Application of GH5GH6 dual-catalytic structural domain compound enzyme in cellulose degradation

Abstract

The invention relates to the technical field of biology, in particular to enzymatic study related to cellulose degradation. Specifically, the invention relates to an application of a GH5GH6 dual-catalytic structural domain compound enzyme in cellulose degradation. According to the application of the GH5GH6 double-catalytic structural domain compound enzyme in cellulose degradation, the GH5GH6 double-catalytic structural domain compound enzyme is derived from ship maggot symbiotic bacteria. The multifunctional enzyme compound provides an efficient and economic solution for green transformation of agricultural waste treatment, biofuel production and paper pulp industry, remarkably improves the utilization efficiency of cellulose, has important economic value and environmental protection significance, and shows a wide industrial application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to enzymology research related to cellulose degradation, and in particular to the application of a GH5_GH6 dual catalytic domain complex enzyme in cellulose degradation. Background Art

[0002] Cellulose is one of the most abundant biomass resources on earth and an important source of biomass energy. It is estimated that the total amount of cellulose produced through photosynthesis in the world can reach 1.0 to 1.1 billion tons per year. However, cellulose is a linear high molecular polymer formed by glucose molecules connected by β-1,4 glycosidic bonds. Its highly crystalline structure makes it difficult to be directly used by microorganisms or other organisms. Therefore, the degradation of cellulose usually requires physical, chemical or enzymatic decomposition into smaller sugar substances before it can be further utilized. Compared with traditional acid-base treatment methods, enzymatic degradation of cellulose has gradually become a more sustainable choice due to its pollution-free and low energy consumption characteristics, and has shown broad application potential in many fields such as food, medicine, and environmental protection.

[0003] Despite this, the application of existing enzymatic methods in cellulose degradation still faces some challenges, especially in industrial applications, where the degradation efficiency and stability of enzymes often cannot meet the needs of efficient cellulose degradation. Cellulose degradation mainly relies on different types of enzymes to work together. For example, the GH5 domain belongs to the GH family of glycoside hydrolases, which can randomly cut the middle part of the cellulose chain to generate oligosaccharides. This process plays a key role in the early stage of cellulose degradation. The GH6 domain belongs to the exoglycosidase family, which can cut from the non-reducing end of the cellulose chain to release monosaccharides (such as glucose) to provide raw materials for the subsequent fermentation process. However, a single GH5 or GH6 enzyme system still has many shortcomings in degradation efficiency, stability, and synergy of the catalytic process. At present, some studies have tried to use a single enzyme system for cellulose degradation. Although GH5-type glycoside hydrolases show good cutting effects in the early stage of cellulose degradation, their stability and degradation efficiency in long-term reactions are still low. Although GH6-type enzymes can effectively release monosaccharides, they have problems with slow catalytic rate and low reaction efficiency during the degradation process. Therefore, a single enzyme system often cannot achieve efficient and continuous degradation effects in industrial applications.

[0004] In order to improve the efficiency of cellulose degradation, in recent years, researchers have proposed the concept of multi-enzyme complex system, which achieves synergistic degradation by combining different types of enzymes. These complex enzyme systems can utilize the characteristics of different enzymes to play a role at different stages of cellulose degradation, thereby improving the overall degradation efficiency. However, the existing complex enzyme system still faces some technical bottlenecks, which limits its effectiveness in large-scale industrial applications. First, the stability of enzymes is particularly prominent. Many complex enzymes lose their activity under high temperature, strong acid and alkali or organic solvent conditions, resulting in a short-lasting degradation effect. Secondly, although complex enzymes can work synergistically at different stages, in some systems, the synergistic effect between different enzymes is not ideal. For example, the catalytic sites of some enzymes may interfere spatially, resulting in their functional performance being limited. Furthermore, the existing complex enzyme system often requires the separation of multiple enzymes from microorganisms from different sources, which not only complicates the production process, but also significantly increases the cost of enzyme preparations. In addition, the expression and purification process of enzymes also faces the problems of limited yield and high cost.

[0005] Therefore, a new solution is urgently needed to improve the degradation of cellulose through a more efficient and stable composite enzyme system, especially in the fields of biomass energy production, agricultural waste treatment and environmental protection, to provide more sustainable and economical technical support for efficient cellulose degradation. Summary of the invention

[0006] The purpose of the present invention is to provide a GH5_GH6 dual catalytic domain complex enzyme (symbiotic bacteria of shipworm Teredinibacter The application of complex enzymes containing GH5, CBM and GH6 domains isolated from the genus in cellulose degradation (cellulose degradation in waste treatment, biofuel production and pulp industry).

[0007] To achieve the above object, the present invention adopts the following technical solutions: Application of a GH5_GH6 dual catalytic domain complex enzyme in cellulose degradation.

[0008] The GH5_GH6 dual catalytic domain complex enzyme is derived from the symbiotic bacteria of shipworms.

[0009] The shipworm symbiotic bacteria are known strains Teredinibacter Species, especially Teredinibacter turnerae strains.

[0010] The specific GH5_GH6 dual catalytic domain complex enzyme is obtained as follows: (1) From the symbiotic bacteria of shipworms ( Teredinibacter Gene sequences for the isolation of GH5, CBM and GH6 domains from the genus Using genome extraction technology, the symbiotic bacteria of shipworms ( Teredinibacter turneraeAs an example, genomic DNA was amplified by PCR using specific primers to obtain the gene sequences of GH5, CBM and GH6 domains. These gene sequences provide the basis for the efficient degradation of cellulose in the present invention.

[0011] (2) Amplify the above gene sequence by PCR technology to obtain the fusion gene: Design the enzyme structure by yourself, such as using 2 to 4 CBMs to combine with the double domains and use the designed specific primers (such as GH5 primers, GH6 primers, CBM primers) to amplify the isolated gene fragments. The template is the genomic DNA of the shipworm symbiotic bacteria, and the reaction system contains high-fidelity enzymes to ensure the accuracy of the amplified fragments. After PCR amplification, a fusion gene sequence containing GH5, CBM and GH6 is obtained. The primers specifically include: The GH5 domain forward primer (GH5-F) sequence: 5′-ATGCGGAAGATAAACAAGCAGAAGCC-3′, located at the beginning of the GH5 domain (near the start position 42 of the sequence).

[0012] The GH5 domain reverse primer (GH5-R) sequence: 5′-ATGGGTGCCGACGAAGATGGAAGCT-3′, is located at the end of the GH5 domain, close to the connecting region with the CBM2 domain.

[0013] The sequence of the CBM2 domain forward primer (CBM2-F) is: 5′-ATGGCTGAAAGACGATTGGAAGGC-3′, which is located in the junction region between GH5 and CBM2.

[0014] The CBM2 domain reverse primer (CBM2-R) sequence: 5′-AGGAAGAGCAGTGAGCTCAG-3′, is located at the end of the CBM2 domain (end of the sequence is 697).

[0015] The sequence of the GH6 domain forward primer (GH6-F) was: 5′-AGATGAAGGTTCCTCCATTATTCAG-3′, located in the junction region between CBM2 and GH6 (start position 793 of the sequence).

[0016] The GH6 domain reverse primer (GH6-R) sequence: 5′-GTAGTGTGTGAAGTGACCTATGTG-3′, located at the end of the GH6 domain (sequence end position 1127).

[0017] (3) Insert the fusion gene into the expression vector: The amplified fusion gene sequence is inserted into a suitable expression vector (such as pET series vector) through enzyme digestion methods (such as EcoRI and BamHI). The vector selection has the characteristics of high-efficiency expression and can achieve high-level protein expression in hosts such as Escherichia coli.

[0018] (4) Transforming the above vector into host cells: Transform the constructed expression vector into E. coli BL21 (DE3) or other suitable host cells. After transformation, screen positive clones to ensure successful insertion and stable expression of the target gene.

[0019] (5) Induce the expression of fusion protein by IPTG at 37°C: Induce the expression of the fusion protein by adding IPTG (isopropyl-β-D-thiogalactopyranoside) at 37°C. The induction concentration and time will be optimized according to the experimental conditions to ensure efficient expression of the fusion protein.

[0020] (6) Purify the fusion protein using affinity chromatography (such as nickel column purification) and gel filtration chromatography to obtain the complex enzyme: The expressed fusion protein is initially purified by affinity chromatography (such as His tag affinity purification using a nickel column), and then further purified using gel filtration chromatography technology to obtain a high-purity GH5_GH6 dual catalytic domain complex enzyme.

[0021] The GH5_GH6 dual catalytic domain complex enzyme is used to degrade waste.

[0022] The waste is agricultural waste, waste paper or pulp from the paper industry.

[0023] A multifunctional enzyme complex for degrading fiber, the complex containing symbiotic bacteria of shipworm ( Teredinibacter The GH5_GH6 dual catalytic domain sequence in the genus) is shown in SEQ ID NO: 7.

[0024] Application conditions of compound enzyme: The composite enzyme of the present invention can still maintain high activity in a wide range of temperatures (5°C to 70°C) and pH ranges (3.0 to 10.0), and can adapt to the use requirements of different industrial environments. Whether under high temperature, strong acid or strong alkali conditions, the composite enzyme can work stably, significantly improve the cellulose degradation efficiency, and can meet the needs of large-scale industrial applications.

[0025] Advantages of the present invention: The present invention is derived from the symbiotic bacteria of shipworm TeredinibacterA multifunctional enzyme complex catalyzed by GH5 and GH6 dual domains is obtained from the genus. The complex is composed of GH5, GH6 and 2 to 4 CBMs arranged and connected, integrating three different functional domains of GH5, CBM and GH6, which significantly improves the efficiency and stability of cellulose degradation. The enzyme complex of the present invention not only has the ability to degrade cellulose efficiently, which is difficult to achieve with a single enzyme system, but also can maintain high stability under industrial conditions and adapt to different production environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The pulse field electrophoresis results of GH5, CBM and GH6 domains show the successful amplification of genes and the verification of their integrity. Among them, 1 is GH6, 2 and 3 are CBM, M is GH5, and 1-M is the electrophoresis result of the complete protein of GH5, CBM and GH6 domains, with a molecular weight of about 128kDa.

[0027] Figure 2 The graph shows the test results of compound enzyme activity under different temperature and pH conditions, showing broad-spectrum adaptability.

[0028] Figure 3 The degradation effects of single enzyme systems GH5 and GH6 on CMC and PASC are shown in Figure 1, where (a) is the degradation effect of GH5 on CMC and PASC; (b) is the degradation effect of GH6 on CMC and PASC. DETAILED DESCRIPTION

[0029] The specific implementation modes of the present invention are further described below in conjunction with examples. It should be noted that the specific implementation modes described here are only for illustrating and explaining the present invention, and are not limited to the present invention.

[0030] The present invention utilizes the fusion protein in the shipworm symbiotic bacteria to contain GH5, CBM and GH6 domains: the GH5 domain has endo-activity and can randomly cut the β-1,4 glycosidic bonds in the cellulose chain; the CBM domain greatly improves the catalytic efficiency by enhancing the binding force between the enzyme and the cellulose substrate; the GH6 domain has exo-activity and can release monosaccharides from the non-reducing end of the cellulose chain. The present invention forms an efficient cellulose degradation system by extracting and arranging the domains by ensuring the synergistic effect of each domain. The fusion protein is efficiently expressed in the host cell by amplification expression technology, and a high-purity enzyme complex is obtained by nickel affinity chromatography and gel filtration chromatography. Enzyme tests show that the complex enzyme exhibits excellent activity and stability under the conditions of a temperature range of 5°C to 70°C and a pH range of 3.0 to 10.0, and the degradation rate of CMC and PASC is increased by more than 80%. Compared with the existing single enzyme system, the enzyme complex of the present invention also improves the efficiency of the degradation of complex cellulose matrices (such as rice straw and corn stalks) by more than 50%. In addition, the present invention significantly reduces the use of chemical reagents and reduces environmental pollution through enzymatic degradation. In the treatment of agricultural waste, the use of the enzyme complex of the present invention can increase the degradation rate by about 60%; at the same time, in the production of biofuels, the conversion rate of glucose generated is increased by about 60%, and the ethanol production is increased by 35%; in the papermaking industry, the amount of bleaching agent is reduced by about 20%, and the bleaching efficiency is increased by more than 30%. The multifunctional enzyme complex of the present invention provides an efficient and economical solution for the green transformation of agricultural waste treatment, biofuel production and pulp industry, which significantly improves the efficiency of cellulose utilization, has important economic value and environmental significance, and shows a wide range of industrial application prospects.

[0031] Example 1: Amplification and expression of complex enzyme (1) Primer design Specific primers were designed for the known sequences of the target genes GH5, CBM2, and GH6 to ensure the efficiency and accuracy of amplification. When designing primers, ensure that the primer length is 18-22 bp, the GC content is about 50%, and the start and stop codons are intact.

[0032] Primers specifically include: The GH5 domain forward primer (GH5-F) sequence is: 5′-ATGCGGAAGATAAACAAGCAGAAGCC-3′ (SEQ ID NO: 1), located at the beginning of the GH5 domain (near the start position 42 of the sequence).

[0033] The GH5 domain reverse primer (GH5-R) sequence: 5′-ATGGGTGCCGACGAAGATGGAAGCT-3′ (SEQ ID NO: 2), is located at the end of the GH5 domain, close to the connection region with the CBM2 domain.

[0034] The CBM2 domain forward primer (CBM2-F) sequence: 5′-ATGGCTGAAAGACGATTGGAAGGC-3′ (SEQ ID NO: 3), located in the connecting region between GH5 and CBM2.

[0035] The CBM2 domain reverse primer (CBM2-R) sequence: 5′-AGGAAGAGCAGTGAGCTCAG-3′ (SEQ ID NO: 4), located at the end of the CBM2 domain (the end of the sequence is 697).

[0036] The GH6 domain forward primer (GH6-F) sequence: 5′-AGATGAAGGTTCCTCCATTATTCAG-3′ (SEQ ID NO: 5), located in the connecting region between CBM2 and GH6 (sequence start position 793).

[0037] The GH6 domain reverse primer (GH6-R) sequence: 5′-GTAGTGTGTGAAGTGACCTATGTG-3′ (SEQ ID NO: 6), located at the end of the GH6 domain (sequence end position 1127).

[0038] (2) PCR amplification and fusion gene construction In this experiment, Phusion high-fidelity DNA polymerase was used for PCR amplification. In the reaction system, the template DNA was the genomic DNA of the shipworm symbiont (ID: GCF_037935975.1), with a concentration of 1-2 ng; the primer concentration was 0.2 μM; the dNTP concentration was 0.2 mM; the amount of high-fidelity DNA polymerase was 1 U, and the concentration of the reaction buffer was 1×. The PCR program was set as follows: the initial denaturation step was performed at 98°C for 30 seconds, followed by 35 cycles, each cycle including denaturation at 98°C for 10 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 1 minute / kb, and finally extension at 72°C for 5 minutes to ensure complete extension of the amplified product. In the experiment, specific primers for GH5, CBM2 and GH6 genes were first designed to amplify these gene fragments respectively. Through PCR amplification, a complete fusion gene containing the GH5, CBM2 and GH6 domains was successfully obtained, with a fragment size of approximately 3.5 kb. Agarose gel electrophoresis showed that the target fragments (GH5: 1.5 kb, CBM2: 0.8 kb, GH6: 1.2 kb) were consistent with the expected size and the bands were clear, verifying the successful construction of the fusion gene ( Figure 1 ).

[0039] (3) Gene cloning and vector construction The amplified fusion gene was inserted into the pET-28a vector using EcoRI and HindIII restriction sites. The experimental steps are as follows: First, the pET-28a vector and the fusion gene fragment were digested with EcoRI and HindIII to obtain compatible restriction ends. Then, the digestion products were connected together using T4 DNA ligase. Next, the ligation products were transformed into DH5α competent cells, and resistant clones were screened for further analysis. Finally, sequencing was used to verify whether the fusion gene was correctly inserted into the vector. The sequencing results showed that the fusion gene had been successfully and accurately inserted into the pET-28a vector.

[0040] (4) Expression and purification After the recombinant vector was transformed into BL21 (DE3) competent cells, the resistant strains were screened and cultured at 37°C until OD600 reached 0.6-0.8. When the cells grew to the logarithmic phase, IPTG (final concentration 0.5 mM) was added to induce protein expression for 4-6 hours. After induction, the cells were collected and broken by ultrasound, followed by centrifugation to obtain the lysate. Affinity purification was performed using a Ni-NTA column, and the target protein was eluted with 300 mM imidazole. Finally, the protein was further purified by gel filtration chromatography to obtain the complex enzyme shown in SEQ ID NO: 7. SDS-PAGE results showed that the purity of the target protein was more than 95%.

[0041] SEQ ID NO: 7:

[0042] Among them, 42-280 are the GH5 domain of the present invention, 380-471 and 612-697 are the CBM domain, and 793-1127 are the GH6 domain.

[0043] Example 2: Enzymatic property test of complex enzyme (1) Temperature adaptability In the experiment, the temperature adaptability of the composite enzyme was determined by testing its activity in the range of 5°C to 70°C to determine its optimal temperature. In the reaction system, 1wt% CMC (sodium carboxymethyl cellulose) was used as the substrate, and 1g of the substrate was obtained by dissolving in 0.1M phosphate buffer (pH 6.0). The composite enzyme was added to the reaction system after purification, and the amount of enzyme added was 1U / mL. The total volume of the reaction system was 1mL. 0.02% (v / v) Triton X-100 was also added to the reaction solution to stabilize the activity of the enzyme. The reaction was carried out at different temperatures ranging from 5°C to 70°C, with one test point every 10°C, and the reaction time was 30 minutes. The amount of reducing sugar generated during the reaction was determined by the DNS method (3,5-dinitrosalicylic acid method) to evaluate the activity of the enzyme. The results are as follows Figure 2 As shown, the composite enzyme exhibited optimal activity at 45°C, but the activity was maintained at a relatively stable level from 5°C to 70°C.

[0044] (2) pH adaptability In order to test the pH adaptability of the composite enzyme, the experiment was carried out in the range of pH 3.0 to 10.0, using 0.1M different pH buffers: citric acid-sodium buffer (pH 3.0-6.0), phosphate buffer (pH 6.0-8.0) and sodium bicarbonate-sodium hydroxide buffer (pH 8.0-10.0). The substrate was 1wt% CMC, the enzyme concentration was 1U / mL, the total amount of the reaction solution was 1mL, and the reaction time was also 30 minutes. The activity of the enzyme was tested at each pH value, and the amount of reducing sugar produced was determined by the DNS method. The results are shown in Figure 2. Figure 2 As shown, the optimum pH of the complex enzyme is 6.5, and the enzyme activity remains above 90% in the range of pH 5.5 to 7.5.

[0045] (3) Substrate degradation efficiency In the substrate degradation experiment, 1% CMC and 1% PASC were used as substrates, respectively, and the reaction of the enzyme and substrate was carried out in the same reaction system. In the reaction system, the substrate was dissolved in 0.1M phosphate buffer (pH 6.5), and 1U / mL of the complex enzyme was added, and the total amount of the system was 1mL. The reaction temperature was controlled at 45°C, the reaction time was 1 hour, and samples were taken every 10 minutes. To avoid substrate aggregation, 0.02% Triton X-100 was added to the reaction system. A system with the same substrate, buffer and Triton X-100 concentrations as the experimental group but without the addition of any enzyme was used as a control. The degradation efficiency of the enzyme was evaluated by measuring the amount of reducing sugar generated in the reaction solution, and the sugar content was determined using the DNS method. Degradation rate:

[0046] The experimental results are shown in Table 1. The degradation efficiency of the composite enzyme in this example for CMC is 91%, and the degradation efficiency for PASC is 88%, which are both increased by more than 80% compared with the control group.

[0047] Table 1 Substrate degradation experimental results

[0048] Example 3: Industrial and agricultural waste treatment (1) Agricultural waste treatment This experiment aims to use composite enzymes to degrade agricultural waste, especially rice straw and corn straw. The waste raw materials include dried rice straw and corn straw, both of which are in the form of fragments with a size of about 1-2 cm. These wastes were added to the buffer solution separately during the treatment process to promote effective degradation. The raw materials of rice straw and corn straw were dried before treatment to ensure that the moisture content was controlled below 5%. Rice straw and corn straw were cut into fragments with a size of 1-2 cm to ensure that the enzyme could effectively contact the substrate during the reaction. In the experiment, rice straw and corn straw were added to 0.1 M phosphate buffer (pH 6.5) at 5wt% (i.e., 5 grams of dry weight of waste dissolved in 100mL phosphate buffer). The use of phosphate buffer ensures the stability of pH during the reaction and avoids the reduction of enzyme activity due to changes in pH. The composite enzyme concentration used in the experiment was 1 U / mL to ensure sufficient enzyme activity for efficient degradation. The reaction system was carried out at 45°C, and the pH value of the reaction was kept at 6.5 to optimize the enzyme activity. The reaction time of the experiment was 48 hours, and samples were taken every 12 hours. At the same time, the control group was set to add the same rice straw and corn straw, phosphate buffer and Triton X-100 as the experimental group, but no compound enzyme was added. The results of the control group can be used to evaluate the degree of natural degradation of the substrate without the addition of enzymes. During the experiment, the temperature of the reaction liquid was constant, and the temperature was kept constant by the temperature control equipment to ensure the stability of the reaction conditions. At the same time, the reaction system was always stirred continuously on the agitator to ensure sufficient contact between the substrate and the enzyme and promote the uniform reaction. In the experiment, the DNS method was used to determine the amount of sugar generated at different reaction times to evaluate the degradation effect of the waste. Before each sampling, the temperature of the reaction solution was maintained at a set 45°C to ensure the consistency and stability of the reaction conditions. The amount of sugar produced by the DNS method can be used to reflect the degree of waste degradation. Specifically, at the sampling time point, 1 mL of the substrate and the mixed enzyme solution is placed in a 45°C constant temperature water bath for saccharification for 60 minutes, then taken out and added with 2 mL of DNS reagent, boiled in a boiling water bath for 10 minutes, diluted to 15 mL with distilled water, cooled in cold water, and the OD550 value is measured. The glucose standard curve is consulted to determine the sugar content and calculate the degradation rate (Table 1). Since 1 mole of cellulose produces 1 mole of glucose, and the molar mass is the same (72.06 g / mol), it can be considered that 1 gram of cellulose will produce 1 gram of glucose. Therefore, this example assumes that the maximum possible sugar concentration is 50 g / L, and the degradation rate is:

[0049] The generated glucose concentration is obtained through a standard curve, and the maximum possible sugar concentration is the maximum sugar concentration that is theoretically generated by all rice straw and corn straw.

[0050] After 48 hours of treatment, the degradation rate of rice straw in this example reached 85%, while the degradation rate of corn straw was 82%. The experimental results are shown in Table 2. The compound enzyme has a significant degradation effect on cellulose and other polysaccharides in rice straw and corn straw, which is 60% and 62% higher than the control group, respectively. The morphology of the substrate after treatment has changed significantly, the fiber structure has become loose, and obvious signs of cleavage and degradation have appeared on the surface, showing the degradation effect of the enzyme.

[0051] Table 2 Results of agricultural waste degradation experiments

[0052] (2) Biofuel production In the experiment, a composite enzyme was used to convert the cellulose in the rice straw into glucose, which was then fermented by yeast to produce ethanol. Specifically, the rice straw was decontaminated and crushed, and then added to the buffer solution so that the concentration of the substrate in the rice straw substrate system was 5wt% (i.e., 5 grams of dry weight of rice straw dissolved in 100mL of phosphate buffer, pH 6.5), and 1 U / mL of the composite enzyme was added to degrade the cellulose. The reaction conditions were set at 45°C and pH 6.5, and the reaction time was 72 hours; then the enzyme was added to the system, and the amount of enzyme added ensured sufficient cellulose hydrolysis, and the generated glucose could be used for yeast fermentation. The yeast used was Saccharomyces cerevisiae ( Saccharomyces cerevisiae ), the addition amount is 10 7 CFU / mL. After yeast inoculation, the fermentation was continued at the same reaction temperature (45°C) for 24 hours to detect the conversion rate of glucose and the yield of ethanol. At the same time, the rice straw was crushed and directly added to the phosphate buffer, adjusted to the same substrate concentration (5 wt%), and then yeast was directly added without adding the complex enzyme for fermentation as a control. The conversion rate was:

[0053] The amount of ethanol produced is determined by the ethanol concentration after yeast fermentation, and the initial amount of glucose is calculated based on the glucose concentration after composite enzyme degradation.

[0054] The experimental results are shown in Table 3. Within 72 hours, after the complex enzyme treatment, the conversion rate of cellulose in rice straw to glucose reached 90%. In addition, after yeast fermentation, the ethanol production increased by 35% compared with the untreated control group. These results show that the complex enzyme has a high efficiency in the process of converting cellulose to glucose, and the subsequent ethanol fermentation effect is significantly improved, demonstrating the effectiveness of the complex enzyme in biofuel production.

[0055] Table 3 Biofuel production experimental results

[0056] (3) Pulp pretreatment In the experiment, the pulp was treated with a composite enzyme to evaluate its effect on the amount of bleaching agent and pulp whiteness. In the experiment, the wood pulp concentration in the pulp raw material was 5 wt%, the concentration of the composite enzyme was set to 1 U / mL, and the reaction conditions were 45℃ and pH 6.0. The treated pulp was bleached, the bleaching agent was chlorine dioxide (ClO2), and the initial bleaching agent dosage was 1000 mL. The pulp that was not treated with the composite enzyme was directly bleached with chlorine dioxide (ClO2), and the other conditions (bleaching agent dosage, reaction temperature, reaction time, etc.) were the same as the experimental group as the control. As the bleaching time increased, the amount of bleaching agent gradually decreased. The experimental results showed that in the 48-hour reaction, the pulp treated with the composite enzyme reduced the amount of bleaching agent by 20%, and the whiteness increased from 56% to 88%. These results show that the composite enzyme treatment can not only effectively improve the bleaching effect of pulp, but also significantly reduce the amount of bleaching agent used and reduce chemical consumption compared with the control group, and has a high potential for industrial application (Table 4).

[0057] Table 4 Pulp pretreatment experimental results

[0058] Comparative Example 1: Degradation effect of GH5 and GH6 alone (1) Experimental design In order to test the effect of GH5 and GH6 on cellulose degradation, this experiment used Thermobifida fusca GH5 (Gene ID: 3760012) and from Coptotermes formosanus (Gene ID: 36987) GH6 enzyme degrades cellulosic materials including substrates such as CMC (carboxymethyl cellulose), PASC (phosphorylated cellulose) and actual rice straw.

[0059] (2) Experimental procedures In this experiment, GH5 enzyme and GH6 enzyme were used to degrade CMC (carboxymethyl cellulose) and PASC (phosphorylated polyglucose) substrates, respectively. In the experimental system, the concentration of the substrate solution was 1 wt%, 0.1 M phosphate buffer (pH 6.0) was used as the reaction medium, and the enzyme concentration was 1U / mL (5 mg enzyme was added to each reaction system). The reaction was carried out at 45°C and the time was set to 1 hour. Samples were taken every 10 minutes, and the amount of reducing sugar generated in the reaction was determined by the DNS method. By analyzing the reducing sugar yield of GH5 enzyme and GH6 enzyme in treating different substrates under the same conditions, the efficiency difference of the two enzymes in degrading CMC and PASC was evaluated. The experimental results help to understand the performance of GH5 and GH6 enzymes extracted from other species in cellulose degradation, and provide a basis for the subsequent optimization of enzyme-catalyzed degradation processes. For 1 wt% CMC solution, there is 1 g CMC in every 100 mL solution, and 1 g cellulose is converted into 1 g glucose. This experiment believes that the maximum amount of sugar that can be completely degraded theoretically is 1 g glucose. Degradation efficiency:

[0060] (3) Results analysis like Figure 3 As shown in the figure, GH5 showed high activity on the PASC substrate during the degradation of PASC and CMC, with a degradation efficiency of about 48%, but lower than the 40% efficiency of the composite enzyme in this application (based on the amount of reducing sugar produced). The degradation efficiency of GH6 on PASC was lower, about 33%. The degradation characteristic of GH5 is that it preferentially attacks the amorphous region and cellulose chains close to the surface. The degradation of cellulose by GH6 depends more on the breakage of long chains, so GH6 shows a stronger advantage on crystalline cellulose.

[0061] Comparative Example 2: Other species obtain the fusion protein effect of random combination of GH5 and GH6 (1) Experimental design In order to further verify the efficacy of the composite enzyme of the present invention, the experiment obtained GH5 and GH6 genes from other species and randomly combined them to form fusion proteins. By testing the cellulose degradation effect of these fusion proteins, it was evaluated whether they had better effects than other individual enzymes or other fusion enzymes.

[0062] (2) Experimental procedures In this experiment, we first Thermobifida fusca (Gene ID: 3760012) and Coptotermes formosanusGH5 and GH6 genes were selected from the strain (Gene ID: 36987), amplified by PCR and cloned into appropriate expression vectors (the target enzymes included multiple GH5 combinations, multiple GH6 combinations, and GH5+GH6 fusion enzyme combinations), and then transformed into Escherichia coli BL21 (DE3) for expression. IPTG (isopropyl-β-D-thiogalactoside) was used to induce the expression of the target enzyme at a concentration of 0.5 mM, and the culture conditions were 37°C (the optimal culture temperature for Escherichia coli) and 180 rpm for 4 hours. The enzyme was extracted by breaking the cells and centrifuging to obtain the supernatant, which contained the target enzyme. Then, the enzyme was purified and prepared for subsequent degradation experiments. In the experiment, three substrates were selected: 1 wt% CMC solution, 1 wt% PASC solution, and actual agricultural waste (such as rice straw powder). The specific reaction system configuration is as follows: the substrate solution is 1 wt% concentration. In each group of experiments, 1 wt% substrate solution (CMC or PASC) or an equal amount of agricultural waste suspension (adjusted to 1 wt%) was added to the reaction system. At the same time, 1U / mL of the target enzyme solution was added to ensure that the enzyme concentration in each reaction system was consistent and complete reaction was ensured. The reaction temperature was set at 45°C, the pH value was 6.0, the reaction time was 72 hours, and samples were taken every 24 hours during the reaction. At the same time, a reaction system without enzyme addition was set up to evaluate the degradation of the substrate under enzyme-free conditions as a control group. The amount of reducing sugar generated at each time point was determined using the DNS method (3,5-dinitrosalicylic acid method). After each sampling, the sample was mixed with 1.5 mL of DNS reagent, heated in a 95°C water bath for 10 minutes, and then cooled to room temperature. The absorbance of the reaction solution at 540nm was measured using a spectrophotometer, and the amount of reducing sugar generated was calculated using the standard glucose curve to evaluate the degradation effect of the enzyme. Through these steps, the degradation efficiency of target enzymes obtained from other species on different substrates was compared, and the activity of cellulose degrading enzymes from different sources and their adaptability to different substrates were analyzed.

[0063] (3) Results analysis By comparing the degradation effects of different combinations of GH5 and GH6 fusion enzymes with those of individual GH5 and GH6 combinations, it was found that some combinations of fusion proteins performed better than individual GH5 or GH6 combinations in the degradation of PASC and agricultural waste. T. fusca ) and GH6 (from C. formosanus ) showed a significant effect in rice straw degradation, but its degradation rate was significantly lower than the dual-domain catalytic degradation effect derived from shipworm in the present invention in all aspects (Table 5).

[0064] Table 5 Degradation effect

[0065] The above GH5 and GH6 sequences from other species are: Coptotermes formosanus GH6:

[0066] Thermobifida fusca GH5:

[0067] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of a GH5_GH6 dual catalytic domain complex enzyme in cellulose degradation.

2. The use according to claim 1, characterized in that: The GH5_GH6 dual catalytic domain complex enzyme is derived from the symbiotic bacteria of shipworms.

3. The use according to claim 2, characterized in that: The shipworm symbiotic bacteria are known strains Teredinibacter Genus species.

4. The use according to any one of claims 1 to 3, characterized in that: (1) Obtain the gene sequence of the GH5_GH6 dual catalytic domain from the shipworm symbiotic bacteria; (2) Based on the above sequence characteristics, 2 to 4 CBMs were designed and combined into artificial catalytic enzymes; (3) amplifying the above gene sequence by PCR technology to obtain the fusion gene; (4) cloning the fusion gene into an expression vector; (5) transforming the above vector into host cells; (6) Induce the expression of fusion protein by IPTG at 37°C; (7) Purifying the fusion protein using affinity chromatography and gel filtration chromatography techniques to obtain the complex enzyme.

5. The use according to claim 1, characterized in that: The GH5_GH6 dual catalytic domain complex enzyme is used to degrade waste.

6. The use according to claim 5, characterized in that: The waste is agricultural waste, waste paper or pulp from the paper industry.

7. A multifunctional enzyme complex for degrading fiber, characterized in that: The complex contains the GH5_GH6 dual catalytic domain sequence in the shipworm symbiotic bacteria, and the sequence is shown in SEQ ID NO:7.

Images