Compound enzyme preparation and hemicellulose enzymolysis method

Abstract

The invention relates to the field of hemicellulase, in particular to a compound enzyme preparation and a hemicellulose enzymolysis method. The compound enzyme preparation comprises exo-xylooligosaccharide enzyme and endoxylanase of which the reducing ends release xylose, and the compound enzyme preparation is used for enzymolysis of hemicellulose. The enzymolysis method comprises the following steps: carrying out delignification pretreatment on a substrate, and carrying out enzymolysis by using the compound enzyme preparation disclosed by the invention. The xylan degradation efficiency is improved through the synergistic effect of the exo-xylooligosaccharide enzyme releasing xylose at the reducing end and the endoxylanase, so that the xylose yield is improved, and the problem that in the prior art, the degradation effect of single xylanase on hemicellulose is poor is solved.

Description

Technical Field

[0001] The present invention relates to the field of hemicellulases, and particularly to a compound enzyme preparation and a method for enzymatically hydrolyzing hemicellulose. Background Art

[0002] Biomass resources have great development potential. In particular, waste forest biomass and crop straws have significant advantages such as low cost and renewability. Agricultural and forestry biomass belongs to lignocellulosic biomass. Lignocellulose has formed a highly dense and resistant structure during the long-term evolution process, and its main components include cellulose, hemicellulose, and lignin. Among them, cellulose, as a natural glycosyl polymer rich in nature, has been widely used in industries such as energy, chemical engineering, and materials. Experiments on degrading the relatively abundant hemicellulose into small molecule compounds by biological and / or chemical means have also been successful and applied in industrial production. However, compared with cellulose, due to the heterogeneity of its structure, the development and utilization of hemicellulose macromolecules have not received enough attention.

[0003] Xylan is widely distributed in the cell walls of higher plants. It is a huge renewable biological resource in nature and is also a type of hemicellulose that is relatively easy to extract, degrade, and utilize. Endo-β-1,4-xylanase and β-xylosidase are currently the main commonly used enzymes for degrading xylan and are also the enzymes that have been studied more. However, the complex structure of hemicellulose requires the combined action of multiple enzymes to complete depolymerization, and the degradation effects of single enzymes reported so far are not ideal. Summary of the Invention

[0004] The purpose of the present invention is to provide a compound enzyme preparation with a high xylose yield and a method for enzymatically hydrolyzing hemicellulose.

[0005] To achieve the above purpose, in the first aspect, the present invention provides the following technical solution: A compound enzyme preparation, comprising an exo-oligoxylanase that releases xylose from the reducing end and an endo-xylanase, and the compound enzyme preparation is used for the enzymatic hydrolysis of hemicellulose.

[0006] Optionally, the endo-xylanase is endo-β-1,4-xylanase, and the sequence is SEQ ID NO: 1.

[0007] Optionally, the sequence of the exo-oligoxylanase that releases xylose from the reducing end is SEQ ID NO: 2.

[0008] Optionally, the mass ratio of the endo-xylanase to the exo-oligoxylanase that releases xylose from the reducing end is any value in (2~10):1.

[0009] Second aspect, the present invention also provides a method for enzymatic hydrolysis of hemicellulose, including: Pretreat the substrate to remove lignin and obtain dry matter; Add buffer solution and enzyme solution to the dry matter, and add water to make up the volume of the reaction system. After reacting at the first temperature for the first time, filter to obtain the hydrolysis solution; The enzyme solution includes the above-mentioned compound enzyme preparation.

[0010] Optionally, the first temperature is any value in the range of 40°C to 55°C.

[0011] Optionally, the pH value of the buffer solution is any value in the range of 5.0 to 6.5.

[0012] Optionally, at least 300 U of the compound enzyme preparation is added for enzymatic hydrolysis of each gram of the dry matter.

[0013] Optionally, the substrate is one or more of corn straw, wheat straw, sweet sorghum straw, sugarcane straw, rice straw and reed straw.

[0014] The beneficial effect of the present invention is that: the exo-oligoxylanase (Rex enzyme) that releases xylose from the reducing end and endo-xylanase produce a synergistic effect, improving the enzymatic hydrolysis ability of hemicellulose. Different from endo-β-1,4-xylanase that degrades glycosides from the middle of the glycoside chain of xylan, the Rex enzyme can release xylose from the reducing end of xylo-oligosaccharide and participate in the degradation process of xylan. When the two cooperate to carry out the enzymatic hydrolysis of hemicellulose, endo-xylanase cuts from the middle of the glycoside chain, thereby converting into glycoside chains with shorter lengths, while the Rex enzyme can release xylose from the reducing end, thus assisting endo-xylanase to promote the degradation of hemicellulose and improving the xylose yield.

[0015] The above description is only an overview of the technical solution of the present invention. In order to be able to understand the technical means of the present invention more clearly and implement it according to the content of the specification, the following takes the preferred embodiments of the present invention and combines with the drawings to describe in detail as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a flowchart of the method for enzymatic hydrolysis of hemicellulose shown in Embodiment 1 of the present invention; Figure 2 It is a graph comparing the activities of BhRXyn and BaRXyn at different pH values shown in Embodiment 1 of the present invention; Figure 3 It is a graph comparing the activities of BhRXyn and BaRXyn reacting for different times at different temperatures shown in Embodiment 1 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0017] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0018] In the description of the present invention, it should be noted that the orientation or positional relationship indicated by the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. is based on the orientation or positional relationship shown in the drawings. It is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus should not be construed as a limitation to the present invention. In addition, the terms "first", "second", "third" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance.

[0019] In the description of the present invention, it should be noted that unless otherwise clearly specified and defined, the terms "mounted", "connected", "coupled" should be understood in a broad sense. For example, it may be a fixed connection, a detachable connection, or an integral connection; it may be a mechanical connection or an electrical connection; it may be directly connected or indirectly connected through an intermediate medium, and it may be the communication inside two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.

[0020] In addition, the technical features involved in different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0021] Term Explanation: Hemicellulose: It is a heterogeneous polymer composed of several different types of monosaccharides, which are pentoses and hexoses, including xylose, arabinose, and galactose, etc. It is widely distributed in the cell walls of higher plants. Xylan is the main component of plant hemicellulose and is a complex poly-pentose.

[0022] Xylose: The monosaccharide obtained by hydrolysis of xylan.

[0023] Xylooligosaccharide: The polysaccharide with a low degree of polymerization obtained by hydrolysis of xylan.

[0024] Endo-xylanase: It includes endo-β-1,4-xylanase, which cleaves the β-1,4 glycosidic bond from the inside of the xylan chain, releasing xylooligosaccharides and a small amount of xylose.

[0025] Exo-oligoxylanase that releases xylose from the reducing end (Rex enzyme): It can release xylose from the reducing end of xylooligosaccharides and belongs to glycoside hydrolase family 8 (GH8).

[0026] The complex structure of hemicellulose results in the need for the combined action of multiple enzymes to complete depolymerization, and the degradation effects of single enzymes reported so far are not satisfactory. The inventors studied the Rex enzyme adapted to endo-xylanase and obtained a compound enzyme preparation.

[0027] The compound enzyme preparation protected by this invention application includes exo-oligoxylanase that releases xylose from the reducing end and endo-xylanase, and the compound enzyme preparation is used for the enzymatic hydrolysis of hemicellulose.

[0028] The exo-oligoxylanase (Rex enzyme) that releases xylose from the reducing end and endo-xylanase produce a synergistic effect, improving the enzymatic hydrolysis ability of hemicellulose. Different from endo-β-1,4-xylanase that degrades glycosides from the middle of the glycoside chain of xylan, the Rex enzyme can release xylose from the reducing end of xylo-oligosaccharide and participate in the degradation process of xylan. When the two cooperate in the enzymatic hydrolysis of hemicellulose, endo-xylanase cuts from the middle of the glycoside chain, thus converting to a glycoside chain with a shorter length, while the Rex enzyme can release xylose from the reducing end, thereby assisting endo-xylanase to promote the degradation of hemicellulose and increasing the xylose yield.

[0029] Optionally, the endo-xylanase is endo-β-1,4-xylanase, and the sequence is SEQ ID NO: 1.

[0030] Optionally, the sequence of the exo-oligoxylanase that releases xylose from the reducing end is SEQ ID NO: 2.

[0031] Optionally, the mass ratio of endo-xylanase to the exo-oligoxylanase that releases xylose from the reducing end is any value in (2-10):1, for example, it can be any value in 4:1, 6:1, 8:1, and 10:1.

[0032] This invention also applies for the protection of a method for enzymatic hydrolysis of hemicellulose, including: S1. Pretreat the substrate to remove lignin and obtain dry matter.

[0033] S2. Add buffer solution and enzyme solution to the dry matter, and add water to make up the volume of the reaction system. After reacting at the first temperature for the first time, filter to obtain the hydrolysis solution.

[0034] The enzyme solution in step S2 includes the above-mentioned compound enzyme preparation.

[0035] Optionally, the first temperature is any value in 40°C - 55°C, for example, it can be any value in 40°C, 45°C, 50°C, and 55°C.

[0036] Optionally, the pH value of the buffer solution is any value in the range of 5.0 to 6.5, for example, it can be any value among 5.0, 5.5, 6.0, and 6.5.

[0037] Optionally, at least 300 U of the compound enzyme preparation is added per gram of dry matter for enzymatic hydrolysis, for example, it can be any value among 300 U, 400 U, 500 U, and 600 U.

[0038] Optionally, the substrate is one or more of corn straw, wheat straw, sweet sorghum straw, sugarcane straw, rice straw, and reed straw.

[0039] For details, please refer to the following examples and comparative examples.

[0040] Example 1: The compound enzyme preparation shown in a preferred embodiment of the present application includes an exo-oligoxylanase (Rex enzyme) that releases xylose at the reducing end and an endo-xylanase, which is used for the enzymatic hydrolysis of hemicellulose. Among them, the endo-xylanase selects the common enzyme "endo-β-1,4-xylanase" that degrades xylan, which is referred to as xylanase b hereinafter. Xylanase b is derived from Penicillium species, and its amino acid sequence is SEQ ID NO: 1. This enzyme is a commonly used enzyme in the art and is directly obtained by purchase. The Rex enzyme selects two types, namely BhRXyn derived from Bacillus halodurans and BaRXyn derived from Bifidobacterium adolescentis. Among them, the amino acid sequence of BhRXyn is SEQID NO: 2, and the amino acid sequence of BaRXyn is SEQ ID NO: 3.

[0041] According to the amino acid sequences of BhRXyn and BaRXyn, the corresponding gene sequences are designed. After synthesis, the nucleotide fragments corresponding to the two Rex enzymes are connected to the pHKA vector by PCR linearization to obtain recombinant plasmids pHKA-BhRXyn and pHKA-BaRXyn. The plasmids are synthesized by Beijing Tsingke Biotechnology Co., Ltd.

[0042] Inoculate Pichia pastoris GS115 into 5 mL of yeast extract peptone dextrose (YPD) liquid medium, and place it in a shaker at 30 °C and 200 rpm for overnight culture. After the culture is completed, transfer it to a 500 mL YPD shake flask and place it in a shaker at 30 °C and 200 rpm again until the OD of the culture solution 600The value is approximately 1.5. Centrifuge at 4000 rpm for 5 min at 4 °C, collect the cell pellet, and resuspend it in 500 mL of pre-cooled sterile water. Centrifuge again and resuspend in 250 mL of pre-cooled sterile water. Centrifuge again, discard the supernatant, and resuspend in 20 mL of pre-cooled 1 mol / L sorbitol to obtain competent cells for storage. 1 L of YPD liquid medium contains 10 g of yeast extract, 20 g of peptone, and 20 g of glucose.

[0043] Take 80 μL of competent cells and mix them with the recombinant plasmids pHKA - BhRXyn and pHKA - BaRXyn respectively, and transfer them into a pre-cooled 0.2 cm electroporation cuvette. Place it on ice for 5 min, perform electroporation using the recommended Pichia pastoris parameters of the device, then immediately add 1 mL of pre-cooled 1 mol / L sorbitol to the cuvette, transfer the contents to a sterile centrifuge tube, incubate at 28 °C for 1 h, and spread on a basal dextrose (MD) solid plate and culture at 30 °C to obtain recombinant strain monoclonal colonies. The MD solid plate is solidified from the MD medium placed in the plate. 1 L of MD medium contains 13.4 g of yeast nitrogen base without amino acids (YNB), 0.0004 g of D-biotin, and 20 g of D-glucose.

[0044] Pick monoclonal colonies of the two recombinant strains respectively and inoculate them into 50 mL of buffered glycerol-complex medium (BMGY) liquid medium. After culturing in a shaker at 30 °C and 200 rpm for 48 h, adjust the temperature to 28 °C for culturing, and add 300 μL of methanol every 24 h for induction. After a total of 144 h of induction, centrifuge at 8000 rpm for 5 min at 4 °C, and collect the supernatant to obtain the crude enzyme solution. 1 L of BMGY liquid medium contains 10 g of yeast extract, 20 g of peptone, 100 mL of 1 mol / L potassium phosphate buffer with a pH of 6.0, 0.0004 g of biotin, 10 mL of glycerol, 0.04 g of histidine, and 13.4 g of YNB.

[0045] Weigh ammonium sulfate and dissolve it in the crude enzyme solution. Add it in small amounts and stir slowly until the final concentration of ammonium sulfate reaches 80%. Centrifuge at 12000 rpm for 20 min at 4 °C and collect the precipitate. Redissolve the precipitate with a 50 mmol / L Tris-HCl buffer with a pH of 7.8 and centrifuge again. Dialyze the supernatant with Tris-HCl buffer at 4 °C. Concentrate the dialysate through an ultrafiltration tube, add glycerol to make the final concentration of glycerol 30% to obtain the BhRXyn enzyme solution and the BaRXyn enzyme solution, and store them at -20 °C for later use.

[0046] Dilute the obtained enzyme solution appropriately and measure the protein concentration through the protein concentration kit SK3041 (Shanghai Sangon Biotech Co., Ltd.) to make the concentrations of the diluted enzyme solutions the same.

[0047] Take seven portions of 500 mmol / L citric acid - disodium hydrogen phosphate buffer, and adjust the pH values to 4, 4.5, 5.2, 5.5, 6, 7, and 8 respectively. Take two portions from each portion of the citric acid - disodium hydrogen phosphate buffer, add 100 μL of BhRXyn enzyme solution and BaRXyn enzyme solution with the same concentration respectively, and then add 100 μL of xylotriose substrate with a concentration of 1% to form a reaction system. Incubate in a water bath at 40 °C for 15 min to obtain the hydrolysates formed by enzymatic hydrolysis at different pH values. Quantitatively determine the xylose concentration in each hydrolysate by liquid chromatography. Define the enzyme amount required to release 1 μmol of xylose per minute as 1 enzyme activity unit U. Taking the activity of the BhRXyn enzyme solution at pH 7 and temperature 40 °C as the benchmark, calculate the relative enzyme activity (%) and draw a line graph for statistics. Please refer to Figure 2 , it can be seen that the optimal pH value of BhRXyn is 7, and the optimal pH value of BaRXyn is 6.

[0048] Construct a reaction system with citric acid - disodium hydrogen phosphate buffer at pH 7 in the same way, and incubate the same reaction system in a water bath at 37 °C, 40 °C, 50 °C, or 60 °C for 15 min and 5 h respectively to obtain the hydrolysates formed by enzymatic hydrolysis at different temperatures for different times. Quantitatively determine the xylose concentration in each hydrolysate by liquid chromatography. Define the enzyme amount required to release 1 μmol of xylose per minute as 1 enzyme activity unit U. Taking the activity of the BhRXyn enzyme solution at pH 7 and temperature 40 °C as the benchmark, calculate the relative enzyme activity (%) and draw a line graph for statistics. Please refer to Figure 3 , it can be seen that the optimal temperature of both BhRXyn and BaRXyn in this application is 40 °C, and both can maintain high thermal stability in the temperature range of 37 °C to 50 °C.

[0049] Compare the specific enzyme activities of xylanase b, BhRXyn, and BaRXyn on xylotriose and beechwood xylan. According to the prior art, the optimal pH value of xylanase b is 5.0, the optimal temperature is 60 °C, it has a relative enzyme activity of more than 70% in the range of 40 °C to 60 °C, and has a relative enzyme activity of more than 85% in the range of pH 4.0 to 7.0. Combining the properties of the three enzymes, select pH 6.0 and 40 °C as the enzymatic hydrolysis conditions.

[0050] Take 100 μL of 1% xylo-oligosaccharide substrate and 100 μL of 1% beechwood xylan, mix them with 100 μL of three enzyme solutions with the same concentration respectively, add them to the citric acid-disodium hydrogen phosphate buffer solution with a pH value of 6.0, and react in a water bath at 40 °C for 15 min. Then, quantitatively determine the xylose concentration in the experimental hydrolysate by liquid chromatography. Calculate the enzyme activity (U / mL) and the crude enzyme solution protein concentration (mg / mL) to obtain the specific enzyme activity (U / mg). The specific enzyme activities of the three enzymes are shown in Table 1 below.

[0051]

[0052] As can be seen from Table 1, both of the two Rex enzymes in this application have relatively high activity towards xylo-oligosaccharide and certain activity towards beechwood xylan. The specific enzyme activities of BhRXyn towards xylo-oligosaccharide and beechwood xylan are slightly higher than those of BaRXyn.

[0053] The two Rex enzymes and xylanase b were respectively compounded at a mass ratio of 1:10, and the enzyme solutions containing the two Rex enzymes, xylanase b, and the two compound enzyme preparations were used to enzymatically hydrolyze the hemicellulose in wheat straw. Please refer to Figure 1 The method for enzymatically hydrolyzing hemicellulose includes: S1. Pretreat the substrate to remove lignin to obtain dry matter.

[0054] S2. Add buffer solution and enzyme solution to the dry matter, and make up the volume of the reaction system with water. After reacting at the first temperature for the first time, filter to obtain the hydrolysate.

[0055] In step S1, the pretreatment method includes: Immerse the substrate containing hemicellulose in a 5.3% ammonium sulfite solution and react with high-temperature steam at 190 °C for 1 hour to obtain dry matter containing hemicellulose.

[0056] In this experiment, wheat straw was used as the substrate, with a mass of 2.616 g, and 1 g of dry matter was obtained after pretreatment.

[0057] In step S2 of this experiment, the buffer solution used was 1 mol / L acetic acid-sodium acetate buffer solution, and the pH value of the buffer solution was 6.0, with an addition amount of 2.5 mL. The addition amount of the enzyme solution was 500 U of enzyme per gram of dry matter. The volume of the reaction system was 50 mL. The reaction system was placed in a water bath shaker at 40 °C for enzymatic hydrolysis for 5 h, and solid-liquid separation was carried out with a 300-mesh mesh bag to obtain the hydrolysate.

[0058] In the present invention, the contents of xylose and its oligosaccharides in the hydrolysate are detected by liquid chromatography, and the sugar yields are calculated respectively. Since liquid chromatography can only detect xylose to xyloheptaose without branches, in order to more accurately compare the degradation effects of different enzymes and compound enzyme preparations, an equal volume of 8% (m / v) sulfuric acid is added to the hydrolysate obtained in step S2, and it is acid-hydrolyzed in an autoclave at a temperature of 121 °C for 60 min. The pH value of the acid-hydrolyzed solution is adjusted to be within the range of 5-7 with sodium hydroxide. After acid hydrolysis, various xylo-oligosaccharides are degraded and converted into xylose. The xylose content after acid hydrolysis is detected and the xylose yield is calculated.

[0059]

[0060] As can be seen from Table 2, the combination of Rex enzyme and endo-β-1,4-xylanase has a significant synergistic effect on the hydrolysis of pretreated wheat straw. The xylose yield of the hydrolysate obtained by the hydrolysis of the two compound enzyme preparations after acid hydrolysis is significantly higher than that of the hydrolysate obtained by single enzyme hydrolysis. The effect of the combination of BhRXyn and xylanase b is better than that of BaRXyn. Therefore, in the subsequent examples, BhRXyn and xylanase b are selected for combination.

[0061] In order to further explore the optimal ratio, pH, temperature and addition amount of the compound enzyme preparation, multiple groups of experiments are carried out by adjusting variables.

[0062] The ratio of the compound enzyme preparation in the enzyme solution is adjusted, and the xylose yield is detected after enzymatic hydrolysis and acid hydrolysis respectively. The ratio of the compound enzyme preparation and the detected xylose yield are shown in Table 3 below.

[0063]

[0064] As can be seen from Table 3, when the ratio of xylanase b to BhRXyn is in the range of (2-10):1, the xylose yield after enzymatic hydrolysis and acid hydrolysis is relatively high. Especially when the ratio of xylanase b to BhRXyn is 5:1, the xylose yield after enzymatic hydrolysis and acid hydrolysis can reach 18.21.

[0065] The pH value of the buffer solution in step S2 is adjusted, and enzymatic hydrolysis is carried out with an enzyme solution containing a compound enzyme preparation with a ratio of xylanase b to BhRXyn of 5:1. The xylose yields after enzymatic hydrolysis and acid hydrolysis are detected respectively. The pH value of the buffer solution and the detection results are shown in Table 4 below.

[0066]

[0067] As can be seen from Table 4, the influence of pH on the hydrolysis system is small. When the hydrolysis pH is maintained at 5.5, the highest xylose yield after acid hydrolysis reaches 19.52%.

[0068] Adjust the water bath temperature in step S2 to make the pH value of the buffer solution 5.5, and perform enzymatic hydrolysis with an enzyme solution containing a compound enzyme preparation with the ratio of xylanase b and BhRXyn being 5:1. Respectively detect the xylose yields after enzymatic hydrolysis and acid hydrolysis. The enzymatic hydrolysis temperature and the detection results are shown in Table 5 below.

[0069]

[0070] As can be seen from Table 5, temperature has a greater impact on enzymatic hydrolysis. When the enzymatic hydrolysis temperature is maintained at 50 °C, the highest xylose yield after acid hydrolysis reaches 23.04%. The possible reason is that the optimal temperature of xylanase b is higher than that of BhRXyn, and increasing the temperature further improves the activity of xylanase b. However, when the temperature exceeds 60 °C, the thermal stabilities of both enzymes decrease, which is not conducive to long-term hydrolysis.

[0071] Adjust the addition amount of the enzyme solution in step S2, thereby adjusting the addition amount of the compound enzyme preparation, make the pH value of the buffer solution 5.5, the enzymatic hydrolysis temperature 50 °C, and perform enzymatic hydrolysis with an enzyme solution containing a compound enzyme preparation with the ratio of xylanase b and BhRXyn being 5:1. Respectively detect the xylose yields after enzymatic hydrolysis and acid hydrolysis. The addition amount of the compound enzyme preparation and the detection results are shown in Table 6 below.

[0072]

[0073] As can be seen from Table 6, when the addition amount of the compound enzyme is between 300 U / g dry matter and 600 U / g dry matter, the xylose yield after acid hydrolysis does not change significantly. Therefore, in order to reduce costs, the addition amount of 300 U / g dry matter is the optimal.

[0074] Comparative Example 1: Use β-xylosidase, arabinofuranosidase and BhRXyn to be compounded with xylanase b at a ratio of 1:5 respectively, and pretreat, enzymatically hydrolyze and acid hydrolyze wheat straw in the same method as in Example 1, and detect the sugar content in the hydrolysis solution by liquid chromatography and calculate the xylose yield. In this comparative example, β-xylosidase and arabinofuranosidase are purchased from Megazyme company. The addition amount of the compound enzyme preparation during the enzymatic hydrolysis process is 300 U / g dry matter, the pH value of the buffer solution is 5.5, and the reaction temperature is 50 °C. The detection results are shown in Table 7 below.

[0075]

[0076] It can be clearly seen from the experimental data that the combination of xylanase b and arabinofuranosidase has little effect on enhancing the ability to hydrolyze xylan, and the increase in xylose yield after combination with β-xylosidase is also small. This proves that not all hemicellulases can cooperate with xylanase b in degrading xylan, and it is confirmed that the complex enzyme preparation obtained by combining Rex enzyme (BhRXyn) with xylanase b has higher efficiency and can convert hemicellulose into xylose more effectively, demonstrating the significant advantage of Rex enzyme in synergistically degrading hemicellulose with xylanase to produce xylose.

[0077] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: The exo-oligoxylanase (Rex enzyme) that releases xylose from the reducing end of the present application can be combined with endo-β-1,4-xylanase to efficiently degrade hemicellulose, showing great potential in the industrial production of xylose and xylo-oligosaccharides. Moreover, the endo-β-1,4-xylanase used in the present invention is just any selected endo-xylanase without screening and modification. Therefore, better hydrolysis effects should be achieved by combining a more efficient endo-xylanase with the Rex enzyme.

[0078] The technical features of the above embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0079] The above embodiments only represent several implementation manners of the present invention, and the description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A composite enzyme preparation, characterized in that: The invention comprises an exo-oligoxylanase and an endo-xylanase for releasing xylose at the reducing end. The composite enzyme preparation is used for enzymolysis of hemicellulose.

2. The composite enzyme preparation according to claim 1, characterized in that The endo-xylanase is endo-β-1,4-xylanase, and its sequence is SEQ ID NO:

1.

3. The composite enzyme preparation according to claim 1, characterized in that The sequence of the exo-oligosaccharidase that releases xylose at the reducing end is SEQ ID NO:

2.

4. The composite enzyme preparation according to any one of claims 1 to 3, characterized in that: The mass ratio of the endo-xylanase to the exo-oligo-xylanase that releases xylose at the reducing end is any value in the range of (2-10):

1.

5. A method for enzymatic hydrolysis of hemicellulose, characterized in that: include: Pre-treating the substrate to remove lignin and obtain dry matter; Adding a buffer solution and an enzyme solution to the dry matter, and adding water to make up the volume of the reaction system, reacting at a first temperature for a first time, and filtering to obtain a hydrolyzate; The enzyme solution comprises the compound enzyme preparation according to any one of claims 1 to 4.

6. The method for enzymatic hydrolysis of hemicellulose according to claim 5, characterized in that: The first temperature is any value between 40°C and 55°C.

7. The method for enzymatic hydrolysis of hemicellulose according to claim 5, characterized in that: The pH value of the buffer solution is any value between 5.0 and 6.

5.

8. The method for enzymatic hydrolysis of hemicellulose according to claim 5, characterized in that: At least 300 U of the composite enzyme preparation is added per gram of the dry matter for enzymatic hydrolysis.

9. The method for enzymatic hydrolysis of hemicellulose according to claim 5, characterized in that: The substrate is one or more of corn straw, wheat straw, sweet sorghum straw, sugarcane straw, rice straw and reed straw.

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