KR20080001334A - A method for preparing carrageenan lyase and oligo galactan from carrageenan using pseudomonas sp. hs5322 - Google Patents

Abstract

A marine microorganism Pseudomonas sp. HS5322 capable of producing a large quantity of carrageenan lyase having high activity in the optimized culture condition is provided to mass produce oligo galactan from carrageenan by using the same enzyme. A carrageenan lyase is produced by culturing a marine microorganism Pseudomonas sp. HS5322(KCTC 18103P) isolated from top shell in a medium containing carrageenan, peptone, yeast extract, seawater and distilled water at 20 deg.C and pH 7.5 for 76 hours. An oligo galactan is produced by reacting carrageenan with the carrageenan lyase produced from Pseudomonas sp. HS5322(KCTC 18103P) at 40 deg.C and pH 8 in the presence of 2.0% NaCl.

Description

Method for preparing carrageenan lyase and oligo galactan from carrageenan using Pseudomonas sp. HS5322}

1 is a graph showing the cell culture time and the activity of carrageenan degrading enzyme.

Figure 2 is a graph showing the results of experiments on enzyme reaction by varying the enzyme reaction temperature of carrageenan degrading enzyme.

3 is a graph showing the results of experiments on enzyme reaction by varying the pH of the carrageenan degrading enzyme.

4 is a graph showing the thermal safety of carrageenan degrading enzyme.

Figure 5 is a graph showing the results of analyzing the residual activity after leaving the pH different in order to determine the pH safety of carrageenan degrading enzyme.

6 is a graph showing the optimum NaCl concentration of the enzyme reaction of carrageenan degrading enzyme.

The present invention relates to a method for producing carrageenan degrading enzyme and oligosaccharide using marine microorganism Pseudomonas sp. More specifically, the present invention is to cultivate a novel marine microorganism Pseudomonas sp . HS5322 KCTC 18103P isolated from the pyramid in an optimized medium to prepare high-efficiency of extracellular carrageenan degrading enzyme and oligo galactan using the degrading enzyme It is about manufacture.

Carrageenan is a seaweed polysaccharide that contains the cell wall of algae manufactured in red algae as a component and contains a large amount of sulfate group, and is used in various fields because of its unique physical properties. Furnaces are widely used in food processing industries such as dairy processing, meat processing and sauces. Carrageenan has a structure in which sulfate groups are bonded to galactose in a large amount, and it is divided into kappa, lambda, and iota carrageenan according to the position of the sulfate group. On the other hand, a series of studies have been conducted to use carrageenan containing a large amount of sulfate as a physiologically active substance, and it has been found to have specific physiological activities such as anticoagulant activity and antibacterial activity.

With the recent westernization of diet, the pharmacological effects of dietary fiber have been demonstrated in the prevention and treatment of various adult diseases such as obesity, arteriosclerosis, angina pectoris and diabetes mellitus. Pharmacological effects such as blood coagulation effect, anticancer effect and nutritional effect were found to be high, and research results showed that the effect of dietary fiber effect and pharmacological effect is increased compared to the original polysaccharide when the degree of polymerization is lowered. It is.

In Korea, research on the production of oligosaccharides using marine resources has been conducted mainly on the acid hydrolysis of chitosan. Especially, studies on the anti-cancer and immuno-activities of oligochitosan (Ryu, 1992) and the function of chitin and chitosan in the digestive tract About in in vitro studies (Chang et al., 1994). As a study on the use of seaweed polysaccharides, a manufacturing method for producing alginic acid oligosaccharides by organic acids has been filed by Yun et al. (Korean Patent Application No. 2001-0018801).

Patents using microbial degrading enzymes as a method for oligosaccharide oligosaccharides of seaweed polysaccharides are not reported, and there is only a method for preparing carrageenan oligosaccharides using organic acids submitted to Korean Patent Application No. 1999-0034910. Despite the progress in the manufacturing process for the production of oligosaccharides using carrageenan, the results of the production of carrageenan degrading enzymes derived from microorganisms are inadequate, and there are few reports of separating microorganisms producing carrageenan degrading enzymes with high efficiency.

Accordingly, an object of the present invention is to isolate and identify marine microorganisms producing carrageenan degrading enzymes.

Another object of the present invention is to provide a method for producing carrageenan lyase using the microorganism with high efficiency.

Still another object of the present invention is to provide a method for preparing carrageenan oligosaccharides using the carrageenan degrading enzyme.

In order to achieve the above object of the present invention, new marine microorganisms having excellent carrageenan degrading enzyme productivity are isolated from the gastrointestinal python's digestive tract, and the optimum conditions for producing carrageenan degrading enzymes from the microorganism with high efficiency are established. The carrageenan degrading enzyme isolated from was achieved by establishing the optimum conditions for producing oligosaccharides from carrageenan.

The present invention is a new marine microorganism isolation step of excellent carrageenan degrading enzyme production ability in the pyramid; Culturing conditions for producing carrageenan degrading enzymes in the microorganism with high efficiency; And establishing optimal conditions for producing oligosaccharides using the carrageenan degrading enzyme.

The present invention provides a method for producing a carrageenan degrading enzyme, characterized by using a novel marine microorganism Pseudomonas sp . HS5322 KCTC 18103P derived from the python.

In order to prepare the carrageenan degrading enzyme of the present invention, the microorganism is added to the carrageenan degrading enzyme production medium consisting of carrageenan 10g / L, peptone 10g / L, yeast extract 5g / L, seawater 750ml / L and distilled water 250ml / L. Inoculate 1 × 10 ³ cells / ml and incubate.

To mass-produce the carrageenan degrading enzyme of the present invention, Pseudomonas sp . HS5322 KCTC 18103P culture medium was carrageenan 10g / L, peptone 10g / L, yeast extract 10g / L, seawater 750ml / L and distilled water 250ml / Carrageenan degrading enzyme production medium consisting of L is characterized in that the fermentation for 76 hours at 20 ℃, pH7.5.

The present invention provides a method for producing carrageenan oligosaccharides by enzymatic reaction of carrageenan degrading enzyme derived from Pseudomonas sp . HS5322 KCTC 18103P at 40 ° C., pH 8, 2.0% NaCl.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are merely illustrative of the present invention and the present invention is not limited thereto.

EXAMPLE

Example  One: Carrageenan  Isolation and Identification of Degrading Enzyme-producing Microorganisms

Liquid medium for separating carrageenan-decomposing microorganisms sterilized at 121 ° C. for 15 minutes to separate the biosynthesis of carrageenan-degrading enzymes from the digestive tract contents of the pythons collected from Chil-Am Village, Busan, Korea (carrageenan 10g / L, yeast extract 2g / L, _{K 2 HPO 4 2g / L,} MgSO 4 · 7H 2 0 1g / L, KCl 1g / L, CaCl 2 · 2H 2 O 0.05g / L) 50㎖ this example, inoculated with gut contents 1g to 250㎖- Erlenmeyer flask Incubated three times each 76 hours in a shake culture (VS-8480SF, Vision Science, Republic of Korea) maintained at 20 ℃, 200rpm to survive only the microorganisms that can decompose carrageenan as a carbon source.

1 × 10 continuously the culture ^{liquid-diluted} from ² to 1 × 10 ^-6 carrageenan degradation microbes solid medium (carrageenan 10g / L, yeast extract 2g / L, NaCl 2g / L , K 2 HPO 4 2g / L, MgSO ₄ · 7H ₂ 0 1g / L, KCl 1g / L, CaCl ₂ · 2H ₂ O 1g / L, agar 15g / L, respectively, inoculated with 100µl well dispersed in a glass rod 20 ℃ incubator (IB-25G, ⁵ hours colonies with carrageenan dissolving ability were isolated from solid medium inoculated with 10 ⁵ dilutions after 48 hours of incubation in JIOTECH. These colonies were inoculated into the carrageenan degrading microbial liquid medium and incubated in a shaker for 24 hours to select the HS5322 strain having the best carrageenan degrading enzyme activity.

For accurate phylogenetic classification of the newly isolated HS5322 strain, 16S rDNA genes were isolated and analyzed for 1,474 nucleotide sequences and compared with nucleotide sequences of various microorganisms in the GenBank database. As a result, more than 97% of Pseudomomas microorganisms were identified. Pseudomonas sp . HS5322, a newly isolated HS5322 strain from the pyramidal gut, showed a similar sequence and was deposited and deposited with the Korea Institute of Bioscience and Biotechnology (KCTC) on June 22, 2004. The number KCTC 18103P was assigned.

Example  2: present invention Carrageenan  High efficiency in lyase-producing microorganisms Carrageenan  Lyase

In Example 1, Pseudomonas sp . (SEO) isolated and identified in order to establish the optimum conditions for the production of carrageenan degrading enzyme of high efficiency.

Experimental Example  One: Carrageenan  On the production of degrading enzymes Carrageenan  Influence of concentration

To determine the effect of carrageenan concentration on carrageenan degrading enzyme production, carrageenan was added 0.6% to carrageenan degrading enzyme production medium (peptone 5g / L, yeast extract 10g, distilled water 250ml / L, seawater 750ml / L). Carrageenan biosynthetic strain Pseudomonas sp., Isolated and identified in 250 ml-triangular flask containing 100 ml of each medium containing carrageenan from 0.1% to 1.3% (concentration of 6-13 g / L) from medium ( Pseudomonas sp .) HS5322 strain was incubated for 72 hours in a shake incubator (Intron Biotechnology, South Korea) maintained at 20 ℃, 200rpm after inoculating 1 × 10 ³ cell / ml, respectively, to measure bacterial counts and carrageenan enzyme activity It was.

Bacterial count was directly counted under a microscope using hematocytometa. Carrageenan degrading enzyme activity was measured by adding 0.1 ml of culture solution to 0.9 ml of 0.5% carrageenan solution as a substrate and reacting at 37 ° C. for 20 minutes, followed by Somogyi-Nelson method. The absorbance (510 nm) was measured to determine the amount of reducing sugar produced. One unit of carrageenan degrading enzyme was defined as the amount of enzyme required to produce 1 mole of reducing sugar during one minute of enzymatic reaction.

Effect of Carrageenan Concentration as a Carbon Source on Cell Growth and Carrageenan Degrading Enzyme Production in Pseudomonas sp . Carrageenan Concentration (g / L) Cell growth (× 10 ⁸ ) Carrageenan Degrading Enzyme Activity (U / 100µl) 6 2.8 16 7 3.2 18 8 3.0 19 9 3.1 22 10 3.8 29 11 3.3 15 12 2.4 11 13 2.3 10

As shown in Table 1, the addition of carrageenan 10g / L at the beginning of the culture showed the highest carrageenan degrading enzyme activity. Therefore, the optimum carrageenan concentration for preparing carrageenan degrading enzyme using Pseudomonas sp . HS5322 was found to be 10 g / L.

Experimental Example  2: Carrageenan  Effect of Nitrogen Sources on the Production of Degrading Enzymes

In the basic medium of Experimental Example 1, the carbon source was replaced with 10 g / L of carrageenan and 10 g / L of various nitrogen sources were added to examine the effect of nitrogen source on carrageenan degrading enzyme production using Pseudomonas sp . HS5322. And cultured under the same conditions as in Experimental Example 1.

As shown in Table 2, when using 10 g / L of peptone as a nitrogen source, 18U of carrageenan degrading enzyme activity was obtained.

Effect of Nitrogen Sources on Cell Growth and Carrageenan Degrading Enzyme Production in Pseudomonas sp . Nitrogen source (10 g / L) Cell growth (× 10 ⁸ ) Carrageenan Degrading Enzyme Activity (U / 100µl) Yeast extract 2.8 6 Beef Extract 2.9 7 peptone 8.1 18 Trypton 2.7 3 Soyton 1.2 One

From the above, in order to examine the effect of the peptone concentration as the optimum nitrogen source, the peptone concentration was incubated by varying from 5 to 20g / L.

As shown in Table 3, the optimum nitrogen source for the production of carrageenan degrading enzyme using Pseudomonas sp . HS5322 was found to be 10 g / L peptone.

Effect of Peptone Concentration as an Optimal Nitrogen Source on Cell Growth and Carrageenan Degrading Enzyme Production in Pseudomonas sp . Peptone concentration (g / L) Cell growth (× 10 ⁸ ) Carrageenan Degrading Enzyme Activity (U / 100µl) 5 4.8 9 10 8.2 17 15 7.0 14 20 3.2 6

Experimental Example  3: Carrageenan  Effect of Yeast Extract on the Production of Degrading Enzymes

In the basic medium for carrageenan degrading enzyme production of Experimental Example 1, 10g / L of carrageenan as a carbon source and 10g / L of peptone as a nitrogen source were used and the effect of yeast extract on carrageenan degrading enzyme production using Pseudomonas sp . In order to examine the yeast extract was incubated by adding 0 ~ 10g / L.

As shown in Table 4, the optimum yeast extract for the production of carrageenan degrading enzyme using Pseudomonas sp . HS5322 was found to be 5g / L.

Effect of Optimal Yeast Extract Concentration on Cell Growth and Carrageenan Degrading Enzyme Production in Pseudomonas sp . Yeast Extract (g / L) Cell growth (× 10 ⁸ ) Carrageenan Degrading Enzyme Activity (U / 100µl) 0 0.1 3 2.5 4.5 12 5 7.0 19 7.5 3.2 17 10 2.7 9

Experimental Example  4: Carrageenan  Effect of Seawater Concentration on the Production of Degrading Enzymes

In order to examine the effect of seawater concentration on carrageenan degrading enzyme production using Pseudomonas sp . HS5322, the seawater concentration was varied from 0 to 90% in the optimum medium obtained from the results of Experiment 3 above. Incubated under the same conditions.

As shown in Table 5, the optimum seawater concentration for the production of carrageenan degrading enzyme using Pseudomonas sp . HS5322 was 75%.

Effect of Seawater Concentration on Cell Growth and Carrageenan Degrading Enzyme Production in Pseudomonas sp . Seawater Concentration (%) Cell growth (× 10 ⁸ ) Carrageenan Degrading Enzyme Activity (U / 100µl) 0 0.1 One 15 2.5 3 30 3.0 8 45 4.2 12 60 6.7 17 75 8.5 22 90 5.5 18

Experimental Example  5: by fermenter culture Carrageenan  Lyase

200 ml of Pseudomonas sp . HS5322 culture broth obtained from the flask culture was optimally prepared for carrageenan degrading enzyme production (10 g / L of peptone, 10 g / L of yeast extract, 10 g / L of carrageenan, 250 ml / L of distilled water and 750 of seawater). ㎖ / L) was inoculated in a 10L fermenter (BG-12, Hanil R & D Co., South Korea) containing 8L was mass culture.

Since it is an aerobic microorganism, the air is supplied at 0.1vvm while stirring at 200rpm, and the pH in the fermenter is automatically controlled to 7.5 by using 2N HCl aqueous solution and 2N NaOH aqueous solution, and Pseudomonas sp is maintained for 120 hours while maintaining the culture temperature at 20 ° C. .) HS5322 was incubated.

As shown in Figure 1, after 76 hours of culture carrageenan degrading enzyme 17U could be prepared.

Example  3: pseudomonas Esp ( Pseudomonas sp .) HS5322  Origin Carrageenan  Biochemical Characterization of Degrading Enzymes

Pseudomonas sp . HS5322 obtained from the fermentor culture of Experimental Example 5 was centrifuged at 12,000 rpm for 30 minutes (SUPRA22K Hanil R & D Co., South Korea) to remove the cells, and a hollow fiber membrane cartridge having an exclusion molecular weight of 100 kDa was removed. The ultrafiltration device (U / F system, Chemicore Incorporated, Republic of Korea) equipped with 5L of supernatant (about 170,000 Units) from which the cells were removed was filtered at a flow rate of 70ml / min to remove the polymer material. A culture solution was obtained. The culture solution was concentrated to 0.5 L (about 150,000 Units) by removing the low molecular weight material from the ultrafiltration device equipped with a hollow fiber membrane cartridge of 10 kDa exclusion molecular weight. 4.5L of 0.2M potassium phosphate buffer (pH 7.0) was added to the concentrate in three portions, and filtered through an ultrafiltration device equipped with a hollow fiber membrane cartridge with an exclusion molecular weight of 10 kDa at a flow rate of 70 ml / min, followed by desalination, followed by 0.5 L. An aqueous solution of desalted carrageenan degrading enzyme (about 110,000 units) was obtained. This was used directly to investigate the biochemical properties of the enzyme.

Experimental Example  One: Carrageenan  Investigation of optimum enzyme reaction temperature

To determine the optimal enzyme reaction temperature of carrageenan degrading enzyme produced by Pseudomonas sp . HS5322, 2 µl of the prepared carrageenan degrading enzyme was diluted in 100 ml of 0.2 M potassium phosphate buffer (pH 7.0) (2U / ml carrageenan). The enzyme was subjected to the enzyme reaction under the same conditions as Experimental Example 1 of Example 2 by varying the enzyme reaction temperature from 5 to 45 ° C.).

As shown in Fig. 2, the optimum enzyme reaction temperature of carrageenan degrading enzyme produced by Pseudomonas sp . HS5322 was 35 ° C.

Experimental Example  2: Carrageenan  Optimal Enzyme Reaction of Degrading Enzyme pH  Research

In order to determine the optimum enzyme reaction pH of carrageenan degrading enzyme produced by Pseudomonas sp . HS5322, the enzyme was changed at 55 ° C. from 4 to 11 at different enzyme reaction pH under the same conditions as in Experiment 1 of Example 2. The reaction was carried out.

As shown in Fig. 3, the optimum pH of the carrageenan degrading enzyme produced by Pseudomonas sp . HS5322 was 7-8.

Experimental Example  3: Carrageenan  Thermal Stability of Degrading Enzymes

In order to determine the thermal stability of the carrageenan degrading enzyme produced by Pseudomonas sp . HS5322, the enzyme dilution was allowed to stand at different temperatures from 0 to 80 ° C. for 30 minutes and then under the same conditions as in Experiment 1 of Example 2 Residual activity was analyzed by enzyme reaction.

As shown in FIG. 4, the carrageenan degrading enzyme produced by Pseudomonas sp . HS5322 had thermal stability in the range of 20-40 ° C., and the optimum thermal stability was 30-40 ° C. FIG.

Experimental Example  4: Carrageenan  Degrading enzyme pH  Stability investigation

To determine the pH stability of Pseudomonas sp . HS5322-produced carrageenan degrading enzyme, the pH was varied from 4 to 11 and left for 60 minutes, followed by enzymatic reaction under the same conditions as in Experiment 1 of Example 2. Activity was analyzed.

As shown in FIG. 5, the carrageenan degrading enzyme produced by Pseudomonas sp . HS5322 had a pH stability within a pH range of 8-9 and an optimum pH stability of pH 8.0.

Experimental Example  5: Carrageenan  Optimal Enzyme Reaction of Degrading Enzyme NaCl Investigation of concentration

Enzymatic Reaction of Carrageenan Degrading Enzyme Produced by Pseudomonas sp . HS5322 NaCl concentration was varied from 0.5 to 3% to determine the residual activity by enzyme reaction under the same conditions as in Experiment 1 of Example 2. Analyzed.

As shown in FIG. 6, carrageenan degrading enzyme produced by Pseudomonas sp . HS5322 showed an optimal activity at 2.0% of NaCl.

From the above results, considering the thermal and pH stability of carrageenan degrading enzyme produced by Pseudomonas sp . HS5322, the optimum temperature of the enzymatic reaction process for the production of carrageenan oligosaccharides by hydrolyzing carrageenan derived from algae was 40 ° C. The optimal pH was 8 and the optimal NaCl concentration was 2.0%.

As described through the above examples, the present invention relates to a method for producing carrageenan degrading enzyme and oligosaccharide using marine microorganism Pseudomonas sp . Etches 5322 strain, and new marine microorganism Pseudomonas sp . By culturing KCTC 18103P in an optimized medium, there is an excellent effect of providing a highly efficient method for producing extracellular carrageenan degrading enzyme. Therefore, the present invention provides a highly efficient carrageenan degrading enzyme that can economically mass-produce carrageenan oligosaccharides by enzymatic engineering of carrageenan derived from algae-derived polysaccharides such as red head of red algae.

Claims (4)

A method for producing a carrageenan degrading enzyme, characterized by using a novel marine microorganism Pseudomonas sp . HS5322 KCTC 18103P derived from the pyramid. According to claim 1, Carrageenan 10g / L, peptone 10g / L, yeast extract 5g / L, seawater 750ml / L and distilled water 250ml / L in the medium for producing a carrageenan degrading enzyme 1 × 10 ³ cell Method for producing a carrageenan degrading enzyme, characterized in that it comprises the step of inoculating each / ml. Pseudomonas sp . HS5322 KCTC 18103P cultured in the culture conditions described in claim 2 was carrageenan 10g / L, peptone 10g / L, yeast extract 10g / L, seawater 750ml / L and distilled water 250ml / L Adding a carrageenan degrading enzyme production medium comprising the step of fermenting at 20 ℃, pH7.5 for 76 hours, characterized in that the mass production method of carrageenan degrading enzyme. In the method for producing carrageenan oligosaccharides, Pseudomonas sp . Pseudomonas sp . HS5322 A method for preparing carrageenan oligosaccharides, characterized in that the carrageenan degrading enzyme derived from KCTC 18103P is obtained by enzymatic reaction at 40 ° C., pH 8 and 2.0% NaCl.

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