Enzyme composition and method for producing 2-ethyl-hexanal by taking wood fiber as raw material

Abstract

The invention relates to the technical field of biology, in particular to an enzyme composition and a method for producing 2-ethyl-hexanal by taking wood fibers as raw materials. The method comprises the following steps: by taking wood fiber hydrolysate as a raw material, optimizing an acid treatment reagent, optimizing the acid treatment time, optimizing the cellulase treatment dosage, screening a fermentation strain, optimizing the dosage of yeast powder in fermentation, and screening and optimizing the dosage of a catalytic enzyme; the biological catalysis method for producing the 2-ethyl-hexanal by taking the wood fiber as the raw material is relatively high; the method is high in conversion efficiency and environment-friendly, and has relatively high popularization and application values.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to an enzyme composition and method for producing 2-ethylhexanal from lignocellulose. Background Technology

[0002] 2-Ethylhexanal, a key fine chemical intermediate, has wide applications in the synthesis of plasticizers, coatings, fragrances, and other fine chemicals. Traditional industrial production routes for 2-ethylhexanal primarily rely on petrochemical processes. This method typically uses non-renewable fossil fuel-derived olefins as starting materials, preparing butyraldehyde via a high-pressure carbonyl synthesis reaction. Subsequently, butyraldehyde undergoes an aldol condensation reaction and a subsequent hydrogenation reduction reaction to ultimately produce 2-ethylhexanal. With increasing global focus on sustainable development and green chemistry, utilizing biomass resources to produce high-value chemicals has become an important direction for the transformation and upgrading of the chemical industry. Lignocellulose, as one of the most abundant renewable biomass resources on Earth, can be hydrolyzed to obtain fermentable sugars, which can then be efficiently produced using microbial fermentation technology. Bio-based butyraldehyde, due to its unique molecular structure and excellent physicochemical properties, is considered a highly promising bio-based platform compound that can be further derived to synthesize a variety of high-value-added chemicals, providing an effective way to reduce dependence on fossil resources. However, the conversion of bio-based butyraldehyde to 2-ethylhexanal still faces technological bottlenecks, especially the immaturity of methods for achieving efficient conversion under mild conditions. Existing chemically catalytic conversion pathways generally require high reaction temperatures. More importantly, to prevent potential impurities in the fermentation broth from poisoning the catalyst, bio-based butanol fermentation broth often requires complex and energy-intensive purification steps, which significantly conflicts with the green and low-carbon principles pursued by bio-based routes. Therefore, developing a novel synthetic method that uses lignocellulose as a raw material to prepare butanol through bio-fermentation, and further utilizes enzyme catalysis to efficiently convert bio-based butanol to 2-ethylhexanal under mild conditions, can not only significantly reduce production costs and environmental impact, but also has significant industrial application value and profound environmental significance. Summary of the Invention

[0003] In view of this, the technical problem to be solved by the present invention is to provide an enzyme composition and method for producing 2-ethylhexanal from lignocellulose.

[0004] The present invention provides an enzyme composition comprising: an alcohol dehydrogenase derived from Clostridium acetobutylicum, a 2-deoxy-D-ribose-5-phosphate aldolase derived from Thermoplasma acidophilum, and an enol reductase derived from Clostridium tyrobutyricum.

[0005] Furthermore, the nucleotide sequence of the alcohol dehydrogenase derived from Clostridium acetobutylicum is shown in SEQ ID NO:1;

[0006] The nucleotide sequence of the 2-deoxy-D-ribose-5-phosphoaldolase derived from Thermoplasma acidophilum is shown in SEQ ID NO:2;

[0007] The nucleotide sequence of the enol reductase derived from Clostridium tyrobutyricum is shown in SEQ ID NO:3.

[0008] This invention, through screening of sources of alcohol dehydrogenase, aldolase, and enol reductase, obtained the optimal combination of the above three enzymes, which are used for the synthesis of 2-ethylhexanal compared to combinations of three enzymes from other sources.

[0009] Furthermore, in the enzyme composition of the present invention,

[0010] The alcohol dehydrogenase derived from Clostridium acetobutylicum also has a heat shock protein fragment attached to its N-terminus or C-terminus; and / or

[0011] The N-terminus or C-terminus of the 2-deoxy-D-ribose-5-phosphoaldolase derived from *Thermoplasma acidophilum* is also linked to a heat shock protein fragment; and / or

[0012] The enol reductase derived from Clostridium tyrobutyricum also has a heat shock protein fragment attached to its N-terminus or C-terminus.

[0013] The amino acid sequence of the heat shock protein fragment is shown in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.

[0014] In a specific embodiment of the present invention, the alcohol dehydrogenase derived from Clostridium acetobutylicum, the 2-deoxy-D-ribose-5-phosphate aldolase derived from Thermoplasma acidophilum, and the enol reductase derived from Clostridium tyrobutyricum were further optimized by linking heat shock protein fragments IDRI (SEQ ID NO:4), IDR2 (SEQ ID NO:5), or IDR3 (SEQ ID NO:6) to their N-terminus or C-terminus, respectively. The experimental results showed that the enzymes with IDRI (SEQ ID NO:4) linked to the N-terminus of the above three enzymes had the highest activity, which was significantly higher than the activity of the enzymes without IDRI.

[0015] This invention provides a biological material comprising at least one of the following: A) to D)

[0016] A) Nucleic acid of the enzyme composition described in this invention;

[0017] B) A recombinant vector containing the nucleic acid described in A);

[0018] C) Transfecting or transforming host cells with the recombinant vector described in B);

[0019] D) Cultures obtained by culturing host cells as described in C)

[0020] The nucleic acid described in this invention can be DNA, RNA, cDNA, or PNA. In embodiments of this invention, the nucleic acid is in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The nucleic acid can include nucleotide sequences with different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g., promoters or transcription terminators). The nucleic acid can be topologically linear or circular. The nucleic acid can be part of a vector (e.g., an expression or cloning vector) or a fragment thereof. The nucleic acid can be obtained directly from natural sources or can be prepared with the assistance of recombinant, enzymatic, or chemical techniques. The RNA form is mRNA obtained by gene transcription, etc.

[0021] In this invention, the nucleic acid may be optimized or unoptimized. These optimizations include, but are not limited to: codon usage bias, elimination of secondary structures that are unfavorable to expression (such as hairpin structures), alteration of GC content, CpG dinucleotide content, mRNA secondary structure, hidden splicing sites, early polyadenylation sites, internal ribosome entry and binding sites, negative CpG islands, RNA unstable regions, repetitive sequences (direct repeats, inverted repeats, etc.), and restriction sites that may affect cloning.

[0022] The present invention also provides a transcription unit containing the nucleic acid, wherein the transcription unit refers to a DNA sequence from the start of a promoter to the end of a terminator. Regulatory fragments may also be included on either side of or between the promoter and terminator, and these regulatory fragments may include a promoter, enhancer, transcription termination signal, polyadenylation sequence, origin of replication, nucleic acid restriction site, and homologous recombination site operatively linked to the nucleic acid sequence, such as an enhancer of the promoter, a poly(A) signal, etc.

[0023] The present invention provides a recombinant vector comprising a vector backbone and the nucleic acid described herein.

[0024] Furthermore, the source of the vector backbone described in this invention includes plants, animals, bacteria, fungi, bacteriophages, or viruses, and this invention does not limit this. The bacterial vectors include, but are not limited to, pET28a, pET16b, pET26b, pET28a, pET31b, pBAD, pBADHis, pTrc99a, pTrcHis, pACYCduet-1, pET duet-1, pCDFduet-1, pColdI, and pColdII. The fungal vectors include, but are not limited to, pYES2, pYES3, pYES6, and pAUR23.

[0025] The recombinant vector described in this invention refers to a recombinant nucleic acid vector, a recombinant DNA molecule containing the desired coding sequence and suitable nucleic acid sequences or elements essential for the expression of an operatively linked coding gene in a specific host organism. Nucleic acid sequences or elements essential for expression in prokaryotic cells include promoters, ribosome binding sites, and possibly other sequences. Prokaryotic cells are known to utilize promoters, enhancers, and terminators. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. In this specification, "plasmid" and "vector" are sometimes used interchangeably because plasmids are currently the most commonly used form of vector. However, this invention intends to include other forms of expression vectors that perform equivalent functions and are known or will become known in the art, including but not limited to: plasmids, phage particles, viral vectors, and / or simply potential genomic inserts. In specific embodiments, the nucleic acid encoding the fusion protein provided by this invention can be constructed in a prokaryotic-eukaryotic expression vector, specifically, the PET series.

[0026] This invention provides one of the following applications in the preparation of 2-ethylhexanal, as shown in I) to II):

[0027] I) The enzyme composition described in this invention;

[0028] II) The biomaterials described in this invention.

[0029] Furthermore, the 2-ethylhexanal is prepared by using wood fiber as raw material.

[0030] This invention provides a method for preparing 2-ethylhexanal, which includes the following steps:

[0031] Step 1: The plants are crushed, acid-treated, and dried to obtain pretreated powder;

[0032] Step 2: The pretreated powder is enzymatically hydrolyzed by cellulase to obtain a hydrolysate;

[0033] Step 3: The hydrolysate is decolorized after fermentation with Clostridium acetone-butanol to obtain a fermentation broth containing butanol;

[0034] Step 4: The butanol-containing fermentation broth is treated with the enzyme composition according to any one of claims 1 to 3 to obtain 2-ethylhexanal.

[0035] Furthermore, the reagent used for acid treatment is 1% H2SO4;

[0036] The acid treatment time is 30 min to 50 min, with 30 min being optimal.

[0037] The amount of cellulase used is 10mg to 20mg per gram of the pretreated powder, with 10mg per gram of the pretreated powder being optimal.

[0038] In the fermentation process, the amount of yeast powder used is 3g / L to 7g / L, with 5g / L being the most common.

[0039] The decolorizing agent is activated carbon, and the amount of activated carbon added is 5 g / L to 10 g / L, with 5 g / L being optimal.

[0040] The amount of alcohol dehydrogenase derived from Clostridium acetobutylicum is 60~120 U / mL, with 80 U / mL being optimal;

[0041] The dosage of the 2-deoxy-D-ribose-5-phosphoaldolase derived from Thermoplasma acidophilum is 40~150U / mL, with 100U / mL being optimal.

[0042] The amount of enol reductase derived from Clostridium tyrobutyricum used is 100 U / mL to 150 U / mL, with 100 U / mL being optimal.

[0043] In the applications described in this invention, the plants include, but are not limited to, bamboo, corn stalks, and / or rice stalks; in a specific embodiment of this invention, the plant is bamboo.

[0044] In the preparation method of the present invention, after the plant is crushed, acid-treated and before drying, the solid is further separated and collected, and the solid is washed with water until it is neutral.

[0045] The cellulase hydrolysis conditions were as follows: hydrolysis at 50°C for 48 hours in 50 mM, pH 4.8 acetic acid buffer.

[0046] The enzymatic hydrolysis is followed by a filtration step, which is not limited in this invention.

[0047] This invention uses lignocellulose hydrolysate as raw material. Through optimization of the acid treatment reagent and time, an intermediate raw material is obtained that can be better hydrolyzed by cellulase. Further optimization of the cellulase dosage, screening of fermentation strains, and optimization of the yeast powder dosage during fermentation yields a fermentation broth with high butanol content. Optimization of enzyme screening and dosage during the enzyme-catalyzed conversion further improves the yield and purity of the final product, 2-ethylhexanal. Therefore, the various parameters in this invention work synergistically to influence the yield and purity of the final product.

[0048] This invention utilizes renewable lignocellulose resources, reducing dependence on non-renewable fossil resources and aligning with the principles of green chemistry and sustainable development. The enzyme-catalyzed reaction conditions are mild, energy-efficient, highly selective, and produce few byproducts. It avoids the high temperatures, high pressures, and strong alkaline conditions required in traditional chemical synthesis, significantly reducing energy consumption and equipment requirements, and minimizing the generation of toxic and harmful byproducts, thereby lowering waste disposal costs and environmental pollution.

[0049] This invention uses lignocellulose hydrolysate as raw material. Through optimization of acid treatment reagents, acid treatment time, cellulase dosage, fermentation strains, yeast powder dosage during fermentation, and catalytic enzymes, a biocatalytic method for producing 2-ethylhexanal from lignocellulose is obtained. This method has high conversion efficiency, is environmentally friendly, and has high application value. Attached Figure Description

[0050] Figure 1 The mass spectrometry identification results of 2-ethylhexanal are shown, where a is the gas chromatogram of the product obtained according to the steps of Example 1 but without enzyme-catalyzed conversion; b is the gas chromatogram of the product obtained according to the steps of Example 1 with enzyme-catalyzed conversion; and c is the mass spectrum of 2-ethylhexanal.

[0051] Figure 2The results show the glucose yield and 2-ethylhexanal production under different acid pretreatments.

[0052] Figure 3 The results show the glucose yield and 2-ethylhexanal production at different acid pretreatment times;

[0053] Figure 4 The results show the glucose yield and 2-ethylhexanal production under different enzyme dosages;

[0054] Figure 5 The fermentation effects of different strains are shown, where a is the liquid chromatogram of the fermentation broth of different strains; b is the concentration of butanol and the yield of 2-ethylhexanal in the fermentation broth of different strains.

[0055] Figure 6 The concentration of butanol and the yield of 2-ethylhexanal in the fermentation broth under different yeast powder dosages are shown.

[0056] Figure 7 The concentration of butanol and the yield of the final product 2-ethylhexanal in the fermentation broth after treatment with different amounts of activated carbon are shown.

[0057] Figure 8 PONDR fraction map of the intrinsic disorder region (IDR) of heat shock proteins and fusion enzymes constructed based on IDR;

[0058] Figure 9 Indicates the activity of different fusion enzymes;

[0059] Figure 10 The concentrations of 2-ethylhexanal produced by the fusion enzyme and the unfusion enzyme at different temperatures are shown, where a is 30℃, b is 40℃, and c is 50℃. Detailed Implementation

[0060] This invention provides an enzyme composition and method for producing 2-ethylhexanal from lignocellulose. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art will clearly be able to modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0061] The nucleotide sequence derived from the fusion of Clostridium acetobutylicum alcohol dehydrogenase and IDR1 is: atg ga agatcctgaaacctgtcaagagcttcaacttttagcagaaagagtgaagaagactttatcacaaagagaaaaggca cctatatcaattaattacgagggagaaagggcaaaaatagaaata

[0062] The nucleotide sequence of the fusion of 2-deoxy-D-ribose-5-phosphoaldolase from Thermoplasma acidophilum with IDR1 is: atg gaagatcctgaaacctgtcaagagcttcaacttttagcagaaagagtgaagaagactttatc acaaagagaaaaggcacctatatcaattaattacgagggagaaagggcaaaaatagaaata (SEQ ID NO:2);

[0063] Nucleotide sequence of enol reductase fused with IDR1 from Clostridium tyrobutyricum:

[0064] atg gaagatcctgaaacctgtcaagagcttcaacttttagcagaaagagtgaagaagactttatcac aaagagaaaaggcacctatatcaattaattacgagggagaaagggcaaaaatagaaata

[0065] IDRI:EDPETCQELQLLAERVKKTLSQREKAPISINYEGERAKIEI (SEQ ID NO:4);

[0066] IDR2: EFTERFGTREENQENVMIKIV (SEQ ID NO:5);

[0067] IDR3:GEAELVLPENLPKNSPVDITFKLNEEGRLEIKAIEAVEKREISTSIETSSVIKGKDLEEAKERNKNIE (SEQ ID NO:6);

[0068] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0069] Example 1: Enzyme composition and method for producing 2-ethylhexanal from lignocellulose.

[0070] An enzyme composition and method for producing 2-ethylhexanal from lignocellulose includes the following steps:

[0071] 1. Preparation of lignocellulose hydrolysate:

[0072] Take 1 kg of dried bamboo, crush it to 40 mesh to obtain bamboo powder, treat it with 1% sulfuric acid solution (the mass ratio of the bamboo powder crushed to 40 mesh to the mass of 1% sulfuric acid solution is 1:10) at 150℃ for 30 min, separate the solid and liquid, wash the solid with water until neutral, and dry it at 50℃ to obtain bamboo pretreated powder.

[0073] Bamboo pretreatment powder was added at 10 wt% to 50 mM, pH 4.8 acetic acid buffer. Cellulase (purchased from Novozymes, Ctec3) was added at a rate of 10 mg per gram of bamboo pretreatment powder. The mixture was then incubated at 50 °C for 48 h and filtered to obtain a hydrolysate containing glucose.

[0074] 2. Fermentation for the production of butanol:

[0075] Add 5 g / L yeast powder to 100 mL of the above hydrolysate, remove oxygen by nitrogen stripping for 20 minutes, sterilize at 115 °C for 20 minutes, cool to room temperature, and add 1 mL of sterilized and deoxygenated 100×P2 basal medium to obtain fermentation medium.

[0076] 100× P2 basal medium: 50 g / L KH2PO4, 50 g / L K2HPO4, 220 g / L CH3COONH4, 20 g / L MgSO4·7H2O, 1 g / L MnSO4·H2O, 1 g / L FeSO4·7H2O, 1 g / L NaCl, 0.1 g / L p-aminobenzoic acid, 0.1 g / L thiamine, and 0.001 g / L biotin;

[0077] Seed culture preparation: Weigh 38g of enhanced Clostridium acetobutylicum culture medium powder (RCM, purchased from Beijing Solarbio Science & Technology Co., Ltd.), add it to 1000mL of distilled water, heat to boiling to dissolve, dispense into 200mL anaerobic bottles, remove nitrogen gas, autoclave at 121℃ for 15 minutes, cool, and use as seed culture medium; after activation, take 100μl of Clostridium acetobutylicum (CICC 8012) stored in glycerol tubes and inoculate it into the seed culture medium. After culturing at 30℃ for 16 hours, the OD reaches 5.2, which is used as seed culture.

[0078] The seed culture was inoculated into the fermentation medium at an inoculation rate of 1% (v / v), and fermented at 30°C under anaerobic conditions for 96 hours. After fermentation, the culture was centrifuged to remove impurities such as bacterial cells, and 5 g / L activated carbon was added for decolorization (with shaking for 30 minutes) to obtain a purified fermentation broth containing butanol.

[0079] 3. Preparation of catalytic enzymes:

[0080] After codon optimization, the IDRI-encoding nucleic acid from the *Clostridium acetobutylicum* alcohol dehydrogenase gene was fused to the N-terminus of the *Clostridium acetobutylicum* alcohol dehydrogenase gene for expression (the fusion nucleotide sequence is shown in SEQ ID NO:1, and the amino acid sequence of IDRI is shown in SEQ ID NO:4). The fusion nucleotide sequence was synthesized by Qingke Biotechnology and inserted into the pET28a plasmid to construct the alcohol dehydrogenase fusion expression vector. The validated alcohol dehydrogenase fusion expression vector was transformed into *E. coli* BL21, and strains expressing alcohol dehydrogenase were screened by plate culture. Single colonies were selected from LB plates and inoculated into liquid LB medium containing kanamycin, cultured at 30°C until the OD reached 0.6, and then IPTG was added to a final concentration of 0.4 mM, and the culture was continued at 25°C for 24 h. The bacterial cells were collected by centrifugation, lysed, and the crude alcohol dehydrogenase enzyme solution was obtained.

[0081] Using the same fusion fragment IDRI, heterologous fusion expression of 2-deoxy-D-ribose-5-phosphate aldolase from Thermoplasma acidophilum (the fusion nucleotide sequence is shown in SEQ ID NO:2) and enol reductase from Clostridium tyrobutyricum (the fusion nucleotide sequence is shown in SEQ ID NO:3) was performed according to the above method, and the corresponding crude enzyme solutions were prepared.

[0082] 4. Enzymatic conversion: Add 80 U / mL alcohol dehydrogenase, 100 U / mL aldolase, and 100 U / mL enol reductase, along with 2 mM NAD, to the purified butanol fermentation broth. + The reaction was carried out at 40℃ for 8 hours. After the reaction was complete, 2-ethylhexanal was extracted three times with 5 mL of ethyl acetate. Gas chromatography analysis showed that the product concentration reached 7.2 g / L, and the yield was 86 g / kg (2-ethylhexanal yield / weight of dried bamboo). Mass spectrometry identification results are as follows. Figure 1 As shown.

[0083] Example 2: Optimization of the Invention

[0084] I. Optimization in the preparation of lignocellulose hydrolysate

[0085] 1. Sugar yield under different acid pretreatment conditions:

[0086] Bamboo powder, pulverized to 40 mesh, was pretreated with 1% H2SO4 (1% sulfuric acid solution), 1% HCl (1% hydrochloric acid solution), and 1% H3PO4 (1% phosphoric acid solution). The mass ratio of the pulverized bamboo powder to the acid solution (1% H2SO4, 1% HCl, and 1% H3PO4) was 1:10. The treatment temperature was 150℃, and the treatment time was 40 minutes. After treatment, the solid and liquid were separated, the solid was washed with water until neutral, and dried at 50℃ to obtain the pretreated bamboo powder.

[0087] Bamboo pretreatment powder was added to 50 mM, pH 4.8 acetic acid buffer at a dosage of 10 wt%. Cellulase (purchased from Novozymes, Ctec3) was added at a dosage of 15 mg per gram of bamboo pretreatment powder. The mixture was incubated at 50 °C for 48 h and then filtered to obtain a hydrolysate containing glucose.

[0088] Glucose concentration was determined by high-performance liquid chromatography (HPLC), and glucose yield was calculated. Glucose yield = glucose in hydrolysate / theoretically convertible glucose from bamboo cellulose.

[0089] The subsequent preparation steps for 2-ethylhexanal were the same as in Example 1. The results are as follows: Figure 2As shown, calculations show that the glucose yields after pretreatment with 1% H2SO4, 1% HCl, and 1% H3PO4 are 85.9%, 73.5%, and 48.0%, respectively. Therefore, 1% H2SO4 pretreatment yields more glucose, and the corresponding yields of the final product 2-ethylhexanal are 43.5 g / kg, 32.1 g / kg, and 22.0 g / kg (yield of 2-ethylhexanal / weight of dried bamboo), respectively. It can be seen that 1% sulfuric acid solution pretreatment provides more raw materials that can be used for the synthesis of 2-ethylhexanal, increasing the yield of the final product.

[0090] 2. Sugar yield under different sulfuric acid pretreatment conditions.

[0091] Bamboo powder pulverized to 40 mesh was pretreated with 1% sulfuric acid solution. The mass ratio of bamboo powder to 1% sulfuric acid solution was 1:10. The treatment temperature was 150℃, and the treatment time was 10, 30, 50, and 80 minutes, respectively. After treatment, solid and liquid were separated, the solid was washed with water until neutral, and dried at 50℃ to obtain pretreated bamboo powder.

[0092] Bamboo pretreatment powder was added at 10 wt% to 50 mM, pH 4.8 acetic acid buffer. Cellulase (purchased from Novozymes, Ctec3) was added at a rate of 15 mg per gram of bamboo pretreatment powder. The mixture was incubated at 50 °C for 48 h and then filtered to obtain a hydrolysate containing glucose.

[0093] Glucose concentration was determined by high-performance liquid chromatography (HPLC), and the glucose concentration after treatment and glucose yield were calculated. Glucose yield = glucose in hydrolysate / theoretically convertible glucose from bamboo cellulose.

[0094] The subsequent preparation steps for 2-ethylhexanal were the same as in Example 1. The results are as follows: Figure 3 As shown, the calculated glucose yields after treatment for 10, 30, 50, and 80 minutes were 64.0%, 90.3%, 76.0%, and 57.4%, respectively. The corresponding yields of the final product, 2-ethylhexanal, were 26.1 g / kg, 46.4 g / kg, 28.4 g / kg, and 23.9 g / kg, respectively. Therefore, the glucose yield after pretreatment with 1% sulfuric acid solution for 30 minutes was significantly higher than that after other treatment times, and the final yield of 2-ethylhexanal was also significantly higher than that of the other groups.

[0095] 3. Optimization of cellulase dosage:

[0096] The bamboo pretreatment powder obtained by treating with 1% sulfuric acid solution at 150℃ for 30 minutes, using the optimal pretreatment conditions in section 2, was used as raw material for enzymatic hydrolysis.

[0097] Cellulase (purchased from Novozymes, Ctec3) was added at concentrations of 2.5, 5, 10, 15, and 20 mg per gram of bamboo pretreated powder, and then placed at 50°C for 48 hours for enzymatic hydrolysis. After filtration, a hydrolysate containing glucose was obtained.

[0098] Glucose concentration was determined by high-performance liquid chromatography (HPLC), and the glucose concentration after treatment and glucose yield were calculated. Glucose yield = glucose in hydrolysate / theoretically convertible glucose from bamboo cellulose.

[0099] The subsequent preparation steps for 2-ethylhexanal were the same as in Example 1. The results are as follows: Figure 4 As shown, the glucose yields were 52.8%, 76.2%, 90.5%, 90.4%, and 90.5%, respectively, and the corresponding yields of 2-ethylhexanal were 25 g / kg, 34 g / kg, 46.8 g / kg, 46.5 g / kg, and 46.5 g / kg, respectively.

[0100] Although the enzyme dosage was increased from 10 mg / g to 20 mg / g, the yield of the corresponding final product 2-ethylhexanal remained unchanged. Considering economic efficiency, 10 mg / g of cellulase was selected (cellulase dosage / gram of bamboo pretreatment powder).

[0101] II. Optimization of the fermentation process for producing butanol

[0102] 1. Screening of fermentation strains:

[0103] The fermentation strains selected were Clostridium acetobutylicum (CICC 8012, purchased from China Industrial Microbial Culture Collection Center), Clostridium beijerinckii (SHMCC D72800, purchased from Shanghai Preservation Microbial Co., Ltd.), and Clostridium saccharobutyricum (SHMCCD24570, purchased from Shanghai Preservation Microbial Co., Ltd.). The preparation of the lignocellulose hydrolysate was the same as in Example 1.

[0104] Add 3 g / L yeast powder to 100 mL of the above hydrolysate, remove oxygen by nitrogen stripping for 20 minutes, sterilize at 115 °C for 20 minutes, cool to room temperature, and add 1 mL of sterilized and deoxygenated 100 × P2 basal medium to obtain fermentation medium.

[0105] The seed culture was inoculated into the fermentation medium at an inoculation rate of 1% (v / v), and fermented at 30°C under anaerobic conditions for 96 hours. After fermentation, the culture was centrifuged to remove impurities such as bacterial cells, and 5 g / L activated carbon was added for decolorization (with shaking for 30 minutes) to obtain a purified fermentation broth containing butanol.

[0106] After fermentation, the concentration of butanol in the fermentation broth was measured by high performance liquid chromatography. The subsequent preparation steps of 2-ethylhexanal were the same as in Example 1.

[0107] like Figure 5 As shown, the butanol concentrations corresponding to treatments with *Clostridium acetone-butanol*, *Clostridium bengalicum*, and *Clostridium succinate* were 8.7 g / L, 6.4 g / L, and 3.7 g / L, respectively, with corresponding butanol yields of 73.2 g / kg, 54.1 g / kg, and 32.5 g / kg bamboo (bamboo powder after 40 mesh grinding, or dried bamboo). The corresponding yields of the final product, 2-ethylhexanal, were 42.4 g / kg, 33.6 g / kg, and 21.3 g / kg bamboo, respectively.

[0108] Therefore, Clostridium acetobutylicum CICC 8012 produces the most butanol, resulting in the highest yield of the corresponding final product.

[0109] 2. Yeast powder dosage:

[0110] Clostridium acetobutylicum CICC 8012 was selected, and the amount of yeast powder used was set to be 1, 3, 5, and 7 g / L. Other steps were the same as in Example 1. After fermentation, the concentration of butanol in the fermentation broth was measured by high performance liquid chromatography. The subsequent preparation steps of 2-ethylhexanal were the same as in Example 1.

[0111] like Figure 6 As shown, the butanol concentrations corresponding to yeast powder dosages of 1, 3, 5, and 7 g / L are 5.5 g / L, 8.4 g / L, 10.6 g / L, and 8.2 g / L, respectively, with corresponding butanol yields of 46.2 g / kg, 71.1 g / kg, 88.3 g / kg, and 65.1 g / kg of bamboo (40-mesh pulverized bamboo powder or dried bamboo); the corresponding yields of the final product 2-ethylhexanal are 29.1 g / kg, 48.2 g / kg, 62.0 g / kg, and 43.0 g / kg of bamboo, respectively. Therefore, 5 g / L of yeast powder is the optimal dosage.

[0112] 3. Decolorization and impurity removal of fermentation broth:

[0113] Activated carbon concentrations of 2 g / L, 5 g / L, 10 g / L, and 20 g / L were used. After shaking for 30 minutes, the activated carbon was removed by filtration, and the butanol concentration in the filtrate was measured. Other steps were the same as in Example 1 to prepare 2-ethylhexanal.

[0114] like Figure 7As shown, the butanol concentrations in the filtrate were 10.4 g / L, 10.0 g / L, 8.4 g / L, and 6.5 g / L, respectively. The corresponding yields of the final product, 2-ethylhexanal, were 42.5 g / kg, 62.1 g / kg, 50.9 g / kg, and 38.0 g / kg; therefore, 5 g / L is the optimal activated carbon dosage. Furthermore, compared to no activated carbon added (21.2 g / kg), the yield increased by 193%.

[0115] III. Screening of Catalytic Enzymes

[0116] 1. Screening of alcohol dehydrogenases

[0117] Five dehydrogenases were selected from *Saccharomyces cerevisiae* (NCBI protein ID: CAA24601.1), *Clostridium acetobutylicum* (NCBI protein ID: AAD04638.1), *Escherichiacoli* (NCBI protein ID: NP_415995.4), *Rhodococcus erythropolis* (NCBI protein ID: AGT94347.1), and *Cupriavidus necator* (NCBI protein ID: CAJ91905.1). Codon optimization software was used to optimize the sequences, and the optimized gene sequences were synthesized by Qingke Biotechnology to obtain the target gene. The target gene was inserted into the pET28a plasmid to construct an alcohol dehydrogenase expression vector. The vector was transformed into *Escherichia coli* BL21, and a strain expressing alcohol dehydrogenase was obtained through plate selection. Single colonies were selected from LB agar plates and inoculated into liquid LB medium containing kanamycin. The culture was carried out at 30°C until the OD reached 0.6. IPTG was then added to a final concentration of 0.4 mM, and the culture was continued at 25°C for 24 h. The cells were collected by centrifugation, lysed, and the crude enzyme solution of alcohol dehydrogenase was obtained. The corresponding product was purified after fermentation according to the method in Example 1, and the enzyme activity was measured.

[0118] Enzyme activity assay: Add 10 mM n-butanol solution and 10 mM NAD⁺ to 50 mL phosphate buffer (50 mM, pH 6.0), add 50 mg alcohol dehydrogenase, react at 30 °C for 1 hour, and stop the reaction by boiling for 5 minutes. Determine the butyraldehyde concentration by high performance liquid chromatography and calculate the specific activity.

[0119] The specific activities of the five enzymes were obtained to be 4.2 U / mg, 8.6 U / mg, 1.3 U / mg, 1.0 U / mg, and 2.1 U / mg, respectively (see Table 1). Therefore, the alcohol dehydrogenase from Clostridium acetobutylicum (NCBI protein ID: AAD04638.1) is the optimal alcohol dehydrogenase, and its nucleotide sequence is shown in SEQ ID NO:1 with the bolded IDR1 sequence removed.

[0120] 2. Aldolase screening:

[0121] Fructose-6-phosphate aldolase from *Escherichia coli* (NCBI protein ID: NP_415346.4), fructose-6-phosphate aldolase from *Saccharomyces cerevisiae* (NCBI protein ID: CAA81897.1), 2-deoxy-D-ribose-5-phosphate aldolase from *Escherichia coli* (NCBI protein ID: NP_418798.1), 2-deoxy-D-ribose-5-phosphate aldolase from *Bacillus subtilis 168* (NCBI protein ID: CAB15978.2), and 2-deoxy-D-ribose-5-phosphate aldolase from *Thermoplasma acidophilum* (NCBI protein ID: CAC11822.1) were selected. The corresponding products were purified after fermentation according to the above method, and enzyme activity was measured.

[0122] Enzyme activity assay: Add 10mM butyraldehyde solution and 50mg aldolase to 50mL phosphate buffer (50mM, pH 6.0), react at 30℃ for 1 hour, and stop the reaction by boiling for 5 minutes. Determine the butyraldehyde concentration by high performance liquid chromatography, and calculate the specific activity based on the amount of butyraldehyde converted.

[0123] The specific activities of the five enzymes were obtained to be 0.60 U / mg, 0.77 U / mg, 1.03 U / mg, 0.40 U / mg, and 3.30 U / mg, respectively (see Table 1). Therefore, the 2-deoxy-D-ribose-5-phosphate aldolase from *Thermoplasma acidophilum* is the optimal choice, and its nucleotide sequence is shown in SEQ ID NO:2 with the bolded IDR1 sequence removed.

[0124] 3. Enol reductase screening:

[0125] 2-enol reductases from Clostridium acetobutylicum (NCBI Protein ID: AAK81302), Clostridium tyrobutyricum (NCBI Protein ID: AND83867.1), Clostridium pasteurianum (NCBI Protein ID: AJA50818.1), Clostridium beijerinckii (NCBI Protein ID: AIU02858), and Clostridium carboxidivorans (NCBI Protein ID: AKN30008) were selected. The corresponding products were purified after fermentation according to the above method, and their enzyme activities were measured.

[0126] Enzyme activity assay: Add 5mM 2-ethyl-2-hexenal, 10mM NADH and 50mg enol reductase to 50mL phosphate buffer (50mM, pH 6.0), react at 30℃ for 1 hour, and stop the reaction by boiling for 5 minutes. Determine the concentration of the product 2-ethyl-hexenal by gas chromatography, and calculate the specific activity based on the generated 2-ethyl-hexenal.

[0127] The specific activities of the five enzymes were obtained as 0.23 U / mg, 1.13 U / mg, 0.19 U / mg, 0.34 U / mg, and 0.10 U / mg, respectively (see Table 1). Therefore, the enol reductase from Clostridium tyrobutyricum was the best, and its nucleotide sequence is shown in SEQ ID NO:3 with the bolded IDR1 sequence removed.

[0128] Table 1. Specific activities of enzymes from different sources

[0129]

[0130] IV. Optimization of the optimal enzyme dosage

[0131] 1. Optimization of alcohol dehydrogenase dosage:

[0132] Add 2-deoxy-D-ribose-5-phosphate aldolase (derived from *Thermoplasma acidophilum*), enol reductase (derived from *Clostridium tyrobutyricum*), alcohol dehydrogenase (derived from *Clostridium acetobutylicum*), and NAD to 10 mL of purified butanol fermentation broth. +The concentrations of aldolase and enol reductase were fixed at 40 U / mL and 60 U / mL, respectively, and NAD+ was... + The concentration of alcohol dehydrogenase was 2 mM, and the dosages were 40 U / mL, 60 U / mL, 80 U / mL, and 120 U / mL, respectively. The reaction was carried out at 30℃ for 24 h. After the reaction was completed, 2-ethylhexanal was extracted three times with 5 mL of ethyl acetate. The concentration of 2-ethylhexanal was determined by gas chromatography. The corresponding product concentrations at different enzyme concentrations were 2.1 g / L, 4.1 g / L, 5.0 g / L, and 4.3 g / L, respectively, and the corresponding yields of the final product 2-ethylhexanal were 19 g / kg, 40 g / kg, 52 g / kg, and 42 g / kg (see Table 2). Therefore, the optimal dosage of alcohol dehydrogenase was 80 U / mL.

[0133] 2. Optimization of aldolase dosage:

[0134] Aldolase, enol reductase, alcohol dehydrogenase, and NAD were added to 10 mL of purified butanol fermentation broth, respectively. + The concentrations of alcohol dehydrogenase and enol reductase were fixed at 80 U / mL and 60 U / mL, respectively, and NAD... + The concentration of aldolase (2-deoxy-D-ribose-5-phosphate aldolase derived from Thermoplasma acidophilum) was 20 U / mL, 40 U / mL, 100 U / mL, and 150 U / mL, respectively. The reaction was carried out at 30℃ for 24 h. After the reaction was complete, 2-ethylhexanal was extracted three times with 5 mL of ethyl acetate. The concentration of 2-ethylhexanal was determined by gas chromatography. The corresponding product concentrations at different enzyme concentrations were 2.6 g / L, 4.9 g / L, 5.5 g / L, and 4.1 g / L, respectively, with corresponding yields of 2-ethylhexanal of 16 g / kg, 49 g / kg, 60 g / kg, and 40 g / kg (see Table 2). Therefore, the optimal aldolase concentration was 100 U / mL.

[0135] 3. Optimization of enol reductase dosage:

[0136] Aldolase, enol reductase, alcohol dehydrogenase, and NAD were added to 10 mL of purified butanol fermentation broth, respectively. + The concentrations of alcohol dehydrogenase and aldolase were fixed at 80 U / mL and 100 U / mL, respectively, and NAD+ was... +The concentration of enol reductase was 2 mM, and the dosages were 20 U / mL, 60 U / mL, 100 U / mL, and 150 U / mL, respectively. The reaction was carried out at 30℃ for 24 h. After the reaction was completed, 2-ethylhexanal was extracted three times with 5 mL of ethyl acetate. The concentration of 2-ethylhexanal was determined by gas chromatography. The corresponding product concentrations at different enzyme concentrations were 2.1 g / L, 5.4 g / L, 6.1 g / L, and 6.0 g / L, respectively, and the corresponding yields of the final product 2-ethylhexanal were 15 g / kg, 56 g / kg, 73 g / kg, and 68 g / kg (see Table 2). Therefore, the optimal dosage of enol reductase was 100 U / mL.

[0137] Table 2. Concentration and yield of 2-ethylhexanal at different enzyme dosages

[0138]

[0139] V. Further Enzyme Optimization - Construction of Fusion Enzymes

[0140] 1. Fragment Filtering

[0141] When wild-type enzymes were used for catalysis, the overall reaction time was long and the space-time conversion rate of the product was low. Therefore, three wild-type enzymes were further modified. The sequence of the heat shock protein (NCBI Protein ID: AAK78452) from Clostridium acetobutylicum was analyzed using the online tool PONDR. Figure 8 In (a), the three highest-scoring Intrinsic Disordered Region (IDR) sequences were obtained.

[0142] They are respectively

[0143] IDRI: EDPETCQELQLLAERVKKTLSQREKAPISINYEGERAKIEI (SEQ ID NO:4);

[0144] IDR2: EFTERFGTREENQENVMIKIV (SEQ ID NO:5);

[0145] IDR3:GEAELVLPENLPKNSPVDITFKLNEEGRLEIKAIEAVEKREISTSIETSSVIKGKDLEEAKERNKNIE (SEQ ID NO:6);

[0146] The three sequences were respectively fused into the N-terminus or C-terminus of the selected optimal alcohol dehydrogenase. Figure 8In section b), different fusion proteins were constructed. The enzymes formed by fusing IDR3 with the N-terminus or C-terminus of the three enzymes all lost their activity. The specific activities of the new alcohol dehydrogenase, aldolase, and enol reductase formed by fusing IDR1 with the N-terminus of the three enzymes were 13.2 U / mg, 5.8 U / mg, and 2.9 U / mg, respectively, significantly higher than the unfused wild-type alcohol dehydrogenase (8.6 U / mg), aldolase (3.3 U / mg), and enol reductase (1.1 U / mg). However, the activities of the three enzymes formed by fusing IDR1 with the C-terminus of the enzymes were 2.0 U / mg, 2.5 U / mg, and 1.3 U / mg, respectively, lower than or not significantly different from the wild-type. The specific activities of alcohol dehydrogenase, aldolase, and enol reductase formed by fusion of IDR2 with the N-terminus of the three enzymes were 7.8 U / mg, 3.1 U / mg, and 1.2 U / mg, respectively. The specific activities of alcohol dehydrogenase, aldolase, and enol reductase formed by C-terminal fusion were 6.4 U / mg, 3.0 U / mg, and 1.6 U / mg, respectively. Therefore, only the enzymes with N-terminal fusion with IDR1 showed a significantly increased specific activity. Figure 9 ).

[0147] 2. Optimization of reaction temperature

[0148] Three enzymes with IDR1 fused to their N-terminus were subjected to catalytic reactions in the same manner as described above, with alcohol dehydrogenase, aldolase, and enol reductase at concentrations of 80 U / mL, 100 U / mL, and 100 U / mL, respectively. The reaction temperatures were set at 30, 40, and 50 °C, and the results are as follows. Figure 10 As shown.

[0149] At 30℃, the yield of 2-ethylhexanal in the fusion enzyme catalytic system reached its maximum at 12 h, with a corresponding concentration and yield of 6.7 g / L and 0.558 g / L / h, respectively. In the unfused enzyme catalytic system, the yield of 2-ethylhexanal reached its maximum at 24 h, with a corresponding concentration and yield of 6.5 g / L and 0.271 g / L / h, respectively.

[0150] At 40℃, the yield of 2-ethylhexanal in the fusion enzyme catalytic system reached its maximum at 8h, with the corresponding concentration and yield increasing to 7.2g / L and 0.90g / L / h, respectively. In the unfused enzyme catalytic system, although the yield of 2-ethylhexanal increased to 0.317g / L / h, the concentration decreased to 3.8g / L.

[0151] At 50°C, in the fusion enzyme catalytic system, the yield of 2-ethylhexanal still reached its maximum at 8 h, but the corresponding concentration and yield decreased to 6.9 g / L and 0.863 g / L / h, respectively. In the unfused enzyme catalytic system, the concentration and yield of 2-ethylhexanal decreased to 0.2 g / L and 0.025 g / L / h, respectively.

[0152] Therefore, compared with the unfused enzyme, the fusion enzyme exhibits better catalytic activity at high temperatures, significantly improving catalytic efficiency and the synthesis efficiency of the final product, 2-ethylhexanal. The optimal catalytic temperature of the fusion enzyme is 40℃, and the yield of 2-ethylhexanal reaches its maximum at 8 hours, with corresponding concentrations, yields, and production rates of 7.2 g / L, 0.90 g / L / h, and 86 g / kg, respectively. Compared with the wild-type enzyme under the same conditions, these represent increases of 47.2%, 183.9%, and 91.1%, respectively.

[0153] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An enzyme composition, characterized in that, include: Alcohol dehydrogenase derived from Clostridium acetobutylicum, 2-deoxy-D-ribose-5-phosphate aldolase derived from Thermoplasma acidophilum, and enol reductase derived from Clostridium tyrobutyricum.

2. The fusion protein composition according to claim 1, characterized in that, The nucleotide sequence of the alcohol dehydrogenase derived from Clostridium acetobutylicum is shown in SEQ ID NO:1; The nucleotide sequence of the 2-deoxy-D-ribose-5-phosphoaldolase derived from Thermoplasma acidophilum is shown in SEQ ID NO:2; The nucleotide sequence of the enol reductase derived from Clostridium tyrobutyricum is shown in SEQ ID NO:

3.

3. The enzyme composition according to claim 2, characterized in that, The alcohol dehydrogenase derived from Clostridium acetobutylicum also has a heat shock protein fragment attached to its N-terminus or C-terminus; and / or The N-terminus or C-terminus of the 2-deoxy-D-ribose-5-phosphoaldolase derived from *Thermoplasma acidophilum* is also linked to a heat shock protein fragment; and / or The enol reductase derived from Clostridium tyrobutyricum also has a heat shock protein fragment attached to its N-terminus or C-terminus. The amino acid sequence of the heat shock protein fragment is shown in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:

6.

4. A biomaterial, characterized in that, Includes at least one of the following: A) to D) A) Nucleic acid encoding the enzyme composition according to any one of claims 1 to 3; B) A recombinant vector containing the nucleic acid described in A); C) Transfecting or transforming host cells with the recombinant vector described in B); D) Cultures obtained by culturing host cells as described in C) 5. An application of one of the following (I) to (II) in the preparation of 2-ethylhexanal: I) The enzyme composition according to any one of claims 1 to 3; II) The biomaterial as described in claim 4.

6. The application according to claim 5, characterized in that, The 2-ethylhexanal is prepared by using wood fiber as raw material. 7.2-Ethylhexanal preparation method, characterized in that, Includes the following steps: Step 1: The plants are crushed, acid-treated, and dried to obtain pretreated powder; Step 2: The pretreated powder is enzymatically hydrolyzed by cellulase to obtain a hydrolysate; Step 3: The hydrolysate is decolorized after fermentation with Clostridium acetone-butanol to obtain a fermentation broth containing butanol; Step 4: The butanol-containing fermentation broth is treated with the enzyme composition according to any one of claims 1 to 3 to obtain 2-ethylhexanal.

8. The preparation method according to claim 7, characterized in that, The reagent used for acid treatment is a 1% sulfuric acid solution; The acid treatment time is 30 min to 50 min; The amount of cellulase used is 10mg to 20mg per gram of the pretreated powder.

9. The preparation method according to claim 8, characterized in that, In the fermentation process, the amount of yeast powder used is 3g / L~7g / L; The decolorizing agent is activated carbon, and the amount of activated carbon added is 5g / L~10g / L.

10. The preparation method according to claim 9, characterized in that, The amount of alcohol dehydrogenase derived from Clostridium acetobutylicum is 60~120 U / mL, with an optimal amount of 80 U / mL; The dosage of the 2-deoxy-D-ribose-5-phosphoaldolase derived from Thermoplasma acidophilum is 40 U / mL to 150 U / mL. The amount of enol reductase derived from Clostridium tyrobutyricum used is 100 U / mL to 150 U / mL.

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