TWI752463B - Oligosaccharide oxidase, preparing method thereof and method of conversion into chito-oligochitosan acid - Google Patents

Abstract

The invention relates to an oligosaccharide oxidase, a preparing method thereof and a method of conversion into chito-oligochitosan acid. The preparing method improves the yield and activity of oligosaccharide oxidase, and the method of conversion into chito-oligochitosan acid can convert chitosan oligosaccharide into chito-oligochitosan acid by the oligosaccharide oxidase under optimal conditions.

Description

Oligosaccharide oxidase and its preparation method and method for converting chitosan oligosaccharide

The present invention relates to an oligosaccharide oxidase, a preparation method thereof, and a method for converting chitosan oligosaccharides, in particular to a novel oligosaccharide oxidase, which can convert chitosan oligosaccharide into chitosan oligosaccharide Sugar acid (chito-oligochitosan acid).

Microorganisms are all over the environment. In order to survive in a competitive environment, microorganisms decompose macromolecular organic matter into small molecular units by secreting digestive enzymes that decompose macromolecular organic matter for absorption. Digestive enzymes can be roughly divided into six categories: oxidoreductases, transferases, hydrolases, isomerases, ligases, and lyases, among which oxidase Reductase is divided into oxidase (oxidase) and dehydrogenase (dehydrogenase), oxidase uses oxygen as electron acceptor; and dehydrogenase uses NAD ⁺ /NADP ⁺ as electron acceptor.

With the understanding of enzymes and the advancement of modern technology, enzymes have been widely used in clinical medicine, industry and biotechnology. For example, using the advantages of high specificity, high sensitivity and rapid response of enzymes, routine tests of blood glucose or cholesterol, or the content of transaminase GPT and GOT in blood during liver function tests; in the food industry, it can be analyzed by analyzing Reductase activity of raw materials, determine whether the raw materials are contaminated by bacteria, or analyze the activity of invertase in milk to confirm whether the low-temperature sterilization process of milk is complete, or to manufacture special formula foods such as lactose-free milk powder.

Oxidative enzymes have their application value in clinical medicine, food industry or cosmetics, so the development of new oxidative enzymes may also be a future trend. For example, U.S. Patent Publication No. US20190127709A1 discloses a " Myrmecridium flexuosum NUK-21 strain and a novel lactose oxidase for its production and a method for converting lactobionic acid", the lactose oxidase is compared to the previous oxidase on the market , has high substrate specificity, catalytic rate and lactose conversion rate, and can effectively oxidize lactose to convert lactobionic acid.

Among the currently published carbohydrate oxidizing enzymes, few literatures discuss the oxidation of chitosan oligosaccharide to chitosan oligosaccharide acid. The enzymes known to act on chitosan oligosaccharide in the published literature only list vanillyl alcohol produced by Fusarium graminearum Vanillyl‐alcohol oxidase (VAO) is a chitosan oligosaccharide oxidase (Heuts, DPHM et al ., 2007), in addition to being reactive with chitobiose, chitotriose and chitotetraose, Also reactive with glucose, cellobiose, lactose and maltose.

Due to the low water solubility of chitosan oligosaccharides, there are still certain limitations in the practical application of chitosan oligosaccharides in medicines, health products, food or cosmetics. Therefore, relevant manufacturers have improved their applicability through different improvement methods. For example, Chinese Patent Publication No. CN100386345C proposes "a preparation method of chitosan oligosaccharide hydrochloride", which is characterized in that: adding chitosan to the reactor, and then according to the amount of amino groups and hydrochloric acid in chitosan. The ratio is 1:0.45-1:0.70, add 0.3-1wt% hydrochloric acid, stir, control the concentration of chitosan to be 2-10wt%, and then add enzyme reagent according to 0.1-5wt% of the chitosan weight to make the reaction The degree of polymerization of the product is 2 to 30, to filter and purify to obtain chitosan oligosaccharide hydrochloride, wherein the enzyme reagent is chitosanase, chitinase, cellulase, hemicellulase, amylase, protease or lysozyme Enzyme, or a mixture of any two of the foregoing enzymes; whereby the above method has the advantages of low cost and the obtained chitooligosaccharide hydrochloride product is baked at 60 ° C for 6 hours without significant color change, and can be used as anti-tumor, Raw materials for medicines and health products for regulating immunity and arthritis, or as additives for cosmetics and food.

Although the above method can improve the lack of low water solubility in the application of chitosan oligosaccharide, the method has the disadvantages of high cost and complicated procedures. Therefore, if the disadvantage of the high cost of the enzyme catalysis method can be improved, and the oligosaccharide oxidizing enzyme can be increased. The production of this enzyme in the production of chitosan oligosaccharide derivatives by enzyme conversion method will enhance the commercial value.

Now, the inventors have created the present invention in view of the fact that the above-mentioned conventionally prepared chitosan oligosaccharide derivatives still have many defects in actual implementation and use.

The main purpose of the present invention is to provide an oligosaccharide oxidase, a preparation method thereof, and a method for converting chitosan oligosaccharide acids, which can not only improve the production of novel oligosaccharide oxidase, but also prove that the novel oligosaccharide oxidase can The chitosan oligosaccharide is converted into chito-oligochitosan acid.

為11.9。對於幾丁寡糖之催化反應速率Km為2.08 mM，Kcat每秒為1.54 S^-1 ，且對於幾丁寡糖之轉化率為52.2%。In order to achieve the above-mentioned purpose of implementation, the present invention provides a method for preparing chitosan oligosaccharide oxidase, comprising the following steps: (a) inoculating an isolated strain of Paraphaeosphaeria sp. NUK-47 strain in a suitable medium, wherein the Paraphaeosphaeria sp. . The NUK-47 strain has an accession number of BCRC 930220; and (b) the NUK-47 strain is cultured at a temperature of 25°C to 35°C for 3 to 6 days to obtain a chitosan oligosaccharide oxidase with a molecular weight of 54 kDa, and the enzyme contains FAD cofactor, calculated as

is 11.9. The catalytic reaction rate Km for chitosan oligosaccharide was 2.08 mM, Kcat was 1.54 S ^-1 per second, and the conversion rate for chitosan oligosaccharide was 52.2%.

The present invention also provides a method for the conversion and production of chitosan oligosaccharide, comprising reacting chitosan oligosaccharide oxidase isolated from Paraphaeosphaeria sp. NUK-47 strain with chitosan oligosaccharide to obtain chitosan oligosaccharide acid; The Paraphaeosphaeria sp. NUK-47 strain was inoculated into a suitable medium, and cultured at 25°C-35°C for 3-6 days to produce chitosan oligosaccharide.

In one embodiment of the present invention, the culture medium is selected from a solid medium consisting of bran and water, or a liquid medium consisting of bran and supplemented with phosphate.

In one embodiment of the present invention, after culturing the solid medium for 3 days, the enzyme activity reaches 0.17 unit/gram of the solid medium, and after culturing the liquid medium for 5 days, the enzyme activity reaches 0.29 unit/ml of the liquid medium.

In one embodiment of the present invention, the chitosan oligosaccharide oxidase is further subjected to a purification process, including ammonium sulfate [(NH ₄ ) ₂ SO ₄ ] precipitation and separation, Toyopearl DEAE-650 anion column, Toyopearl phenyl-650M hydrophobic chromatographic column, HW-50 molecular sieve column, MX-Trp-650M hydrophobic column and Ultrogel-hydroxyapatite column chromatography.

Thereby, the present invention can improve the yield and activity of the novel oligosaccharide oxidase, and can effectively convert chitosan oligosaccharides into chitosan oligosaccharides.

The purpose of the present invention and its structural and functional advantages will be described based on the structure shown in the following drawings, together with specific embodiments, so that the examiners can have a more in-depth and specific understanding of the present invention.

The present invention provides a method for preparing chitosan oligosaccharide oxidase, comprising the following steps: (a) inoculating an isolated strain of Paraphaeosphaeria sp. NUK-47 strain on a suitable medium, wherein the Paraphaeosphaeria sp. NUK-47 strain is registered The number is BCRC 930220; and (b) the Paraphaeosphaeria sp. NUK-47 strain is cultivated at a temperature of 25°C-35°C for 3 to 6 days to obtain chitosan oligosaccharide oxidase; wherein the culture medium is selected from the group consisting of bran and water solid medium, or a liquid medium containing bran and supplemented with phosphate. Preferably, the chitosan oligosaccharide oxidase is further subjected to a purification procedure, including ammonium sulfate [(NH ₄ ) ₂ SO ₄ ] precipitation and partition, Toyopearl DEAE-650 anion column, Toyopearl phenyl-650M hydrophobic column, HW-50 molecular sieve column, MX-Trp-650M hydrophobic column and Ultrogel-hydroxyapatite column chromatography.

為11.9。對於幾丁寡糖之催化反應速率Km為2.08 mM，Kcat每秒為1.54 S^-1 ，且對於幾丁寡糖之轉化率為52.2%。The present invention also provides a chitosan oligosaccharide oxidase, which is prepared by the above-mentioned preparation method, and the molecular weight of the chitosan oligosaccharide oxidase is 54 kDa, and the enzyme contains FAD cofactor.

is 11.9. The catalytic reaction rate Km for chitosan oligosaccharide was 2.08 mM, Kcat was 1.54 S ^-1 per second, and the conversion rate for chitosan oligosaccharide was 52.2%.

The present invention also provides a method for the conversion and production of chitosan oligosaccharide, comprising reacting chitosan oligosaccharide oxidase isolated from Paraphaeosphaeria sp. NUK-47 strain with chitosan oligosaccharide to obtain chitosan oligosaccharide acid; The Paraphaeosphaeria sp. NUK-47 strain is inoculated into a suitable medium, and cultured at 25°C-35°C for 3 to 6 days to transform and produce chitosan oligosaccharide; preferably, the medium is selected from bran-containing A solid medium consisting of water, or a liquid medium consisting of bran supplemented with phosphate.

In addition, the following specific examples can further prove the scope of practical application of the present invention, but are not intended to limit the scope of the present invention in any form.

The inventor screened and isolated a NUK-47 strain capable of transforming chitosan oligosaccharide acid in the soil of Nanzi District, Kaohsiung. The strain identification result showed that the strain was Paraphaeosphaeria , and its species name strain (hereinafter referred to as NUK-47) could not be established. strains).

NUK-47 was cultured in MEA (Malt extract agar) at 30°C for 2 to 5 days. Please refer to the first picture, the appearance of the cells is in the MEA medium, the new hyphae are white, some areas are gray-yellow to olive-brown, and the colony diameter is 40-47 mm; observed under an optical microscope (NIKON ECLIPSE E100), its meristems The spores (conidia) are smooth in color and transparent in the early stage of growth, brown when mature, ellipsoidal in shape and 5.4-7.3 x 2.8-3.8 μm in size.

The NUK-47 strain was subjected to serial analysis of LSU rDNA D1/D2 fragments (such as SEQ ID NO: 1 in the sequence listing) , with a total sequence length of 573 bp, which was aligned with the NCBI GeneBank database, and had 99.27% with Paraphaeosphaeria CBS 978.95 (550/554 ) similarity. The rDNA ITS1-5.8S-ITS2 fragment (such as SEQ ID NO: 2 in the sequence listing) was then analyzed serially. The total length of the sequence was 575 bp, and it was aligned with the NCBI GeneBank database, which was 98.07% with Paraphaeosphaeria CBS 978.95 (560/571) similarity. Although the strain NUK-47 was the closest to CBS 978.95, it was only identified as Paraphaeosphaeria by molecular biological comparison, and its species name could not be established. The strain in this case was named Paraphaeosphaeria sp. NUK-47 strain, and was deposited in the biological material of the Food Industry Research Institute, a consortium. The application was filed on March 19, 2020, and the number is BCRC 930220.

Embodiment 1: bacterial culture and its relationship with enzyme activity

(1) Strain culture: NUK-47 strain was grown in MY solid medium (containing 0.3% malt extract, 0.3% yeast extract, 0.5% peptone, 1% glucose and 1.5% agar), and cultured at 30°C 2~6 days. After that, cut out 4 square centimeters of MY solid medium, inoculate it into a 250-ml conical flask containing 5 g of bran and 5 ml of water, and cultivate at 30°C for 2 to 6 days, until the mycelium is covered with bran. Add 30 ml of 50 mM Tris-HCl (pH 7.8) buffer (referred to as "A buffer") containing 0.1% Triton X-100 for enzyme extraction on the surface of the bran, filter with gauze to remove the residual bran, and heat at 4°C with After centrifugation at 9000 rpm for 30 minutes to remove bacterial cells, the supernatant enzyme solution was obtained and the enzyme activity was measured.

(2) Solid State Fermentation (SSF): After culturing the bacteria in MY solid medium for 2 days, cut the MY solid medium with an area of about 4 square centimeters and inoculate it on the bran medium (bran: water = 1:4). ), stirred evenly and cultured at 30°C for 1, 2, 3, 3.5, 4, 4.5, 5, 5.5 and 6 days, respectively, extracted with 30 ml of A buffer, and then centrifuged at 13,000 rpm for 15 minutes to remove bacteria The supernatant was obtained from the body, and the supernatant was aspirated for retention, and then the enzyme activity was tested by 4-aminoantipyrine (4-AA) coloration method.

Please refer to the second picture, the best days for oligosaccharide oxidase activity are 3 days, and its enzyme activity reaches 0.12 units per ml. If the incubation time is increased, the enzyme activity begins to gradually decrease, and the enzyme activity has declined by the 5th day. to 0.09 units of activity per milliliter.

(3) After culturing the strains in MY solid medium for 2 days, cut the MY solid medium with an area of about 4 square centimeters and inoculate it into the bran medium, where the bran is 5 grams, and the water volume is from 4 to 28. The test was carried out in milliliters. The strain and bran were stirred evenly and then cultured at 30°C. After 3 days of incubation, 30 ml of A buffer was used for extraction. Then, the bacteria were removed by centrifugation at 13,000 rpm for 10 minutes to obtain the supernatant, and the supernatant was aspirated. The solution was retained, and then the enzyme activity was analyzed by a spectrophotometer.

Please refer to the third figure. Under the same incubation time, the best enzyme activity was obtained when the solid medium was supplemented with 20 ml of water, and the enzyme activity reached 0.11 units per ml.

(4) Liquid fermentation (SmF): After culturing the bacteria in MY solid medium for 2 days, cut the MY solid medium with an area of about 4 square centimeters and inoculate it in liquid medium (containing 0.5% NaCl, 1.25% K _{2 )} HPO ₄ , 0.02% MgSO ₄ .7H ₂ O), and then added 4% bran or soybean flour respectively, and cultured at 30°C at 110 rpm for 2, 3, 3.5, 4, 4.5, 5, and 5.5 days, respectively. After centrifugation at 13,000 rpm for 15 minutes, the supernatant was taken, and the enzyme activity was analyzed by a spectrophotometer.

Please refer to Figure 4. In liquid fermentation production, on the 4.5th day, the activity of enzymes produced with bran as nitrogen source can reach a maximum of about 0.10 units per milliliter of activity. On the other hand, the activity of enzymes produced with soybean flour is nearly The activity is maintained at 0 units. Therefore, in terms of nitrogen source selection, bran is a better nitrogen source, which can improve the enzyme activity compared with other nitrogen sources.

(5) After culturing the strains in MY solid medium for 2 days, cut the MY solid medium with an area of about 4 square centimeters and inoculate it in liquid bran medium (containing 0.2% K ₂ HPO ₄ , 0.02% MgSO ₄ . 7H ₂ O, 4% bran and 0-2% NaCl; or containing 0.05% NaCl, 0.02% MgSO _{4 .} 7H ₂ O, 4% bran and 0-2% K ₂ HPO ₄ ) at 30°C or less After culturing at 110 rpm for 4.5 days, 1 ml of the enzyme solution was centrifuged at 13,000 rpm for 15 minutes, and the supernatant was continuously monitored by spectrophotometer for the effect of NaCl concentration and K ₂ HPO ₄ content on enzyme production.

The results are shown in Figure 5. The best enzyme activity was 0.08 units per ml when 0.5% NaCl was added, and the enzyme activity decreased with the increase of NaCl concentration. In addition, the best enzyme activity was achieved when 1.25% K ₂ HPO ₄ was added. The enzymatic activity was 0.31 units per ml, but decreased to 0.29 units per ml _{as the K 2} HPO _{4 concentration increased to 1.5%.}

(6) In order to compare the effect of solid and liquid fermentation on the production of oligosaccharide oxidase, after culturing the strains in MY solid medium for 2 days, cut the MY solid medium with an area of about 4 square centimeters and inoculate it on the bran Bark medium (bran: water = 1:4), stirred evenly and cultured at 30°C for 1 to 6 days, extracted with 30 ml of A buffer, and then centrifuged at 13,000 rpm for 15 minutes to remove bacterial cells to obtain a supernatant. , absorb the supernatant for retention, and then test the enzyme activity by 4-aminoantipyrine (4-AA) coloration method; in addition, after culturing the strains in MY solid medium for 2 days, cut out about 4 square centimeters of MY solid medium, and added to the measured optimum bran liquid medium (0.5% NaCl, 1.25% K 2 HPO 4, 0.02% MgSO 4 .7H 2 O, 4% wheat bran), and at a rotation speed of 110 rpm 30 ℃ The samples were sampled at 1-6 days, centrifuged at 13,000 rpm for 15 minutes, and the supernatant was collected for enzyme activity analysis by spectrophotometer.

The results are shown in Figure 6. The best enzyme activity of solid-state fermentation is only 0.12 units per ml, and the best enzyme activity of liquid fermentation can reach 0.82 units per ml. Therefore, the use of liquid fermentation can significantly improve the activity of enzymes; from the third It is found in the optimal production conditions in Figure 6 and Figure 6 that this strain prefers water, and the enzyme activity produced by liquid culture is higher than that of solid-state culture. If industrialized mass production is required in the future, liquid culture will be more efficient than solid-state culture. Production advantage.

(7) The basic composition of liquid medium is 0.5% NaCl, 0.02% MgSO ₄ . 7H ₂ O and 4% bran, and add 1.25% K ₂ HPO ₄ and KH ₂ PO ₄ respectively, test the pH value of the overall medium _{containing 1.25 % K 2} HPO ₄ and 1.25 % KH ₂ PO _{4, respectively, will contain} The medium with 1.25 % KH ₂ PO ₄ was divided into 2 groups, one was pH adjusted with 1N NaOH to be _{the same as the medium containing K 2} HPO ₄ , and the other was not changed its pH value.

After culturing the strains in MY solid medium for 2 days, cut the MY solid medium with an area of about 4 square centimeters and inoculate it into three kinds of liquid bran. After centrifugation at 13,000 rpm for 15 minutes, the supernatant was continuously monitored with a spectrophotometer, and the enzyme activity test was performed to analyze the effect of pH on the enzyme activity under the condition of phosphoric acid. In addition, the effect of pH value on enzyme activity was investigated in the absence of phosphate. The medium of the control group was composed of 0.5% NaCl, 1.25% K ₂ HPO ₄ and 0.02% MgSO ₄ . 7H ₂ O and 4% bran, and the medium composition of the other two experimental groups was 0.5% NaCl, 0.02% MgSO ₄ . 7H ₂ O and 4% bran, the medium was tested for pH value, and the pH value of one experimental group (without phosphate) was adjusted from the original 7.5 with 1 N sodium hydroxide to about 9.0, the same as the control group , and then the cultured strains were cut, and the MY solid medium with an area of about 4 square centimeters was inoculated into three kinds of liquid bran. After centrifugation at rpm for 15 minutes, the supernatant was continuously monitored with a spectrophotometer, and the enzyme activity test was performed to analyze the effect of pH on the enzyme activity in the absence of phosphate.

Please refer to the seventh figure, in the condition of containing phosphate, K ₂ HPO ₄ and KH ₂ PO ₄ with pH adjusted to 9.1 only differed by 0.15 units per ml of active enzyme activity, compared with KH without pH adjustment. ₂ PO ₄ (0.04 units of activity per ml) has a significant increase. From this result, it is known that the pH value is one of the reasons that affect the activity of the enzyme. The bacteria can be cultured in an alkaline (pH 9.1) medium to obtain higher enzymes. active. In addition, since the _{pH value of KH 2} PO ₄ was adjusted to 9.1 with NaOH, the enzyme activity could not be recovered to the same level as that of _{K 2} HPO _{4 group.} The same trend shows that this strain is not suitable for environments with high sodium content.

Please refer to the eighth figure, if the phosphate is removed, regardless of whether the pH value is changed, the activity of the enzyme produced falls at 0.03 units per ml, which is significantly different from the 0.52 units per ml of the control group containing phosphate , the experimental results prove that this strain has a preference for phosphate and must be maintained in an alkaline (pH 9.1) environment.

Example 2: Enzyme purification and property analysis

Enzyme purification: After culturing the strains in MY solid medium for 2 days, cut the MY solid medium with an area of about 4 square centimeters and inoculate it in liquid bran medium (containing 0.5% NaCl, 1.25% K2HPO4, 0.02% MgSO4.7H2O and 4% bran), a total of 20 bottles, cultured at 30°C at 110 rpm for 5 days, then centrifuged at 4°C at 9000 rpm for 30 minutes, the supernatant was tested for enzyme activity, and the supernatant enzyme solution was obtained. Enzyme activity assays were performed.

Continue, use ammonium sulfate [(NH ₄ ) ₂ SO ₄ ] precipitation and partition, Toyopearl DEAE-650 anion column (purchased from Merck), Toyopearl phenyl-650M hydrophobic column (purchased from TOSOH), HW-50 molecular sieve column (purchased from Merck), MX-Trp-650M hydrophobic column (purchased from TOSOH) and Ultrogel-hydroxyapatite column chromatography (purchased from IBF Biotechnics) and other steps to obtain relatively purified enzymes.

Ammonium sulfate precipitation: measure the volume of the enzyme solution, put the enzyme solution on ice and slowly add ammonium sulfate to make its saturation reach 35%, let it stand for 4-6 hours after it is completely dissolved, and centrifuge at 9000 rpm at 4°C for 30 minutes Remove the precipitate, measure the volume of the supernatant, put it on ice and slowly add ammonium sulfate to make its saturation reach 90%. Centrifuge at 9000 rpm for 30 minutes, and test the enzyme activity of the precipitate and supernatant. It is found that the enzyme is active in the precipitate. The active precipitate is redissolved with 50 mM Tris-HCl; After the volume of enzyme has been redissolved, cut a suitable size dialysis bag, add the enzyme solution to the dialysis bag, put it into 2 liters of buffer A, stir slowly at 4°C, and dialyze until the next day, at least 12 hours;

Toyopearl DEAE-650 Anion Column Chromatography: Fill Toyopearl DEAE-650 anion colloid column (2.5 × 30 cm) with A buffer solution. Elute with buffer A, collect the enzymes that cannot be adsorbed but have activity (0.4 activity units per ml), and then elute the buffer A containing NaCl with a linear gradient of 0-0.25 M, the total volume of the gradient is 1000 ml, and the operation The flow rate is 66 ml per hour, and a tube is collected every 10 minutes;

Toyopearl phenyl-650M hydrophobic column chromatography: Calculate the total volume of the enzyme solution collected above, slowly add ammonium sulfate on ice to a concentration of 3.5 M, stir at low speed until completely dissolved, and fill the column with A buffer solution (2.5×30 centimeter), pass the 3.5 M ammonium sulfate enzyme solution into the column, and then use the buffer to flush out the non-adsorbed impurity protein, and then use the 3.5 M ammonium sulfate buffer solution to elute with a linear gradient from 3.5 M to 0 M. The total gradient is The volume is 1000 ml, the operating flow rate is 72 ml per hour, and one tube is collected every 10 minutes. The enzyme is about 0.65~1.27 M in concentration. Next, calculate the total volume of the enzyme solution after Toyopearl phenyl-650 M and its ammonium sulfate. content, slowly add ammonium sulfate on ice, make up the enzyme concentration to 3.5 M, stir at low speed until it is completely dissolved; fill a concentrated phenyl-650 M column (1×6 cm) with buffer solution, and after the column is equilibrated, add the enzyme Then, the enzyme is directly extracted with A buffer solution, and the operating flow rate is 66 ml per hour, reaching the concentration of the enzyme volume for molecular sieve column use;

Fractogel HW-50 molecular sieve column chromatography: Pass the enzyme solution collected in the previous step into a Fractogel HW-50 molecular sieve column (2.5×115 cm) equilibrated with A buffer, and then use A containing 0.5 N sodium chloride The buffer is flushed, the operating flow rate is 12 ml per hour, and one tube is collected every 10 minutes;

Mx-Trp-650M Hydrophobic column chromatography: Equilibrate the Mx-Trp-650M column (1.5×15 cm) with A buffer containing 3.5 M ammonium sulfate, and add ammonium sulfate to the enzyme solution to make it contain 3.5 M ammonium sulfate, after it was completely dissolved, was passed into the column at a flow rate of 72 ml per hour. After the enzyme completely entered the column, it was washed with A buffer containing 3.5 M ammonium sulfate, and then the enzyme containing 3.5 M ammonium sulfate was used. A buffer solution is eluted with a linear gradient of 3.5 M ~ 0 M, the total gradient volume is 1000 ml, the flow rate is 72 ml per hour, and one tube is collected every 8 minutes;

Ultrogel-hydroxyapatite column chromatography: All enzymes were collected and _{dialyzed against 10 mM KH 2} PO ₄ -Na ₂ HPO ₄ (pH 7.0) buffer solution, and the colloid was filled with 10 mM of the same buffer solution (2.5 × 15 cm), the dialyzed enzyme into the end of the column, and then using the hybrid protein (pH 7.0) buffer no adsorption of _{10 mM K 2 HPO 4 -NaH 2} PO 4 out, followed by _{K 2 HPO 4 -NaH 2 PO 4} (pH 7.0) The buffer is eluted with a linear gradient of 10 mM to 250 mM, and the total volume of the gradient is 1000 ml, and then the active part is collected. The operating flow rate is 70 ml per hour, and one tube is collected every 5 minutes.

The results are shown in Table 1. After each step of purification, the specific activity of the oxidative enzyme increased from 0.40 activity units per mg to 2.8 activity units, the purification fold was 7.0 times, and the recovery rate was yield) was 2.3%.

Table I Total volume (ml) total protein Total activity (U) Specific activity (U/mg) Purification ratio Recovery rate(%) Crude extract 920 955.7 385.6 0.4 1.0 100 Ammonium sulfate precipitation 115 450.6 248.6 0.6 1.4 64.5 Toyopearl DEAE-650 String 350 46.3 141.5 3.1 7.6 36.7 Toyopearl phenyl-650M column 192 27.6 24.8 0.9 2.2 6.4 HW-50 molecular sieve column 30.2 9.2 3.3 0.4 0.9 0.9 Mx-Trp-650M Column 104.5 3.4 6.3 1.9 4.6 1.6 Ultrogel-hydroxyapatite column chromatography 110.5 3.1 8.8 2.9 7.0 2.3

Determination of protein content: Bradford protein assay (Bradford, 1976) method was used. Put 0.8 ml samples containing different concentrations of bovine serum albumin (2-10μg) or enzymes in the test tubes respectively, then add 0.2 ml Bio-Rad Protein Assay reagent, mix well and let stand for 10 minutes, and measure the change of OD 595nm absorbance, and determine the difference. After the concentration of bovine serum albumin standard protein curve, the protein concentration is calculated from the OD 595nm absorbance value of the enzyme sample, and the R value must be greater than 0.99.

Protein electrophoresis analysis: The molecular weight of the enzyme was determined by the SDS-PAGE (polyacrylamide colloid) electrophoresis method of Chrambach and Rodbard. The Amersham casting system was used to make the glue. The stacking gel was 5% polyacrylamide colloid and the running gel was 10% polyacrylamide colloid. The protein sample was first mixed with sample buffer at 6:1 and heated at 100°C for 10 minutes. Electrophoresis was performed at 4°C, and the stacking was run at 80 volts to the running gel interface and then raised to 120 volts; after that, the cells were stained with Coomassie solution for 20 minutes, and then decolorized with 10% aqueous acetic acid.

The protein was identified by SDS-PAGE. As shown in Figure 9, the molecular weight of oligosaccharide oxidase was determined by 10% SDS-PAGE, and the molecular weight of oligosaccharide oxidase was estimated from the molecular weight marker. The molecular weight of oligosaccharide oxidase was estimated to be about 54kDa by interpolation method.

N-terminal sequencing: The purified enzyme was sent to Mingxin Biotechnology Co., Ltd. for N-terminal sequencing, and the sequence shown in Table 2 was obtained. Its amino acid sequence is shown in SEQ ID NO: 3, in which carbohydrate: acceptor oxidoreductase (COX) is another N-terminal sequence that can produce lactose oxidase (Xu, F. et al ., 2001), and its amino acid sequence is shown in SEQ ID NO: 4. Although the obtained sequence similarity is high, it is not completely the same. Another oxidative enzyme PCOX (amino acid sequence is SEQ ID NO: 5) (Kiryu, T. et al ., 2008) also has a similar sequence but is still not completely the same. . At present, there are still few literatures discussing the contribution of enzymes to the oxidation of chitosan oligosaccharides. The known enzymes that can act on chitosan oligosaccharides in the published literature only list Chitooligosaccharide oxidase (ChitO) produced by Fusarium graminearum ( Heuts, DPHM et al ., 2007), but its N-terminal sequence (please refer to SEQ ID NO: 6 in the sequence listing) was compared with the N-terminal sequence of the oligosaccharide oxidase in this case, and the N-terminal sequences of the two were found. Completely different, it can be seen that this enzyme is special.

的吸光值為11.9mM^-1cm^-1，439nm之吸收峰與FAD之吸收峰相似，因此可以推測酵素含有FAD。曲線B的部分為添加了基質後的結果，可以看到在添加了基質後在439nm的吸收峰消失了，這是因為酵素所帶的輔基(prosthetic group)由氧化態型式轉變成還原態。 Enzyme coenzyme (prosthetic group) analysis: take 5.4 mg of protein per ml oligosaccharide oxidase for UV/VIS spectral scanning, the results are shown in Figure 10, there are obvious absorption peaks at 276nm, 372nm and 439nm, the

The absorbance of the enzyme is 11.9mM ^-1 cm ^-1 , and the absorption peak at 439 nm is similar to that of FAD, so it can be speculated that the enzyme contains FAD. The part of curve B is the result after adding the substrate. It can be seen that the absorption peak at 439 nm disappears after adding the substrate. This is because the prosthetic group carried by the enzyme is changed from an oxidized state to a reduced state.

Optimum reaction pH value test: configure 50mM buffer solutions of different types and pH values, including acetate buffer solution (Acetate buffer, CH ₃ COOH-CH ₃ COONa, pH 4.5-5.5), phosphate buffer solution (Phosphate buffer, K ₂ HPO _4- NaH ₂ PO ₄ , pH 5.5-8.0), Tris-HCl buffer solution (Tris-HCl buffer, pH 7.0-9.0), Carbonate buffer solution (Carbonate buffer, Na ₂ CO ₃ -NaHCO ₃ , pH 9.0- 11), and the oligosaccharide oxidase activity was measured using a dissolved oxygen electrode instrument (HQ 430d/HACH Company, USA). First, deionized water was used as a control group, and 2.8 ml of different buffer solutions and 1.5 ml of 3.75mM matrix were added to a 5 ml reaction tank. After the temperature was stabilized at 30 °C, 0.2 ml of deionized water was added. Use a dissolved oxygen electrode detector to test (every 10 seconds, 3 minutes in total), record the data, clean the reaction tank, add 2.8 ml of buffer solution and 1.5 ml of 3.75mM matrix to mix, and finally add 0.2 ml of enzyme solution , under the same conditions, use a dissolved oxygen electrode detector to test (detect once every 10 seconds, a total of 3 minutes) and record the data. When calculating the data, first take the dissolved oxygen change of the control group and the experimental group at 1 minute, and then subtract the value of the control group from the data of the experimental group. relative activity.

Please refer to Figure 11 for the results. There are two optimum pH values in the present invention, namely Tris-HCl 8.5 and phosphate 6.0. The effect on the activity is not much different at pH 7 to 8, but after pH 10 , the relative activity of enzymes began to decline rapidly. Oligosaccharide oxidase is best suited for pH values between approximately 6 and 9. The optimum pH value of the oligosaccharide oxidase of the present invention is similar to that of catalase. In the future, using catalase to remove hydrogen peroxide to produce sugar acid has advantages in commercial production.

Optimum reaction temperature test: use a dissolved oxygen electrode detector for testing, add 4.5 ml of the reaction solution to a 5 ml reaction tank at different temperatures, and the composition of the reaction solution in the control group is 2.8 ml of 50 mM Tris-HCl (pH 8.0) Buffer solution, 1.5 ml of 3.75 mM matrix and 0.2 ml of deionized water, except that the experimental group changed the deionized water to 0.2 ml of enzyme solution, the other operating conditions and composition remained unchanged, and the remaining dissolved oxygen was detected every 10 seconds. , the total length of detection is 3 minutes. When calculating the data, first take the dissolved oxygen change of the control group and the experimental group at 1 minute, and then subtract the value of the control group from the data of the experimental group. relative activity.

The results are shown in Figure 12. The optimum temperature of the oligosaccharide oxidase of the present invention is about 40°C. From the results, the relative activity can be maintained at more than 90% at 30°C to 50°C. At 60 °C, its activity can still be maintained at 80%, but it drops to about 60% at a low temperature of 20 °C.

pH value stability test: configure 50mM buffer solutions of different types and pH values, including acetate buffer solution (Acetate buffer, CH ₃ COOH-CH ₃ COONa, pH 4.5-5.5), phosphate buffer solution (Phosphate buffer, K ₂ HPO _4- NaH ₂ PO ₄ , pH 5.5-8.0), Tris-HCl buffer solution (Tris-HCl buffer, pH 7.0-9.0), Carbonate buffer solution (Carbonate buffer, Na ₂ CO ₃ -NaHCO ₃ , pH 9.0- 11); Mix each buffer solution and enzyme (0.95 units of activity per ml) in a ratio of 9:1, the total volume is 0.1 ml, react at 30 ° C for 1 hour, and then add 0.5 ml of A buffer solution, 0.2 ml of 3.75mM alcohol Butyl oligosaccharide and 0.2 ml of 4-aminoantipyrine (4-AA) color solution were used for OD 500nm kinetic measurement at room temperature using a spectrophotometer (detected once every 20 seconds, a total of 120 seconds), determined by 4-AA The enzyme activity was measured by the method (Allain et al., 1974).

Please refer to Figure 13, oligosaccharide oxidase is relatively stable in Tris-HCl buffer solution and phosphate buffer solution, and the relative stability in phosphate buffer solution can reach 94%. 9.5 are all above 80%, which means that the enzyme is quite stable between pH 4.5-9.5 (the optimum reaction pH is about 8.5), and it begins to decline after pH 10.

Temperature stability test: take 0.02 ml of enzyme (0.95 units of activity per ml) and react at different temperatures for 1 hour, then add 0.58 ml of 50mM Tris-HCl (pH 8.0), 0.2 ml of 3.75mM substrate and 0.2 ml of 4-AA for coloration _{The OD 500} kinetics was measured using a spectrophotometer (detection once every 20 seconds for a total of 120 seconds), and the relative activity of each group was calculated.

Please refer to Figure 14. After the oligosaccharide oxidase acts at the specified temperature for 1 hour, the temperature stability of the enzyme can be as high as 50°C, but when the temperature rises to 60°C, the activity decreases to 1.4%. The temperature is about 40°C-50°C.

Example 3: Detection of enzyme substrate reactivity

35mM of different carbohydrates were prepared for the reaction and diluted to 10 times the Km reaction concentration of 0.7mM and 100 times the Km reaction concentration of 7mM. OD 500nm kinetic measurements were performed at room temperature using a spectrophotometer (detection once every 20 seconds for a total of 120 seconds, and the relative activity of each group was calculated.

Please refer to Table 3. If the relative activity of oligosaccharide oxidase on lactose is taken as 100%, the relative activities of cellobiose, maltose, chitosan oligosaccharide and glucose are 76.2%, 4.8%, 52.2% and 2.9%, respectively. Compared with monosaccharides, this enzyme has higher relative activity to disaccharides, especially to trisaccharides (Chitotriose) chitosan oligosaccharides with a relative activity of up to 52.2%.

Also, please refer to Table 4. K _m represents the affinity of the enzyme to the substrate. The lower the K _m value, the higher the affinity between the enzyme and the substrate. The better the affinity, the better the product conversion rate. The results show that, The best K _m value for the affinity effect of lactose was 0.11 mM, followed by 0.15 mM of cellobiose. The relative activity of the enzyme used in this experiment can reach 52.2%, which is 16 times faster than the 3.17% of the oxidative enzyme in the previously published patent case announcement No. TWI649420B. Butyl oligosaccharides are not reactive.

Table 4: Kinetic analysis of oligosaccharide oxidase on different substrates

Please refer to Table 5 to compare the molecular weight, prosthetic group and substrate specificity of this enzyme with the carbohydrate oxidative enzymes mentioned in the known literature. ), except that Myrmecridium flexuosum NUK-21 is not FAD in the composition of the prosthetic group, there is little difference with each other, but in the reactivity of chitin oligosaccharides, it has not been found in other literatures.

Example 4: Detecting the influence of metal ions on enzyme activity

Mix the enzyme solution and 10mM metal ion solution at a ratio of 9:1 so that the total reaction concentration of each metal ion is 1mM, and the total volume is 0.2ml. 3.75mM lactose and post-reaction enzyme solution were added to the reaction tank, and the relative activity was measured with a dissolved oxygen electrode meter (HQ 430d/HACH Company, USA) (detected once every 10 seconds for a total of 3 minutes); under the same ratio and conditions In the following, deionized water is used to replace the enzyme, the data of water is used as the control group, and the enzyme is used as the experimental group. Take the change of dissolved oxygen in the control group and the experimental group in 1 minute, and then subtract the data of the experimental group from the value of the control group. , the data obtained, and then calculate the relative activity of different metal ions for enzymes in percentage with the results of each group.

Please refer to Table 6, CaCl ₂ and PbCl ₂ slightly increased the enzyme activity by 109% and 101% respectively, while FeCl ₃ and FeSO ₄ inhibited the enzyme activity, making the relative activity drop to 65.2% and 61%, respectively. Metal ions showed no significant change.

Example 5: Influence of test compounds on enzyme activity

Mix the enzyme solution with 10mM chemicals at a ratio of 9:1, the total volume is 0.2ml, and after reacting at 30°C for 1 hour, add 2.8ml A buffer solution, 1.5ml 3.75mM lactose and the post-reaction enzyme solution to the reaction tank In the test, the dissolved oxygen electrode detector was used to test (detected once every 10 seconds for a total of 3 minutes); under the same ratio and conditions, deionized water was used to replace the enzyme, the data of water was used as the control group, and the enzyme was used as the experimental group. Take the change of dissolved oxygen in the control group and the experimental group at 1 minute, and then subtract the data of the experimental group from the value of the control group to obtain the data, and calculate the relative activity of different compounds in percentage with the results of each group.

Please refer to Table 7. Only EDTA increased the enzyme activity by 2.5%. Except for 2-bromo-4-nitroacetophenone, which inhibited the enzyme activity by 29.3%, the inhibitory effects of other chemical reagents were all within about 15%.

Embodiment 6: the production of chitosan oligosaccharide acid

From the structure of chitosan oligosaccharide, it is found that after the first carbon position is converted into acid, the whole chitosan oligosaccharide may become a new type of amino acid. In order to develop the application of chitosan oligosaccharide, try This oligosaccharide oxidase is used for the production of chitosan oligosaccharide.

Take a microcentrifuge tube, add 4mM chitosan oligosaccharide, and then add 0.05 μl of oligosaccharide oxidase (0.96 units of activity per ml), seal the mouth of the centrifuge tube with parafilm, and poke several small holes to ensure that the air is free from air. In and out, placed at 30°C for reaction for 2 hours, after the reaction, about 7.5 microliters of reaction solution was taken for silica gel thin layer chromatography (Silicone Thin Layer Chromatography, TLC); TLC silica 60 was cut into 2 × 8 square centimeters, Take an appropriate amount of chitosan oligosaccharide and spot it at about 0.5 cm below the TLC to prepare 2 ml of developing solution. The ratio is deionized water: ammonia water: methanol: n-butanol = 1:2:4:5, and the total volume is 2.4 ml. When the developing solution is about 0.5 cm away from the top of the TLC, it can be finished. After the TLC is dried, spray it evenly on the TLC with 50% sulfuric acid, bake it at 150 °C to develop color, and observe the color development position.

Please refer to Figure 15, the position of 4mM chitosan oligosaccharide at the beginning of the reaction, two points can be seen on the silica gel thin layer chromatography analysis, from top to bottom are disaccharide and trisaccharide respectively, after 1.5 hours of reaction It can be clearly seen that the polarity becomes larger, which represents the conversion of alcohol group to carboxylic acid, which proves the production of chitosan oligosaccharide. After 0.5 hours, it can be seen that it is completely converted into chitosan oligosaccharide.

As can be seen from the above-mentioned implementation description, compared with the prior art, the present invention is Ming has the following advantages:

1. The oligosaccharide oxidase of the present invention has particularity, not only has the function of producing lactobionic acid, but also can produce chitosan oligosaccharide.

2. The present invention can improve the yield and activity of oligosaccharide oxidase, and convert chitosan oligosaccharides into chitosan oligosaccharides by oligosaccharide oxidase under optimum reaction conditions.

To sum up, the oligosaccharide oxidase of the present invention and its preparation method and the method for converting chitosan oligosaccharide acid can indeed achieve the expected use effect through the above disclosed embodiments, and the present invention has not disclosed Before the application, Cheng has fully complied with the provisions and requirements of the Patent Law. It is indeed a virtue to file an application for an invention patent in accordance with the law.

However, the above-mentioned illustrations and descriptions are only preferred embodiments of the present invention, and are not intended to limit the scope of protection of the present invention; those who are familiar with the art, based on the characteristic scope of the present invention, do other Equivalent changes or modifications should be considered as not departing from the design scope of the present invention.

【Biological Material Deposit】

The Food Industry Development Research Institute, a consortium, applied on March 19, 2020, and the deposit number is BCRC 930220.

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The first figure: the schematic diagram of the Paraphaeosphaeria sp. NUK-47 strain of the present invention.

Figure 2: Analysis of the effect of culture time of solid state fermentation on the activity of oligosaccharide oxidase.

Figure 3: Analysis of the effect of water in solid state fermentation on the activity of oligosaccharide oxidase.

Figure 4: Analysis of the effect of the nitrogen source of the liquid fermentation medium on the yield of oligosaccharide oxidase.

Figure 5: Analysis of the effect of salt on the activity of oligosaccharide oxidase.

Figure 6: Analysis of the effect of oligosaccharide oxidase activity on solid and liquid fermentation at different times.

Figure 7: The effect of phosphates at different pH values on the activity of oligosaccharide oxidase.

Figure 8: Analysis of the effect of different pH values on the activity of oligosaccharide oxidase without phosphate.

Figure 9: SDS-PAGE analysis of the novel oligosaccharide oxidase.

Figure 10: UV/VIS spectral scanning analysis of the novel oligosaccharide oxidase.

Figure 11: Analysis of the optimal pH value of the novel oligosaccharide oxidase.

Figure 12: Analysis of the optimal reaction temperature of the novel oligosaccharide oxidase.

Figure 13: Analysis of pH stability of the novel oligosaccharide oxidase.

Figure 14: Analysis of the temperature stability of the novel oligosaccharide oxidase.

Figure 15: TLC analysis of the conversion of chitosan oligosaccharides into chitosan oligosaccharides by novel oligosaccharide oxidase.

<110> National Kaohsiung University

<120> Oligosaccharide oxidase and its preparation method and method for converting chitosan oligosaccharide

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 573

<212> DNA

<213> Paraphaeosphaeria sp.NUK-47

<223> LSU rDNA D1/D2

<400> 1

<210> 2

<211> 575

<212> DNA

<213> Paraphaeosphaeria sp.NUK-47

<223> rDNA ITS1-5.8S-ITS2

<400> 2

<210> 3

<211> 15

<212> PRT

<213> Paraphaeosphaeria sp.NUK-47

<223> Novel oligosaccharide oxidase N-terminal sequence

<400> 3

<210> 4

<211> 15

<212> PRT

<213> Microdochium nivale

<223> carbohydrate: acceptor oxidoreductase

<400> 4

<210> 5

<211> 15

<212> PRT

<213> Paraconiothyrium sp.

<223> PCOX

<400> 5

<210> 6

<211> 15

<212> PRT

<213> Fusarium graminearum

<223> Chitooligosaccharide oxidase

<400> 6

Claims (5)

A method for preparing chitosan oligosaccharide oxidase, comprising the steps of: (a) inoculating an isolated strain of Paraphaeosphaeria sp.NUK-47 strain in a suitable medium, wherein the Paraphaeosphaeria sp.NUK-47 strain has a deposit number of BCRC 930220, and wherein the culture medium is selected from a solid medium consisting of bran and water, or a _{liquid medium consisting of bran and supplemented with dipotassium hydrogen phosphate (K 2} HPO ₄ ); and (b) the Paraphaeosphaeria sp. NUK-47 strain was cultured at 25℃-35℃ for 3~6 days to obtain chitosan oligosaccharide oxidase. The method as claimed in claim 1, wherein the chitosan oligosaccharide oxidase is further subjected to a purification procedure, including ammonium sulfate [(NH4)2SO4] precipitation and separation, Toyopearl DEAE-650 anion column, Toyopearl phenyl-650M hydrophobicity Column, HW-50 molecular sieve column, MX-Trp-650M hydrophobic column and Ultrogel-hydroxyapatite column chromatography. A chitosan oligosaccharide oxidase, which is prepared by the method described in claim 1, and the molecular weight of the chitosan oligosaccharide oxidase is 54kDa, and the enzyme contains FAD cofactor, calculated to obtain

was 11.9, the catalytic reaction rate Km for chitosan oligosaccharide was 2.08 mM, Kcat was 1.54 S ^-1 per second, and the conversion rate for chitosan oligosaccharide was 52.2%. A method for the transformation and production of chitosan oligosaccharide acid, comprising inoculating a Paraphaeosphaeria sp.NUK-47 strain in a culture medium, and culturing at 25°C-35°C for 3 to 6 days, wherein the culture medium is selected from the group consisting of bran and A solid medium composed of water, or a liquid medium composed of bran and supplemented with dipotassium hydrogen phosphate (K ₂ HPO ₄ ); a chitosan oligosaccharide oxidase produced by the Paraphaeosphaeria sp.NUK-47 strain is derived from the medium isolation; and reacting the isolated chitosan oligosaccharide oxidase with chitosan oligosaccharide to obtain chitosan oligosaccharide acid, wherein the Paraphaeosphaeria sp. NUK-47 strain accession number is BCRC 930220. The method of claim 4, wherein the enzyme activity reaches 0.12 units/g solid medium after culturing the solid medium for 3 days, _{and the liquid medium containing 1.25% dipotassium hydrogen phosphate (K 2} HPO ₄ ) is cultured for 5 days After that, the enzyme activity reached 0.31 units/ml liquid medium.

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