TW202138557A - 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 acid

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

Microorganisms are all over the environment. In order to survive in a competitive environment, microorganisms secrete digestive enzymes that decompose macromolecular organic matter and break them down into small molecular units for absorption. Digestive enzymes can be roughly divided into six categories: oxidoreductase, transferases, hydrolases, isomerases, ligases, and lyases. Reductase is divided into oxidase and dehydrogenase. Oxidase uses oxygen as an electron acceptor; while dehydrogenase uses NAD ⁺ /NADP ⁺ as an 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, taking advantage of the high specificity, high sensitivity and rapid response of enzymes, routine tests of blood sugar or cholesterol levels, or the levels of transaminases GPT and GOT in the blood during liver function tests, can be analyzed in the food industry. The reductase activity of the raw material is used to determine whether the raw material is contaminated by bacteria, or analyze the activity of the invertase in the milk, to confirm whether the milk low-temperature sterilization process is complete, or to manufacture special formula foods such as lactose-free milk powder.

Oxidase has its application value no matter in medical clinic, food industry or cosmetics. Therefore, the development of new type of oxidase may also be the future trend. For example, U.S. Patent Publication No. US20190127709A1 discloses a " Myrmecridium flexuosum NUK-21 strain and its novel lactose oxidase and method for inverting lactobionic acid". The lactose oxidase is compared with the previous oxidases on the market. , It has high specificity of substrate, catalytic rate and lactose conversion rate, and can effectively oxidize lactose to convert it into lactobionic acid.

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

Due to the low water solubility of chitosan oligosaccharides, there are still certain limitations when actually applied to medicines, health care products, foods or cosmetics. Therefore, relevant industries have improved their applicability through different improved methods. For example, Chinese Patent Publication No. CN100386345C proposes "a method for preparing chitosan oligosaccharide hydrochloride", which is characterized in that chitosan is added to the reactor, and then the amount of amino acid and hydrochloric acid in the chitosan is The ratio is 1:0.45-1:0.70, add 0.3-1wt% hydrochloric acid, stir to control the concentration of chitosan to 2-10wt%, and then add enzyme reagent at 0.1-5wt% of the weight of chitosan to make the reaction The product has a degree of polymerization of 2 to 30, which can be filtered and purified to obtain chito-oligosaccharide hydrochloride, wherein the enzyme reagent is chitosanase, chitinase, cellulase, hemicellulase, amylase, protease or lysis Enzyme, or a mixture of any two of the aforementioned enzymes; thereby, the above method has the advantages of low cost and the obtained chitooligosaccharide hydrochloride product at 60°C for 6 hours without significant color change, and can be used as an anti-tumor, It is used as a raw material for medicines and health products for regulating immunity and arthritis, or as an additive for cosmetics and food.

Although the above method can improve the lack of low water solubility of chitin oligosaccharides in application, the method has the disadvantages of high cost and complicated procedures. Therefore, if the disadvantages of higher cost of the enzyme catalysis method can be improved, and the oligosaccharide oxidase can be added The production of this enzyme will be applied to the production of chitin-oligosaccharide derivatives by the enzyme conversion method, which will increase the commercial value.

Today, the inventors have developed the present invention based on the fact that the above-mentioned existing prepared chitosan oligosaccharide derivatives still have many defects in actual application.

The main purpose of the present invention is to provide an oligosaccharide oxidase and its preparation method and a method for converting chitosan oligosaccharide acid, which can not only increase the production of novel oligosaccharide oxidase, but also the novel oligosaccharide oxidase is the first time it has been proved to be able to 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 objectives, the present invention provides a method for preparing chitin oligosaccharide oxidase, which comprises the following steps: (a) inoculating an isolate of Paraphaeosphaeria sp. NUK-47 strain in a suitable medium, wherein the Paraphaeosphaeria sp The deposit number of NUK-47 strain is BCRC 930220; and (b) The NUK-47 strain is cultured at a temperature of 25℃-35℃ for 3~6 days to obtain chitin oligosaccharide oxidase, which has a molecular weight of 54 kDa and contains enzymes. FAD cofactor, calculated

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

The present invention also provides a method for conversion and production of chito -oligosaccharides, which comprises reacting chito-oligosaccharide oxidase isolated from Paraphaeosphaeria sp. NUK-47 strain with chito-oligosaccharides to obtain chito-oligosaccharides; Among them, the Paraphaeosphaeria sp. NUK-47 strain was inoculated in a suitable medium and cultured at 25℃-35℃ for 3~6 days to transform and produce chitosan oligosaccharide.

In an embodiment of the present invention, the culture medium is selected from a solid culture medium composed of bran and water, or a liquid medium composed of bran and added with phosphate.

In an embodiment of the present invention, the enzyme activity reached 0.17 units/g of solid medium after 3 days of culture in the solid medium, and 0.29 units/ml of liquid medium after 5 days of culture in the liquid medium.

In an embodiment of the present invention, the chitin oligosaccharide oxidase is further subjected to a purification procedure, including ammonium sulfate [(NH ₄ ) ₂ SO ₄ ] precipitation division, Toyopearl DEAE-650 anion column, Toyopearl phenyl-650M hydrophobic Chromatography with flexible column, HW-50 molecular sieve column, MX-Trp-650M hydrophobic column and Ultrogel-hydroxyapatite column.

Thus, the present invention can increase the yield and activity of the novel oligosaccharide oxidase, and can effectively convert chito-oligosaccharides into chito-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 and specific embodiments, so that the review committee can have a deeper and specific understanding of the present invention.

The present invention provides a method for preparing chitin oligosaccharide oxidase, which comprises the following steps: (a) inoculating an isolate of Paraphaeosphaeria sp. NUK-47 strain into a suitable medium, wherein the Paraphaeosphaeria sp. NUK-47 strain is deposited Numbered as BCRC 930220; and (b) Culture the Paraphaeosphaeria sp. NUK-47 strain at a temperature of 25℃-35℃ for 3~6 days to obtain chito-oligosaccharide oxidase; the medium is selected from the composition containing bran and water The solid medium, or the liquid medium containing bran and added with phosphate. Preferably, the chitin oligosaccharide oxidase is further subjected to a purification procedure, including ammonium sulfate [(NH ₄ ) ₂ SO ₄ ] precipitation division, Toyopearl DEAE-650 anion column, Toyopearl phenyl-650M hydrophobic column, Chromatography with HW-50 molecular sieve column, MX-Trp-650M hydrophobic column and Ultrogel-hydroxyapatite column.

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 chitin oligosaccharide oxidase, which is prepared by the above-mentioned preparation method, and the molecular weight of chitin oligosaccharide oxidase is 54 kDa, and the enzyme contains FAD cofactor, which is calculated

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

The present invention also provides a method for conversion and production of chito -oligosaccharides, which comprises reacting chito-oligosaccharide oxidase isolated from Paraphaeosphaeria sp. NUK-47 strain with chito-oligosaccharides to obtain chito-oligosaccharides; Among them, the Paraphaeosphaeria sp. NUK-47 strain is inoculated in a suitable medium, and cultured at 25°C-35°C for 3~6 days to transform and produce chitosan oligosaccharide; preferably, the medium is selected from the group containing bran A solid medium composed of water, or a liquid medium composed of bran and added with phosphate.

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

The inventors screened and isolated a NUK-47 strain that can transform chitosan oligosaccharides in the soil of Nanzi District, Kaohsiung. The strain identification results showed that it was Paraphaeosphaeria , and its species name strain (hereinafter referred to as NUK-47) could not be established. Strain).

NUK-47 is cultured in MEA (Malt extract agar) based on 30°C culture for 2 to 5 days. Please refer to the first picture. The appearance of the bacteria is white under the MEA medium, and some areas are grayish yellow to olive brown, and the colony diameter is 40-47 mm. Observed under an optical microscope (NIKON ECLIPSE E100), the meristems are The surface of the spore (conidia) is smooth and transparent in the early growth stage, and brown after maturity, with an ellipsoidal shape and a size of 5.4-7.3 x 2.8-3.8 μm.

The NUK-47 strain was subjected to serial analysis of LSU rDNA D1/D2 fragments (such as SEQ ID NO: 1 in the sequence list). The total length of the sequence was 573 bp. It was compared in the NCBI GeneBank database. It was 99.27% (550/554) with Paraphaeosphaeria CBS 978.95. ) Similarity. Then perform serial analysis of rDNA ITS1-5.8S-ITS2 fragments (such as SEQ ID NO: 2 in the sequence listing). The total length of the sequence is 575 bp. It is compared in the NCBI GeneBank database. It is 98.07% (560/571) with Paraphaeosphaeria CBS 978.95.的similarity. Although the NUK-47 strain is closest to the CBS 978.95 strain, it is only identified as Paraphaeosphaeria through molecular biological comparison, and its species name cannot be established. The strain in this case was named Paraphaeosphaeria sp. NUK-47 strain, and it was deposited with the biological materials of the Food Industry Research Institute of the Consortium, and the application was applied on March 19, 2020, with the number BCRC 930220.

Example 1: Strain culture and its relationship with enzyme activity

(1) Strain culture: MY solid medium (containing 0.3% malt extract, 0.3% yeast extract, 0.5% peptone, 1% glucose and 1.5% agar) grown on NUK-47 strain, cultured at 30°C 2~6 days. After that, cut 4 cm² of MY solid medium, inoculate it in a 250 ml Erlenmeyer flask containing 5 g of bran and 5 ml of water, and incubate at 30°C for 2 to 6 days, and wait until the hypha becomes full of bran. Add 30 ml of 50 mM Tris-HCl (pH 7.8) buffer ("A buffer") containing 0.1% Triton X-100 to the surface of the skin for enzyme extraction. Filter with gauze to remove residual bran. After centrifugation at 9000 rpm for 30 minutes to remove the bacteria, the supernatant enzyme solution was obtained and the enzyme activity was measured.

(2) Solid State Fermentation (SSF): After culturing the strain on MY solid medium for 2 days, cut off the MY solid medium with a bacterial cell area of about 4 cm², and inoculate it on the bran medium (bran: water = 1:4 ), stir and incubate at 30°C for 1, 2, 3, 3.5, 4, 4.5, 5, 5.5 and 6 days respectively, extract with 30 ml of buffer A, and then centrifuge at 13000 rpm for 15 minutes to remove bacteria Obtain the supernatant, absorb the supernatant for retention, and then use the 4-aminoantipyrine (4-AA) color method to test the enzyme activity.

Please refer to the second figure. The optimal number of days for oligosaccharide oxidase activity is 3 days, and its enzyme activity reaches 0.12 unit activity per milliliter. If the incubation time is increased, the enzyme activity begins to decrease gradually, and the enzyme activity has already decreased by the 5th day. To 0.09 unit activity per milliliter.

(3) After culturing the strains on MY solid medium for 2 days, cut out the MY solid medium with a bacterial cell area of about 4 cm² and inoculate it on the bran medium. The bran is 5 grams and the water volume is from 4 to 28. Test in milliliters. Stir the bacteria and bran evenly and culture at 30°C. After culturing for 3 days, extract with 30 mL of buffer A, then centrifuge at 13000 rpm for 10 minutes to remove the bacteria to obtain the supernatant, and then absorb the supernatant. Keep the solution, and analyze the enzyme activity with a spectrophotometer.

Please refer to the third figure. Under the same cultivation time, the best enzyme activity is obtained when 20 ml of solid medium is added with water, and the enzyme activity reaches 0.11 unit activity per ml.

(4) Liquid fermentation (SmF): After culturing the bacteria in MY solid medium for 2 days, cut out the MY solid medium with a bacterial cell area of about 4 cm², and inoculate it in a liquid medium (containing 0.5% NaCl, 1.25% K ₂ HPO ₄ , 0.02% MgSO 4.7H ₂ O), and then add 4% bran or soybean flour respectively, and incubate at 30°C at 110 rpm for 2, 3, 3.5, _{4, 4.5, 5, and 5.5 days.} After centrifugation at 13000 rpm for 15 minutes, the supernatant was taken, and the enzyme activity was analyzed by a spectrophotometer.

Please refer to the fourth figure. In the liquid fermentation production, on the 4.5th day, the enzyme activity produced with bran as the nitrogen source can reach about 0.10 unit activity per milliliter. On the contrary, the enzyme activity produced with soy flour is almost Maintained at 0 unit activity, therefore, in terms of nitrogen source selection, bran is a better source of nitrogen source, which can improve enzyme activity compared with other different kinds of nitrogen sources.

(5) After culturing the strains in MY solid medium for 2 days, cut out the MY solid medium with a bacterial cell area of about 4 cm², and inoculate it in a 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 ℃ After culturing at 110 rpm for 4.5 days, take 1 ml of enzyme solution and centrifuge at 13000 rpm for 15 minutes. The supernatant was used to continuously monitor _{the influence of NaCl concentration and K 2} HPO ₄ content on enzyme production by spectrophotometer.

For the results, please refer to the fifth figure. When 0.5% NaCl is added, the best enzyme activity is 0.08 unit activity per milliliter. As the concentration of NaCl increases, the enzyme activity decreases; on the other hand, when 1.25% K ₂ HPO ₄ is added, the best enzyme activity is reached. The enzyme activity is 0.31 unit activity per ml, but as the K ₂ HPO ₄ concentration increases to 1.5%, it drops to 0.29 unit activity per ml.

(6) In order to compare the effects of solid and liquid fermentation on the activity of oligosaccharide oxidase production, after culturing the bacteria in MY solid medium for 2 days, cut off the MY solid medium with a bacterial cell area of about 4 cm², and inoculate it in bran. Husk culture medium (bran: water = 1:4), stir well and incubate at 30℃ for 1~6 days, extract with 30ml buffer A, then centrifuge at 13000 rpm for 15 minutes to remove the bacteria to obtain the supernatant , Absorb the supernatant for retention, and then use the 4-aminoantipyrine (4-AA) coloring method to test the enzyme activity; in addition, after culturing the bacteria in the MY solid medium for 2 days, cut off the bacteria area of about 4 cm² 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 ℃ After incubating, samples were taken at 1 to 6 days, and centrifuged at 13000 rpm for 15 minutes, and the supernatant was taken for analysis of enzyme activity with a spectrophotometer.

Please refer to Figure 6 for the results. The best enzyme activity for solid-state fermentation is only 0.12 unit activity per milliliter, and the best enzyme activity for liquid fermentation can reach 0.82 unit activity per milliliter. Therefore, the use of liquid fermentation can significantly increase the activity of enzymes; It is found in the optimal production conditions in the figure and the sixth figure that this strain prefers water, and the enzyme activity produced by liquid culture is higher than that of solid culture. If industrial mass production is required in the future, liquid culture will be better than solid culture. Production advantage.

(7) The basic composition of liquid medium is 0.5% NaCl and 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 whole medium _{containing 1.25% K 2} HPO ₄ and 1.25% KH ₂ PO _{4, which will contain} The 1.25% KH ₂ PO ₄ medium is divided into 2 groups, one group is adjusted with 1N NaOH to the same pH value as _{the medium containing K 2} HPO ₄ , and the other group is not changed its pH value.

After cultivating the strains in MY solid medium for 2 days, cut out the MY solid medium with an area of about 4 cm² and inoculate it into three kinds of liquid bran. After culturing at 30°C at 110 rpm for 5 days, take 1 ml of each Centrifuge at 13000 rpm for 15 minutes, and monitor the supernatant continuously with a spectrophotometer to test the enzyme activity to analyze the effect of pH on the enzyme activity under the condition of phosphoric acid. In addition, to explore the effect of pH on enzyme activity in the absence of phosphate, the control medium consisted of 0.5% NaCl, 1.25% K ₂ HPO ₄ , and 0.02% MgSO ₄ . 7H ₂ O and 4% bran, the other two experimental groups consist of 0.5% NaCl and 0.02% MgSO ₄ . The _{pH value of 7H 2} O and 4% bran and the culture medium were tested, and the pH value of one of the experimental groups (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 bacteria, cut out the MY solid medium with an area of about 4 cm², and inoculate it in three kinds of liquid bran. After incubating at 30℃ at 110 rpm for 5 days, take 1 ml of each at 13000 Centrifuge at rpm for 15 minutes, and the supernatant was continuously monitored with a spectrophotometer to test the enzyme activity to analyze the effect of pH on the enzyme activity without phosphate.

Please refer to the seventh figure. Under the condition of containing phosphate, the _{difference between K 2} HPO ₄ and KH ₂ PO ₄ adjusted to pH 9.1 is only 0.15 units of active enzyme activity per milliliter, which is compared with KH without pH adjustment. ₂ PO ₄ (0.04 unit activity per milliliter) has a significant increase. From this result, it is known that the pH value is one of the reasons that affect the enzyme activity. The bacteria can be cultured in alkaline (pH 9.1) medium to obtain higher enzymes. active. In addition, because KH ₂ PO ₄ is adjusted to pH 9.1 with NaOH, the enzyme activity cannot be restored to the _{same level as the K 2} HPO ₄ group. This result is also similar to the result of the decrease in the enzyme activity as the NaCl concentration in the fifth graph increases. 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 will fall at 0.03 unit activity per milliliter, which is a significant difference compared with the 0.52 unit activity per milliliter of the control group containing phosphate. This experimental result proves 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 bacteria in MY solid medium for 2 days, cut out the MY solid medium with a bacterial cell area of about 4 cm², and inoculate it in a 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, centrifuged at 4°C at 9000 rpm for 30 minutes, the supernatant was tested for enzyme activity, the supernatant enzyme solution was obtained and Perform enzyme activity determination.

Next, use ammonium sulfate [(NH ₄ ) ₂ SO ₄ ] precipitation division, 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, place the enzyme solution on ice and slowly add ammonium sulfate to make the saturation reach 35%. After it is completely dissolved, let it stand for 4-6 hours, and centrifuge at 9000 rpm at 4°C for 30 minutes Remove the precipitate, measure the volume of the supernatant, place it on ice and slowly add ammonium sulfate to make the saturation reach 90%. After it is completely dissolved, let it stand overnight (12 hours) at 4°C, and at 4°C the next day Centrifuge at 9000 rpm for 30 minutes. Test the enzyme activity of the precipitate and supernatant. It is found that the enzyme activity is in the precipitate. The active precipitate is re-dissolved with 50 mM Tris-HCl; continue, calculate as A buffer After re-dissolving the volume of the enzyme, cut a dialysis bag of appropriate size, add the enzyme solution to the dialysis bag, put the enzyme solution into 2 liters of buffer A, stir slowly at 4°C, and dialyze until the next day for 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. After equilibration, pass the dialysis enzyme solution into the column. The A buffer solution is used for extraction, and the enzymes that cannot be adsorbed but are active (0.4 activity unit per ml) are collected, and then the A buffer solution containing NaCl is extracted with a linear gradient of 0~0.25 M. The total volume of the gradient is 1000 ml. The flow rate is 66 milliliters per hour, and one tube is collected at 10 minute intervals;

Toyopearl phenyl-650M hydrophobic column chromatography: Calculate the total volume of the enzyme solution collected above, slowly add ammonium sulfate to 3.5 M on ice, stir at low speed until it is completely dissolved, and fill the column with buffer A (2.5×30 Cm), pass the 3.5 M ammonium sulfate enzyme solution into the column, then use the buffer to flush out the non-adsorbed impurities, and then use the 3.5 M ammonium sulfate buffer solution to extract with a 3.5 M~0 M linear gradient. The gradient is total The volume is 1000 ml, the operating flow rate is 72 ml per hour, and one tube is collected at 10 minutes intervals. The enzyme is about to fall in the part with a concentration of 0.65 to 1.27 M; continue, 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 the concentrated phenyl-650 M column (1×6 cm) with buffer solution. After the column is equilibrated, remove the enzyme The solution is introduced, and then the A buffer solution is used to directly extract the enzyme. The operating flow rate is 66 ml per hour, reaching the volume of the concentrated enzyme for use in the molecular sieve column;

Fractogel HW-50 molecular sieve column chromatography: Pass the enzyme solution collected in the previous step into Fractogel HW-50 molecular sieve column (2.5×115 cm) equilibrated with buffer A, and then use A containing 0.5 N sodium chloride The buffer solution is used to flush the embankment, 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 buffer A containing 3.5 M ammonium sulfate, and add ammonium sulfate to the enzyme solution to make it contain 3.5 After the ammonium sulfate of M is completely dissolved, it is passed into the column at a flow rate of 72 ml per hour. After the enzyme has completely entered the column, it is extracted with buffer A containing 3.5 M ammonium sulfate, and then the ammonium sulfate containing 3.5 M A buffer solution is extracted with a linear gradient of 3.5 M ~ 0 M, the total volume of the gradient is 1000 ml, the flow rate is 72 ml per hour, and a tube is collected every 8 minutes;

Ultrogel-hydroxyapatite column chromatography: After all the enzymes are collected, they _{are dialyzed with 10 mM KH 2} PO ₄ -Na ₂ HPO ₄ (pH 7.0) buffer solution, and the colloid is filled with the same 10 mM buffer solution (2.5 × 15 cm). Pass the enzyme after the dialysis into the column, then use 10 mM K ₂ HPO ₄ -NaH ₂ PO ₄ (pH 7.0) buffer to flush out the non-adsorbed protein, and then flush the K ₂ HPO ₄ -NaH ₂ PO ₄ (pH 7.0) The buffer is extracted with a linear gradient of 10 mM ~ 250 mM, 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 a tube is collected every 5 minutes.

Please refer to Table 1 for the results. After purification of each step, the specific activity of oxidase increased from 0.40 activity unit per mg to 2.8 activity unit, the purification fold was 7.0 times, and the recovery rate (Recovery) yield) is 2.3%.

Table I Total volume (ml) Total protein Total activity (U) Specific activity (U/mg) Purification rate 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 column 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

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

Protein electrophoresis analysis: Use Chrambach and Rodbard's SDS-PAGE (polypropylene amide colloid) electrophoresis method to determine the molecular weight of the enzyme. The glue is made by Amersham casting system. The stacking gel is 5% polypropylene amide colloid and the running gel is 10% polypropylene amide colloid. The protein sample is first mixed with sample buffer at a ratio of 6:1 and heated at 100°C for 10 minutes. The electrophoresis was carried out at 4°C, the stacking was moved to the running gel interface at 80 volts and then raised to 120 volts; after that, staining was performed with Coomassie solution for 20 minutes, and then decolorized with 10% acetic acid aqueous solution.

Use SDS-PAGE to identify the protein. As shown in Figure 9, 10% SDS-PAGE is used to determine the molecular weight of oligosaccharide oxidase, and it is estimated from the molecular weight marker. The interpolation method can infer that the molecular weight of oligosaccharide oxidase is about 54 kDa. .

N-terminal sequencing: send the purified enzyme to Mingxin Biotechnology Co., Ltd. for N-terminal sequencing to obtain the sequence shown in Table 2, where carbohydrate: acceptor oxidoreductase (COX) is another N-terminal that can produce lactose oxidase Sequence (Xu, F. et al ., 2001), after comparison, it can be found that the similarity with the sequence obtained in this study is high but not completely identical, another oxidase PCOX (Kiryu, T. et al ., 2008) Although there are similar sequences, they are still not exactly the same. At present, there are still few literatures discussing the contribution of enzymes to the oxidation of chito-oligosaccharides. The only known enzymes in the published literature on chito-oligosaccharides are listed as Chitooligosaccharide oxidase (Chitooligosaccharide oxidase, ChitO) produced by Fusarium graminearum ( Heuts, DPHM et al ., 2007), but comparing its N-terminal sequence with the N-terminal sequence of oligosaccharide oxidase in this case, it is found that the N-terminal sequence of the two is completely different, which shows that the enzyme is unique.

Table 2: N-terminal amino acid sequence of oligosaccharide oxidase Novel oligosaccharide oxidase AAIDKXLTDAGVPID COX ( Microdochium nivale ) G A I EAC L SA A G V P I D PCOX ( Paraconiothyrium sp.) A V I D K X L T D D G V P V D Fusarium graminearum VPTKREAVNSCLTQA

的吸光值為11.9 mM^-1 cm^-1 ，439 nm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439 nm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 protein per milliliter of oligosaccharide oxidase and perform UV/VIS spectral scanning. The result is shown in Figure 10. There are obvious absorption peaks at 276 nm, 372 nm and 439 nm.

The absorbance value is 11.9 mM ^-1 cm ^-1 , and the absorption peak at 439 nm is similar to that of FAD, so it can be inferred 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 the addition of the substrate. This is because the prosthetic group of the enzyme changes from an oxidized form to a reduced form. .

Optimal reaction pH test: configure 50 mM buffer solutions of different types and pH values, including acetate buffer (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 (Tris-HCl buffer, pH 7.0-9.0), Carbonate buffer (Na ₂ CO ₃ -NaHCO ₃ , pH 9.0) -11), and use the dissolved oxygen electrode instrument (HQ 430d/HACH company, USA) to measure the oligosaccharide oxidase activity. First, with deionized water as the control group, add 2.8 ml of different buffer solutions and 1.5 ml of 3.75 mM substrate to a 5 ml reaction tank. After the temperature stabilizes at 30°C, add 0.2 ml of deionized water at 30°C. Use the dissolved oxygen electrode detector to test (detect every 10 seconds, 3 minutes in total), record the data and clean the reaction tank, then add 2.8 ml of buffer solution and 1.5 ml of 3.75 mM substrate to mix, and finally add 0.2 ml of enzyme solution , Use the dissolved oxygen electrode detector to test under the same conditions (detect every 10 seconds, 3 minutes in total) 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. The relative activity.

Please refer to Figure 11 for the results. There are two optimal pH values for the present invention, namely Tris-HCl 8.5 and phosphate 6.0. There is no significant difference in the effect on activity at pH 7 to 8, but after pH 10 , The relative activity of enzymes began to decline rapidly. Oligosaccharide oxidase is most suitable for pH between 6-9. The optimal reaction pH value of the oligosaccharide oxidase of the present invention is similar to the optimal pH value of catalase. In the future, catalase can be used with catalase to remove hydrogen peroxide to produce sugar acid, which has advantages in commercial production.

Optimal reaction temperature test: Use a dissolved oxygen electrode detector to test, add 4.5 ml of the reaction solution to a 5 ml reaction tank at different temperatures. 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 3.75 mM substrate and 0.2 ml deionized water. Except for changing the deionized water to 0.2 ml of enzyme solution, the other operating conditions and composition of the experimental group are not changed. The remaining dissolved oxygen is detected every 10 seconds. The total length is detected for 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. The relative activity.

Please refer to Figure 12 for the results. The optimal temperature of the oligosaccharide oxidase of the present invention is about 40°C. From the results, the relative activity can be maintained above 90% at 30 to 50°C, and its activity can still be maintained at 60°C. Maintained at 80%, but dropped to about 60% at a low temperature of 20°C.

pH stability test: configure 50 mM buffer solutions of different types and pH values, including acetate buffer (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 (Tris-HCl buffer, pH 7.0-9.0), Carbonate buffer (Na ₂ CO ₃ -NaHCO ₃ , pH 9.0) -11); Mix each buffer solution and enzyme (0.95 unit activity per ml) in a ratio of 9:1, with a total volume of 0.1 ml, react at 30°C for 1 hour, then add 0.5 ml buffer A, 0.2 ml 3.75 mM Chitosan oligosaccharides and 0.2ml 4-aminoantipyrine (4-AA) coloring solution were used to measure OD 500nm kinetics using a spectrophotometer at room temperature (detection once in 20 seconds, 120 seconds in total), by 4-AA The assay method (Allain et al., 1974) was used to measure enzyme activity.

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%. Overall, the pH value is stable at 4.5 to It is above 80% at 9.5, which means that the enzyme is quite stable between pH 4.5-9.5 (the optimal reaction pH is about 8.5), and it begins to decrease after pH 10.

Temperature stability test: Take 0.02 ml of enzyme (0.95 unit activity per ml) and react for 1 hour at different temperatures, then add 0.58 ml of 50 mM Tris-HCl (pH 8.0), 0.2 ml of 3.75 mM substrate and 0.2 ml of 4-AA. For the color solution, use a spectrophotometer to _{measure OD 500} kinetics (detect once every 20 seconds, 120 seconds in total), and calculate the relative activity of each group.

Please refer to Figure 14. After the oligosaccharide oxidizing enzyme 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 is increased to 60°C, the activity drops to 1.4%. This enzyme is the most suitable The temperature is about 40℃-50℃.

Example 3: Detection of enzyme substrate reactivity

Configure 35 mM different carbohydrates for the reaction, and dilute to 10 times the Km reaction concentration of 0.7 mM, and 100 times the Km reaction concentration of 7 mM. At room temperature, use a spectrophotometer to perform OD 500nm kinetic measurement (detect once every 20 seconds for a total of 120 seconds, and calculate the relative activity of each group.

Please refer to Table 3. If the relative activity of oligosaccharide oxidase to 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 a higher relative activity for disaccharides, especially for chitotriose (Chitotriose), which has a relative activity of up to 52.2%.

Table 3: Relative activity of oligosaccharide oxidase to substrate Matrix Bonding type Relative activity (%) lactose Gal-Glc, β-1,4 100.0 Cellobiose Glc- Glc, β-1,4 76.2 maltose Glc- Glc, α-1,4 4.8 Chitosan Gs-Gs-Gs, β-1,4 52.2 glucose - 2.9 sucrose Glc- Frc, α-1,2 0

In addition, 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 the enzyme has with the substrate. The better the affinity, the better the product conversion rate. The results show that, The best affinity effect of lactose has a K _m value of 0.11 mM, followed by cellobiose of 0.15 mM. 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 oxidase of the previously published patent notice number TWI649420B. The test of the oxidase of the previous case shows that it is more Butyl oligosaccharides are not reactive.

Table 4: Kinetic analysis of oligosaccharide oxidase on different substrates Matrix K _m (mM) K _cat (S ^-1 ) K _cat / K _m (S ^-1 mM ^-1 ) lactose 0.11 0.59 5.23 Cellobiose 0.15 1.02 6.76 Chitosan 2.08 1.54 0.74 maltose 33.52 0.45 0.01 glucose 103.32 1.00 0.01

Please refer to Table 5 to compare the molecular weight, prosthetic group and substrate specificity of this enzyme with the carbohydrate oxidase mentioned in the known literature. The molecular weight, prosthetic group and substrate specificity ), except for Myrmecridium flexuosum NUK-21 in the prosthetic group composition non-FAD, there is little difference from each other, but the reactivity of chitin oligosaccharides is not seen in other literature.

Table 5: Comparison of the properties of various oxidases Enzyme Strains Molecular weight (kDa) Auxiliary Matrix specificity (%) lactose Cellobiose maltose Chitosan Oligosaccharide Oxidase NUK-47 54 FAD 100 76.2 4.8 52.2 LOD Myrmecridium flexuosum 47 Non-FAD 100 83 4 3.2 COX Microdochium nivale 55 FAD 52 100 ND - PCOX Paraconniothyrium sp 54 FAD - - - - COOX Sarocladium oryzae 51 FAD 83 100 13 - GOOX Acremonium strictum 61 FAD 64 47 100 0 CBG Phanerochaete chrysosporium 58 FAD - - - -

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

Mix the enzyme solution with 10 mM metal ion solution in a ratio of 9:1 so that the total reaction concentration of each metal ion is 1 mM, and the total volume is 0.2 ml. After reacting at 30°C for 1 hour, add 2.8 ml of buffer A , 1.5 ml 3.75 mM lactose and the enzyme solution after the reaction were added to the reaction tank, and the relative activity was measured with a dissolved oxygen electrode instrument (HQ 430d/HACH Company, USA) (detected every 10 seconds for a total of 3 minutes); in the same ratio Under the conditions, deionized water is used instead of enzymes, water data is used as the control group, and the enzymes are used as the experimental group. Take the dissolved oxygen changes of the control group and the experimental group at 1 minute respectively, and then subtract the data of the experimental group from the control group Calculate the relative activity of different metal ions to enzymes by percentage with the results of each group.

Please refer to Table 6. CaCl ₂ and PbCl ₂ increased the enzyme activity by 109% and 101% respectively, while FeCl ₃ and FeSO ₄ inhibited the enzyme activity, reducing the relative activity to 65.2% and 61%, respectively. There is no significant change in metal ions.

Table 6: The effect of metal ions on the activity of oligosaccharide oxidase Metal ion (1 mM) Relative activity (%) Control group 100.0 AgNO ₃ 87.2 CaCl ₂ 109.4 CdSO ₄ 91.4 CoCl ₂ 95.4 CuSO ₄ 91.2 PbCl ₂ 101.1 SnCl ₂ 96.4 FeSO ₄ 61.0 FeCl ₃ 65.2 HgCl ₂ 82.2 AlSO ₄ 93.7 MnSO ₄ 87.0 MgSO ₄ 71.5 NiCl ₂ 82.8

Example 5: Testing the influence of compounds on enzyme activity

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

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

Table 7: Effects of compounds on the activity of oligosaccharide oxidase Compound Concentration (mM) Relative activity (%) Control group 1 100.0 2,4'-dibromoacetophenone 1 96.9 2-bromo-4'-nitroacetophenone 1 70.7 NaN ₃ 1 92.2 EDTA 1 102.5 N-ethylmaleimide 1 82.4 1,10-phenanthroline 1 85.3 H ₂ O ₂ 1 85.9 PMSF 1 96.6 2-bromo-4'-nitroacetophenone 4 72.7

Example 6: Production of Chitosan Oligosaccharide

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

Take a microcentrifuge tube and add 4 mM chitin oligosaccharides, then add 0.05 μl oligosaccharide oxidase (0.96 unit activity per milliliter), seal the tube of the centrifuge tube with a parafilm, and poke a few small holes to ensure air In and out, place the reaction at 30℃ for 2 hours. After the reaction, take about 7.5 microliters of the reaction solution for Silicon Thin Layer Chromatography (TLC); cut the TLC silica 60 into 2×8 cm² , Take an appropriate amount of chitosan oligosaccharide and place it at a position about 0.5 cm below the TLC, and prepare 2 ml of developing solution. The ratio is deionized water: ammonia: 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 will be finished. After the TLC is dried, spray it with 50% sulfuric acid evenly on the TLC, bake it at 150℃ to make it develop color, and observe its colored position.

Please refer to the fifteenth figure, the position of 4 mM chitosan oligosaccharides at the beginning of the reaction. Two spots can be seen on the silica gel thin layer chromatography analysis. From top to bottom, they are disaccharides and trisaccharides. After 1.5 hours of reaction Later, the points of increased polarity can be clearly seen, which represents the conversion from alcohol groups to carboxylic acids, which proves the production of chitosan oligosaccharides. After another 0.5 hours, the complete conversion into chitosan oligosaccharides can be seen.

As can be seen from the above implementation description, compared with the prior art, the present invention 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 acid.

2. The present invention can increase the yield and activity of oligosaccharide oxidase, and convert chito-oligosaccharides into chito-oligosaccharides by oligosaccharide oxidase under optimal reaction conditions.

In summary, the oligosaccharide oxidase and the preparation method thereof and the method for converting chitosan oligosaccharide acid of the present invention can indeed achieve the expected use effect through the embodiments disclosed above, and the present invention has not been disclosed. Before the application, Cheng has fully complied with the provisions and requirements of the Patent Law. If you file an application for a patent for invention in accordance with the law, you are kindly requested to review and grant a quasi-patent.

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. Anyone familiar with the art will do other things based on the characteristic scope of the present invention. Equivalent changes or modifications should be regarded as not departing from the design scope of the present invention.

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Figure 1: Schematic diagram of the type of Paraphaeosphaeria sp. NUK-47 strain of the present invention.

Figure 2: Analysis of the influence of the incubation time of solid-state fermentation on the activity of oligosaccharide oxidase.

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

Figure 4: Analysis of the influence of the nitrogen source of the liquid fermentation medium on the production of oligosaccharide oxidase.

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

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

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

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

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

Figure 10: UV/VIS spectrum scanning analysis image of novel oligosaccharide oxidase.

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

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

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

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

Figure 15: Thin-layer chromatographic analysis of the conversion of chito-oligosaccharides into chito-oligosaccharides by novel oligosaccharide oxidase.

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

Claims (8)

A method for preparing chitin oligosaccharide oxidase, which comprises the following steps: (a) Inoculating an isolate 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 (b) culture the Paraphaeosphaeria sp. NUK-47 strain at a temperature of 25° C.-35° C. for 3 to 6 days to obtain chitin oligosaccharide oxidase. The method according to claim 1, wherein the medium is selected from a solid medium containing bran and water, or a liquid medium containing bran and added with phosphate. The method according to claim 1 or 2, wherein the chitin oligosaccharide oxidase is further subjected to a purification procedure, including ammonium sulfate [(NH ₄ ) ₂ SO ₄ ] precipitation division, Toyopearl DEAE-650 anion column, Toyopearl Chromatography with phenyl-650M hydrophobic column, HW-50 molecular sieve column, MX-Trp-650M hydrophobic column and Ultrogel-hydroxyapatite column. A chitin oligosaccharide oxidase, which is prepared by the method described in claim 1 or 2, and the molecular weight of the chitin oligosaccharide oxidase is 54 kDa, and the enzyme contains FAD cofactors, calculated

It is 11.9, the catalytic reaction rate Km for chitin oligosaccharides is 2.08 mM, Kcat is 1.54 S ^-1 per second, and the conversion rate for chitin oligosaccharides is 52.2%. A method for the conversion and production of chito -oligosaccharides, which comprises reacting chito-oligosaccharide oxidase isolated from Paraphaeosphaeria sp. NUK-47 strain with chito-oligosaccharides to obtain chito -oligosaccharides, wherein the Paraphaeosphaeria sp . The deposit number of NUK-47 strain is BCRC 930220. The method according to claim 5, wherein the Paraphaeosphaeria sp. NUK-47 strain is inoculated in a suitable medium and cultured at 25°C-35°C for 3-6 days to transform and produce chitosan oligosaccharides. The method according to claim 6, wherein the medium is selected from a solid medium containing bran and water, or a liquid medium containing bran and added with phosphate. The method according to claim 7, wherein the enzyme activity reaches 0.17 units/g of the solid medium after culturing the solid medium for 3 days, and the enzyme activity reaches 0.29 units/ml of the liquid medium after culturing the liquid medium for 5 days.

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