KR101235265B1 - Novel Pseudoalteromonas sp. and Agarase Produced by the Same - Google Patents

Abstract

The present invention relates to a novel pseudoalteromonas genus microorganism and the agarase secreted by it.
The agarase produced by the microorganism of the present invention not only has very excellent salt stability, but also directly decomposes woodworms without pretreatment, thereby providing a saccharification technique that utilizes woodworms directly as a substrate.

Description

Novel Pseudoalteromonas sp. and Agarase Produced by the Same}

The present invention relates to a novel pseudoalteromonas genus microorganism and the agarase secreted by it.

More specifically, the present invention relates to a novel Pseudo-Altermonas genus microorganism that produces agarase capable of directly decomposing locus in high salt environment and the agarase secreted by it.

Bioenergy refers to renewable energy obtained through processes from biomass, which is organic matter of life. The key step in the production of eco-friendly and economical bioenergy is securing biomass, which is a raw material of bioenergy, and developing saccharification technology. In order to develop eco-friendly and economical biomass and its pretreatment technology, various approaches have been tried. Recently, many researches and developments have been made to utilize algae called third generation biomass as biomass.

Locust is a kind of marine red algae, and since most of its dry weight is composed of carbohydrates, it has recently attracted attention as a biomass raw material for producing bioenergy. Since about 60% of the barley is composed of agar formed by combining monosaccharides in the form of galactose, it is essential to develop a technology to secure monosaccharides by pretreating agar to use it as a biomass for bioenergy production. .

So far, the technology for saccharifying algae is in the early stages of technology development. Like the first and second generation biomass, research and development is being conducted in terms of chemical pretreatment and biological pretreatment. The chemical pretreatment method has the advantage that the process is simple and relatively more technical development has been achieved than the biological pretreatment method, but it is difficult to control the pretreatment product, and the toxic by-products are generated in the saccharification process, causing further ethical and environmental problems. There are disadvantages. In the case of biological pretreatment methods, research is being conducted mainly in terms of utilizing enzymes produced by living organisms, and does not generate toxic by-products generated during chemical pretreatment, reacts specifically with the substrate of the enzyme, and the final product of each enzyme. In this clear, it has a lot of potential.

In the case of agarase that decomposes agar, it is traditionally attracting attention from the viewpoint of securing biomass for bioenergy production. Attention has been paid in terms of producing additives and the like. In recent years, attention has been drawn from the viewpoint of biomass pretreatment for bioenergy production, but until now, the technology is limited to the decomposition of agarose or agar, or the development of commercially available methods is insignificant. In the case of using agarose or agar as a substrate for the enzymatic reaction, the pretreatment process of producing agarose or agar from the wood must first be performed, and thus, it is less economical than using the wood starch as the substrate. Therefore, in the present invention, efforts have been made to develop a glycosylation technology that utilizes the locust as a substrate for oligosaccharide production.

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.

The present inventors earnestly researched to develop a saccharification technique that directly utilizes woodworm as a substrate without pretreatment. As a result, a novel Pseudo-Altermonas genus microorganism is produced that produces agarase that can directly degrade woodworm in high salt environments. By this, the present invention has been completed.

It is therefore an object of the present invention to provide a novel Pseudo-Altermonas microorganism which produces agarase.

Another object of the present invention to provide an agarase produced by the microorganism.

It is another object of the present invention to provide a method for directly saccharifying a starfish without pretreatment.

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 invention there is provided the pseudo Alteromonas J. wayibi CL producing an agarase (agarase) 1 (Pseudoalteromonas sp . JYBCL 1 ) provides KCCM11139P.

The present inventors earnestly researched to develop a saccharification technique that directly utilizes woodworm as a substrate without pretreatment. As a result, a novel Pseudo-Altermonas genus microorganism is produced that produces agarase that can directly degrade woodworm in high salt environments. By this, the present invention has been completed.

The agarase-producing microorganism Pseudo-Altermonas J. YB. C. 1 of the present invention is newly isolated from the intestine of a conch that feeds on woodworms. The present inventors deposited the Pseudo-Altermonas J. YB. C 1 to the Korea Microorganism Conservation Center, which is a depository institution, on November 30, 2010, and received the accession number KCCM11139P.

The base sequence of the 16s rRNA of the novel microorganism of the present invention is represented by SEQ ID NO: 1, and the microorganism of the present invention belongs to the genus Pseudoaltermonas.

The agarase produced by Pseudo-Altermonas J.W.B.CL 1 of the present invention is characterized by being able to directly decompose loaves without pretreatment, since the locus is a substrate. In the case of known agarases, agarose or agar is used as a substrate for the enzymatic reaction, and thus, pretreatment for producing agarose or agar from locus is essential, but the agarase produced by the microorganism of the present invention is agarose. Alternatively, it is economical because it is directly decomposed without the pretreatment to convert to agar.

As used herein, the term "woom sari" is a seaweed of red algae belonging to the family of worms, the scientific name is Gelidium amansii and refers to sea horses used as the main raw material of agar as a kind of sea horse .

The term "agar" refers to a viscous, indigestible complex polysaccharide that is a cell wall component of red algae. Agar consists of agarose and agaropectin, and is mainly composed of about 70% agarose and about 30% agalopectin.

The Pseudo-Altermonas JYBCI 1 of the present invention produces an agarase having an ion stability significantly superior to other known agarases. Therefore, the agarase produced by the microorganism shows excellent enzymatic activity even in a high salt environment.

In one embodiment of the present invention, the agarase produced by Pseudo-Altermonas J.W.B.CL 1 of the present invention has an enzyme activity at a sodium chloride concentration of 2000 mM of 70% to 100% of an enzyme activity at a sodium chloride concentration of 0 mM.

In another embodiment, the agarase has an enzymatic activity at sodium chloride concentration of 4000 mM of 50% to 80% of enzymatic activity at sodium chloride concentration of 0 mM.

According to another aspect of the present invention, the present invention provides agarase, characterized in that the enzyme activity at sodium chloride concentration of 2000 mM is 70% to 100% of the enzyme activity at sodium chloride concentration of 0 mM.

According to another aspect of the invention, the present invention provides agarase, characterized in that the enzyme activity at sodium chloride concentration 4000 mM is 50% to 80% of the enzyme activity at sodium chloride concentration 0 mM.

Since the agarase having excellent stability in the high salt environment of the present invention is an enzyme produced by the above-mentioned Pseudo-Altermonas J.W.B.C.L.1, the common contents in the relationship with the Pseudo-Alteronas. In order to avoid complexity, the description thereof is omitted.

In one embodiment, the agarase is directly based on the locus. Buttfish is derived from the sea and contains a large amount of salt, but since the agarase of the present invention shows high enzymatic activity even in a high salt environment, it is more economical because it does not need a pretreatment step for completely removing the salt.

According to another aspect of the invention, the present invention comprises the steps of (a) obtaining a starfish; And (b) treating the agarase of claim 6 to the obtained woodfish.

According to another aspect of the invention, the present invention comprises the steps of (a) obtaining a starfish; And (b) providing a method for saccharifying a saccharin comprising the step of treating the obtained saccharin with Pseudo-Altermonas J. YB CI of any one of claims 1 to 5.

In the case of using agarose or agar as a substrate for the enzymatic reaction, a pretreatment process for producing agarose or agar from locus must be preceded. However, according to the method of the present invention, the locus is used as a substrate for oligosaccharide production. Therefore, algae can be saccharified with high efficiency.

In one embodiment of the present invention, the agarase is treated with Agarase produced by Pseudo-Altermonas J.W.B.C.L.1, and the used agarase can be purified and reused, in which case a series of more efficient and economical glycosylation systems are employed. It can be established.

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

(Iii) The present invention provides novel Pseudo-Altermonas microorganisms and agarases secreted by them.

(Ii) The agarase produced by the microorganism of the present invention not only has a very good salt stability, but also can directly decompose woodworms without pretreatment, thereby providing a saccharification technique that utilizes woodworms directly as a substrate.

Figure 1 shows the colony colony form and golgol staining results of the isolated microorganisms.
2 is a culture curve showing the effect of sodium chloride on the growth of isolated microorganisms (■; growth curve in NaCl 5g / l, ●; growth curve in NaCl 15g / l, ▲; growth curve in NaCl 25g / l ).
Figure 3 shows the phylogenetic tree prepared for the comparative analysis of homology of the isolated microorganism of the present invention.
Figure 4 is the result of measuring the growth of the bacteria and the viscosity in the medium to confirm the agar resolution of the isolated microorganisms (■; control viscosity, ●; experimental group viscosity, ▲; control absorbance ▼; experimental group absorbance).
FIG. 5 shows the results of a temperature optimization experiment of agarase secreted by Pseudo-Altermonas J.W.B.CL1.
FIG. 6 shows the results of pH optimization experiments of agarase secreted by Pseudo-Altermonas J.Y.B.CL1.
Figure 7 is a graph showing the decrease in relative activity as the NaCl concentration of agarase secreted by Pseudo-Altermonas J. Y. B. CL 1 increases.
8 is a graph showing a decrease in relative activity with increasing NaCl concentration of the known agarase.
9 is agarase extract SDS PAGE electrophoresis was performed by covering the electrophoretic gel on the aga gel prepared separately, and then reacted with arugel solution stained agarase band.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not to be construed as limiting the scope of the present invention. It will be self-evident.

Example

In the present invention, an effort was made to find a microorganism that secretes agarase in the intestine of a conch that feeds on the larvae. In order to select microorganisms having agar resolution, two selection conditions were used. First, it was confirmed whether erosion occurred around the colonies formed by the microorganisms on the solid agar medium. Second, the agar decomposition was confirmed by staining the solid medium with a rugial solution.

The DNS method was used to quantitatively determine the amount of reducing sugars present in the culture medium and the reducing sugars produced in the determination of enzyme activity, and more precisely, the reduced terminal groups. DNS reagent was prepared by dissolving 0.25 g of 3,5-dinitrosalicylic acid and 75 g of sodium potassium tartrate in 50 ml of 2M NaOH and diluting with water to a volume of 250 ml. The amount of reducing sugar produced was mixed with 200 μl of sample and 900 μl of DNS reagent, reacted at 99 ° C. for 10 minutes, cooled to room temperature, and absorbed at 546 nm using an UV / Vis spectrophotometer. It was. The calibration curve was prepared using galactose.

For experiments measuring the activity of agarase, 0.25% agarose dissolved in 20mM Tris-cl buffer was used as a substrate solution, and the agarase extract obtained in the procedure of Example 5 to be described below was used as an enzyme solution. The mixture was mixed at a ratio of 1 and allowed to react at 120 rpm.

< Example  1> New Agarase  Isolation of Secretory Marine Microorganisms

The intestines of Sora, which were purchased from Noryangjin Fish Market in Seoul, were treated and used as inoculum sample solution. Inoculation medium was subcultured four times at intervals of about 4 days, to prepare a culture medium with the composition shown in Table 1 below.

ingredient Primary medium (g / l) Secondary medium (g / l) Tertiary badge (g / l) 4th badge (g / l) NaCl 5 5 5 5 (NH ₄ ) ₂ SO ₄ One One One One Yeast extract 0.5 0.2 0 0 K ₂ HPO ₄ 2 2 2 2 MgCl ₂ 6H ₂ O 0.2 0.2 0.2 0.2 CaCl ₂ 2H ₂ O 0.02 0.02 0.02 0.02 Agar One One One One

Since yeast extract used as a nitrogen source contains many nutrients, yeast extract was not added from the tertiary culture in order to select agar decomposition strains more selectively.

After 4 passages, NaCl 5 g / l, (NH ₄ ) ₂ SO ₄ 1 g / l, K ₂ HPO ₄ Diluted solutions were prepared at 2 g / l and diluted in multiples of 10 ⁻⁴ and 10 ⁻⁵ and spread on the same solid medium (18 g / l only) as the primary passage medium. As a result of the culture of the solid medium, microorganisms showing distinct erosion were found around the colonies, and purely separated, and confirmed by the Lugol solution, it was confirmed that the agarase was secreted in vitro. Colony colony form and golgol staining results of the isolated microorganisms are shown in FIG.

< Example  2> Separation of Microorganisms Physiological Biochemical  characteristic

The physiological and biochemical characteristics of the isolated microorganisms were investigated. To investigate the physiological and biochemical properties of the isolated microorganisms, API 20NE and API ZYM kits of bioMerieux were used. The sample solution was prepared in two cases, sterile saline solution and seawater solution. Physiological and biochemical properties of the isolated microorganisms are shown in Table 2 below.

characteristic Saline Seawater
Typical experiment API  20 NE ) Reduction of Nitrate to Nitrite + + Reduction of Nitrate to Nitrogen + + Indole production (tryptophan) - - Fermentation (Glucose) - - Arginine dehydrolase - - Urease - - Hydrolysis (β-glucosidase) (esculin) - - Hydrolysis (Protease) (Gelatin) - + ? -galactosidase - -
Assimilation experiment API  20 NE ) D-glucose + + L-Arabinose - - D-Mannos - - D-mannitol - + N-acetyl-glucosamine - - D-Maltose + + Potassium Gluconate + + Capric Acid - - Adipic acid - - Malic acid + + Trisodium Citrate - + Phenylacetic acid - -
Enzyme activity API zym ) Alkaline phosphatase + + Esterase (C4) + + Esterase lipase (C8) + + Lipase (C 14) - - Leucine arylamidase + + Valine arylamidase + + Cystine Arulamidase - - Trypsin - - α-chymotrypsin - - Acid phosphatase - + Naphthol-AS-BI-phosphohydrolase + + α-galactosidase - - ? -galactosidase - - β-glucuronidase - - α-glucosidase - - β-glucosidase - - N-acetyl-β-glucosaminidase - - α-mannosidase - - α-fucosidase - -

For isolated microorganisms, the activity of some enzymes was higher in seawater than in saline, suggesting that sodium chloride could affect the activity of the enzyme.

In addition, in order to confirm the effect of sodium chloride on the growth of isolated microorganisms, the culture experiment was performed by adjusting the concentration of sodium chloride to 15 g / l, 25 g / l in the primary passage culture medium, the culture curve is shown in Figure 2 It was.

Example 3 Identification of Isolated Microorganisms

For accurate identification of isolated microorganisms, 16s rRNA identification was performed. After extracting the chromosomal DNA of the isolated microorganism, 16s rRNA fragment was amplified by polymerase chain reaction (PCR), and its nucleotide sequence was determined and shown in the first sequence list. The 16s rRNA sequences were compared to GenBank's database using the BLASTN program of the National Center for Biotechnology Information (NCBI). As a result, there was no existing microorganism having a nucleotide sequence 100% identical to that of the 16s rRNA sequence. The isolated microorganism of the present invention was found to be new.

Homology comparative analysis was performed using ClastalW and Mega 4 program and the generated phylogenetic tree is shown in FIG. 3. Separating microorganisms pseudo Alteromonas were identified as belonging to the genus (Pseudoalteromonas), and finally pseudo Alteromonas Jay wayibi CL 1 (Pseudoalteromonas sp. JYBCL 1) as named by November 30, 2010 it to the depository Korea Culture Center of Microorganisms Date Was deposited with the accession number KCCM11139P.

Example 4 Determination of Agar Resolution of Isolated Microorganisms

Further culture experiments were performed to confirm the agar resolution of Pseudo-Altermonas J.Y.B.CL1. The culture medium was prepared in the same manner as the primary passage medium (only 2 g / l of agar), and the growth of bacteria and the viscosity in the medium were measured and shown in FIG. 4. Over time after incubation, the viscosity in the medium was confirmed by the naked eye, and the actual viscosity measurement results were confirmed that the viscosity in the medium decreases as the bacteria grow.

Example 5 Securing Agarase Extract for Saccharification

In the case of Pseudo-Altermonas J. YBCI 1, it was confirmed that the secretion of agarase in vitro, thereby securing the agarase extract by the following method. After incubating the strain, the cells were centrifuged for 30 minutes at an acceleration of 7000 g at 4 ° C. after 12 hours. After centrifugation, the supernatant was collected and dissolved (NH ₄ ) ₂ SO ₄ to 70% (g / mL). Thereafter, the mixture was centrifuged for 30 minutes at an acceleration of 14500 g at 4 ° C., and the supernatant was discarded and the protein precipitate was resuspended in 20 mM Tris-cl buffer (pH 8) to obtain an agarase extract. If necessary, it was purified by dialysis in the same buffer.

Example 6 Agarase Activity Optimization for Glycation

Temperature and pH optimization experiments were performed to optimize the activity of Agarase secreted by Pseudo-Altermonas J.Y.B.CL1. Temperature optimization experiments were carried out by measuring the amount of reducing sugars formed in the reaction solution after one day of reaction at five temperatures of 15, 25, 35, 45 and 55 ℃. pH optimization experiments were performed by measuring the amount of reducing sugars produced in the reaction solution after six days of reaction at six different pHs of pH 4, 5, 6, 7, 8 and 9, and the results were shown in FIG. 6 is shown.

< Example  7> Agarase  Structural Stability Measurement

An experiment was conducted to determine how stable the three-dimensional protein structure of Agarase secreted by Pseudo-Altermonas J.Y.B.C.I.I. (Hiroshi Xavier Chiura et al., Purification and Characterization of a Novel Agarase Secreted by a Marine Bacterium, Pseudoalteromonas sp.Strain CKT1, Microbes and Environments 15 (1): 11-22 (2000)).

Sodium chloride was added to the agarose substrate solution by concentration, and the relative activity decrease as the NaCl concentration of the agarase secreted by Pseudo-Altermonas J.Y.B.C.I. 1 was measured and shown in FIG. 7. See also Hiroshi Xavier Chiura et al., Purification and Characterization of a Novel Agarase Secreted by a Marine Bacterium, Pseudoalteromonas sp.Strain CKT1, Microbes and Environments The decrease in relative activity as the NaCl concentration of the conventional agarase described in 15 (1): 11-22 (2000)) is shown in FIG. 8.

Comparing FIGS. 7 and 8, it can be seen that the agarase secreted by Pseudo-Altermonas J.W.B.C.L. 1 has significantly superior ion stability than other known agarases.

< Example  8> Agarase  Confirm

Zymogram analysis was performed to confirm the presence of agarase in the secreted protein. SDS electrophoresis was performed with the agarase extract obtained in Example 5, and the agar gel was prepared by reacting with an electrophoretic gel, and then stained with lugol solution to confirm the agarase band.

< Example  9> Agarase  Direct use of woodworm Glycation

Experiments were performed to directly saccharin the locus with the obtained agarase extract. Agarase extract was added to a buffer containing 0.25% (w / v) of agar and fine ash powder, respectively. Relative activity of the two substrates in the following is shown in Table 3 below.

temperament Reducing Sugar Production (mM) Relative activity (%) Baby 0.875 100 Mugwort 0.408 46.6

As a result, it was confirmed that agarase secreted by Pseudo-Altermonas J.W.B.Ciel 1 can be directly used as a substrate without pretreatment to produce agarose or agar from woodworms, unlike other known agarases. .

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. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Korea Microorganism Conservation Center (overseas) KCCM11139P 20101130

<110> Industry-University Cooperation Foundation Yonsei University <120> Novel Pseudoalteromonas sp. and Agarase Produced by the Same <160> 1 <170> Kopatentin 1.71 <210> 1 <211> 947 <212> DNA Pseudoalteromonas sp. JYBCL 1, 16s rRNA <400> 1 ggtacgtcct cccgagggtt agactatcta cttctggagc aacccactcc catggtgtga 60 cgggcggtgt gtacaaggcc cgggaacgta ttcaccgcgt cattctgata cgcgattact 120 agcgattccg acttcatgga gtcgagttgc agactccaat ccggactacg acgcacttta 180 agtgattcgc ttaccttcgc aggttcgcag cactctgtat gcgccattgt agcacgtgtg 240 tagccctaca cgtaagggcc atgatgactt gacgtcgtcc ccaccttcct ccggtttatc 300 accggcagtc tccttagagt tctcagcatt acctgctagc aactaaggat aggggttgcg 360 ctcgttgcgg gacttaaccc aacatctcac aacacgagct gacgacagcc atgcagcacc 420 tgtatcagag ttcccgaagg caccaaacca tctctggtaa gttctctgta tgtcaagtgt 480 aggtaaggtt cttcgcgttg catcgaatta aaccacatgc tccaccgctt gtgcgggccc 540 ccgtcaattc atttgagttt taaccttgcg gccgtactcc ccaggcggtc tacttaatgc 600 gttagctttg aaaaacagaa ccgaggctcc gagcttctag tagacatcgt ttacggcgtg 660 gactaccggg gtatctaatc ccgtttgctc cccacgcttt cgtacatgag cgtcagtgtt 720 gacccaggtg gctgccttcg ccatcggtat tccttcagat ctctacgcat ttcaccgcta 780 cacctgaaat tctaccaccc tctatcacac tctagtttgc cagttcgaaa tgcagttccc 840 aggttgagcc cggggctttc acatctcgct taacaaaccg cctgcgtacg ctttacgccc 900 agtaatttcc gattaacgct cgcaccctcc gtattaccgc ggctgct 947

Claims (11)

Pseudoalteromonas sp. JYBCL 1 ) KCCM11139P, which produces agarase (Agarase) directly as a substrate and the nucleotide sequence of 16S rRNA is represented by SEQ ID NO: 1 .
delete The method of claim 1, wherein the agarase is Pseudo-Altermonas J. YB CI, characterized in that the enzyme activity at sodium chloride concentration of 2000 mM is 70% to 80% of the enzyme activity at sodium chloride concentration of 0 mM.
The method of claim 1, wherein the agarase is Pseudo-Altermonas J. YB CI, characterized in that the enzyme activity at sodium chloride concentration of 4000 mM is 50% to 60% of the enzyme activity at sodium chloride concentration of 0 mM.
delete delete delete delete delete delete The method of saccharifying a starfish includes the following steps:
(a) obtaining a starfish; And
(b) treating the obtained wormwood with Pseudo-Altermonas J. YBCL 1 of claim 1.

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