Preparation method of xylooligosaccharide and xylose based on compound enzyme

Abstract

The invention discloses a preparation method of xylooligosaccharide and xylose based on a compound enzyme, and relates to the technical field of xylanase catalytic enzymolysis. The preparation method comprises the following steps: adding a compound enzyme into fiber pulp to prepare an enzymolysis mixed solution, and carrying out enzymolysis reaction on the enzymolysis mixed solution to prepare xylose and xylooligosaccharide, wherein the compound enzyme is a composition of xylanase Xyl1 and xylanase Xyl2, the sequence of the xylanase Xyl1 is as shown in SEQ ID NO: 1, the sequence of the xylanase Xyl2 is as shown in SEQ ID NO: 2, and the enzyme activity unit ratio of the xylanase Xyl1 to the xylanase Xyl2 is any one of 1: 10-10: 1. The xylanase Xyl1 and the xylanase Xyl2 are used for preparing the xylooligosaccharide and the xylose through catalytic enzymolysis, and the enzymolysis efficiency of xylan production through catalysis of the compound enzyme is improved.

Description

Technical Field

[0001] The invention relates to the technical field of xylanase-catalyzed enzymolysis, and in particular to a method for preparing xylooligosaccharides and xylose based on a composite enzyme. Background Art

[0002] Xylan is an abundant hemicellulose component in plant cell walls, and its degradation products such as oligoxylose and xylose have wide application value in food, medicine, fermentation and bioenergy. Xylan is mainly prepared by acid hydrolysis, enzymatic hydrolysis or their combination, among which the enzymatic method has become a research hotspot due to its mild reaction conditions and controllable product structure.

[0003] Xylanases are mainly divided into two families, GH10 and GH11, according to their structure and mode of action. GH10 xylanases have a wide substrate adaptability and can hydrolyze xylan more thoroughly to produce more xylose, while GH11 xylanases often show selective cutting at specific sites on the main chain and are often used to prepare xylooligosaccharides. Previous studies have shown that a reasonable combination of GH10 and GH11 enzymes can achieve a balance between hydrolysis efficiency and product distribution. However, most enzymatic hydrolysis systems currently still have insufficient enzyme source diversity, poor industrial controllability, and lack of systematic optimization of enzyme ratios, enzymatic pH, time, temperature and other parameters, resulting in difficulty in regulating product composition and conversion competition between xylose and oligosaccharides, making it difficult to achieve technical problems such as directional product enrichment.

[0004] Therefore, there is an urgent need to develop a composite enzymatic hydrolysis method with adjustable product composition, good tolerance for reaction conditions, and a clear enzyme combination design strategy to improve the synergistic preparation efficiency of oligosaccharides and xylose and meet the needs of different application scenarios. Summary of the invention

[0005] One object of the present invention is to provide a method for preparing xylooligosaccharides and xylose based on a composite enzyme, so as to solve the technical problems in the prior art of low efficiency of single xylanase catalytic hydrolysis and poor efficiency of polyxylanase synergistic hydrolysis.

[0006] Another object of the present invention is to further improve the enzymatic hydrolysis rate and the yield of enzymatic hydrolysis products of the composite enzymatic hydrolysis system.

[0007] According to the purpose of the present invention, the present invention provides a method for preparing xylooligosaccharides and xylose based on a composite enzyme, comprising: preparing a substrate containing hemicellulose to obtain fiber pulp; Adding a complex enzyme into the fiber pulp to prepare an enzymatic mixed solution, and subjecting the enzymatic mixed solution to an enzymatic reaction to prepare xylose and xylooligosaccharides; The complex enzyme is a combination of xylanase Xyl1 and xylanase Xyl2, the sequence of the xylanase Xyl1 is such as SEQ ID NO: 1, the sequence of the xylanase Xyl2 is such as SEQ ID NO: 2, the enzyme activity unit ratio of the xylanase Xyl1 to the xylanase Xyl2 is any value between 1:10 and 10:1, the enzymolysis temperature is any value between 30°C and 60°C, the enzymolysis pH value is any value between 4.0 and 7.0, and the enzymolysis time is any value between 4h and 12h.

[0008] Optionally, the enzyme activity unit ratio of the xylanase Xyl1 to the xylanase Xyl2 is any value between 2:1 and 1:2.

[0009] Optionally, the added amount of the complex enzyme is any value between 200U / g and 1000U / g.

[0010] Optionally, the enzymolysis temperature is any value between 40°C and 50°C, the enzymolysis pH is any value between 4.5 and 5.5, and the enzymolysis time is any value between 5h and 8h.

[0011] Optionally, the hemicellulose-containing substrate is any one of oat xylan, birch xylan, beech xylan and straw fiber pulp.

[0012] Optionally, the raw material of the straw fiber pulp is any one of corn straw, wheat straw, sweet sorghum straw, sugarcane straw, rice straw or reed straw.

[0013] Optionally, the raw material is pretreated to prepare the straw fiber pulp, and the pretreatment method is to soak the raw material in a sodium hydroxide solution for a preset time.

[0014] Optionally, the mass volume concentration of the sodium hydroxide solution is any value between 10% and 30%.

[0015] Optionally, the buffer solution of the complex enzyme is any one of acetic acid-sodium acetate buffer, citric acid-phosphate buffer or phosphate buffer.

[0016] The present invention adds a composite enzyme formed by compounding GH10 type xylanase Xyl1 and GH11 type xylanase Xyl2 derived from the same microorganism into fiber pulp, and controls the enzyme activity unit ratio of GH10 type xylanase Xyl1 and GH11 type xylanase Xyl2 in the composite enzyme, and the enzymatic hydrolysis temperature, enzymatic hydrolysis pH and enzymatic hydrolysis time of an enzymatic hydrolysis mixed solution formed by the composite enzyme and the fiber pulp, so that the enzymatic hydrolysis reaction conditions are simultaneously applicable to the GH10 type xylanase Xyl1 and the GH11 type xylanase Xyl2, thereby improving the maximum enzyme activity of the composite enzyme within the above reaction conditions, thereby improving the enzymatic hydrolysis efficiency of the composite enzyme on the fiber pulp, and making the GH10 type xylanase Xyl1 and GH11 type xylanase Xyl2 have a higher enzymatic activity than the GH10 type xylanase Xyl1. Type 10 xylanase Xyl1 and GH11 xylanase Xyl2 work synergistically to enzymatically hydrolyze fiber pulp to produce xylooligosaccharides and xylose, and the ratio of xylooligosaccharides to xylose in the enzymatic hydrolysis products and the xylose yield are regulated by adjusting the enzyme activity unit ratio of GH10 xylanase Xyl1 and GH11 xylanase Xyl2 and the reaction conditions, thereby constructing a synergistic, efficient, and product composition-controllable composite enzymatic hydrolysis system, breaking through the limitations of traditional single enzyme systems in reaction efficiency and target product yield, achieving precise regulation of xylan enzymatic hydrolysis products, and improving the purity and yield of the target product, so as to meet the differentiated needs for xylooligosaccharides and xylose in different application scenarios in industrial production.

[0017] Furthermore, the present invention sets the added amount of the complex enzyme in each gram of substrate to any value between 200U and 1000U, thereby adjusting the enzyme dosage, regulating the depth of the hydrolysis reaction and the product composition, and improving the complex enzyme hydrolysis rate and the yield of the hydrolysis product.

[0018] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Hereinafter, some specific embodiments of the present invention will be described in detail in an exemplary and non-limiting manner with reference to the accompanying drawings. The same reference numerals in the accompanying drawings indicate the same or similar components or parts. It should be understood by those skilled in the art that these drawings are not necessarily drawn to scale. In the accompanying drawings: Figure 1 is a schematic flow chart of a preparation method according to one embodiment of the present invention; Figure 2 is a pH response curve diagram of xylanase Xyl1 according to one embodiment of the present invention; Figure 3 is a pH response curve diagram of xylanase Xyl2 according to one embodiment of the present invention; Figure 4 is a temperature response curve diagram of xylanase Xyl1 according to one embodiment of the present invention; Figure 5 1 is a temperature response curve of xylanase Xyl2 according to an embodiment of the present invention. DETAILED DESCRIPTION

[0020] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0021] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail below in conjunction with the accompanying drawings. It is to be understood that the specific embodiments described herein are only used to explain the present application, rather than to limit the present application. It should also be noted that, for ease of description, only some structures related to the present application are shown in the accompanying drawings, rather than all structures. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0022] The terms "including" and "having" and any variations thereof in this application are intended to cover non-exclusive inclusions. For example, a process, method, system, product or device comprising a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to these processes, methods, products or devices.

[0023] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0024] Figure 1 is a schematic flow chart of a preparation method according to one embodiment of the present invention, Figure 2 is a pH response curve diagram of xylanase Xyl1 according to one embodiment of the present invention, Figure 3 is a pH response curve diagram of xylanase Xyl2 according to one embodiment of the present invention, Figure 4 is a temperature response curve of xylanase Xyl1 according to one embodiment of the present invention, Figure 5 1 is a temperature response curve of xylanase Xyl2 according to an embodiment of the present invention.

[0025] like Figure 1 As shown, the present invention provides a method for preparing xylooligosaccharides and xylose based on a composite enzyme, comprising: Step S100: preparing a substrate containing hemicellulose to obtain fiber pulp; Step S200: adding a complex enzyme to the fiber pulp to prepare an enzymatic hydrolysis mixed solution, and subjecting the enzymatic hydrolysis mixed solution to an enzymatic hydrolysis reaction to prepare xylose and xylooligosaccharides; The complex enzyme is a combination of xylanase Xyl1 and xylanase Xyl2, the sequence of xylanase Xyl1 is such as SEQ ID NO: 1, the sequence of xylanase Xyl2 is such as SEQ ID NO: 2, the enzyme activity unit ratio of xylanase Xyl1 to xylanase Xyl2 is any value between 1:10 and 10:1, the enzymolysis temperature is any value between 30°C and 60°C, the enzymolysis pH value is any value between 4.0 and 7.0, and the enzymolysis time is any value between 4h and 12h.

[0026] In this embodiment, a substrate containing hemicellulose is first pretreated to obtain a fiber pulp, and a composite enzyme is added to the fiber pulp to obtain an enzymatic hydrolysis mixed solution, and an enzymatic hydrolysis reaction is performed under controlled reaction conditions to obtain xylooligosaccharides and xylose. The composite enzyme is a composition formed by compounding xylanase Xyl1 and xylanase Xyl2 with an enzyme activity unit ratio of any value in the range of 1:10-10:1, that is, the enzyme activity unit ratio of xylanase Xyl1 and xylanase Xyl2 can be 1:10, 1:5, 1:3, 1:2, 1:1, 2:1, 3:1, 5:1 or 10:1, or any value in the range of 1:10-10:1, and the enzymatic hydrolysis temperature can be 30 ℃, 35℃, 40℃, 45℃, 50℃, 55℃ or 60℃, or any value between 30℃ and 60℃, the enzymatic hydrolysis pH value can be 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 or 7.0, or any value between 4.0 and 7.0, and the enzymatic hydrolysis time can be 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h or 12h, or any value between 4h and 12h. Here, xylanase Xyl1 and xylanase Xyl2 are both derived from Penicillium decumbens, the amino acid sequence of xylanase Xyl1 is shown in SEQ ID NO: 1, and the amino acid sequence of xylanase Xyl2 is shown in SEQ ID NO: 2.

[0027] In this embodiment, a composite enzyme formed by compounding GH10 xylanase Xyl1 and GH11 xylanase Xyl2 derived from the same microorganism is added to the fiber pulp, and the enzymatic activity unit ratio of GH10 xylanase Xyl1 and GH11 xylanase Xyl2 in the composite enzyme, as well as the enzymatic hydrolysis temperature, enzymatic hydrolysis pH and enzymatic hydrolysis time of the enzymatic hydrolysis mixed solution formed by the composite enzyme and the fiber pulp are controlled so that the enzymatic hydrolysis reaction conditions are simultaneously applicable to both GH10 xylanase Xyl1 and GH11 xylanase Xyl2, thereby improving the maximum enzyme activity of the composite enzyme within the above reaction conditions, thereby improving the enzymatic hydrolysis efficiency of the composite enzyme on the fiber pulp, and making G10 xylanase Xyl1 and GH11 xylanase Xyl2 reach the maximum enzyme activity within the above reaction conditions. H10 type xylanase Xyl1 and GH11 type xylanase Xyl2 work synergistically to enzymatically hydrolyze fiber pulp to produce xylooligosaccharides and xylose, and the ratio of xylooligosaccharides to xylose in the enzymatic hydrolysis products and the xylose yield are regulated by adjusting the enzyme activity unit ratio of GH10 type xylanase Xyl1 and GH11 type xylanase Xyl2 and the reaction conditions, thereby constructing a synergistic, efficient, and product composition-controllable composite enzymatic hydrolysis system, breaking through the limitations of traditional single enzyme systems in reaction efficiency and target product yield, achieving precise regulation of xylan enzymatic hydrolysis products, and improving the purity and yield of the target product, so as to meet the differentiated needs for xylooligosaccharides and xylose in different application scenarios in industrial production.

[0028] It should be noted that in the prior art, a single type of xylanase is usually used to enzymatically hydrolyze the xylan substrate, and the enzymes used are mostly single GH10 or GH11 xylanases. Any type of xylanase alone cannot act efficiently on a complex natural substrate system. In addition, due to the poor synergy of various enzymes in the reaction system and the fundamental problem of mismatched synergistic efficiency of different GH family enzymes, multiple variables such as enzyme activity expression, reaction condition adaptation, and substrate structure response are involved. That is, simply combining different types of xylanases to hydrolyze fiber pulp will lead to uncontrollable ratios of xylooligosaccharides to xylose in the hydrolyzate and reduced enzymatic efficiency of a single xylanase in the complex enzyme. In addition, there is a nonlinear coupling relationship between the ratio of the reaction products and the parameters of the reaction conditions, and the optimal product combination cannot be obtained by simple adjustment.

[0029] To this end, the applicant conducted a large number of experiments and found in the specific experimental process that by regulating the enzyme activity unit ratio of GH10 type xylanase Xyl1 and GH11 type xylanase Xyl2 and combining the regulation of enzymatic hydrolysis pH, temperature, and time, the two different types of enzymes can reach a high activity range under the same reaction conditions, realizing a synergistic enzymatic hydrolysis reaction system, and by systematically regulating the enzyme activity ratio and reaction conditions, the ratio of oligoxylose and xylose products can be controlled and adjusted, while greatly improving the xylose yield, providing a technological basis for the high-value utilization of xylan resources.

[0030] In this embodiment, the enzyme activity unit ratio of xylanase Xyl1 to xylanase Xyl2 is any value in the range of 1:10-10:1. The combination of enzyme activity units in different ratios can adjust the leading role of GH10 type xylanase Xyl1 and GH11 type xylanase Xyl2, and realize the directional regulation of the ratio of xylooligosaccharides and xylose. That is, when the enzyme activity unit ratio of GH10 type xylanase Xyl1 is higher, the composite enzymatic hydrolysis system tends to produce xylose, and when the enzyme activity unit ratio of GH11 type xylanase Xyl2 is higher, the product is more inclined to xylooligosaccharides. By regulating the enzyme activity unit ratio, the GH10 type xylanase Xyl1 and GH11 type xylanase Xyl2 can complement each other and optimize their efficiency, thereby improving the overall substrate conversion rate and the yield of the target product, thereby optimizing the synergistic mechanism and avoiding the situation where a certain type of enzyme is inhibited or the enzyme activity resources are wasted due to unsuitable conditions or substrate competition.

[0031] In a preferred embodiment, the enzyme activity unit ratio of xylanase Xyl1 to xylanase Xyl2 is any value in the range of 2:1-1:2, that is, the enzyme activity unit ratio of xylanase Xyl1 to xylanase Xyl2 can be 2:1, 1.5:1, 1:1, 1:1.5 or 1:2, or any value in the range of 2:1-1:2. By setting the enzyme activity unit ratio of xylanase Xyl1 to xylanase Xyl2 within the above range, the synergistic catalytic effect of xylanase Xyl1 and xylanase Xyl2 in the same reaction system can be effectively promoted, and the degradation efficiency of hemicellulose substrates can be improved. At the same time, by adjusting the enzyme activity unit ratio of xylanase Xyl1 to xylanase Xyl2, the product composition can be controlled and adjusted, the purity and yield of the target product can be improved, and a more adaptable and flexible process scheme can be provided for industrial applications such as functional sugar production. Here, the product composition is the ratio of oligoxylose to xylose.

[0032] like Figure 4 and Figure 5 As shown, in this embodiment, the enzymatic hydrolysis temperature is any value between 30℃-60℃, that is, the GH10 type xylanase Xyl1 and GH11 type xylanase Xyl2 derived from Penicillium decumbens maintain high enzyme activity within the range of 30℃-60℃, showing good medium-temperature heat resistance, and can change the affinity and cleavage mode of the enzyme and substrate segment by regulating the enzymatic hydrolysis temperature, thereby adjusting the product chain length and composition. Specifically, the enzymatic hydrolysis temperature at 30℃-40℃ is more conducive to the production of oligoxylose, and the enzymatic hydrolysis temperature at 50℃-60℃ will accelerate the complete hydrolysis of oligoxylose and promote xylose production.

[0033] In a preferred embodiment, the enzymolysis temperature is any value between 40°C and 50°C, that is, the enzymolysis temperature of the composite enzymolysis system can be 40°C, 42°C, 44°C, 46°C, 48°C or 50°C, or any value between 40°C and 50°C. By setting the enzymolysis temperature interval within the above temperature interval, the GH10 type xylanase Xyl1 and the GH11 type xylanase Xyl2 are both in a highly active state, which significantly improves the enzymolysis efficiency and reaction stability of the composite enzyme system. At the same time, the temperature regulation can achieve controllable adjustment of the ratio of oligoxylose to xylose products, meeting the personalized requirements of product structure for different industrial uses. In addition, in most bioreactor systems, the above temperature interval belongs to the medium and low energy consumption interval, which is easy to maintain and can be connected with the temperature control range of conventional fermentation pretreatment, saccharification stage and other processes to reduce energy consumption and equipment costs. Here, the xylanase Xyl1 reacts for 0.5h and 5h at an enzymolysis temperature of 40°C-50°C, respectively (refer to Figure 4 ), xylanase Xyl2 was reacted at an enzymatic temperature of 40℃-50℃ for 0.5h and 5h respectively (refer to Figure 5 ).

[0034] like Figure 2 and Figure 3 As shown, in the present embodiment, the enzymolysis pH value is any value in 4.0-7.0, because GH10 type xylanase Xyl1 and GH11 type xylanase Xyl2 both maintain high catalytic activity in a neutral and acidic environment, by adjusting the enzymolysis pH, the conformational state of the enzyme molecule can be regulated, thereby affecting the enzyme activity peak and substrate affinity, that is, when the enzymolysis pH is 4.0-5.0, the enzyme activity of GH10 type xylanase Xyl1 is higher, which is conducive to xylose generation, and when the enzymolysis pH is 5.0-6.0, the enzyme activity of GH11 type xylanase Xyl2 is higher, which is conducive to retaining oligoxylose. By setting the enzymolysis pH value within the above range, the optimal catalytic activity region of GH10 type xylanase Xyl1 and GH11 type xylanase Xyl2 can be taken into account, ensuring that the complex enzyme can react efficiently under the same system, and realizing synergistic and efficient enzymolysis.

[0035] In a preferred embodiment, the enzymatic hydrolysis pH is any value in the range of 4.5-5.5, that is, the enzymatic hydrolysis pH can be 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4 or 5.5, or any value in the range of 4.5-5.5. By setting the enzymatic hydrolysis pH to any value in the above range, the GH10 type xylanase Xyl1 and the GH11 type xylanase Xyl2 are both in a highly active state, which significantly improves the enzymatic hydrolysis efficiency and reaction stability of the composite enzyme system. Here, the xylanase Xyl1 is reacted at an enzymatic hydrolysis pH of 4.5-5.5 for 0.5h and 5h, respectively (refer to Figure 2), xylanase Xyl2 was reacted at pH 4.5-5.5 for 0.5h and 5h respectively (ref. Figure 3 ).

[0036] In this embodiment, the enzymolysis time is any value in 4h-12h, and the adjustability of the reaction time is determined by different substrate types and target product requirements, that is, the enzymolysis time is 4h-6h, which is conducive to the generation of a high proportion of xylooligosaccharides and prevents further hydrolysis of xylooligosaccharides. When the enzymolysis time is 8h-12h, it helps the xylooligosaccharides to be further hydrolyzed into xylose, which is suitable for the preparation of fermentation base materials or high-xylose products. By setting the enzymolysis time within the above range, the ratio of xylooligosaccharides and xylose in the product can be controlled by regulating the reaction time, avoiding problems such as incomplete conversion due to too short a reaction time or accumulation of by-products due to too long a reaction time, and improving process flexibility and stability.

[0037] In a preferred embodiment, the enzymolysis time is any value between 5h and 8h, that is, the enzymolysis time in the composite enzymolysis system can be 5h, 5.5h, 6h, 6.5h, 7h, 7.5h or 8h, or any value between 5h and 8h. By setting the enzymolysis time within the above range, the hydrolysis conversion of xylan can be effectively achieved, and the dynamic control of the hydrolyzate structure can be achieved by regulating the enzymolysis time to meet different application requirements.

[0038] In a further embodiment, the amount of the complex enzyme added is any value in the range of 200U / g-1000U / g, that is, the amount of the complex enzyme added per gram of substrate is any value in the range of 200U-1000U. In this embodiment, the amount of the complex enzyme added per gram of substrate can be 200U, 300U, 400U, 500U, 600U, 700U, 800U, 900U or 1000U, or any value in the range of 200U-1000U. When the compound enzyme addition amount is 200U-300U, the enzyme activity concentration of the compound enzyme is low, the hydrolysis rate is slow, and the main product is short-chain oligosaccharides. When the compound enzyme addition amount is 400U-600U, the hydrolysis rate is accelerated, and the reaction equilibrium point is more inclined to xylose production. When the compound enzyme addition amount is 700U-1000U, the compound enzymatic hydrolysis system can quickly complete the complete hydrolysis of large molecular xylan, and the proportion of xylose is high, which is conducive to subsequent fermentation or sugar alcohol conversion, but may usually bring about cost increase and by-product problems. By setting the addition amount of compound enzymes per gram of substrate within the above range, the depth of the hydrolysis reaction and the product composition can be regulated by adjusting the enzyme dosage, thereby improving the compound enzyme hydrolysis rate and the yield of the hydrolysis product.

[0039] In a preferred embodiment, the amount of the complex enzyme added per gram of substrate is any value between 400U-600U, that is, the amount of the complex enzyme added per gram of substrate can be 400U, 450U, 500U, 550U or 600U, or any value between 400U-600U. By setting the amount of the complex enzyme added per gram of substrate to any value between 400U-600U, it is possible to ensure that the enzyme activity concentration in the complex enzymatic hydrolysis system is high to increase the enzymatic hydrolysis rate, while also preventing an increase in by-products caused by excessive addition of the complex enzyme.

[0040] In a further embodiment, the substrate containing hemicellulose is any one of oat xylan, birch xylan, beech xylan and straw fiber pulp. In this embodiment, the substrate containing hemicellulose in the composite enzymatic hydrolysis system can be any one of the above xylans or straw fiber pulp, and the composite enzymatic hydrolysis system can enzymatically hydrolyze any one of oat xylan, birch xylan, beech xylan and straw fiber pulp to produce xylo-oligosaccharides and xylose, that is, the composite enzymatic hydrolysis system has adaptability to multiple reaction substrates and broad-spectrum hydrolysis ability.

[0041] In a further embodiment, the raw material of the straw fiber pulp is any one of corn straw, wheat straw, sweet sorghum straw, sugarcane straw, rice straw or reed straw, and the raw material is pretreated to prepare the straw fiber pulp, and the pretreatment method is to soak the raw material in a sodium hydroxide solution for a preset time. In this embodiment, the corn straw, wheat straw, sweet sorghum straw, sugarcane straw, rice straw or reed straw is pretreated with a sodium hydroxide solution to remove lignin in the straw, and the straw fiber pulp that can be directly enzymatically hydrolyzed by the composite enzymatic hydrolysis system is prepared to prevent the lignin present in the straw from reducing the enzymatic activity of the xylanase in the composite enzymatic hydrolysis system.

[0042] In a further embodiment, the mass volume concentration of the sodium hydroxide solution is any value between 10% and 30%, that is, the mass volume concentration of the sodium hydroxide solution can be 10%, 15%, 20%, 25% or 30%, or any value between 10% and 30%. In this embodiment, by setting the sodium hydroxide solution in the pretreatment within the above range, the looseness and accessibility of the substrate structure can be adjusted, and the lignin can be partially removed or softened, reducing the nonspecific adsorption of the complex enzyme and lignin, thereby improving the conversion efficiency of xylan to oligosaccharides or xylose.

[0043] In a further embodiment, the buffer solution of the complex enzyme is any one of acetic acid-sodium acetate buffer, citric acid-phosphate buffer or phosphate buffer. In the present embodiment, in the acetic acid-sodium acetate buffer system, the complex enzyme is in the most suitable acidic environment, which is conducive to maximizing the catalytic efficiency and is suitable for oligosaccharide product enrichment process. In the citric acid-phosphate buffer, the pH adjustment range is wide, which is suitable for multiple enzyme synergistic systems and dynamic reaction processes, and in the phosphate buffer, the enzyme activity is maintained stable, which is helpful for product structure control, and is particularly suitable for xylose retention and subsequent fermentation requirements. The above buffer solutions can effectively support the efficient conduct of the complex enzymolysis reaction in the present invention.

[0044] The present application will be further described in detail below with reference to specific embodiments.

[0045] Example 1 The composite enzyme is added to corn straw fiber pulp to prepare an enzymatic hydrolysis mixed solution, and the enzymatic hydrolysis mixed solution is subjected to an enzymatic hydrolysis reaction to prepare xylose and xylo-oligosaccharides, wherein the enzyme activity unit ratio of xylanase Xyl1 (GH10, Penicilliumdecumbens, SEQ ID NO:1) and xylanase Xyl2 (GH11, Penicillium decumbens, SEQ ID NO:2) in the composite enzyme is 10:1, the addition amount of the composite enzyme per gram of fiber pulp is 500U, the enzymatic hydrolysis temperature is 45°C, the enzymatic hydrolysis pH value is 5.0, the enzymatic hydrolysis time is 7h, and the buffer solution of the composite enzyme is an acetic acid-sodium acetate buffer with a final concentration of 1mM.

[0046] Example 2 The only difference between Example 2 and Example 1 is that the ratio of the enzyme activity units of xylanase Xyl1 and xylanase Xyl2 in the composite enzyme is 5:1.

[0047] Example 3 The only difference between Example 3 and Example 1 is that the ratio of the enzyme activity units of xylanase Xyl1 and xylanase Xyl2 in the composite enzyme is 3:1.

[0048] Example 4 The only difference between Example 4 and Example 1 is that the ratio of the enzyme activity units of xylanase Xyl1 and xylanase Xyl2 in the composite enzyme is 2:1.

[0049] Example 5 The only difference between Example 5 and Example 1 is that the ratio of the enzyme activity units of xylanase Xyl1 and xylanase Xyl2 in the composite enzyme is 1:1.

[0050] Example 6 The only difference between Example 6 and Example 1 is that the ratio of the enzyme activity units of xylanase Xyl1 and xylanase Xyl2 in the composite enzyme is 1:2.

[0051] Example 7 The only difference between Example 7 and Example 1 is that the ratio of the enzyme activity units of xylanase Xyl1 and xylanase Xyl2 in the composite enzyme is 1:3.

[0052] Example 8 The only difference between Example 8 and Example 1 is that the ratio of the enzyme activity units of xylanase Xyl1 and xylanase Xyl2 in the composite enzyme is 1:5.

[0053] Example 9 The only difference between Example 9 and Example 1 is that the ratio of the enzyme activity units of xylanase Xyl1 and xylanase Xyl2 in the composite enzyme is 1:10.

[0054] Comparative Example 1 The only difference between Comparative Example 1 and Example 1 is that the complex enzyme does not include xylanase Xyl2.

[0055] Comparative Example 2 The only difference between Comparative Example 2 and Example 1 is that the complex enzyme does not include xylanase Xyl1.

[0056] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that the complex enzyme only includes xylanase Xyl3 (GH11, Penicillium decumbens, SEQ ID NO: 3).

[0057] Comparative Example 4 The only difference between Comparative Example 4 and Example 1 is that the complex enzyme includes xylanase Xyl1 and xylanase Xyl3, and the ratio of the enzyme activity units of xylanase Xyl1 and xylanase Xyl3 is 1:1.

[0058] Comparative Example 5 The only difference between Comparative Example 5 and Example 1 is that the complex enzyme includes xylanase Xyl2 and xylanase Xyl3, and the ratio of the enzyme activity units of xylanase Xyl2 and xylanase Xyl3 is 1:1.

[0059] Comparative Example 6 The only difference between Comparative Example 6 and Example 1 is that the complex enzyme includes xylanase Xyl1, xylanase Xyl2 and xylanase Xyl3, and the enzyme activity unit ratio of xylanase Xyl1, xylanase Xyl2 and xylanase Xyl3 is 1:1:1.

[0060] The enzymatic hydrolyzates in Examples 1-9 and Comparative Examples 1-6 were subjected to high performance liquid chromatography to test the ratio of xylooligosaccharides to xylose in the enzymatic hydrolyzates, and an equal volume of 8% sulfuric acid (m / v) was added to the enzymatic hydrolyzed solution, and acid hydrolyzed at 121°C for 60 min in an autoclave, the pH of the acid hydrolysis treatment was 6, and the sugar content in the hydrolyzate obtained above was detected according to the method in GB / T 35545-2017, and the xylose yield was calculated as shown in Table 1. Here, xylooligosaccharides are xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexose.

[0061]

[0062] As shown in Table 1, in Examples 1-9, as the enzyme activity unit ratio of xylanase Xyl1 to xylanase Xyl2 decreases, that is, the content of G11 type xylanase Xyl2 increases, the ratio of xylooligosaccharides to xylose increases, and the amount of xylooligosaccharides produced by enzymatic hydrolysis increases. In Examples 1-4, the xylose yield increases as the enzyme activity unit ratio of xylanase Xyl1 to xylanase Xyl2 decreases. As the enzyme activity unit ratio of xylanase Xyl1 to xylanase Xyl2 continues to decrease, the xylose yield decreases, indicating that the ratio of xylooligosaccharides to xylose in the product can be regulated by regulating the enzyme activity unit ratio of xylanase Xyl1 to xylanase Xyl2. When the enzyme activity unit ratio of xylanase Xyl1 to xylanase Xyl2 is between 2:1-1:2, the proportion of xylooligosaccharides and xylose in the enzymatic hydrolysis products increases, and the xylose yield is higher, indicating that the enzymatic hydrolysis efficiency of xylanase Xyl1 and xylanase Xyl2 with the enzyme activity unit ratio within this range is the highest, and the yield of xylooligosaccharides and xylose produced by hydrolysis is the highest.

[0063]

[0064] Here, Xyl3 is a G11-type xylanase derived from Penicillium decumbens, and the amino acid sequence is shown in SEQ ID NO:3.

[0065] As shown in Table 2, the composite enzyme formed by the combination of xylanase Xyl1 and xylanase Xyl2, xylanase Xyl1 and xylanase Xyl3, xylanase Xyl2 and xylanase Xyl3, or xylanase Xyl1, xylanase Xyl2 and xylanase Xyl3 was used for the enzymatic hydrolysis of straw fiber pulp. Even if the above four composite enzymes were hydrolyzed under the same pH conditions, the xylose yield of the enzymatic hydrolysis products of the composite enzymes formed by the combination of xylanase Xyl1 and xylanase Xyl3, xylanase Xyl2 and xylanase Xyl3, or xylanase Xyl1, xylanase Xyl2 and xylanase Xyl3 was significantly lower than the xylose yield of the enzymatic hydrolysis products of the composite enzymes formed by the combination of xylanase Xyl1 and xylanase Xyl2, indicating that the enzymatic hydrolysis efficiency of the composite enzyme formed by the combination of xylanase Xyl1 and xylanase Xyl2 is the highest between pH 4.0 and 5.5.

[0066]

[0067] As shown in Table 3, with the increase in the enzyme addition amount of xylanase Xyl1, the xylose yield increased significantly, while the xylooligosaccharides / xylose ratio remained at a low level, indicating that it has a strong main chain hydrolysis ability and mainly produces monosaccharides. Xylanase Xyl2 maintains a high proportion of xylooligosaccharides release ability at different addition amounts, reflecting its good oligosaccharide enrichment characteristics. The xylooligosaccharides and xylose ratio and xylose yield in the product of xylanase Xyl3 are significantly lower than those of the products of xylanase Xyl2 and xylanase Xyl1.

[0068] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0069] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing xylooligosaccharides and xylose based on a composite enzyme, characterized in that: include: preparing a substrate containing hemicellulose to obtain fiber pulp; Adding a complex enzyme into the fiber pulp to prepare an enzymatic mixed solution, and subjecting the enzymatic mixed solution to an enzymatic reaction to prepare xylose and xylooligosaccharides; The complex enzyme is a combination of xylanase Xyl1 and xylanase Xyl2, the sequence of the xylanase Xyl1 is such as SEQ ID NO: 1, the sequence of the xylanase Xyl2 is such as SEQ ID NO: 2, the enzyme activity unit ratio of the xylanase Xyl1 to the xylanase Xyl2 is any value between 1:10 and 10:1, the enzymolysis temperature is any value between 30°C and 60°C, the enzymolysis pH value is any value between 4.0 and 7.0, and the enzymolysis time is any value between 4h and 12h.

2. The preparation method according to claim 1, characterized in that: The enzyme activity unit ratio of the xylanase Xyl1 to the xylanase Xyl2 is any value between 2:1 and 1:

2.

3. The preparation method according to claim 2, characterized in that: The added amount of the complex enzyme is any value between 200U / g and 1000U / g.

4. The preparation method according to claim 3, characterized in that: The enzymolysis temperature is any value between 40° C. and 50° C., the enzymolysis pH is any value between 4.5 and 5.5, and the enzymolysis time is any value between 5 h and 8 h.

5. The preparation method according to claim 4, characterized in that: The substrate containing hemicellulose is any one of oat xylan, birch xylan, beech xylan and straw fiber pulp.

6. The preparation method according to claim 5, characterized in that: The raw material of the straw fiber pulp is any one of corn straw, wheat straw, sweet sorghum straw, sugarcane straw, rice straw or reed straw.

7. The preparation method according to claim 6, characterized in that: The raw material is pretreated to prepare the straw fiber pulp, and the pretreatment method is to soak the raw material in a sodium hydroxide solution for a preset time.

8. The preparation method according to claim 7, characterized in that: The mass volume concentration of the sodium hydroxide solution is any value between 10% and 30%.

9. The preparation method according to any one of claims 1 to 8, characterized in that: The buffer solution of the complex enzyme is any one of acetic acid-sodium acetate buffer, citric acid-phosphate buffer or phosphate buffer.

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