TWI738358B - Method for preparing xylo-oligosaccharide - Google Patents

Abstract

The preparation method of xylo-oligosaccharides of the present invention comprises: (a) adding a solvent to pineapple peel residue to obtain The pineapple peel residue liquid mixture is subjected to a hydrothermal reaction to obtain a hydrothermally reacted pineapple peel residue liquid mixture; (b) the pineapple peel residue liquid mixture after the hydrothermal reaction is subjected to a debranching reaction in an acidic environment to obtain The liquid mixture of pineapple peel residue after debranching reaction; (c) adding endo-xylanase to the liquid mixture of pineapple peel residue after debranching reaction, and performing an enzyme reaction to obtain xylo-oligosaccharides. The present invention also provides a protein composition comprising the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 7, which has endo-xylanase activity. The preparation method of xylo-oligosaccharides of the present invention can obtain high-yield xylo-oligosaccharides, reduce pineapple peel agricultural waste, and further reduce environmental pollution.

Description

Method for preparing xylo-oligosaccharide

The invention relates to a method for preparing oligosaccharides, in particular to a method for preparing xylo-oligosaccharides. It also relates to a protein composition, especially a protein composition with endo-xylanase enzyme activity.

Xylooligosaccharide (XOS), also known as xylo-oligosaccharides and xylo-oligosaccharides, refers to the general term for oligosaccharides composed of 2 to 7 xyloses connected by glycosidic bonds. Xylooligosaccharides are equivalent to low-digestible oligosaccharides with fructo-oligosaccharides, galactose-oligosaccharides, and isomalto-oligosaccharides. Although human Called bifidobacteria or bifidobacteria) hyperplasia, not easy to cause dental caries. Because it can promote the proliferation of Bifidella and Lactobacillus, it belongs to prebiotics. In addition to improving the function of the gastrointestinal tract, xylo-oligosaccharides can also reduce the risk of colorectal cancer, significantly improve type II diabetes, and inhibit tumor growth.

Xylooligosaccharides are often added to foods to help activate and grow gastrointestinal probiotics due to their acid and alkali resistance and temperature resistance. Therefore, the market demand for xylo-oligosaccharides is increasing day by day. The market price of xylo-oligosaccharides is about 22-50 US dollars per kilogram, and its price is related to purity. Generally, for food-related applications, the optimal polymerization degree range for xylo-oligosaccharides is 2-4. In addition to being helpful to human health, xylo-oligosaccharides also have similar functions to animals and can be used as feed additives.

Xylooligosaccharides are molecular chains of xylose linked to β1-4 chains, which can be produced by hydrolyzing polyxylose by enzyme methods, chemical methods, or a combination of both. Xylose, also known as xylan, is a kind of heteropolymer, mainly composed of D-xylose connected by β-1,4-xylopyranose residues. There is always Allah on the xylose main chain of Xylose The branched chain of primary furanosyl and glucuronic acid, and the xylanase hydrolysate after most of the arabinose units are removed is generally called xylo-oligosaccharide. Commercial plant sources of xylose are generally based on agricultural wastes such as corn cobs (also called corn cobs).

Pineapple peel is a large agricultural waste in Taiwan. With the popularity of pineapple cakes in recent years, pineapple peel waste has been increasing year by year, resulting in an increasingly serious environmental pollution problem. Therefore, if xylo-oligosaccharides can be produced from pineapple peel agricultural waste, it will reduce environmental pollution and increase the added value of pineapple.

There are very few research literatures on the production of xylo-oligosaccharides using pineapple peel as a raw material. The only literature uses alkali treatment and enzyme reaction as a method to produce xylo-oligosaccharides. Among them, Zhang Boyao’s master's thesis (2018) showed that 1g of pineapple peel powder contains 35.55% of hemicellulose. After alkali treatment and alcohol precipitation, 0.2996g of hemicellulose can be obtained, which means that pineapple peel Treatment and alcohol precipitation can extract 84.3% of the raw material hemicellulose, but the content of xylose is low. Impurities in this hemicellulose accounted for 50.87%. Because it contains components that inhibit endoxylase hydrolysis, the production of xylo-oligosaccharides is not ideal. Only 1.02g of xylo-oligosaccharides can be obtained per 20g of hemicellulose. Sugar and xylotriose), the raw material pineapple peel residue can only get 0.018g of xylo-oligosaccharides (xylobiose and xylotriose) (1.8% by weight) in terms of unit dry weight. If the xylo-oligosaccharides with a degree of polymerization of 2 to 10 are calculated, the raw material pineapple peel residue per dry weight can obtain 0.067g xylo-oligosaccharides (6.7% by weight) at most (Zhang Boyao, 2018). It can be seen that the prior art methods for producing xylo-oligosaccharides from pineapple peel have low yields and require the use of high-concentration alkali, which is not friendly to the environment. Therefore, improving the yield of xylo-oligosaccharides prepared from pineapple peel and reducing the use of high-concentration alkali are important issues to be solved.

In view of the low yield of xylo-oligosaccharides prepared from pineapple peel in the prior art and the need to use high-concentration alkali, the purpose of the present invention is to effectively increase the yield of xylo-oligosaccharides and reduce the use of high-concentration alkali.

In order to achieve the above-mentioned purpose of the invention, the present invention provides a method for preparing xylo-oligosaccharides, which comprises: (a) Add a solvent to the pineapple peel residue to obtain a liquid mixture of pineapple peel residue, wherein the solid-liquid ratio of the pineapple peel residue to the solvent is 1:1 to 1:30 (w:v), and the temperature is between 115°C and 160°C. Perform a hydrothermal reaction at ℃ to obtain a liquid mixture of pineapple peel residue after hydrothermal reaction; (b) Debranching reaction of the pineapple peel residue liquid mixture after the hydrothermal reaction is carried out at 30°C to 80°C in an acid environment To obtain a liquid mixture of pineapple peel residues after debranching reaction; (c) adding endo-xylanase to the liquid mixture of pineapple peel residues after debranching reaction, and perform an enzyme reaction to obtain xylo-oligosaccharides.

The invention improves the yield of xylo-oligosaccharides by controlling the parameters of specific hydrothermal reaction, debranching reaction, and enzyme reaction. Therefore, the present invention provides a method for preparing xylo-oligosaccharides from pineapple peel which can obtain a higher yield than the prior art, and the method for preparing xylo-oligosaccharides of the present invention can reduce the use of high-concentration alkali and is more environmentally friendly.

The xylo-oligosaccharides referred to in the present invention include xylobiose, xylotriose, xylo-oligosaccharides with a degree of polymerization of four or more, and the xylo-oligosaccharide solution obtained by the method of the present invention may still contain a small amount of xylose and other monomers. sugar.

Preferably, a purification step is further performed on the xylo-oligosaccharides to obtain purified xylo-oligosaccharides. The purification step includes, but is not limited to, adsorption and purification using activated carbon, anion exchange resin and/or cation exchange resin.

Preferably, the step (a) includes: adding a solvent to the pineapple peel residue to obtain a liquid mixture of the pineapple peel residue, wherein the solid-liquid ratio of the pineapple peel residue to the solvent is 1:1 to 1:30 (w: v), and conduct a hydrothermal reaction at 115°C to 160°C, and then separate to remove the pineapple peel residue to obtain a liquid mixture of pineapple peel residue after the hydrothermal reaction.

Preferably, the step (b) includes: debranching the pineapple peel residue liquid mixture after the hydrothermal reaction at 30°C to 80°C in an acidic environment, and then separating to remove the pineapple peel residue to obtain Liquid mixture of pineapple peel residue after debranching reaction.

Preferably, the step (c) includes: adding endo-xylanase to the liquid mixture of pineapple peel residue after the debranching reaction, performing an enzyme reaction, and then separating to remove the pineapple peel residue to obtain wood Oligosaccharides.

Preferably, the separation step includes, but is not limited to, filter paper filtration separation and centrifugal separation.

Preferably, the solid-liquid ratio of the pineapple peel residue to the solvent for the hydrothermal reaction in step (a) is 1:5 to 1:20 (w:v), more preferably, the hydrothermal reaction is performed in step (a) The solid-liquid ratio of the pineapple peel residue to the solvent is 1:7 to 1:15 (w:v). More preferably, the solid-liquid ratio of the pineapple peel residue and the solvent for the hydrothermal reaction in step (a) is 1 : 10 (w: v). Under this solid-liquid ratio, the hydrothermal treatment has better reaction efficiency and can obtain higher yield of xylo-oligosaccharides. Preferably, the solvent is water.

Preferably, the hydrothermal reaction is carried out at 120°C to 140°C, and more preferably, the hydrothermal reaction is carried out at 125°C.

Preferably, the hydrothermal reaction time is 0.5 to 6 hours, more preferably, the hydrothermal reaction reaction time is 1 to 4 hours, and more preferably, the hydrothermal reaction reaction time is 1.5 to 6 hours. 3 hours.

Preferably, the pressure of the hydrothermal reaction is 0.2 MPa to 10 MPa, and more preferably, the pressure of the hydrothermal reaction is 0.45 MPa.

Preferably, the liquid mixture of pineapple peel residue in step (a) further adds 0.1% to 3% by weight of the acid of the pineapple peel residue to promote the progress of the hydrothermal reaction. Preferably, the pineapple peel residue is added 0.5% by weight to 2% by weight of the acid, more preferably 1% by weight to 1.7% by weight of the pineapple peel. The acid includes but is not limited to acetic acid, hydrochloric acid, phosphoric acid, oxalic acid and the like. In some embodiments, 1.5% by weight of acetic acid of the pineapple peel residue can be added to promote the reaction.

Preferably, the acidic environment in step (b) is pH 0.5 to pH 5.0, and more preferably, the acidic environment is pH 1.0 to pH 3.0.

Preferably, the debranching reaction in step (b) is carried out at 55°C to 80°C, more preferably, the debranching reaction is carried out at 50°C to 75°C, and even more preferably, The debranching reaction is carried out at 70°C.

Preferably, the time for the debranching reaction step is 1 to 5 hours, more preferably, the time for the debranching reaction is 1.5 to 3.5 hours, and even more preferably, the debranching reaction time is 2 hours.

Preferably, the enzyme reaction is carried out at a concentration of endo-xylanase from 50 U/L to 50,000 U/L.

Preferably, the enzyme reaction is carried out at pH 4.0 to pH 11.0, and more preferably, the enzyme reaction is carried out at pH 8.0.

Preferably, the enzyme reaction is carried out at 30°C to 80°C, and more preferably, the enzyme reaction is carried out at 55°C to 65°C.

Preferably, the reaction time of the enzyme is 20 hours to 30 hours, and more preferably, the reaction time of the enzyme is 22 hours to 26 hours.

Preferably, the endo-xylanase is one of the endo-xylanases Xyn45 and Xyn23 of Bacillus halodurans BCRC 910501, or a gene fusion protein of a combination of the two. In one embodiment, a protein composition of inclusion bodies from a recombinant expression system is used, which includes the fusion protein of the endo-xylanase Xyn45 and Xyn23 of Bacillus halodurans BCRC 910501, such as the amine shown in SEQ ID NO:1 Base acid sequence. Preferably, the endo-xylanase system is composed of the amino acid sequence shown in SEQ ID NO:1.

Preferably, the endo- xylanase is a fusion protein of the endo-xylanase Xyn45 and Xyn23 of Bacillus halodurans BCRC 910501, which is encoded by the nucleotide sequence shown in SEQ ID NO: 2, After being expressed by E. coli, it stays inside the cell. Preferably, the endo-xylanase is a protein expressed by E. coli in the inclusion body. Preferably, the E. coli is E. coli BL21 (DE3).

Preferably, the source of the endo-xylanase is Bacillus halodurans BCRC 910501, Bacillus halodurans C-125 or other strains of Bacillus halodurans . The endo-xylanase gene is genetically recombined or DNA shuffling. After the expression of E. coli, it is secreted to the outside of the cell or remains inside the cell with endo-xylanase activity protein. In one embodiment, an extracellular protein solution from a recombinant expression system is used, which contains a fusion protein of Bacillus halodurans BCRC 910501 and Bacillus halodurans C-125 endo-xylanase Xyn45, as shown in SEQ ID NO: 5 The amino acid sequence.

Compared with the prior art, the xylo-oligosaccharide preparation method of the present invention has a better yield of xylo-oligosaccharides, and can reduce the use of high-concentration acids and reduce environmental pollution caused by agricultural waste-pineapple peel.

In order to increase the production of xylo-oligosaccharides, another object of the present invention is to provide a protein composition with higher endo-xylanase enzyme activity, which comprises an amino acid sequence as shown in SEQ ID NO: 5 Protein, and the protein is an extracellular protein solution from a recombinant expression system. Preferably, the endo-xylanase enzyme activity per milligram of protein of the protein composition is 80 U to 200 U. More preferably, the endo-xylanase enzyme activity per milligram of protein is 100 U to 190 U.

Preferably, the protein composition comprises a protein composed of the amino acid sequence of SEQ ID NO:5.

Preferably, the protein composition comprises a protein encoded by the nucleotide sequence shown in SEQ ID NO: 6 and secreted to the outside of the cell after being expressed by E. coli. Preferably, the E. coli is E. coli BL21 (DE3).

In order to increase the production of xylo-oligosaccharides, another object of the present invention is to provide a protein composition with higher endo-xylanase enzyme activity, which comprises an amino acid sequence as shown in SEQ ID NO: 7 Protein, and the protein is an extracellular protein solution from a recombinant expression system. Preferably, the endo-xylanase enzyme activity per milligram of protein of the protein composition is 80 U to 250 U. More preferably, the endo-xylanase enzyme activity per milligram of protein is 100 U/mg to 230 U/mg.

Preferably, the protein composition comprises a protein composed of the amino acid sequence of SEQ ID NO:7.

Preferably, the protein composition comprises a protein encoded by the nucleotide sequence shown in SEQ ID NO: 8 and secreted to the outside of the cell after being expressed by E. coli. Preferably, the E. coli is E. coli BL21 (DE3).

The protein composition provided by the present invention has excellent endo-xylanase enzyme activity, and has higher endo-xylanase Xyn45 extracellular protein composition than the known Bacillus halodurans BCRC 910501. Xylase enzyme activity, suitable for the production of xylo-oligosaccharides.

S1: Step

S2: Step

S3: steps

Figure 1 is a flow chart of the method for preparing xylo-oligosaccharides of the present invention.

Hereinafter, several examples will be used to illustrate the specific implementation of the xylo-oligosaccharide preparation method of the present invention. Those who are familiar with this technique can easily understand the advantages and effects that can be achieved by this creation through the content of this manual. Various modifications and changes are made to implement or apply the content of the present invention under the spirit of this creation.

Example 1

First, add water to the pineapple peel residue in an autoclave to perform the hydrothermal treatment in step S1 as shown in FIG. 1. Specifically, take 50g of pineapple peel and 500 milliliters (mL) of solvent-water, that is, the ratio of pineapple peel to water (solid-liquid ratio w/v) is 1:10 and mix, and then add 0.714 ml of acetic acid (density About 1.05 g/mL), the added amount is about 1.5% by weight of the pineapple peel slag, soaked in the autoclave at a pressure of 0.45MPa and the temperature is set at 125°C (the actual temperature varies between 118°C and 143°C) After two hours, a liquid mixture of pineapple peel residue after liquid hot water pretreatment was obtained.

The aforementioned pineapple skin residue is in powder form. Specifically, the golden diamond pineapple skin is collected from the Zhengxing Pineapple Wholesale Market in Minxiong Township, Chiayi County. The pineapple skin is separated from the pineapple juice and pineapple skin residue by a juicer, and then the pineapple skin is washed with tap water. Slag until the washing liquid is clear. Then, put the washed pineapple peel residue in an oven at 50°C and dry it to a constant weight. Finally, the dried pineapple skin residue is crushed into powder by a pulverizer, and sieved with a 40 mesh (mesh 40) screen to obtain the pineapple skin residue in powder form, that is, the size of the pineapple skin residue is less than 380 microns.

Then, the liquid mixture of pineapple peel residue after the hydrothermal reaction is subjected to the debranching reaction in step S2 as shown in FIG. 1. In detail, the liquid mixture of pineapple peel residue after hydrothermal reaction was adjusted to pH 1.0 with 12N hydrochloric acid, and reacted for 6 hours in a water bath at 70°C and shaking at 120 rpm to remove the branched arabinose to obtain debranching. Liquid mixture of pineapple peel residue after chain reaction.

Next, the liquid mixture of pineapple peel residue after debranching reaction is subjected to the enzyme reaction in step S3 as shown in FIG. 1. Specifically, the pineapple peel liquid mixture after debranching reaction was adjusted to pH 8.0 with 5N sodium hydroxide, and 179 mL of endo-xylanase solution (total activity 5000 U) was added to make the liquid mixture The enzyme concentration was 7800U/L, and then the enzyme reaction was carried out at pH 8.0, 60°C, and shaking at 120 rpm. It is separated by filter paper. In detail, it is filtered with TOYO NO.5A filter paper and then filtered with TOYO NO.5C filter paper to obtain 640 mL of xylo-oligosaccharide solution.

The aforementioned endo-xylanase solution uses Escherichia coli to express a large amount of the gene of the endo-xylanase Xyn45 of Bacillus halodurans BCRC 910501. According to the Republic of China Invention Patent No. I418634, the aforementioned Xyn45 enzyme is composed of 396 amino acids. The corresponding DNA fragment is inserted into the plastid pET-29a(+) (Quantum Biotechnology), and then E. coli BL21(DE3) is inserted. , Inoculate the constructed genetically recombinant bacteria in PM liquid medium (20g/L glycerol, 3g/L diammonium hydrogen phosphate, 7g/L potassium dihydrogen phosphate, 0.8g/L citric acid, 1g/L magnesium sulfate heptahydrate, 2g /L yeast extract, 3mL/L trace metal solution, pH adjusted to 6.8), incubate at 37°C until OD _600nm reaches 0.8 to 1.0, add 0.1mM IPTG (isopropyl thiogalactoside, Isopropyl β- d-1-thiogalactopyranoside) was induced and cultured at 25°C for 18 hours to allow the genetically recombinant E. coli to express the recombinant protein. Afterwards, the bacterial solution was centrifuged at 10,000×g for 20 minutes at 4°C, and the supernatant obtained was the extracellular protein solution containing the genetically recombined protein Xyn45, which was the endo-xylanase solution required for the enzyme reaction. The enzyme activity is defined as 1 U of reducing sugar released by 1 μmole per minute. The specific activity of this extracellular protein solution is 75.8 U/mg, that is, 75.8 U endo-xylanase activity per milligram of protein. Since Xylanases can hydrolyze polyxylose into xylo-oligosaccharides, and some oligosaccharides and monosaccharides are reducing sugars, and will interact with 3,5-Dinitrosalicylic acid (DNS) A reaction occurs, so the DNS method is used to determine the reducing sugar content. Take 1% (w/v) beechwood polyxylose (dissolved in 100mM pH 8.0 phosphate buffer) as the matrix, mix with the extracellular protein solution and react at 60°C for 5 minutes. The reacted product is mixed with DNS reagent, React in a water bath at 100°C for 7 minutes, measure the absorbance at OD _540nm with a spectrophotometer, and substitute it into the xylose standard calibration line to quantify the amount of reducing sugar in the sample, and use the increased amount of reducing sugar to convert the enzyme activity. The DNS reagent formula is shown in Table 1 below:

Use High Performance Liquid Chromatography (HPLC) to further analyze the concentration of xylobiose, xylotriose, glucose, xylose, arabinose and other components in the xylo-oligosaccharide solution, using Aminex HPX-87H The column (BioRad) 300mm×7.8mm, 65°C column temperature, the mobile phase is 5mM sulfuric acid with a flow rate of 0.6mL/min, and the detection is performed with an RI detector (Refractive Index Detector).

The total concentration of xylobiose and xylotriose in the 640 mL xylo-oligosaccharide solution obtained in step S3 is 12.13 g/L. The yield of xylo-oligosaccharides (xylobiose and xylotriose) per unit dry weight of pineapple peel residue was 15.53% by weight (the calculation method is the total weight of xylobiose and xylotriose in the enzyme hydrolysate/dry weight of pineapple peel residue × 100%).

In some embodiments, the obtained xylo-oligosaccharide solution can be further adsorbed and purified through activated carbon, anion exchange resin, and cation exchange resin to obtain purified xylo-oligosaccharide syrup.

Example 2

The preparation method of Example 2 is similar to the method of Example 1. The difference is only in the hydrothermal treatment of step S1. Although the temperature setting is the same at 125°C, the actual temperature varies between 116°C and 150°C; the other difference is The liquid mixture of pineapple peel residues after the debranching reaction obtained in step S2 is filtered and separated with filter paper. Specifically, it is filtered with TOYO NO.1 filter paper and then filtered with TOYO NO.5C filter paper. Take the filtrate, and then use 130mL The endo-xylanase liquid mixture (total activity 1602U) is subjected to the enzyme reaction of step S3 on the aforementioned filtrate to obtain the liquid mixture of xylo-oligosaccharides. Example 2 has been filtered after step S2, so the enzyme reaction of step S3 After that, no filtration was performed, and the xylo-oligosaccharide solution prepared by the preparation method of Example 2 was 630 mL. After detection by the HPLC detection method as described in Example 1, the total concentration of xylobiose and xylotriose was 10.14g/L, and the yield of xylo-oligosaccharides (xylobiose and xylotriose) per unit dry weight of pineapple peel residue It is 12.78% by weight.

Compared with Example 1, Example 2 only needs to add less than 1/3 of the total activity of endo-xylanase to achieve a similar yield of xylo-oligosaccharide per dry weight of pineapple peel residue.

Example 3 Optimal conditions for hydrothermal reaction

Example 3 is divided into Examples 3-1 to 3-7. The preparation method is similar to that of Example 2. However, the pineapple peel residue used in the hydrothermal reaction of step S1 is adjusted to only 2g, and the water is adjusted separately. The conditions of the three parameters of the thermal reaction, the solid-liquid ratio, the immersion time, and the amount of acetic acid added, are shown in Table 2 below, and the temperature of the hydrothermal reaction is still the same as that of Example 2 at 125°C.

In addition, the difference from Example 2 is that after the hydrothermal reaction in step S1, filter paper is used for filtration and separation, the filtrate is taken, and then the filtrate is subjected to the debranching reaction in step S2 and the enzyme reaction in step S3. The debranching reaction of Examples 3-1 to 3-7 is different from that of Example 2 in that the reaction temperature is adjusted to 60°C; the enzyme reaction of Examples 3-1 to 3-7 is different from that of Example 2 in that the reaction The temperature and the vibration frequency were adjusted to 60℃, 100rpm vibration, and the added enzyme was the same as the endo-xylanase of Examples 1 and 2, but the enzyme concentration of the added liquid mixture was adjusted to 500U /L, and the matrix solution used in the activity analysis is 1% (w/v) birchwood polyxylose (dissolved in 100mM pH 8.0 phosphate buffer). Examples 3-1 to 3-7 have been heated in S1 The reaction step is followed by filtration separation, so the filtration separation step after the debranching reaction in step S2 as in Example 2 is not performed. The yields and yields of xylo-oligosaccharides in Examples 3-1 to 3-7 are shown in Table 3 below:

From the results in the above table, it can be seen that the yield of xylo-oligosaccharides in Example 3-3 is the best, which is 6.3%. The conditions of the hydrothermal reaction are solid-liquid ratio 1:20, 2 hours immersion time and 1.5% acetic acid environment. . However, considering the solid-liquid ratio, the yield of xylo-oligosaccharides in Example 3-2 is 6.1%, which is close to the 6.3% in Example 3-3. However, the amount of liquid required for the hydrothermal reaction is only 1/2, so the efficiency is relatively higher. Example 3-3 is much higher and can reduce energy consumption. Therefore, Example 3-2 is taken as the preferred hydrothermal conditions, that is, the solid-to-liquid ratio is 1:10, and the acetic acid is soaked for 2 hours at 1.5% for subsequent testing. Examples 4-1 to 4 of the preferred debranching reaction conditions -4. In addition, the yields of xylo-oligosaccharides (xylobiose and xylotriose) in Examples 3-2, 3-3, and 3-5 are higher than those of the prior art (xylobiose and xylotriose). The sum of sugar) 1.8%.

Example 4 Optimal conditions for debranching reaction

Example 4 is divided into Example 4-1 to Example 4-4. In Example 4-1 to Example 4-4, the same method of preparing xylo-oligosaccharides as in Example 3-2 was carried out until the hydrothermal reaction in step S1 was completed, and then the filtrate was used to carry out the debranching of step S2. Chain reaction or perform the debranching reaction of step S2 first, then filter and compare the debranching reaction temperature of step S2 (60°C or 70°C), as shown in Table 4 below, and the remaining steps of S3 are the same as in Example 3-2 Method steps.

As shown in Table 4 above, the yield of xylo-oligosaccharides in Example 4-4 is the highest. Therefore, it can be seen that the debranching reaction of step S2 is better performed at 70°C, and it is carried out after the debranching reaction of step S2. The yield of xylo-oligosaccharides obtained by filtration is better. That is, the same method as in Example 2 can obtain the highest yield of xylo-oligosaccharides. And compared with the yield of xylo-oligosaccharides (xylobiose and xylotriose) in the prior art, Examples 4-2 and 4-3 are higher than the yield of xylo-oligosaccharides in the prior art (the sum of xylobiose and xylotriose). ) 1.8%.

Example 5 Preparation method of endo-xylanase solution (1)

In addition to the method of Example 1, the preparation method of the endo-xylanase solution can also use genetically recombinant bacteria containing plastids expressing the Xyn45-Xyn23 fusion protein. The first is the two endo-xylanases Xyn45 and Xyn23 (proteins containing 396 and 210 amino acids, respectively) from Bacillus halodurans BCRC 910501, according to their amino acid sequence (for example, the Republic of China Invention Patent No. 1418634 (Shown), design a fusion protein (containing 597 amino acids) whose amino acid sequence is as SEQ ID NO:1. Aiming at the amino acid sequence of the fusion protein, a DNA sequence suitable for expression in E. coli (SEQ ID NO: 2) was designed to produce the xyn45-xyn23 fusion gene by chemical synthesis, and then the plastid 16ACBZOP containing this fusion gene -xyn45-xyn23 (using the plastids custom-synthesized ^{from Thermo Scientific TM} entrusted by Quantum Biotechnology Co., Ltd. ), sent into the E.coli DH5α host strain by the Heat Shock method, and then extracted the bacteria's substance As a template, design a forward primer as shown in SEQ ID NO: 3 and a reverse primer as shown in SEQ ID NO: 4 to perform a PCR reaction, and insert the target DNA fragment amplified by PCR into pET-29a (+) In the plastid, then transform it to E.coli DH5α to save the plastid. The plastids of the bacteria were extracted into E.coli BL21(DE3) expressing host strains by heat shock method, and the transformation results were confirmed by Colony PCR. The transformed E. coli BL21(DE3)-pET29a(+)-xyn45-xyn23 was cultured at 37°C, induced by 1mM IPTG, and 4 hours later, a large amount of the fusion protein Xyn45-( with endo-xylanase activity could be expressed G ₄ S) ₂ -Xyn23 is shown in SEQ ID NO:1. Centrifuge the bacterial solution at 8000 rpm and 4°C for 20 minutes, and separate the supernatant and bacterial clumps. The supernatant contains the proteins secreted to the outside of the cell, and the bacterial clumps are re-dissolved in deionized water, and are subjected to high-pressure sterilization in a French high-pressure sterilizer under a pressure of 20 kpsi, and then centrifuged at 4°C and 12000 rpm for 20 minutes. The supernatant The fluid contains intracellular soluble proteins. The solid part is washed and then suspended in deionized water, and the resulting suspension solution contains intracellular insoluble proteins from inclusion bodies. Measure the total activity of the endo-xylanase enzyme in the protein produced by recombinant E. coli. 25% of the protein is soluble in the cell, and 60% is contributed by the insoluble protein in the cell. The enzyme specific activity of intracellular insoluble protein is 79.1U/mg, that is, 79.1U endo-xylanase activity per milligram of protein, which is much higher than that of intracellular soluble protein (3.9U/mg) or extracellular The endo-xylanase activity of protein (32.0U/mg) is expected to be high. This insoluble protein suspended in an aqueous solution can be used as an endo-xylanase solution for decomposing the xylose of pineapple peel residue to produce xylo-oligosaccharides. When the Xyn45-Xyn23 fusion protein of this example is expressed, the main enzyme activity is in the inclusion bodies. Although the specific activity is equivalent to that of the protein of Example 1, because it exists in the form of inclusion bodies, it has the better effect of easy recovery and convenient application.

Example 6 Preparation method of endo-xylanase solution (2)

In addition to the method in Example 1, the preparation method of the endo-xylanase solution can also use genetic recombination bacteria. The amino acid sequence of Xyn45 contained therein is referenced to Bacillus halodurans BCRC 910501 and Bacillus halodurans C-125. The sequence characteristics of Xyn45 of the strain are determined. The amino acid sequence of this protein is shown in SEQ ID NO:5. The total number of amino acids such as Xyn45 of Bacillus halodurans BCRC 910501 maintains 396, and is almost the same as the amino acid sequence of the protein, but 364D is changed to G and 393G is changed to R to correspond to the Xyn45 sequence of Bacillus halodurans C-125. The DNA sequence corresponding to the amino acid sequence of the protein is inserted into the plastid pET-29a(+) as shown in SEQ ID NO: 6, and transformed into E. coli BL21 (DE3) to obtain genetically recombinant bacteria. Culture the previously constructed genetically recombinant bacteria in PM liquid medium until the OD _600nm reaches 0.8-1.0, add 0.1 mM IPTG for induction culture at 25°C for 18 hours, centrifuge to collect the supernatant, which is the extracellular protein solution And its enzyme activity per milligram of protein is 171U, which is higher than the specific enzyme activity (75.8U/mg) of the endomeric polymerase solution in Example 1. Different from Example 5, the specific activity of the endonuclease polyxylanase enzyme of the intracellular soluble protein in this example is 13.5 U/mg, which accounts for the percentage of the endonuclease polyxylanase enzyme produced by the genetically recombined bacteria. 13.8% of the total activity, and the specific activity of the endonuclease poly-xylanase enzyme of the insoluble intracellular protein is 11.4 U/mg, which accounts for the total activity of the endo-xylanase enzyme of the protein produced by the genetically recombined bacteria 0.6%. The total enzyme activity of the two endo-xylanase enzymes is far less than that of the extracellular protein solution endo-xylanase enzyme activity. The extracellular protein solution accounts for 85.6% of the total enzyme activity of the protein produced by the genetically recombined bacteria. This cell The exoprotein solution can be used as an endo-xylanase solution for decomposing xylo-oligosaccharides from pineapple peel residues.

Example 7 Preparation method of endo-xylanase solution (3)

The method of making genetically recombinant bacteria in this example is similar to that in Example 6, but the amino acid sequence of the protein shown in this example is shown in SEQ ID NO: 7, and the total number of amino acids is Xyn45 of Bacillus halodurans BCRC 910501. It maintains 396 and is almost the same as the amino acid sequence of the protein. Only 393G is changed to R to correspond to the Xyn45 sequence of Bacillus halodurans C-125. The DNA sequence corresponding to the amino acid sequence of this protein is inserted into the plastid pET-29a(+) as shown in SEQ ID NO: 8, and transformed into E. coli BL21 (DE3) to obtain genetically recombinant bacteria. The induction culture was carried out under the conditions set out in Example 6, and the resulting extracellular protein solution had an enzyme activity of 217 U per milligram of protein, which was higher than the specific enzyme activity of the endomeric polymerase solution in Example 1 (75.8 U/ mg) high. In addition, the extracellular protein solution accounts for 90% of the total enzyme activity produced by the genetically recombined bacteria. This extracellular protein solution can be used as an endo-xylanase solution for decomposing the xylose of pineapple peel residue to produce xylo-oligosaccharides.

Based on the results of the foregoing Examples 1 to 7, it can be seen that the preparation method of the present invention can effectively increase the yield of xylo-oligosaccharides compared with the prior art method for preparing xylo-oligosaccharides, and is an excellent method for preparing xylo-oligosaccharides.

The above-mentioned embodiments are only examples to illustrate the creation, and do not limit the scope of rights claimed by the creation in any respect. The scope of rights claimed in this creation should be subject to the scope of the patent application, rather than limited to the specific embodiments described above.

                                  Sequence Listing
          <![CDATA[<110> National Chung Cheng University]]>
          <![CDATA[<120> Method for preparing xylo-oligosaccharide and protein composition used in it]]>
          <![CDATA[<160> 8 ]]>
          <![CDATA[<170> PatentIn Version 3.5]]>
          <![CDATA[<210> 1]]>
          <![CDATA[<211> 597]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> Manual Sequence]]>
          <![CDATA[<400> 1]]>
          Met Met Ile Thr Leu Phe Lys Lys Pro Phe Val Ala Gly Leu Ala Ile
          1 5 10 15
          Ser Leu Leu Val Gly Gly Gly Leu Gly Asn Val Ala Ala Ala Gln Gly
                      20 25 30
          Gly Pro Pro Lys Ser Gly Val Phe Gly Glu Asn Gln Lys Arg Asn Asp
                  35 40 45
          Gln Pro Phe Ala Trp Gln Val Ala Ser Leu Ser Glu Arg Tyr Gln Glu
              50 55 60
          Gln Phe Asp Ile Gly Ala Ala Val Glu Pro Tyr Gln Leu Glu Gly Arg
          65 70 75 80
          Gln Ala Gln Ile Leu Lys His His Tyr Asn Ser Leu Val Ala Glu Asn
                          85 90 95
          Ala Met Lys Pro Val Ser Leu Gln Pro Arg Glu Gly Glu Trp Asn Trp
                      100 105 110
          Glu Gly Ala Asp Lys Ile Val Glu Phe Ala Arg Lys His Asn Met Glu
                  115 120 125
          Leu Arg Phe His Thr Leu Val Trp His Ser Gln Val Pro Glu Trp Phe
              130 135 140
          Phe Ile Asp Glu Asn Gly Asn Arg Met Val Asp Glu Thr Asp Pro Glu
          145 150 155 160
          Lys Arg Lys Ala Asn Lys Gln Leu Leu Leu Glu Arg Met Glu Asn His
                          165 170 175
          Ile Lys Thr Val Val Glu Arg Tyr Lys Asp Asp Val Thr Ser Trp Asp
                      180 185 190
          Val Val Asn Glu Val Ile Asp Asp Gly Gly Gly Leu Arg Glu Ser Glu
                  195 200 205
          Trp Tyr Gln Ile Thr Gly Thr Asp Tyr Ile Lys Val Ala Phe Glu Thr
              210 215 220
          Ala Arg Lys Tyr Gly Gly Glu Glu Ala Lys Leu Tyr Ile Asn Asp Tyr
          225 230 235 240
          Asn Thr Glu Val Pro Ser Lys Arg Asp Asp Leu Tyr Asn Leu Val Lys
                          245 250 255
          Asp Leu Leu Glu Gln Gly Val Pro Ile Asp Gly Val Gly His Gln Ser
                      260 265 270
          His Ile Gln Ile Gly Trp Pro Ser Ile Glu Asp Thr Arg Ala Ser Phe
                  275 280 285
          Glu Lys Phe Thr Ser Leu Gly Leu Asp Asn Gln Val Thr Glu Leu Asp
              290 295 300
          Met Ser Leu Tyr Gly Trp Pro Pro Thr Gly Ala Tyr Thr Ser Tyr Asp
          305 310 315 320
          Asp Ile Pro Glu Glu Leu Phe Gln Ala Gln Ala Asp Arg Tyr Asp Gln
                          325 330 335
          Leu Phe Glu Leu Tyr Glu Glu Leu Ser Ala Thr Ile Ser Ser Val Thr
                      340 345 350
          Phe Trp Gly Ile Ala Asp Asn His Thr Trp Leu Asp Asp Arg Ala Arg
                  355 360 365
          Glu Tyr Asn Asn Gly Val Gly Val Asp Ala Pro Phe Val Phe Asp His
              370 375 380
          Asn Tyr Arg Val Lys Pro Ala Tyr Trp Gly Ile Ile Asp Gly Gly Gly
          385 390 395 400
          Gly Ser Gly Gly Gly Gly Ser Asn Thr Tyr Trp Gln Tyr Trp Thr Asp
                          405 410 415
          Gly Gly Gly Thr Val Asn Ala Thr Asn Gly Pro Gly Gly Asn Tyr Ser
                      420 425 430
          Val Thr Trp Arg Asp Thr Gly Asn Phe Val Val Gly Lys Gly Trp Glu
                  435 440 445
          Ile Gly Ser Pro Asn Arg Thr Ile His Tyr Asn Ala Gly Val Trp Glu
              450 455 460
          Pro Ser Gly Asn Gly Tyr Leu Thr Leu Tyr Gly Trp Thr Arg Asn Gln
          465 470 475 480
          Leu Ile Glu Tyr Tyr Val Val Asp Asn Trp Gly Thr Tyr Arg Pro Thr
                          485 490 495
          Gly Thr His Arg Gly Thr Val Val Ser Asp Gly Gly Thr Tyr Asp Ile
                      500 505 510
          Tyr Thr Thr Met Arg Tyr Asn Ala Pro Ser Ile Asp Gly Thr Gln Thr
                  515 520 525
          Phe Gln Gln Phe Trp Ser Val Arg Gln Ser Lys Arg Pro Thr Gly Asn
              530 535 540
          Asn Val Ser Val Thr Phe Ser Asn His Val Asn Ala Trp Arg Asn Ala
          545 550 555 560
          Gly Met Asn Leu Gly Ser Ser Trp Ser Tyr Gln Val Leu Ala Thr Glu
                          565 570 575
          Gly Tyr Gln Ser Ser Gly Arg Ser Asn Val Thr Val Trp Leu Glu His
                      580 585 590
          His His His His His
                  595
          <![CDATA[<210> 2]]>
          <![CDATA[<211> 1794]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> Manual Sequence]]>
          <![CDATA[<400> 2]]>
          atgatgatta ccctgttcaa aaaaccgttt gttgcaggtc tggcaattag cctgctggtt 60
          ggtggtggtc tgggtaatgt tgcagcagca cagggtggtc cgcctaaaag cggtgttttt 120
          ggtgaaaatc agaaacgtaa tgatcagccg tttgcatggc aggttgcaag cctgagcgaa 180
          cgttatcaag aacagtttga tattggtgca gcagttgaac cgtatcagct ggaaggtcgt 240
          caggcacaga ttctgaaaca tcattataac agcctggttg ccgaaaatgc aatgaaaccg 300
          gttagcctgc agcctcgtga aggtgaatgg aattgggaag gtgcagataa aattgttgag 360
          tttgcccgta aacacaatat ggaactgcgt tttcataccc tggtttggca tagccaggtt 420
          ccggaatggt tttttatcga tgaaaatggt aatcgcatgg tggatgaaac cgatccggaa 480
          aaacgtaaag caaataaaca gctgctgctg gaacgtatgg aaaaccatat caaaaccgtt 540
          gtggaacgct ataaagatga tgttaccagc tgggatgttg tgaacgaagt tattgatgat 600
          ggtggtggcc tgcgtgaaag cgaatggtat cagattaccg gcaccgatta tatcaaagtt 660
          gcatttgaaa ccgcacgcaa atatggtggt gaagaagcaa aactgtatat caacgattat 720
          aacaccgaag tgccgagcaa acgtgatgat ctgtataatc tggttaaaga cctgctggaa 780
          cagggtgttc cgattgatgg tgttggtcat cagagccata ttcagattgg ttggccgagc 840
          attgaagata cccgtgcaag ctttgaaaaa ttcaccagcc tgggtctgga taatcaggtt 900
          accgaactgg atatgagcct gtatggttgg cctccgaccg gtgcatatac cagctatgat 960
          gatattccgg aagaactgtt tcaggcccag gcagatcgtt atgatcagct gttcgaactg 1020
          tatgaagaac tgagcgcaac cattagcagc gttacctttt ggggtattgc agataatcat 1080
          acctggctgg atgatcgtgc acgtgaatat aacaatggtg tgggtgttga tgcaccgttt 1140
          gtgtttgatc ataattatcg tgtgaaaccg gcatattggg gcattattga tggcggtggc 1200
          ggtagcggtg gtggcggttc aaatacctat tggcagtatt ggaccgatgg tggcggaacc 1260
          gttaatgcaa ccaatggtcc gggtggtaat tatagcgtga cctggcgtga taccggcaat 1320
          tttgttgttg gtaaaggttg ggaaattggt agcccgaatc gtaccattca ctataatgcc 1380
          ggtgtttggg aaccgagcgg taatggttat ctgaccctgt atggctggac ccgcaaccag 1440
          ctgattgaat attatgttgt tgataactgg ggcacctatc gtccgaccgg cacccatcgt 1500
          ggcaccgttg ttagtgatgg tggcacctat gatatctata ccacgatgcg ttataatgca 1560
          ccgtcaattg atggcaccca gacctttcag cagttttgga gcgttcgtca gagtaaacgt 1620
          ccgacaggta ataatgttag tgtgaccttt agcaatcatg tgaatgcatg gcgtaatgca 1680
          ggtatgaatc tgggtagcag ctggtcatat caggttctgg caaccgaagg ttatcagagc 1740
          agcggtcgta gcaatgttac cgtttggctc gagcaccacc accaccacca ctga 1794
          <![CDATA[<210> 3]]>
          <![CDATA[<211> 57]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> Manual Sequence]]>
          <![CDATA[<400> 3]]>
          ggaattccat atgatgatta ccctgttcaa aaaaccgttt gttgcaggtc tggcaat 57
          <![CDATA[<210> 4]]>
          <![CDATA[<211> 33]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> Manual Sequence]]>
          <![CDATA[<400> 4]]>
          cgcctcgagc caaacggtaa cattgctacg acc 33
          <![CDATA[<210> 5]]>
          <![CDATA[<211> 396]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> Manual Sequence]]>
          <![CDATA[<400> 5]]>
          Met Val Thr Leu Phe Lys Lys Pro Phe Val Ala Gly Leu Ala Ile Ser
          1 5 10 15
          Leu Leu Val Gly Gly Gly Leu Gly Asn Val Ala Ala Ala Gln Gly Gly
                      20 25 30
          Pro Pro Lys Ser Gly Val Phe Gly Glu Asn Gln Lys Arg Asn Asp Gln
                  35 40 45
          Pro Phe Ala Trp Gln Val Ala Ser Leu Ser Glu Arg Tyr Gln Glu Gln
              50 55 60
          Phe Asp Ile Gly Ala Ala Val Glu Pro Tyr Gln Leu Glu Gly Arg Gln
          65 70 75 80
          Ala Gln Ile Leu Lys His His Tyr Asn Ser Leu Val Ala Glu Asn Ala
                          85 90 95
          Met Lys Pro Val Ser Leu Gln Pro Arg Glu Gly Glu Trp Asn Trp Glu
                      100 105 110
          Gly Ala Asp Lys Ile Val Glu Phe Ala Arg Lys His Asn Met Glu Leu
                  115 120 125
          Arg Phe His Thr Leu Val Trp His Ser Gln Val Pro Glu Trp Phe Phe
              130 135 140
          Ile Asp Glu Asn Gly Asn Arg Met Val Asp Glu Thr Asp Pro Glu Lys
          145 150 155 160
          Arg Lys Ala Asn Lys Gln Leu Leu Leu Glu Arg Met Glu Asn His Ile
                          165 170 175
          Lys Thr Val Val Glu Arg Tyr Lys Asp Asp Val Thr Ser Trp Asp Val
                      180 185 190
          Val Asn Glu Val Ile Asp Asp Gly Gly Gly Leu Arg Glu Ser Glu Trp
                  195 200 205
          Tyr Gln Ile Thr Gly Thr Asp Tyr Ile Lys Val Ala Phe Glu Thr Ala
              210 215 220
          Arg Lys Tyr Gly Gly Glu Glu Ala Lys Leu Tyr Ile Asn Asp Tyr Asn
          225 230 235 240
          Thr Glu Val Pro Ser Lys Arg Asp Asp Leu Tyr Asn Leu Val Lys Asp
                          245 250 255
          Leu Leu Glu Gln Gly Val Pro Ile Asp Gly Val Gly His Gln Ser His
                      260 265 270
          Ile Gln Ile Gly Trp Pro Ser Ile Glu Asp Thr Arg Ala Ser Phe Glu
                  275 280 285
          Lys Phe Thr Ser Leu Gly Leu Asp Asn Gln Val Thr Glu Leu Asp Met
              290 295 300
          Ser Leu Tyr Gly Trp Pro Pro Thr Gly Ala Tyr Thr Ser Tyr Asp Asp
          305 310 315 320
          Ile Pro Glu Glu Leu Phe Gln Ala Gln Ala Asp Arg Tyr Asp Gln Leu
                          325 330 335
          Phe Glu Leu Tyr Glu Glu Leu Ser Ala Thr Ile Ser Ser Val Thr Phe
                      340 345 350
          Trp Gly Ile Ala Asp Asn His Thr Trp Leu Asp Gly Arg Ala Arg Glu
                  355 360 365
          Tyr Asn Asn Gly Val Gly Val Asp Ala Pro Phe Val Phe Asp His Asn
              370 375 380
          Tyr Arg Val Lys Pro Ala Tyr Trp Arg Ile Ile Asp
          385 390 395
          <![CDATA[<210> 6]]>
          <![CDATA[<211> 1191]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> Manual Sequence]]>
          <![CDATA[<400> 6]]>
          atggttacac tttttaaaaa gccttttgtt gctggactag cgatctcttt attagttgga 60
          ggggggctag gcaatgtagc tgctgctcaa ggaggaccac caaaatctgg agtctttgga 120
          gaaaatcaaa aaagaaatga tcagcctttt gcatggcaag ttgcttctct ttctgagcga 180
          tatcaagagc agtttgatat tggagctgcg gttgagccct atcaattaga aggaagacaa 240
          gcccaaattt taaagcatca ttataacagc cttgtggcgg aaaatgcaat gaaacctgta 300
          tcactccagc caagagaagg tgagtggaac tgggaaggcg ctgacaaaat tgtggagttt 360
          gcccgcaaac ataacatgga gcttcgcttc cacacactcg tttggcatag ccaagtacca 420
          gaatggtttt tcatcgatga aaatggcaat cggatggttg atgaaaccga tccagaaaaa 480
          cgtaaagcga ataaacaatt gttattggag cgaatggaaa accatattaa aacggttgtt 540
          gaacgttata aagatgatgt gacttcatgg gatgtggtga atgaagttat tgatgatggc 600
          gggggcctcc gtgaatcaga atggtatcaa ataacaggca ctgactacat taaggtagct 660
          tttgaaactg caagaaaata tggtggtgaa gaggcaaagc tgtacattaa tgattacaac 720
          accgaagtac cttctaaaag agatgacctt tacaacctgg tgaaagactt attagagcaa 780
          ggagtaccaa ttgacggggt aggacatcag tctcatatcc aaatcggctg gccttccatt 840
          gaagatacaa gagcttcttt tgaaaagttt acgagtttag gattagacaa ccaagtaact 900
          gaactagaca tgagtcttta tggctggcca ccgacagggg cctatacctc ttatgacgac 960
          attccagaag agctttttca agctcaagca gaccgttatg atcagctatt tgagttatat 1020
          gaagaattaa gcgctactat cagtagtgta accttctggg gaattgctga taaccataca 1080
          tggcttgatg gccgcgctag agagtacaat aatggagtag gggtcgatgc accatttgtt 1140
          tttgatcaca actatcgagt gaagcctgct tactggagaa ttattgatta a 1191
          <![CDATA[<210> 7]]>
          <![CDATA[<211> 396]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> Manual Sequence]]>
          <![CDATA[<400> 7]]>
          Met Val Thr Leu Phe Lys Lys Pro Phe Val Ala Gly Leu Ala Ile Ser
          1 5 10 15
          Leu Leu Val Gly Gly Gly Leu Gly Asn Val Ala Ala Ala Gln Gly Gly
                      20 25 30
          Pro Pro Lys Ser Gly Val Phe Gly Glu Asn Gln Lys Arg Asn Asp Gln
                  35 40 45
          Pro Phe Ala Trp Gln Val Ala Ser Leu Ser Glu Arg Tyr Gln Glu Gln
              50 55 60
          Phe Asp Ile Gly Ala Ala Val Glu Pro Tyr Gln Leu Glu Gly Arg Gln
          65 70 75 80
          Ala Gln Ile Leu Lys His His Tyr Asn Ser Leu Val Ala Glu Asn Ala
                          85 90 95
          Met Lys Pro Val Ser Leu Gln Pro Arg Glu Gly Glu Trp Asn Trp Glu
                      100 105 110
          Gly Ala Asp Lys Ile Val Glu Phe Ala Arg Lys His Asn Met Glu Leu
                  115 120 125
          Arg Phe His Thr Leu Val Trp His Ser Gln Val Pro Glu Trp Phe Phe
              130 135 140
          Ile Asp Glu Asn Gly Asn Arg Met Val Asp Glu Thr Asp Pro Glu Lys
          145 150 155 160
          Arg Lys Ala Asn Lys Gln Leu Leu Leu Glu Arg Met Glu Asn His Ile
                          165 170 175
          Lys Thr Val Val Glu Arg Tyr Lys Asp Asp Val Thr Ser Trp Asp Val
                      180 185 190
          Val Asn Glu Val Ile Asp Asp Gly Gly Gly Leu Arg Glu Ser Glu Trp
                  195 200 205
          Tyr Gln Ile Thr Gly Thr Asp Tyr Ile Lys Val Ala Phe Glu Thr Ala
              210 215 220
          Arg Lys Tyr Gly Gly Glu Glu Ala Lys Leu Tyr Ile Asn Asp Tyr Asn
          225 230 235 240
          Thr Glu Val Pro Ser Lys Arg Asp Asp Leu Tyr Asn Leu Val Lys Asp
                          245 250 255
          Leu Leu Glu Gln Gly Val Pro Ile Asp Gly Val Gly His Gln Ser His
                      260 265 270
          Ile Gln Ile Gly Trp Pro Ser Ile Glu Asp Thr Arg Ala Ser Phe Glu
                  275 280 285
          Lys Phe Thr Ser Leu Gly Leu Asp Asn Gln Val Thr Glu Leu Asp Met
              290 295 300
          Ser Leu Tyr Gly Trp Pro Pro Thr Gly Ala Tyr Thr Ser Tyr Asp Asp
          305 310 315 320
          Ile Pro Glu Glu Leu Phe Gln Ala Gln Ala Asp Arg Tyr Asp Gln Leu
                          325 330 335
          Phe Glu Leu Tyr Glu Glu Leu Ser Ala Thr Ile Ser Ser Val Thr Phe
                      340 345 350
          Trp Gly Ile Ala Asp Asn His Thr Trp Leu Asp Asp Arg Ala Arg Glu
                  355 360 365
          Tyr Asn Asn Gly Val Gly Val Asp Ala Pro Phe Val Phe Asp His Asn
              370 375 380
          Tyr Arg Val Lys Pro Ala Tyr Trp Arg Ile Ile Asp
          385 390 395
          <![CDATA[<210> 8]]>
          <![CDATA[<211> 1191]]>
          <![CDATA[<212> DNA]]>
          <![CDATA[<213> Manual Sequence]]>
          <![CDATA[<400> 8]]>
          atggttacac tttttaaaaa gccttttgtt gctggactag cgatctcttt attagttgga 60
          ggggggctag gcaatgtagc tgctgctcaa ggaggaccac caaaatctgg agtctttgga 120
          gaaaatcaaa aaagaaatga tcagcctttt gcatggcaag ttgcttctct ttctgagcga 180
          tatcaagagc agtttgatat tggagctgcg gttgagccct atcaattaga aggaagacaa 240
          gcccaaattt taaagcatca ttataacagc cttgtggcgg aaaatgcaat gaaacctgta 300
          tcactccagc caagagaagg tgagtggaac tgggaaggcg ctgacaaaat tgtggagttt 360
          gcccgcaaac ataacatgga gcttcgcttc cacacactcg tttggcatag ccaagtacca 420
          gaatggtttt tcatcgatga aaatggcaat cggatggttg atgaaaccga tccagaaaaa 480
          cgtaaagcga ataaacaatt gttattggag cgaatggaaa accatattaa aacggttgtt 540
          gaacgttata aagatgatgt gacttcatgg gatgtggtga atgaagttat tgatgatggc 600
          gggggcctcc gtgaatcaga atggtatcaa ataacaggca ctgactacat taaggtagct 660
          tttgaaactg caagaaaata tggtggtgaa gaggcaaagc tgtacattaa tgattacaac 720
          accgaagtac cttctaaaag agatgacctt tacaacctgg tgaaagactt attagagcaa 780
          ggagtaccaa ttgacggggt aggacatcag tctcatatcc aaatcggctg gccttccatt 840
          gaagatacaa gagcttcttt tgaaaagttt acgagtttag gattagacaa ccaagtaact 900
          gaactagaca tgagtcttta tggctggcca ccgacagggg cctatacctc ttatgacgac 960
          attccagaag agctttttca agctcaagca gaccgttatg atcagctatt tgagttatat 1020
          gaagaattaa gcgctactat cagtagtgta accttctggg gaattgctga taaccataca 1080
          tggcttgatg accgcgctag agagtacaat aatggagtag gggtcgatgc accatttgtt 1140
          tttgatcaca actatcgagt gaagcctgct tactggagaa ttattgatta a 1191
          
      

S1: Step

S2: Step

S3: steps

Claims (6)

A preparation method of xylo-oligosaccharides, which consists of the following steps: (a) adding a solvent to pineapple peel residue to obtain a liquid mixture of pineapple peel residue, wherein the solid-liquid ratio of the pineapple peel residue to the solvent is 1:1 to 1:30 (w: v), and conduct a hydrothermal reaction at 115°C to 160°C to obtain a liquid mixture of pineapple peel residue after hydrothermal reaction, the solvent is water, and the pineapple peel residue liquid mixture is further added 0.1% to 3% by weight of the acid of the pineapple peel residue; (b) debranching the liquid mixture of the pineapple peel residue after the hydrothermal reaction at 30°C to 80°C in an acidic environment to obtain debranching The reacted pineapple peel residue liquid mixture; and (c) adding endo-xylanase to the pineapple peel residue liquid mixture after the debranching reaction to perform an enzyme reaction to obtain xylo-oligosaccharides. Such as the preparation method of xylo-oligosaccharide of claim 1, wherein the solid-liquid ratio of the pineapple peel residue to the solvent for the hydrothermal reaction in step (a) is 1:7 to 1:15 (w:v). The method for preparing xylo-oligosaccharides according to claim 2, wherein the hydrothermal reaction time is 1.5 to 3 hours. The method for preparing xylo-oligosaccharides according to claim 1, wherein the debranching reaction in step (b) is carried out at 70°C. A preparation method of xylo-oligosaccharides, which consists of the following steps: (a) adding a solvent to pineapple peel residue to obtain a liquid mixture of pineapple peel residue, wherein the solid-liquid ratio of the pineapple peel residue to the solvent is 1:1 to 1:30 (w: v), and conduct a hydrothermal reaction at 115°C to 160°C to obtain a liquid mixture of pineapple peel residue after hydrothermal reaction, the solvent is water, and the pineapple peel residue liquid mixture is further added 0.1% to 3% by weight of the acid of the pineapple peel residue; (b) Debranching reaction of the pineapple peel residue liquid mixture after the hydrothermal reaction is carried out at 30°C to 80°C in an acidic environment, and then separated to remove Pineapple peel residue to obtain a liquid mixture of pineapple peel residue after debranching reaction; and (c) adding endo-xylanase to the liquid mixture of pineapple peel residue after the debranching reaction, and performing an enzyme reaction to obtain xylo-oligosaccharides. A preparation method of xylo-oligosaccharides, which consists of the following steps: (a) adding a solvent to pineapple peel residue to obtain a liquid mixture of pineapple peel residue, wherein the solid-liquid ratio of the pineapple peel residue to the solvent is 1:1 to 1:30 (w: v), and conduct a hydrothermal reaction at 115°C to 160°C to obtain a liquid mixture of pineapple peel residue after hydrothermal reaction, the solvent is water, and the pineapple peel residue liquid mixture is further added 0.1% to 3% by weight of the acid of the pineapple peel residue; (b) debranching the liquid mixture of the pineapple peel residue after the hydrothermal reaction at 30°C to 80°C in an acidic environment to obtain debranching The reacted pineapple peel residue liquid mixture; and (c) adding endo-xylanase to the pineapple peel residue liquid mixture after the debranching reaction, perform an enzyme reaction, and then separate to remove the pineapple peel residue to obtain Xylooligosaccharides.

Images