the cells.

The isoprene-based rubber in the present invention is mainly cis-
A rubber having a 1,4-polyisoprene structure, which includes natural rubber and synthetic isoprene rubber. The rubber-decomposing activity in the present invention refers to the activity of oxidatively cleaving the double bond in the cis-1,4-polyisoprene molecular chain. The chemical structure of the oligomer produced from natural rubber by this decomposition reaction is as follows.

Synthetic isoprene rubber and natural rubber contain solvent-insoluble gel particles of 1 μ to 0.1 μ or less, and the degrading enzyme produced by this bacterium can also act on such a gel to decompose it. If the rubber latex is allowed to act for a short period of time, an oligomer having an average molecular weight of about tens of thousands can be mainly obtained, but if it is allowed to act for a longer period of time and decomposed decisively,
It is also possible to obtain mainly low molecular weight oligomers of about 3 to 5 or less. The contact with the isoprene-based rubber may be in any state of cultured bacterial cells, culture filtrate, or a treated product thereof.

The present invention will be specifically described below with reference to examples.

〔Example〕

Example 1 Xanthomonas NR-35Y strain (Ministry of Industrial Science and Technology No. 9640) 1
Platinum loop, medium of Table 1 with 25 mg of natural rubber latex added
In addition to 50 m, static culture was carried out at 30 ° C. for 7 days. After the culturing, the cells were separated into a culture solution by centrifugation, 10 mg of natural rubber latex was newly added to each of them, and the reaction was carried out at 30 ° C. for 1 day. After the reaction, the rubber was subjected to solvent extraction and analyzed by GPC (gel permeation chromatography). As a result, it was found that the molecular weight was lowered.

Example 2 Bacteria obtained in the same manner as in Example 1 were supplemented with natural rubber latex.
5 g was added and reacted at 30 ° C. for 7 days. As a result of GPC analysis, it was shown that about half of the rubber had a low molecular weight, as shown in FIG.

Example 3 Except for using synthetic isoprene rubber latex as a growth substrate, 25 mg of synthetic rubber latex was added to the extract obtained by subjecting the cells obtained in the same manner as in Example 1 to ultrasonic treatment to 30
The reaction was carried out at 0 ° C for 2 days. After solvent extraction, it was analyzed by GPC and NMR. The results are shown in Table-3.

Example 4 To the culture filtrate obtained in the same manner as in Example 1, 25 mg of synthetic isoprene rubber latex or natural rubber latex was added, reacted at 30 ° C. for 2 days, and then analyzed by GPC and NMR.
From the results of analysis by GPC, the product from the synthetic isoprene rubber latex shows a wide molecular weight distribution centered on a molecular weight of about 10,000 (A in the figure), as shown in FIG.
The product from natural rubber latex contains a considerable amount of oligomers with n = 3 to 5 in addition to the same broad distribution (B in the figure). By NMR analysis, the number average molecular weights are 1,500 and 600, respectively.

〔The invention's effect〕

The method of the present invention has a strong activity for decomposing particularly isoprene-based rubber in the form of latex, and the culture filtrate exhibits a strong activity, so that it is possible to easily recover the isoprene-based rubber decomposing liquid without side reaction, and various industrial processes. Expected to be used in the field.

[Brief description of drawings]

Both FIG. 1 and FIG. 2 show the molecular weight distribution of the rubber decomposition product, the vertical axis shows the value of the differential refractometer, and the horizontal axis shows the amount of eluate.

Claims (1)

[Claims] 1. A cultured cell obtained by culturing a microorganism belonging to the genus Xanthomonas and capable of degrading isoprene rubber,
A method for degrading isoprene-based rubber by a microorganism, which comprises contacting a culture filtrate or a treated product thereof with isoprene-based rubber.