KR101063195B1 - How to Dispose of Food Waste Using Micrococcus Alkanovora SL-010 Strains - Google Patents

Abstract

The present invention relates to a lipolytic strain, and more particularly, as a novel micrococcus alkanovora SL-010 showing lipolytic ability, a strain capable of surviving under various stresses, for example, high temperature, acid, and high salinity. It is about.

Lipolysis Strain, Micrococcus alkanovora SL-010, Stress Resistant Strain

Description

The treatment method of food waste using Micrococcus alkanovora SL-010}

The present invention relates to a lipolytic strain, and more particularly, novel micrococcus alkanobora ( Micrococcus) showing lipolytic ability. alkanovora SL-010) relates to strains that can survive in a variety of stresses, such as high temperature, acidic, high salt conditions.

Lipase degrading enzymes (lipases) catalyze various reactions such as ester synthesis, hydrolysis and transesterification, and are important enzymes in the detergent industry, pharmaceutical industry, fine chemical industry, environmental industry, and food industry. In the detergent industry, Humicolar , Novo Nordisk, 1988 Since lanuginosa lipolytic enzymes have been mass-produced in Aspergillus oryza and sold on the market for enzyme cleaners, many types of detergents have been developed.

In Korea, lipolytic enzymes have been used as enzyme catalysts for flavor development in the food industry since the 1970s. Currently, about 200 kinds of lipolytic enzymes are being studied biochemically, among which 40 species are commercial and research enzymes. It is marketed by.

Recently, as the importance of chiral materials in the pharmaceutical industry has emerged, research on the synthesis of ester compounds using lipolytic enzymes that are stable to organic solvents and has positional and stereospecific properties has been actively conducted. For example, expensive chiral drugs such as ibuprofen and ketoprofen are synthesized using lipolytic enzymes. However, considering the low activity and high cost of currently used optical isomeric lipolytic enzymes, the development of high functional lipolytic enzymes that maximize the optical isomeric separation activity is expected to create high added value.

In this regard, the development of lipolytic strains that produce excellent lipolytic enzymes is very useful. Since lipolytic strains can be used as source materials for lipolytic enzymes and lipolytic enzyme coding genes, they can be used as good microbial candidates for effective treatment (composting or degradation) of food waste. However, since the food contains a variety of components, preferably the strain must be resistant to the acidity and high salinity contained in the food, and the heat generated during decomposition can be said to be industrial.

Accordingly, it is an object of the present invention to provide a strain that exhibits high activity of lipid degradation and shows excellent resistance under various stress conditions. The activity of these strains or lipolytic enzyme coding genes (DNA) and lipolytic enzymes (proteins) derived therefrom can be utilized in a variety of applications, including food degradation.

To this end, the inventors of the present invention seek to be utilized as a source material of lipolytic enzymes and lipolytic enzyme coding genes by searching for a group of bacteria having a lipolytic ability to select excellent lipolytic bacteria and identifying selected bacteria. .

In addition, it is intended to provide excellent lipolytic bacteria having practical utility by showing high survival under stress conditions such as acidity, high salinity, or high temperature.

A strain according to the present invention for achieving the above object is, Micrococcus alkanobora SL-010 ( Micrococcus alkanovora SL-010, Accession No .: KCTC 18130P).

It is stress resistant and has good lipid resolution.

The present invention also provides a micrococcus alkanobora SL-010 ( Micrococcus alkanovora Lipolytic enzyme coding gene (DNA) derived from SL-010, Accession No .: KCTC 18130P) and Micrococcus alkanobora SL-010 ( Micrococcus) alkanovora SL-010, Accession No .: KCTC 18130P), characterized in that it is a lipolytic enzyme (protein) derived from.

In addition, it is characterized by a method of treating food waste using the above strains, genes or enzymes.

The novel micrococcus alkanobora SL-010 according to the present invention shows high activity of lipolysis and also shows resistance under various stress conditions. Therefore, the bacterium, as well as lipolytic enzyme coding gene (DNA) and lipolytic enzyme (protein) derived therefrom, have practical and useful utility in various uses such as food degradation.

Hereinafter, the present invention will be described in detail with an experimental example.

1. Search for Excellent Lipolytic Bacteria

1-1. Various bacteria isolated from insects (centipedes) were examined for lipase resolution. The medium for bacteria used for this purpose is as follows, E. coli ( Esherichia) as a lipolysis negative control coli ) was inoculated together and compared.

Lipolytic agar (Difco Co.): 10 g peptone per liter of distilled water; 5 g sodium chloride; 0.1 g calcium chloride; 20 g Bacto agar; 3.5% Polysolbate-80 10ml, pH 7.2 ± 0.2

The SL-010 strain, which is an excellent lipolytic strain, was finally selected by comparing the diameter of the polysolbate-80 around the colony of each bacterium grown on the Lipolytic agar and swelling. Grown Selected Strains (SL-010) and Escherichia coli ) see photo].

1-2. Finally selected SL-010 strain and control bacteria ( Esherichia coli ), the enzyme activity of the lipolysis emitted by them was investigated.

After incubating the selected SL-010 strain and the control bacteria, respectively, the supernatant, ie, extracellular lipolytic enzyme, was obtained by centrifugation (14,000 rpm, 4 ° C., 10 min) and filter sterilization. Enzyme activity was investigated (Ref. Winkler and Stuchmann. 1979. J. Bacteriol. 138: 663-670). The reaction solution (10 mM p-nitrophenyl palmitate: ethanol: 50mM Tris-HCl (pH8.5) [1: 4: 95]) and the supernatant were mixed 1: 1, and then reacted at 37 ° C. for 30 min. Activity was measured by a spectrophotometer of OD 405 nm, and this value was corrected to a bacterial density of OD 630 nm.

The result is shown in Fig. 2 [lipolytic enzyme activity of lipolytic strain SL-010 and control bacterium Escherichia coli ].

2. Selected SL Excellent Lipid Degradation of -010 Strains

For comparison with the strains contained in the product marketed in 2002 (Daewoo Electronics Co, Green Chip for Blue World Food Processor), 10 g of green chips were extracted with 50 ml of R2A broth (Bacto yeast extract 0.05%; Bacto proteose peptone No. 3 0.05%; Bacto casamino acids 0.05%; Bacto dextrose 0.05%; Soluble starch 0.05%; Sodium pyruvate 0.03%; Potassium phosphate, dibasic 0.03%; Magnesium sulfate 0.005%), followed by enrichment culture After 1 and 9 days, the appropriate amount of suspension was spread on the R2A agar plate and a total of 102 bacterial isolates were obtained in pure culture. Among them, 35 kinds of bacteria (GC-series) were inoculated in Lipolytic agar and then cultured at each temperature to compare their degradation.

As a result, the degree of degradation of each nutrient (the diameter of net clearing zone [= diameter of total clearing zone-diameter of bacterial colony] observed on each agar medium) was compared. The highest resolution was found to be 14 mm (Table 1).

Table 1. Lipolysis SL -010 and other bacteria Lipid resolution  compare

Strain Net cleared zone (mm) GC-1 0 GC-2 0 GC-3 0 GC-4 0 GC-5 0 GC-6 0 GC-7 5 GC-8 0 GC-9 0 GC-10 0 GC-11 0 GC-12 0 GC-13 0 GC-14 0 GC-15 0 GC-16 0 GC-17 0 GC-18 0 GC-19 0 GC-20 0 GC-21 0 GC-22 0 GC-23 2 GC-24 0 GC-25 0 GC-26 5 GC-27 0 GC-28 4 GC-15-3 5 GC-15-2 0 GC-1-1 0 GC-1-3 0 GC-1-10 0 GC-1-11 8 GC-1-12 0 SL -010 14

3. Identification of Excellent Lipolytic Bacteria

3-1. Identification method using 16S rDNA sequence of SL-010 strain was performed. To this end, after purifying total DNA of the SL-010 strain, using this as a template, a DNA portion encoding 16S rRNA was amplified by PCR. The PCR primers used were 27F and 1492R (27F: 5'- AGA GTT TGA TCM TGG CTC AG -3 '; 1492R: 5'- GGY TAC CTT GTT ACG ACT T -3'). And PCR reaction conditions are as follows.

PCR reaction condition:

94C 5 min;

30 cycles (94C 1 min; 50C 1 min; 72C 1min 30sec);

72C 10 min

PCR reaction mixture (total 50ul):

Bacterial total DNA: 0.5ul

10X Taq buffer: 5ul

10mM dNTP: 5ul

primer (27F): 0.5ul

primer (1492R): 0.5ul

5U / ul Taq: 0.5ul

DW: 38ul

The product was ligation into the pGEMT vector and transformed into E. coli DH5a. After plasmid DNA was purified from the transformant growing in the presence of the antibiotic ampicillin, the nucleotide sequence of the insert DNA in the vector was read. The 1,384 nucleotide sequences read were as follows (see SEQ ID NO: 1).

GCCCAGCTTGCTGGGTGGATTAGTGGCGAACGGGTGAGTAACACGTGAGTAACCTGCCCTTGACTCTGGGATAAGCCTGGGAAACTGGGTCTAATACCGGATAGGAACGTCCACCGCATGGTGGGTGTTGGAAAGATTTATCGGTCATGGATGGACTCGCGGCCTATCAGCTTGTTGGTGAGGTAATGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGTAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTATCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCTGTCGTGAAAGTCCGGGGCTTAACCCCGGATCTGCGGTGGGTACGGGCAGACTAGAGTGCAGTAGGGGAGACTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATATCAGGAGGAACACCGATGGCGAAGGCAGGTCTCTGGGCTGTAACTGACGCTGAGGAGCGAAAGCATGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGTTGGGCACTAGGTGTGGGGACCATTCCACGGTTTCCGCGCCGCAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCAAGGCTTGACATGTTCTCGATCGCCGTAGAGATACGGTTTCCCCTTTGGGGCGGGATCACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGT CCCGCAACGAGCGCAACCCTCGTTCCATGTTGCCAGCACGTGATGGTGGGGACTCATGGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGTCTTGGGCTTCACGCATGCTACAATGGCCGGTACAATGGGTTGCGATACTGTGAGGTGGAGCTAATCCCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCACGAAAGTCGGTAACACCCGAAGCCGGTGGCCTAACCCTTGTGGGGGGAGCCGTCGAAGGTGGGA

3-2. Micrococcus results from database and BLAST searches in Robosomal Database Project II (http://rdp.cme.msu.edu/html/) It showed 99.9% identity with 16S rDNA sequence of alkanovora (see control table in Table 2 below). Accordingly, the SL-010 strain was named Microcaucus Alkanovoa SL-010, and the strain was deposited with KCTC 18130P as an accession number to Korea Biotechnology Research Institute.

SL-010 GCCCAGCTTGCTGGGTGGATTAGTGGCGAACGGGTGAGTAACACGTGAGT

Micrococcus_alkanovora GCCCAGCTTGCTGGGTGGATTAGTGGCGAACGGGTGAGTAACACGTGAGT

**************************************************

SL-010 AACCTGCCCTTGACTCTGGGATAAGCCTGGGAAACTGGGTCTAATACCGG

Micrococcus_alkanovora AACCTGCCCTTGACTCTGGGATAAGCCTGGGAAACTGGGTCTAATACCGG

**************************************************

SL-010 ATAGGAACGTCCACCGCATGGTGGGTGTTGGAAAGATTTATCGGTCATGG

Micrococcus_alkanovora ATAGGAACGTCCACCGCATGGTGGGTGTTGGAAAGATTTATCGGTCATGG

**************************************************

SL-010 ATGGACTCGCGGCCTATCAGCTTGTTGGTGAGGTAATGGCTCACCAAGGC

Micrococcus_alkanovora ATGGACTCGCGGCCTATCAGCTTGTTGGTGAGGTAATGGCTCACCAAGGC

**************************************************

SL-010 GACGACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACACTGGGACTGAGA

Micrococcus_alkanovora GACGACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACACTGGGACTGAGA

**************************************************

SL-010 CACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGG

Micrococcus_alkanovora CACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGG

**************************************************

SL-010 GCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTT

Micrococcus_alkanovora GCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTT

**************************************************

SL-010 GTAAACCTCTTTCAGTAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAG

Micrococcus_alkanovora GTAAACCTCTTTCAGTAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAG

**************************************************

SL-010 AAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGA

Micrococcus_alkanovora AAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGA

**************************************************

SL-010 GCGTTATCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCG

Micrococcus_alkanovora GCGTTATCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCG

**************************************************

SL-010 TCTGTCGTGAAAGTCCGGGGCTTAACCCCGGATCTGCGGTGGGTACGGGC

Micrococcus_alkanovora TCTGTCGTGAAAGTCCGGGGCTTAACCCCGGATCTGCGGTGGGTACGGGC

**************************************************

SL-010 AGACTAGAGTGCAGTAGGGGAGACTGGAATTCCTGGTGTAGCGGTGGAAT

Micrococcus_alkanovora AGACTAGAGTGCAGTAGGGGAGACTGGAATTCCTGGTGTAGCGGTGGAAT

**************************************************

SL-010 GCGCAGATATCAGGAGGAACACCGATGGCGAAGGCAGGTCTCTGGGCTGT

Micrococcus_alkanovora GCGCAGATATCAGGAGGAACACCGATGGCGAAGGCAGGTCTCTGGGCTGT

**************************************************

SL-010 AACTGACGCTGAGGAGCGAAAGCATGGGGAGCGAACAGGATTAGATACCC

Micrococcus_alkanovora AACTGACGCTGAGGAGCGAAAGCATGGGGAGCGAACAGGATTAGATACCC

**************************************************

SL-010 TGGTAGTCCATGCCGTAAACGTTGGGCACTAGGTGTGGGGACCATTCCAC

Micrococcus_alkanovora TGGTAGTCCATGCCGTAAACGTTGGGCACTAGGTGTGGGGACCATTCCAC

**************************************************

SL-010 GGTTTCCGCGCCGCAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGG

Micrococcus_alkanovora GGTTTCCGCGCCGCAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGG

**************************************************

SL-010 CCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGG

Micrococcus_alkanovora CCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGG

**************************************************

SL-010 AGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCAAGGCTTGAC

Micrococcus_alkanovora AGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCAAGGCTTGAC

**************************************************

SL-010 ATGTTCTCGATCGCCGTAGAGATACGGTTTCCCCTTTGGGGCGGGATCAC

Micrococcus_alkanovora ATGTTCTCGATCGCCGTAGAGATACGGTTTCCCCTTTGGGGCGGGATCAC

**************************************************

SL-010 AGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGT

Micrococcus_alkanovora AGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGT

**************************************************

SL-010 CCCGCAACGAGCGCAACCCTCGTTCCATGTTGCCAGCACGTGATGGTGGG

Micrococcus_alkanovora CCCGCAACGAGCGCAACCCTCGTTCCATGTTGCCAGCACGTCATGGTGGG

***************************************** ********

SL-010 GACTCATGGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGT

Micrococcus_alkanovora GACTCATGGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGT

**************************************************

SL-010 CAAATCATCATGCCCCTTATGTCTTGGGCTTCACGCATGCTACAATGGCC

Micrococcus_alkanovora CAAATCATCATGCCCCTTATGTCTTGGGCTTCACGCATGCTACAATGGCC

**************************************************

SL-010 GGTACAATGGGTTGCGATACTGTGAGGTGGAGCTAATCCCAAAAAGCCGG

Micrococcus_alkanovora GGTACAATGGGTTGCGATACTGTGAGGTGGAGCTAATCCCAAAAAGCCGG

**************************************************

SL-010 TCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGC

Micrococcus_alkanovora TCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGC

**************************************************

SL-010 TAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGT

Micrococcus_alkanovora TAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGT

**************************************************

SL-010 ACACACCGCCCGTCAAGTCACGAAAGTCGGTAACACCCGAAGCCGGTGGC

Micrococcus_alkanovora ACACACCGCCCGTCAAGTCACGAAAGTCGGTAACACCCGAAGCCNGTGGC

******************************************** *****

SL-010 CTAACCCTTGTGGGGGGAGCCGTCGAAGGTGGGA

Micrococcus_alkanovora CTAACCCTTGTGGGGGGAGCCGTCGAAGGTGGGA

**********************************

Table 2. 16S rDNA Sequences and Micrococus of Superior Lipolytic Strain SL-010 Comparison with 16S rDNA Sequence of alkanovora

4. Stress Response of Superior Lipolytic Bacteria

The growth and survival of the novel Micrococcus alkanobora SL-010 according to the present invention was investigated.

4-1. To determine the survival of Micrococcus alkanobora SL-010 in nutrient depletion, the first inoculation was maintained during the 20-day experiment after inoculation of bacteria in saline (0.9% NaCl solution). .

4-2. When inoculated with LBS (10 g tryptone per 1 L; yeast extract 5 g; NaCl 25 g) inoculated with Micrococcus alkanobora SL-010 and incubated at 37 ° C., 43 ° C. and 47 ° C., all temperature ranges 37 ° C.-43 All growth was possible at ℃.

4-3. Micrococcus SL-001 was inoculated in LB containing salt concentrations of 2.5, 5.0, and 7.5% NaCl (tryptone 10g per 1L; yeast extract 5g), followed by incubation at 30 ° C for 3 days to compare the growth of each bacteria. . As a result, the growth was even from 2.5% to 7.5%.

4-4. To determine the response to acidity, pH5.0. Growth of 5.5, 6.0 in 30 ° C. LBS medium was investigated. As a result, all growth was possible.

1 is a strain selected (SL-010) and E. coli grown on the agar Lipolytic (Escherichia coli )

Figure 2 is lipolysis strain SL-010 and control bacteria Escherichia coli ( Esherichia coli ) lipolytic enzyme activity.

<110> Lee Sang Hwan; Hankuk University Of Foreign Studies Research and Industry-University Cooperation Foundation <120> Stress-resistant lipolytic bacterium, Micrococcus alkanovora          SL-010 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1384 <212> DNA <213> Micrococcus alkanovora SL-010 <400> 1 gcccagcttg ctgggtggat tagtggcgaa cgggtgagta acacgtgagt aacctgccct 60 tgactctggg ataagcctgg gaaactgggt ctaataccgg ataggaacgt ccaccgcatg 120 gtgggtgttg gaaagattta tcggtcatgg atggactcgc ggcctatcag cttgttggtg 180 aggtaatggc tcaccaaggc gacgacgggt agccggcctg agagggtgac cggccacact 240 gggactgaga cacggcccag actcctacgg gaggcagcag tggggaatat tgcacaatgg 300 gcgaaagcct gatgcagcga cgccgcgtga gggatgacgg ccttcgggtt gtaaacctct 360 ttcagtaggg aagaagcgaa agtgacggta cctgcagaag aagcaccggc taactacgtg 420 ccagcagccg cggtaatacg tagggtgcga gcgttatccg gaattattgg gcgtaaagag 480 ctcgtaggcg gtttgtcgcg tctgtcgtga aagtccgggg cttaaccccg gatctgcggt 540 gggtacgggc agactagagt gcagtagggg agactggaat tcctggtgta gcggtggaat 600 gcgcagatat caggaggaac accgatggcg aaggcaggtc tctgggctgt aactgacgct 660 gaggagcgaa agcatgggga gcgaacagga ttagataccc tggtagtcca tgccgtaaac 720 gttgggcact aggtgtgggg accattccac ggtttccgcg ccgcagctaa cgcattaagt 780 gccccgcctg gggagtacgg ccgcaaggct aaaactcaaa ggaattgacg ggggcccgca 840 caagcggcgg agcatgcgga ttaattcgat gcaacgcgaa gaaccttacc aaggcttgac 900 atgttctcga tcgccgtaga gatacggttt cccctttggg gcgggatcac aggtggtgca 960 tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct 1020 cgttccatgt tgccagcacg tgatggtggg gactcatggg agactgccgg ggtcaactcg 1080 gaggaaggtg gggacgacgt caaatcatca tgccccttat gtcttgggct tcacgcatgc 1140 tacaatggcc ggtacaatgg gttgcgatac tgtgaggtgg agctaatccc aaaaagccgg 1200 tctcagttcg gattggggtc tgcaactcga ccccatgaag tcggagtcgc tagtaatcgc 1260 agatcagcaa cgctgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcaagtca 1320 cgaaagtcgg taacacccga agccggtggc ctaacccttg tggggggagc cgtcgaaggt 1380 ggga 1384  

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delete delete delete delete A method of treating food waste using a Micrococcus alkanovora SL-010 (Accession No .: KCTC 18130P) strain having excellent lipid resolution.

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