KR101979067B1 - Novel Acinetobacter sp. KFCC11687P Using Methyl Acetate as a Sole Carbon Source - Google Patents

Abstract

The present invention provides a novel strain of the genus Acinetobacter KFCC11687P which utilizes methyl acetate as a single carbon source. The strain KFCC11687P, which is a novel strain of the genus Acinetobacterium, has a carboxyl esterase activity, thereby decomposing methyl acetate in the culture broth into acetic acid and methanol and simultaneously using it as a carbon source of methyl acetate. According to the present invention, the novel strain of the genus Ashentanobacter KFCC11687P can easily and economically decompose methyl acetate, which is a by-product of chemical processes, through a biological method.

Description

A novel Acinetobacter strain KFCC11687P {Novel Acinetobacter sp. KFCC11687P Using Methyl Acetate as a Sole Carbon Source}

The present invention relates to a novel Acinetobacter strain KFCC11687P which utilizes methyl acetate as a single carbon source.

In recent years, the production of shale gas is increasing rapidly in the United States using horizontal boring and plating techniques. As a result, the shale gas, which has an advantage in abundant production and price, is a substance having a possibility as a C1 carbon source because its main component is methane. However, the low solubility of methane gas causes slow growth of microorganisms and difficulties in fermentation.

Methyl acetate and methyl formate can be used as feed materials as materials that can be derived from methanol and carbon monoxide. In particular, methyl acetate can be produced from methanol and carbon monoxide using a metal catalyst such as cobalt from BASF, a Cativa process using an iridium catalyst, or a rhodium catalyst from Monsanto. Therefore, methyl acetate has potential as a liquid raw material to be used in place of the Cl gas, and research is being conducted. Methyl acetate is a less toxic, volatile organic compound and has the advantage that it is not considered as an element of air pollution. Acetobacterium < / RTI > It is known that U. rumerium can hydrolyze methyl acetate to acetate and methanol under anaerobic conditions.

Ashinobacterium strains belong to the gamma proteobacteria, Gram-negative bacteria. The strain has non-motile, oxidase negative, and aerobic conditions. This strain is a soil organism that contributes to the mineralization of aromatic compounds. Ashinotobacter kalloacetics was used for the production of emulsan from ethanol as a raw material, and Ashinotobacter strain PD12 for biodegradation of phenol. However, no bacteria have been reported that can utilize methyl acetate in the Ashinobacterium species.

High concentrations of methyl acetate are used as auxiliary fuel for the boiler. Most low-concentration methyl acetate is hydrolyzed by liquid acid catalyst to acetic acid and methanol or sent to the wastewater treatment plant. The hydrolysis of methyl acetate is reversible and the equilibrium constant is low, so a large amount of water is used to increase conversion. Accordingly, a plurality of distillation columns must be continuously installed, resulting in low economical efficiency. As a method to overcome this, there is a reactive distillation method, which is a method which improves the economical efficiency by simultaneously performing reaction and separation.

Domestic patents related to methyl acetate decomposition include Patent No. 10-0888065 (improved hydrolysis method of methyl acetate), Patent No. 10-0339973 (method and apparatus for the hydrolysis of methyl acetate), Patent No. 10-2015 -0096792 (an integrated processor for the production of acetic acid) and the like are known and are described in Choo S et al., Engineering of Corynebacterium glutamicum to utilize methyl acetate, a potential feedstock derived by carbonylation of methanol with CO (J Biotechnol. 224 2016 47 ≪ / RTI > -50) using methyl acetate as a single carbon source.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

Patent No. 10-0888065 Patent No. 10-0339973 Published Japanese Patent Application No. 10-2015-0096792

 Burnham A, Han J, Clark CE, Wang M, Dunn JB, Palou-Rivera I. 2011. Life cycle greenhouse gas emissions of shale gas, natural gas, coal, and petroleum. Environ. Sci. Technol. 46: 619-627.  DuP., Eikmanns, B. J., 2015. C1-carbon sources for chemical and fuel productionby microbial gas fermentation. Curr. Opin. Biotechnol. 35, 6372.  Kwangmin Kim, Jaewon Lee, Kyeongjun Seo, Myung-Gil Kim, Kyoung-Su Ha, Choongik Kim. 2016. Enhancement of methanewater volumetric mass transfer coefficient by inhibiting bubble coalescence with electrolyte, Journal of Industrial and Engineering Chemistry 33, 326329.  Morris, G. 2005. Carbonylation of methanol to acetic acid and methyl acetate toacetic anhydride. In: Heaton, B. (Ed.), Mechanisms in Homogeneous Catalysis: A Spectroscopic Approach. Wiley-VCH Verlag GmbH, Weinheim, pp. 195230.  Thomas, C. M., Suss-Fink, G. 2003. Ligand effects in the rhodium-catalyzedcarbonylation of methanol. Coord. Chem. Rev. 243, 125142.  Le Berre, C., Kalck, P., Serp, P., Layeillon, L., Thiebaut, D. 2000. Method forpreparing acetic acid and methyl acetate in the presence of iridium andplatinum. Acetex Chimie. US Patent US6916952 B1.  Uhm, S. J., Han, S. H., Oh, J. W., Joo, O.S. 1996. Processes for the carbonylation of methanol to form acetic acid, methyl acetate and acetic anhydride. Korea Institute of Science and Technology. US Patent US5488143 A.  Lessner D. J., Lhu L., Wahal C. S., Ferry J. G. 2010. An Engineered Methanogenic Pathway Derived from the Domains Bacteria and Archaea, MBio., 1 (5) e0024310.  Liu S. and Suflita J. M. 1994. Anaerobic biodegradation of methyl esters by Acetobacterium woodii and Eubacterium limosu ,. J. Ind. Microbiol., 13 (5), 321-7.  Choi J. W., Choi H. G., Lee K. S. and Lee W. H. 1996. A proprietary learning algorithm for acetylobacter using a fed-batch cultivation of ethanol. J. Biotechnol., 49, 29-43.  Wang Y., Tian Y., Han B., Zhao H., Bi J. and Cai B. 2007. Biodegradation of phenol by free and immobilized Acinetobacter sp. strain PD12, Journal of Environmental Sciences 19, 222-225.  Choo S et al. Engineering of Corynebacterium glutamicum to utilize methyl acetate, a potential feedstock derived by carbonylation of methanol with CO J Biotechnol. 224 2016 47-50.

The present inventors have sought to isolate and identify a novel strain capable of degrading methyl acetate, which is a byproduct of chemical processing. As a result, the present invention was accomplished by isolating and identifying an Acinetobacter sp. Strain KFCC11687P using methyl acetate as a single carbon source.

Accordingly, an object of the present invention is to provide strain KFCC11687P belonging to the genus Acinetobacter.

Another object of the present invention is to provide a method for decomposing methyl acetate.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a strain of the genus KFCC11687P in the genus Acinetobacter using methyl acetate as a single carbon source.

The present inventors have sought to isolate and identify a novel strain capable of degrading methyl acetate, which is a byproduct of chemical processing. As a result, the strain KFCC11687P belonging to the genus Ashtona bacterium, which uses methyl acetate as a single carbon source, was isolated and identified.

The novel strain KFCC11687P of the present invention was identified as a microorganism belonging to the genus Ashinabor through 16S rRNA sequencing analysis. It was deposited on August 23, 2016 with the Korean Society for Microbiological Conservation (KCCM) and received the deposit number KFCC11687P.

According to an embodiment of the present invention, the strain KFCC11687P having the Asn Thorac bacterium has the 16S rRNA sequence of the first sequence of the sequence listing. The 16S rRNA sequence of the strain KFCC11687P of the genus Ashitto Bacterium was registered in NCBI GenBank and was given accession number KX608736.

Acinetobacter baumannii DSM 30007 was detected as the closest strain through analysis of the phylogeny of 16S rRNA of strain KFCC11687P of the genus Asnitto bacterium.

A major feature of the strain KFCC11687P of the invention is the use of methyl acetate as a single carbon source.

The strain KFCC11687P, which has the carboxyl esterase activity, can hydrolyze methyl acetate to acetic acid and methanol.

The strain KFCC11687P of the present invention has an activity of degrading methyl acetate under aerobic and anaerobic conditions.

According to one embodiment of the present invention, the strain KFCC11687P has a methyl acetate decomposition rate of 2 to 10 mM / h under an aerobic condition.

According to another embodiment of the present invention, said strain KFCC11687P has a methyl acetate decomposition rate of 2 to 8 mM / h, 2 to 6 mM / h or 2 to 4 mM / h under aerobic conditions.

As demonstrated in the Examples below, the strain KFCC11687P has a methyl acetate decomposition rate of 3.65 mM / h under aerobic conditions.

According to one embodiment of the present invention, the strain KFCC11687P has a methyl acetate decomposition rate of 2 to 10 mM / h under anaerobic conditions.

According to another embodiment of the present invention, the strain KFCC11687P has a methyl acetate decomposition rate of 2 to 8 mM / h, 2 to 6 mM / h or 2 to 4 mM / h under anaerobic conditions.

As demonstrated in the Examples below, the strain KFCC11687P has a methyl acetate decomposition rate of 5.43 mM / h under anaerobic conditions.

The strain KFCC11687P of the present invention has a temperature of 15 ° C to 38 ° C; And (ii) pH 4.5-8.5.

According to one embodiment of the present invention, the strain KFCC11687P is cultivated at a temperature of 15 to 36 캜, 17 to 36 캜, 19 to 36 캜 or 20 to 35 캜.

As demonstrated in the following examples, the strain KFCC11687P exhibited a normal microorganism growth curve at a temperature of 20 ° C to 35 ° C, and the optimum temperature was 30 ° C.

According to one embodiment of the present invention, the strain KFCC11687P, which is a strain belonging to the genus Ashinabota, grows at pH 4.5-8.5.

According to another embodiment of the present invention, the strain KFCC11687P is cultivated under the conditions of pH 5.0-8.5, pH 5.0-8.0, pH 6.0-8.0, or pH 6.5-7.5.

As demonstrated in the following examples, the strain KFCC11687P exhibited an absorbance of at least 0.5 at pH 5.0-8.0 (660 nm), and the optimum pH was pH 7.

According to another aspect of the present invention, there is provided a method for degrading methyl acetate, comprising contacting said strain of the genus Ashthinobacter KFCC11687P.

According to one embodiment of the present invention, the methyl acetate has a concentration of 0.05 to 0.5 mol / L.

According to another embodiment of the present invention, the methyl acetate is 0.1 to 0.5 mol / L.

Since the method of the present invention utilizes the strain KFCC11687P, the description common to both is omitted in order to avoid the excessive complexity of the present specification.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a novel strain of the genus Acinetobacter KFCC11687P using methyl acetate as a single carbon source.

(b) KFCC11687P, a novel strain of the genus Acinetobacterium, has a carboxyl esterase activity, thereby decomposing methyl acetate in the culture broth into acetic acid and methanol and simultaneously using it as a carbon source of methyl acetate.

(c) The novel strain of the genus Ashentanobacter KFCC11687P can easily and economically decompose methyl acetate, which is a byproduct of the chemical process, through a biological method.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows an electron microscope image of the strain KFCC11687P in the genus Ashitto Bacterium.
Fig. 2 shows the phylogeny of the 16s rRNA of the strain KFCC11687P of the genus Ashentin bacterium.
Figure 3 shows the biodegradation process of methyl acetate with methyl acetate hydrolase (EC 3.1.1.).
Figs. 4A to 4C show the growth characteristics of the strain KFCC11687P in the genus Ashimoto. Figures 4A-4C show the effect of temperature, pH and initial concentration of methyl acetate on the growth of the strain KFCC11687P, respectively, on the strain Ashkenobacter.
Figure 5 shows the effect of aerobic conditions on the activity of the strain KFCC11687P, the strain of the genus Ashtona bambuse, in the methyl acetate decomposition.
Figure 6 shows the effect of anaerobic conditions on the activity of degrading the methyl acetate of the strain KFCC11687P.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Methyl acetate is obtained as a byproduct of chemical processes such as acetic acid production as well as carbonylation of methanol and carbon monoxide. A microorganism having methyl acetate as a single carbon source was identified in the soil, and a novel Acinetobacter sp. Strain KFCC11687P was obtained which was able to use methyl acetate as a single carbon source for the first time. Samples were obtained at the Dongducheon Environment Office in order to identify strains capable of using methyl acetate as a single carbon source. Three grams of the sample was added to the third order, followed by culturing. NMS (Nitrate Minimal Salt) medium and 100 mM methyl acetate were added and cultured. In addition, screening was performed on solid medium (NMS medium and 100 mM methyl acetate) to obtain a single colony, thereby obtaining a 16s rRNA sequence.

The strain KFCC11687P, which was found through identification of microorganisms, was confirmed by 16s rRNA sequencing ( Acinetobacter sp. SWSWS 1), Acinetobacter sp. DSM 30007 ( Acinetobacter baumannii strain DSM 30007) (Figs. 1 and 2). Accession number KX608736 from NCBI Genbank.

new Ashton Bautter  Biodegradation of Methyl Acetate with Strain

Carboxylesterase (methyl acetate hydrolase, EC 3.1.1.) Is required for biodegradation of methyl acetate in microorganisms. Carboxyl esterase acts to decompose methyl acetate into acetate and methanol. Methacetate-derived acetate and methanol are converted to acetyl-CoA and converted to metabolites or cell mass, respectively (Figure 3).

Growth Characteristics of New Asymptomatic Bacteria

In order to analyze the growth characteristics of the strain of Ashitto Bacterium in the NMS medium (Table 1), experiments were carried out by temperature, pH and initial methyl acetate concentration.

To optimize the temperature of strains in the genus Ashinobacterium, 100 ml of the filtered methyl acetate was added to 50 ml of NMS medium at pH 7, and the strains were inoculated at 1 v / v% and incubated at 20, 25, 30, 35, The shaking incubator was set up. In the pH optimization experiment, 100 ml of filter-treated methyl acetate was added to 50 ml of NMS medium at pH 4, 5, 6, 7, 8, 9 and 10, and the cells were inoculated with 1 v / v% of the strain and cultured in a shaking incubator Respectively. In the initial methyl acetate concentration test, filtered methyl acetate was added to 50 ml of NMS medium at pH 7, and the cells were inoculated with 1 v / v% of strain and cultured in a 30 ° C stirred incubator.

The temperature was in the range of 20-40 ℃ and the pH was in the range of pH 4-10. In addition, initial methyl acetate concentrations proceeded at 0.1, 0.5, 1.0 and 2.0 mol / L. The strain showed the highest growth at 30 ° C, pH 7, and showed the highest growth when the initial methyl acetate concentration was 0.1 mol / L (FIGS. 4a to 4c). It is known that excess methyl acetate acts as a toxic substance to bacteria and acts as a strain growth inhibitor.

Aerobic culture

When methyl acetate was supplied as a single carbon source in the NMS medium, growth of a new strain of the genus Ashinobacterium and analysis of culture broth were carried out. The conditions were as follows: inoculated with the strain under aerobic conditions and into a flask not inoculated with the strain.

In a 250 ml plain-flask, 50 ml of NMS medium at pH 7 and 100 mM of filter-treated methyl acetate were added, followed by inoculation with 1 v / v% of strains belonging to the genus Ashtona bacterium . The culture was carried out in a shaking incubator at 30 ° C. A stopper was used for the experiment under aerobic conditions. The concentration of methyl acetate in the culture was assayed using WATERS HPLC.

Methyl acetate was highly volatile and decreased to 1.48 mM / h in the strain-free condition and decreased to 3.65 mM / h in the inoculated state (FIG. 5). When methyl acetate is hydrolyzed, acetate and methanol should be produced. When the strain was inoculated, acetate was not measured and methanol was measured and then disappears. Although the metabolic pathway of a new strain of Acinetobacter spp. Has not been elucidated, it can be assumed that acetonitrile and methanol are used as a cell mass or a metabolite in a strain of the genus Ashinobacterium.

Anaerobic culture

Anaerobic culture is an experiment to confirm whether SWSWS1 in Ashinotobacter can convert methylacetate to methanol and acetate using esterase. Because of the decrease of methyl acetate, the growth of the strain, and the disappearance of methanol after the measurement under aerobic condition, the conversion experiment using the enzyme activity in the strain was carried out under anaerobic conditions. Methyl acetate is converted to methanol and acetate by esterase. When acetate is formed and accumulated, the pH of the culture solution is lowered, which affects the enzyme activity. Experimentation under anaerobic conditions is not an important factor because the growth of the strain is not the objective. 150 mM phosphate buffer was used because 1 mM phosphate buffer was present in the NMS medium but no buffering effect due to the accumulation of acetate. Strain 10 g / l (OD ₆₀₀ 15.2) and 250 mM methyl acetate were placed in a serum bottle, capped with a rubber cap, and incubated in an aluminum seal crimp cap and vial crimper under anaerobic conditions . Samples were collected every 2 hours using a syringe and measured by HPLC and GC-FID.

In an anaerobic condition, whole cell conversion was performed by adding DCW (Dry Cell Weight) of 10 g / L of Asinobacterium strain and 250 mM of methyl acetate to 150 mM phosphate buffer. As a result of 44 hours of experiment, 251.7 mM and 180.0 mM accumulation of methanol and acetate produced by biodegradation of 250 mM of methyl acetate were measured by GC-FID (FIG. 6). When methyl acetate is hydrolyzed, methanol and acetate should be produced at a ratio of 1: 1. However, oxygen and acetate in the upper space may be presumed to be consumed as cell masses or metabolites since the anaerobic conditions are not made into vacuum conditions. On the other hand, in the anaerobic condition, the concentration of methyl acetate remained constant in the experimental group which did not contain the strain belonging to the genus Ashinobacterium.

Experiments in anaerobic conditions are experiments to determine if methyl acetate is converted to methanol and acetate using esterase in the strain and accumulated without being consumed. Therefore, a 150 mM phosphate buffer solution was used that could buffer the pH buffering activity of the acetate when the NMS medium containing the components required for growth of the strain was used. Phosphoric acid buffer solution may be limited in strain growth because there is no added substance other than weak acid and conjugate base for pH buffering action.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Korea Microorganism Conservation Center (Domestic) KFCC11687P 20160823

<110> SOGANG UNIVERSITY RESEARCH FOUNDATION <120> Novel Acinetobacter sp. KFCC11687P Using Methyl Acetate as a Sole          Carbon Source <130> PN160274 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 1483 <212> DNA <213> Acinetobacter sp. <400> 1 attgacgctg gcggcaggct taacacatgc aagtcgagcg ggggaaggta gcttgctacc 60 ggacctagcg gcggacgggt gagtaatgct taggaatctg cctattagtg ggggacaaca 120 tctcgaaagg gatgctaata ccgcatacgt cctacgggag aaagcagggg atcttcggac 180 cttgcgctaa tagatgagcc taagtcggat tagctagttg gtggggtaaa ggcctaccaa 240 ggcgacgatc tgtagcgggt ctgagaggat gatccgccac actgggactg agacacggcc 300 cagactccta cgggaggcag cagtggggaa tattggacaa tggggggaac cctgatccag 360 ccatgccgcg tgtgtgaaga aggccttatg gttgtaaagc actttaagcg aggaggaggc 420 tactttagtt aatacctagg gatagtggac gttactcgca gaataagcac cggctaactc 480 tgtgccagca gccgcggtaa tacagagggt gcgagcgtta atcggattta ctgggcgtaa 540 agcgtgcgta ggcggcttat taagtcggat gtgaaatccc cgagcttaac ttgggaattg 600 cattcgatac tggtgagcta gagtatggga gaggatggta gaattccagg tgtagcggtg 660 aaatgcgtag agatctggag gaataccgat ggcgaaggca gccatctggc ctaatactga 720 cgctgaggta cgaaagcatg gggagcaaac aggattagat accctggtag tccatgccgt 780 aaacgatgtc tactagccgt tggggccttt gaggctttag tggcgcagct aacgcgataa 840 gtagaccgcc tggggagtac ggtcgcaaga ctaaaactca aatgaattga cgggggcccg 900 cacaagcggt ggagcatgtg gtttaattcg atgcaacgcg aagaacctta cctggccttg 960 acatactaga aactttccag agatggattg gtgccttcgg gaatctagat acaggtgctg 1020 catggctgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc 1080 cttttcctta cttgccagca tttcggatgg gaactttaag gatactgcca gtgacaaact 1140 ggaggaaggc ggggacgacg tcaagtcatc atggccctta cggccagggc tacacacgtg 1200 ctacaatggt cggtacaaag ggttgctaca cagcgatgtg atgctaatct caaaaagccg 1260 atcgtagtcc ggattggagt ctgcaactcg actccatgaa gtcggaatcg ctagtaatcg 1320 cggatcagaa tgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca 1380 tgggagtttg ttgcaccaga agtagctagc ctaactgcaa agagggcggt taccacggtg 1440 tggccgatga ctggggtgaa gtcgtaacag gggaaacccg taa 1483

Claims (9)

A method for degrading methyl acetate, comprising contacting an Acinetobacter sp. Strain KFCC11687P using methyl acetate as a single carbon source,
Wherein said step is carried out at a pH of from 6.5 to 7.5, wherein methyl acetate is present in a concentration of from 0.1 to 0.5 mol / L.
2. The method according to claim 1, wherein the strain KFCC11687P has the 16S rRNA sequence of SEQ ID NO: 1.
2. The method according to claim 1, wherein the strain KFCC11687P has a carboxylesterase activity.
4. The method of claim 3, wherein the carboxylic esterase hydrolyzes methyl acetate to acetic acid and methanol.
2. The method according to claim 1, wherein the strain KFCC11687P has a methyl acetate decomposition rate of 2 to 10 mM / h under an aerobic condition.
2. The method according to claim 1, wherein the strain KFCC11687P has a methylacetate decomposition rate of 2 to 10 mM / h under anaerobic conditions.
The strain according to claim 1, wherein the strain KFCC11687P is selected from the group consisting of (i) a temperature of 15 to 38 DEG C; And (ii) a pH of 4.5-8.5. delete delete

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