Method for promoting enzymolysis and fermentation of straw cellulose by using fresh straw wall protein

Abstract

The invention belongs to the technical field of enzymolysis and fermentation of lignocellulose, and particularly relates to a method for promoting enzymolysis and fermentation of straw cellulose by using fresh straw wall protein, which comprises the following steps: firstly, extracting straw cell wall protein and culturing engineering bacteria to obtain expansive-like protein; the method comprises the following steps: mixing the fresh straw cell wall protein and the expansion-like protein with steam explosion straw, carrying out pretreatment under proper pH and concentration, carrying out cellulose enzymolysis and synchronous saccharification fermentation, and finally converting into ethanol. The method effectively improves the straw conversion efficiency, is suitable for bioethanol production, combines the fresh straw cell wall protein and the expansion-like protein from microorganisms, and improves the bioethanol yield. The expansive-like protein and the fresh straw cell wall protein have a remarkable synergistic effect, the expansive-like protein can destroy a cellulose crystal structure in a non-hydrolytic manner and increase accessibility of cellulose, and the fresh straw cell wall protein provides multiple enzyme activities to jointly promote the enzymolysis process.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of enzymatic hydrolysis and fermentation of lignocellulose, and particularly relates to a method for promoting enzymatic hydrolysis and fermentation of straw cellulose by using fresh straw wall protein. BACKGROUND

[0002] Straw lignocellulose is the most abundant renewable resource on earth. Effective utilization of straw can not only alleviate the problems of food shortage, energy crisis and environmental pollution faced by mankind, but also realize high-value utilization of resources. Conversion of lignocellulose raw materials into fuel ethanol is one of the important ways to develop renewable energy, and has been increasingly valued worldwide. However, enzymatic hydrolysis of straw into fermentable sugars is the central link of conversion, but the current enzymatic hydrolysis process still has problems such as low enzymatic hydrolysis efficiency, low concentration of fermentable sugars, and high cost of cellulase.

[0003] The patent CN101633939B entitled "Method for promoting enzymatic hydrolysis and fermentation of straw cellulose by using fresh straw wall protein" by Chen Hongzhang et al. of Institute of Process Engineering, Chinese Academy of Sciences proposes a method for promoting enzymatic hydrolysis and conversion of straw lignocellulose by using fresh straw cell wall protein. This method extracts abundant and inexpensive endogenous cellulase and its synergistic factor from fresh straw and applies it to straw conversion, which achieves the purposes of improving cellulose conversion efficiency, reducing cellulase cost, and saving the cost of fresh straw pretreatment, but still has certain limitations. Specifically, this patent mainly relies on the promotion of endogenous cell wall protein in fresh straw to the enzymatic hydrolysis process, but does not fully consider how to further destroy the structure of straw and improve the enzymatic hydrolysis efficiency.

[0004] Therefore, it is urgent to develop a new type of straw enzymatic hydrolysis and fermentation process that can not only utilize endogenous cell wall protein in fresh straw, but also combine the synergistic effect of microbial-derived expansin-like protein to further improve enzymatic hydrolysis efficiency, reduce enzyme cost, and realize efficient utilization of straw resources. SUMMARY

[0005] In view of the deficiencies in the prior art, the purpose of the present application is to provide a method for promoting enzymatic hydrolysis and conversion of straw lignocellulose by using the synergistic effect of fresh straw cell wall protein and microbial-derived expansin-like protein. The present application not only utilizes abundant endogenous cell wall protein in fresh straw, but also introduces microbial-derived expansin-like protein as a synergistic factor, which significantly improves enzymatic hydrolysis efficiency, reduces enzyme dosage, saves pretreatment cost, and realizes efficient utilization of straw resources.

[0006] To achieve the above-mentioned purpose, the present application provides a method for promoting enzymatic hydrolysis and fermentation of straw cellulose by using fresh straw wall protein, characterized in that it comprises the following steps:

[0007] A. Preparation of fresh straw cell wall protein and expansin-like protein: fresh straw powder with 40-100 mesh is placed in cell wall protein extraction solution, the solid-liquid ratio is 1 / 4-1 / 10, and the cell wall protein extraction solution is added, and the mixture is placed at 4°C for 12-24 hours for extraction of cell wall protein; meanwhile, the engineering bacteria expressing the expansin-like protein are cultured, the fermentation broth is collected, and the ammonium sulfate precipitation is used to enrich the expansin-like protein;

[0008] B. Straw pretreatment and mixing: the pH of the cell wall protein solution is adjusted to 4.2-5.2, the adjusted cell wall protein solution is mixed with the steam explosion treated straw at a fresh steam explosion straw dry weight ratio of 1 / 3-3 / 1, the expansin-like protein solution is added to the mixture, the addition amount is 0.5-2.0 mg / g substrate, the acetic acid buffer solution with a pH of 4.2-5.2 is added, and the substrate concentration is adjusted to 20-100 mg / ml;

[0009] C. Pretreatment stage: the mixture obtained in step B is pretreated at 30-40°C for 4-12 hours;

[0010] D. Enzymatic hydrolysis stage: cellulase is added at 8-25 FPU / g substrate for enzymatic hydrolysis, the temperature is 45-50°C, and the time is 48-72 hours;

[0011] E. Simultaneous saccharification and fermentation of ethanol: cellulase is added at 8-35 FPU / g substrate, and activated yeast is added, the temperature is 30-37°C, and the time is 72-96 hours.

[0012] Preferably, the cell wall protein extraction solution is 20-35 mM phosphate buffer, 0.6-1.8 M NaCl, 0.6-2.5 mM benzylsulfonyl fluoride, and the pH is 4.8-7.2.

[0013] Preferably, the engineering bacteria expressing the expansin-like protein are Aspergillus or Trichoderma microorganisms, which are cultured in a culture medium containing a carbon source and a nitrogen source at 28-30°C for 48-72 hours.

[0014] In an embodiment of the present application, the enrichment of the expansin-like protein uses 60%-80% saturation ammonium sulfate precipitation, centrifugal collection of the precipitate after standing at 4°C for 6-12 hours, dissolution in 20 mM phosphate buffer with a pH of 5.0, and desalting by dialysis.

[0015] Further, in step B, the pH adjustment is performed using acetic acid, and the acetic acid buffer solution has a concentration of 20-100 mM.

[0016] In addition, in step C, the pretreatment aims to allow the expansin-like protein to fully act on the straw, destroy the crystal structure of cellulose, and increase the accessibility of cellulose.

[0017] In another embodiment of the present application, the fresh straw is corn straw, wheat straw, rice straw, sorghum straw or yellow bamboo grass.

[0018] Preferably, the steam explosion treatment is carried out at 180-220℃ for 3-15 minutes.

[0019] Further, in step D, the cellulase is a commercial cellulase preparation comprising endoglucanase, exoglucanase and beta-glucosidase.

[0020] In a preferred embodiment of the present application, in step E, the activated yeast is Saccharomyces cerevisiae, and the inoculation amount is 0.5-2.0 g / L. The yeast converts glucose to ethanol during the saccharification and fermentation process.

[0021] The present application has the following advantages:

[0022] 1. Synergistic effect: The present application combines fresh straw cell wall protein and microbially derived expansin-like protein, which produces significant synergistic effect. The expansin-like protein can non-hydrolytically destroy the cellulose crystal structure, increasing the accessibility of cellulose, while the fresh straw cell wall protein provides various enzyme activities, which together promote the enzymatic hydrolysis process. Experimental results show that the sugar conversion rate can be increased by 30%-45% compared with using fresh straw cell wall protein alone.

[0023] 2. Reducing enzyme dosage: Through synergistic effect, the present application can reduce the cellulase dosage by 25%-40% while maintaining the same or higher saccharification rate and ethanol yield, greatly reducing the production cost.

[0024] 3. Reducing pretreatment cost: The present application uses fresh straw as the source of endogenous cell wall protein. The fresh straw does not undergo pretreatment process, saving the pretreatment cost. At the same time, it reduces the production of inhibitors during the pretreatment process, which is beneficial to the subsequent fermentation process.

[0025] 4. Wide source: Fresh straw is abundant in source, which not only ensures sufficient raw material supply, but also realizes efficient utilization of straw resources, achieving the purpose of efficient degradation using plant's own resources.

[0026] 5. Temperature synergy: The optimal temperature of the endogenous cellulase in fresh straw is consistent with that of the yeast, to some extent solving the problem of inconsistent temperature between enzymolysis and fermentation, and simplifying the process flow. DETAILED DESCRIPTION

[0027] The present application will be further described in detail below in conjunction with specific embodiments, but the embodiments of the present application are not limited thereto.

[0028] Example 1: Utilization of fresh corn stalk cell wall protein and swollenin to synergistically improve the enzymatic hydrolysis and fermentation efficiency of corn stalk

[0029] In this example, first, the preparation of fresh corn stalk cell wall protein and swollenin was performed. The freshly harvested corn stalk was fully air-dried and then crushed to 60 mesh, and then placed in a cell wall protein extraction solution (25 mM phosphate buffer, 1.0 M NaCl, 1.0 mM benzylsulfonyl fluoride, pH 5.0). The protein extraction solution was added according to a solid-liquid ratio of 1 / 5, and the cell wall protein extraction was performed at 4°C for 15 hours.

[0030] Meanwhile, the Trichoderma engineered bacteria expressing swollenin (a recombinant strain of Aspergillus expressing Trichoderma swollenin gene) were inoculated in a culture medium containing 30 g / L sucrose, 5 g / L yeast extract, 1 g / L KH2PO4, and 0.5 g / L MgSO4·7H2O at an inoculation amount of 10 6 spores / mL, and then cultured at 28°C and 180 rpm for 60 hours. The fermentation broth was collected, precipitated with 70% saturated ammonium sulfate, and then centrifuged at 10,000 g for 20 minutes after being placed at 4°C for 8 hours. The precipitate was dissolved in 20 mM phosphate buffer (pH 5.0), and then dialyzed at 4°C for 24 hours. The protein content was determined to be 1.2 mg / mL using the Bradford method.

[0031] A Trichoderma engineered bacteria expressing swollenin was constructed using molecular cloning technology. The specific method is as follows:

[0032] The Trichoderma reesei QM9414 strain was selected as the host bacteria and the source strain of the swollenin gene. The strain can be obtained from the United States Department of Agriculture Microbial Strain Preservation Center (NRRL). The plasmids pUC19 and pAN7-1 were used for intermediate cloning and Trichoderma transformation, respectively, and the pAN7-1 contains the hygromycin B resistance gene (hph) as a selection marker.

[0033] Trichoderma reesei strains were cultured on potato dextrose agar (PDA) plates at 28°C for 5–7 days until spore formation. For liquid culture, Mandels medium containing 2% cellulose or 2% sucrose was used (components: KH₂PO₄ 2.0 g / L, (NH₄)₂SO₄ 1.4 g / L, MgSO₄·7H₂O 0.3 g / L, CaCl₂·2H₂O 0.3 g / L, FeSO₄·7H₂O 5 mg / L, MnSO₄·H₂O 1.6 mg / L, ZnSO₄·7H₂O 1.4 mg / L, CoCl₂·6H₂O 2.0 mg / L, yeast extract 0.25 g / L, pH 5.0). Liquid culture was carried out at 28°C and 180 rpm, with an induction time of 72–96 hours.

[0034] First, total RNA was extracted from *Trichoderma reesei* strain QM9414. Approximately 100 mg of *Trichoderma reesei* mycelium cultured for 48 hours was added to 1 mL of TRIzol reagent (Invitrogen), and RNA extraction was performed according to the kit instructions. A reverse transcription kit (Takara's PrimeScript) was used. TM The RT Reagent Kit reverse transcribes total RNA into cDNA.

[0035] Based on the swollenin gene sequence of *Trichoderma reesei* registered in the GenBank database, specific primers were designed:

[0036] Forward primer: 5'-GCCGAATTCATGAAGTTCTCCAGCGTCG-3' (containing EcoRI restriction site);

[0037] Reverse primer: 5'-GCCGCGGCCGCTTAGTTGACACTGGCGTTATC-3' (containing NotI restriction site);

[0038] Use high-fidelity DNA polymerase (e.g.) PCR amplification was performed using High-Fidelity DNA Polymerase (NEB). The reaction conditions were: 98°C pre-denaturation for 2 minutes; 98°C denaturation for 10 seconds, 60°C annealing for 30 seconds, 72°C extension for 90 seconds, for 35 cycles; and a final extension at 72°C for 10 minutes. The PCR product was detected by 1% agarose gel electrophoresis, and the expected product size was approximately 1.5 kb.

[0039] The obtained PCR product was cut from the gel and purified using a gel recovery kit (such as TIANgel Midi Purification Kit). The purified PCR product and pUC19 plasmid were digested with EcoRI and NotI restriction enzymes, respectively, and incubated at 37°C for 3 hours. The digested PCR product and vector were ligated using T4 DNA ligase at 16°C overnight, and then transformed into E. coli DH5α competent cells.

[0040] The plasmid was extracted from the transformed colonies, and the recombinant plasmid was verified by PCR and enzyme digestion, and the correctness of the swollenin gene sequence was confirmed by DNA sequencing.

[0041] The verified correct swollenin gene was cut from the intermediate vector pUC19-SWO and cloned into the Trichoderma expression vector pAN7-1. First, a primer pair containing the strong cbh1 (cellulase I) promoter of Trichoderma was designed:

[0042] cbh1-F: 5'-GCCAAGCTTGCATGCCTGCAGGTCGACTCTAG-3' (containing a HindIII site);

[0043] cbh1-R: 5'-GCCGAATTCTGTTTAGCGCTTGTTCTCG-3' (containing an EcoRI site);

[0044] The cbh1 promoter region of about 1.2 kb was amplified from the genomic DNA of T. reesei using the above primers. The amplified product was digested by HindIII and EcoRI, and then ligated with the EcoRI-NotI fragment containing the swollenin gene, and the pAN7-1 vector digested by HindIII and NotI, to obtain the expression vector pAN 7-1-cbh1p-SWO. The vector contains the cbh1 promoter-controlled swollenin gene and the hygromycin B resistance gene (hph) as a selection marker.

[0045] PEG-mediated protoplast transformation method was used. The specific steps are as follows:

[0046] (1) Protoplast preparation: Trichoderma reesei QM9414 spores were cultured in 20 mL PDB medium at 28°C, 200 rpm for 16-20 hours. Mycelia were collected and washed twice with 0.7 M NaCl, then resuspended in enzyme solution containing 10 mg / mL Novozyme 234 (or equivalent amount of Lysing Enzymes, Sigma-Aldrich) (enzyme solution composition: 0.7 M NaCl, 10 mM Na-phosphate buffer, pH 6.0). Mild shaking at 28°C for 3-4 hours until a large number of protoplasts were observed under microscope. Un-digested mycelia were removed by filtration through sterile nylon cloth (pore size 30 μm). The filtrate was centrifuged at 3000 g for 10 minutes at 4°C, and the protoplast pellet was collected and washed twice with STC buffer (1.2 M sorbitol, 10 mM Tris-HCl, pH 7.5, 10 mM CaCl2), and finally resuspended in STC buffer to a concentration of 10 8

[0047] (2) Transformation: 100 μL of protoplast suspension was taken, and 5-10 μg of linearized pAN7-1-cbh1p-SWO plasmid (fully digested by appropriate restriction enzymes such as Sail or BamHI) was added, mixed gently, and incubated at room temperature for 20 minutes. 1 mL of PEG solution (60% PEG4000, 10 mM Tris-HCl, pH 7.5, 10 mM CaCl2) was added dropwise, mixed gently, and incubated at room temperature for 20 minutes. Dilution was performed by adding 5 mL of STC buffer, and the protoplasts were collected by centrifugation at 3000 g for 10 minutes at 4°C. The protoplasts were resuspended in 1 mL of STC buffer, and aliquots were spread on selective regeneration medium containing 100 μg / mL hygromycin B (regeneration medium composition: sucrose 342.3 g / L, yeast extract 0.5 g / L, casein hydrolysate 0.5 g / L, (NH4)2SO4 1 g / L, KH2PO4 1 g / L, MgSO4-7H2O 0.5 g / L, agar 15 g / L). The plates were incubated at 28°C for 5-7 days until resistant colonies appeared.

[0048] Well-grown single colonies from the selective medium containing hygromycin B were picked and transferred to new PDA plates containing hygromycin B. Genomic DNA was extracted from the stably growing transformants, and PCR was performed using specific primers to verify the integration of the swollenin gene. Meanwhile, the transformants were inoculated in Mandels liquid medium containing 2% cellulose, and the culture supernatant was collected after 72 hours of induction of expression. The expression of swollenin protein was verified by SDS-PAGE and Western blot analysis.

[0049] ​For Western blot analysis, polyclonal antibodies against the C-terminal of swollenin or commercial antibodies against His-tag (if the expressed swollenin is fused with His-tag) can be used.

[0050] The transformants with the highest expression efficiency are selected for expression condition optimization. The effects of different carbon sources (e.g. cellulose, sucrose, cellobiose, etc.), nitrogen sources and culture time on swollenin expression are studied. Generally, a higher expression level can be obtained by culturing at 28°C, 180 rpm for 96 hours in Mandels medium containing 2% microcrystalline cellulose (Avicel).

[0051] For large-scale culture, the supernatant of the fermentation broth cultured under the optimized expression conditions is collected by centrifugation (6000 g, 4°C, 20 minutes), and the supernatant contains the secreted and expressed swollenin protein. The swollenin protein can be preliminarily enriched by ammonium sulfate fractionation (60%-80% saturation), and the precipitate is dissolved in 20 mM sodium phosphate buffer (pH 5.0) and dialyzed against water at 4°C for desalination before use.

[0052] Further purification is performed by ion exchange chromatography (e.g. DEAE-Sepharose) combined with gel filtration chromatography (e.g. Sephadex G-75). The final swollenin protein can be determined for protein concentration by the Bradford method, and its purity and specificity can be confirmed by SDS-PAGE and Western blot.

[0053] The Trichoderma engineering strain expressing swollenin constructed by the above method can stably and efficiently express biologically active swollenin protein, which can be used for subsequent straw cellulase hydrolysis and fermentation experiments.

[0054] Next, straw pretreatment and mixing are performed. The pH of the cell wall protein solution is adjusted to 4.8 using acetic acid. The above mixture and steam exploded corn straw (treated by steam explosion at 200°C for 10 minutes) are mixed at a ratio of fresh to steam exploded corn straw dry weight of 1 / 1, and the prepared swollenin-like protein solution (1.0 mg / g substrate) is added to the mixture, and acetic acid buffer (50 mM, pH 4.8) is added to adjust the substrate concentration to 50 mg / ml.

[0055] Subsequently, the pretreatment stage is entered, and the mixture is pretreated at 35°C for 8 hours to allow the swollenin-like protein to fully act on the straw, destroy the crystalline structure of cellulose, and increase the accessibility of cellulose. After the pretreatment, the enzyme hydrolysis stage is entered, and commercial cellulase (Novozymes Enzymatic hydrolysis was carried out at 45°C for 48 hours. The glucose content was analyzed. Compared with the control group (without adding swollenin-like protein), the glucose yield was increased from 5.8 mg / ml to 8.2 mg / ml, an increase of 41.4%.

[0056] Finally, the simultaneous saccharification and fermentation experiment was carried out. The cellulase was added at 20 FPU / g substrate, and the activated yeast (Saccharomyces cerevisiae, Angel active dry yeast, inoculation amount 1.0 g / L) was added. The temperature was 35°C, and the time was 72 hours. Compared with the control group (without adding swollenin-like protein), the ethanol yield was increased from 2.85 mg / ml to 4.05 mg / ml, an increase of 42.1%.

[0057] Example 2: Synergistic effect of fresh corn stalk cell wall protein and swollenin-like protein on improving the enzymatic hydrolysis and fermentation efficiency of wheat straw

[0058] In this embodiment, first, the preparation of fresh corn stalk cell wall protein and swollenin-like protein was carried out. The freshly harvested corn stalks were fully air-dried and crushed to 40 mesh, and then placed in a cell wall protein extraction solution (20 mM phosphate buffer, 0.8 M NaCl, 0.8 mM benzylsulfonyl fluoride, pH 5.5). The protein extraction solution was added at a solid-liquid ratio of 1 / 6, and the cell wall protein was extracted at 4°C for 18 hours.

[0059] At the same time, the Aspergillus engineering bacteria expressing swollenin (recombinant strain of Aspergillus expressing Trichoderma swollenin gene) were inoculated in a culture medium containing 25 g / L sucrose, 6 g / L yeast extract, 1.2 g / L KH2PO4, and 0.6 g / L MgSO4·7H2O at an inoculation amount of 2×10 6 Spores / mL, 29°C, 200 rpm for 55 hours. The fermentation broth was collected, precipitated with 65% saturated ammonium sulfate, and then centrifuged at 12000 g for 25 minutes after standing at 4°C for 10 hours. The precipitate was dissolved in 20 mM phosphate buffer (pH 5.0), and then dialyzed at 4°C for 36 hours. The protein content was 1.5 mg / mL, which was determined by the Bradford method.

[0060] Next, the straw pretreatment and mixing were carried out. The pH of the cell wall protein solution was adjusted to 4.5 with acetic acid. The above mixture and steam exploded wheat straw (steam exploded at 190°C for 8 minutes) were mixed at a fresh and steam exploded wheat straw dry weight ratio of 1 / 2. The swollenin-like protein solution prepared (1.5 mg / g substrate) was added to the mixture, and the substrate concentration was adjusted to 60 mg / ml with acetic acid buffer (40 mM, pH 4.5).

[0061] Subsequently, the mixture was pretreated at 32°C for 10 hours. After the pretreatment, enzymatic hydrolysis was performed at 48°C for 60 hours by adding commercial cellulase (Novozymes CTec3) at 12 FPU / g substrate. The glucose content was analyzed by sampling. Compared with the control group (without the addition of swollenin-like protein), the glucose yield was increased from 5.2 mg / ml to 7.1 mg / ml, an increase of 36.5%.

[0062] Finally, simultaneous saccharification and fermentation of ethanol was performed by adding cellulase at 18 FPU / g substrate and adding activated yeast (Saccharomyces cerevisiae, Angel active dry yeast, inoculation amount 1.2 g / L) at 33°C for 84 hours. The ethanol yield was increased to 3.35 mg / ml.

[0063] Example 3: Synergistic improvement of enzymatic hydrolysis and fermentation efficiency of rice straw by fresh corn stalk cell wall protein and swollenin-like protein

[0064] In this example, first, the preparation of fresh corn stalk cell wall protein and swollenin-like protein was performed. Freshly harvested corn stalks were fully air-dried and ground to 80 mesh, and then placed in a cell wall protein extraction solution (30 mM phosphate buffer, 1.2 M NaCl, 1.2 mM benzylsulfonyl fluoride, pH 6.0). The protein extraction solution was added at a solid-liquid ratio of 1 / 7, and the cell wall protein was extracted by standing at 4°C for 20 hours.

[0065] Meanwhile, the Trichoderma sp. engineering bacteria expressing swollenin were inoculated in a culture medium containing 35 g / L sucrose, 4 g / L yeast extract, 1.5 g / L KH2PO4, 0.8 g / L MgSO4·7H2O, at an inoculation amount of 1.5 x 10 6 Spores / mL, 30°C, 220 rpm for 65 hours. The fermentation broth was precipitated with 75% saturated ammonium sulfate, and the precipitate was collected by centrifugation at 11000 g for 22 minutes after standing at 4°C for 7 hours. The precipitate was dissolved in 20 mM phosphate buffer (pH 5.0), and dialyzed at 4°C for 30 hours. The protein content was determined by the Bradford method to be 1.8 mg / mL.

[0066] Next, the straw was pretreated and mixed. The pH of the cell wall protein solution was adjusted to 5.0 using acetic acid. The above mixture and steam exploded rice straw (treated by steam explosion at 210°C for 6 minutes) were mixed at a fresh and steam exploded rice straw dry weight ratio of 1 / 3. Swollenin-like protein solution (0.8 mg / g substrate) prepared was added to the mixture, and acetic acid buffer (60 mM, pH 5.0) was added to adjust the substrate concentration to 40 mg / ml.

[0067] Then the mixture was pretreated at 38°C for 6 hours. After the pretreatment, enzymatic hydrolysis was carried out at 50°C for 55 hours with 10 FPU / g substrate of commercial cellulase. The glucose content was analyzed. Compared with the control group, the glucose yield was increased from 4.6 mg / ml to 6.5 mg / ml, an increase of 41.3%.

[0068] Finally, simultaneous saccharification and fermentation (SSF) was carried out at 32°C for 90 hours with 15 FPU / g substrate of cellulase and 0.8 g / L of activated yeast. The ethanol yield was increased to 2.95 mg / ml.

[0069] Example 4: Synergistic effect of fresh corn stalk cell wall protein and swollenin on the enzymatic hydrolysis and fermentation efficiency of sorghum stalk

[0070] In this example, first, the fresh corn stalk cell wall protein and swollenin were prepared. The freshly harvested corn stalks were fully air-dried and ground to 100 mesh, and then placed in a cell wall protein extraction solution (35 mM phosphate buffer, 1.5 M NaCl, 1.5 mM benzylsulfonyl fluoride, pH 6.5). The protein extraction solution was added at a solid-liquid ratio of 1 / 4, and the cell wall protein was extracted at 4°C for 22 hours.

[0071] Meanwhile, the Aspergillus engineering bacteria expressing swollenin were inoculated in a culture medium containing 40 g / L sucrose, 3 g / L yeast extract, 1.8 g / L KH2PO4, 1.0 g / L MgSO4·7H2O, at an inoculation amount of 2.5 x 10 6 Spores / mL, 28°C, 210 rpm for 70 hours. The fermentation liquid was collected, precipitated with 80% saturated ammonium sulfate, and then centrifuged at 13,000 g for 30 minutes at 4°C to collect the precipitate. The precipitate was dissolved in 20 mM phosphate buffer (pH 5.0), and then dialyzed at 4°C for 40 hours. The protein content was determined by the Bradford method to be 2.0 mg / mL.

[0072] Next, the stalk pretreatment and mixing were carried out. The pH of the cell wall protein solution was adjusted to 4.2 with acetic acid. The above mixture and steam exploded sorghum stalk (treated by steam explosion at 220°C for 5 minutes) were mixed at a fresh and steam exploded sorghum stalk dry weight ratio of 2 / 1. The swollenin solution (2.0 mg / g substrate) prepared above was added to the mixture, and the substrate concentration was adjusted to 80 mg / ml with acetic acid buffer (80 mM, pH 4.2).

[0073] Then the mixture was pretreated at 40°C for 5 hours. After the pretreatment, enzymatic hydrolysis was carried out at 47°C for 65 hours with 20 FPU / g substrate of commercial cellulase. The glucose content was analyzed. Compared with the control group, the glucose yield was increased from 5.5 mg / ml to 7.9 mg / ml, an increase of 43.6%.

[0074] Finally, the simultaneous saccharification and fermentation experiment was carried out. The cellulase was added at 25 FPU / g substrate, and activated yeast was added (inoculation amount was 1.5 g / L), and the temperature was 36°C, and the time was 80 hours. The ethanol yield reached 3.80 mg / ml.

[0075] Example 5: Use of fresh corn stalk cell wall protein and swollenin to synergistically improve the enzymatic hydrolysis and fermentation efficiency of yellow bamboo grass

[0076] In this embodiment, first, the preparation of fresh corn stalk cell wall protein and swollenin was carried out. The freshly harvested corn stalks were fully air-dried and crushed to 90 mesh, and were placed in a cell wall protein extraction solution (22 mM phosphate buffer, 0.6 M NaCl, 0.6 mM benzylsulfonyl fluoride, pH 4.8). The protein extraction solution was added at a solid-liquid ratio of 1 / 8, and the cell wall protein was extracted at 4°C for 16 hours.

[0077] At the same time, the Trichoderma engineering bacteria expressing swollenin were inoculated in a culture medium containing 28 g / L sucrose, 4.5 g / L yeast extract, 1.3 g / L KH2PO4, and 0.7 g / L MgSO4·7H2O, and the inoculation amount was 1.8×10 6 Spores / mL, 29°C, 190 rpm for 58 hours. The fermentation liquid was collected, precipitated with 60% saturated ammonium sulfate, and centrifuged at 9500 g for 18 minutes after standing at 4°C for 9 hours. The precipitate was dissolved in 20 mM phosphate buffer (pH 5.0), and dialyzed at 4°C for 28 hours. The protein content was 1.3 mg / mL, which was determined by the Bradford method.

[0078] Next, the straw pretreatment and mixing were carried out. The pH of the cell wall protein solution was adjusted to 5.2 with acetic acid. The above mixture and steam exploded yellow bamboo grass (steam exploded at 185°C for 12 minutes) were mixed at a fresh and steam exploded yellow bamboo grass dry weight ratio of 3 / 1. The swollenin solution prepared (0.5 mg / g substrate) was added to the mixture, and the substrate concentration was adjusted to 100 mg / ml with acetic acid buffer (100 mM, pH 5.2).

[0079] Then the mixture was pretreated at 30°C for 12 hours. After the pretreatment, the enzymatic hydrolysis was carried out at 46°C for 72 hours with the addition of commercial cellulase at 8 FPU / g substrate. The glucose content was analyzed. Compared with the control group, the glucose yield was increased from 4.2 mg / ml to 5.9 mg / ml, an increase of 40.5%.

[0080] Finally, the simultaneous saccharification and fermentation experiment was carried out at 30°C for 96 hours with the addition of cellulase at 12 FPU / g substrate and activated yeast (inoculation amount of 0.5 g / L). Compared with the control group, the ethanol yield reached 3.25 mg / ml.

[0081] Comparative Example 1: Enzymolysis and fermentation of corn stalks promoted by fresh stalk cell wall protein only

[0082] The preparation method of the present comparative example is basically the same as that of Example 1, except that no expansin-like protein is added, and only fresh stalk cell wall protein is used to promote the enzymolysis and fermentation of corn stalks.

[0083] Specifically, the freshly harvested corn stalks were fully air-dried and crushed to 60 mesh, and then placed in a cell wall protein extraction solution (25 mM phosphate buffer, 1.0 M NaCl, 1.0 mM benzylsulfonyl fluoride, pH 5.0). The protein extraction solution was added at a solid-liquid ratio of 1 / 5, and the cell wall protein was extracted at 4°C for 15 hours. The pH of the cell wall protein solution was adjusted to 4.8 with acetic acid. The above mixture and steam exploded corn stalks (treated at 200°C for 10 minutes) were mixed at a fresh and steam exploded corn stalk dry weight ratio of 1 / 1, and the substrate concentration was adjusted to 50 mg / ml with acetic acid buffer (50 mM, pH 4.8).

[0084] Without the synergistic effect of expansin-like protein, the present comparative example omitted the pretreatment stage and directly entered the enzymolysis stage. Enzymolysis was carried out at 45°C for 48 hours with the addition of commercial cellulase at 15 FPU / g substrate. The analysis results showed that the glucose yield was 5.8 mg / ml.

[0085] In the simultaneous saccharification and fermentation experiment, cellulase was added at 20 FPU / g substrate, and activated yeast was added (inoculation amount of 1.0 g / L) at 35°C for 72 hours. The final ethanol yield was 2.85 mg / ml.

[0086] Comparative Example 2: Enzymolysis and fermentation of corn stalks promoted by expansin-like protein only

[0087] The preparation method of the present comparative example is basically the same as that of Example 1, except that no fresh stalk cell wall protein is used, and only expansin-like protein is used to promote the enzymolysis and fermentation of corn stalks.

[0088] Specifically, the Trichoderma engineering bacteria expressing swollenin were inoculated in a culture medium containing 30 g / L sucrose, 5 g / L yeast extract, 1 g / L KH2PO4, 0.5 g / L MgSO4·7H2O at an inoculation amount of 10 6 Spores / mL, 28°C, 180 rpm for 60 hours, the fermentation liquor was collected, precipitated with 70% saturated ammonium sulfate, and centrifuged at 10,000 g for 20 minutes after standing at 4°C for 8 hours to collect the precipitate, which was dissolved in 20 mM phosphate buffer (pH 5.0) and dialyzed at 4°C for 24 hours for standby. The protein content was determined by the Bradford method to be 1.2 mg / mL.

[0089] The steam-exploded corn stalks (treated by steam explosion at 200°C for 10 minutes) were directly used, and the prepared swollenin-like protein solution (1.0 mg / g substrate) was added, and the substrate concentration was adjusted to 50 mg / ml by adding acetic acid buffer (50 mM, pH 4.8).

[0090] The mixture was pretreated at 35°C for 8 hours to allow the swollenin-like protein to fully act on the stalks. After the pretreatment, the enzymatic hydrolysis stage was entered, and commercial cellulase was added at 15 FPU / g substrate for enzymatic hydrolysis at a temperature of 45°C for 48 hours. The analysis result showed that the glucose yield was 6.5 mg / ml.

[0091] In the simultaneous saccharification and fermentation ethanol experiment, cellulase was added at 20 FPU / g substrate, and activated yeast bacteria were added (inoculation amount was 1.0 g / L), and the temperature was 35°C, and the time was 72 hours. The final ethanol yield was 3.18 mg / ml.

[0092] Comparative Example 3: Enzymatic hydrolysis and fermentation of corn stalks without using any synergistic protein

[0093] This comparative example does not use fresh stalk cell wall protein or swollenin-like protein, and only uses commercial cellulase for enzymatic hydrolysis and fermentation of corn stalks.

[0094] Specifically, the steam-exploded corn stalks (treated by steam explosion at 200°C for 10 minutes) were directly used, and the substrate concentration was adjusted to 50 mg / ml by adding acetic acid buffer (50 mM, pH 4.8). Then commercial cellulase was added at 15 FPU / g substrate for enzymatic hydrolysis at a temperature of 45°C for 48 hours. The analysis result showed that the glucose yield was 4.2 mg / ml.

[0095] In the simultaneous saccharification and fermentation ethanol experiment, cellulase was added at 20 FPU / g substrate, and activated yeast bacteria were added (inoculation amount was 1.0 g / L), and the temperature was 35°C, and the time was 72 hours. The final ethanol yield was 3.18 mg / ml.

[0096] Comparative Example 4: Enzymatic hydrolysis and fermentation of yellow grass using only fresh straw cell wall protein

[0097] The preparation method of this comparative example is basically the same as that of Example 5, except that no swelling-like protein is added, and only fresh straw cell wall protein is used to promote the enzymatic hydrolysis and fermentation of yellow grass.

[0098] Specifically, the freshly harvested corn straw was fully air-dried and crushed to 90 mesh, and then placed in a cell wall protein extraction solution (22 mM phosphate buffer, 0.6 M NaCl, 0.6 mM benzylsulfonyl fluoride, pH 4.8). The protein extraction solution was added at a solid-liquid ratio of 1 / 8, and the cell wall protein was extracted by standing at 4°C for 16 hours. The pH of the cell wall protein solution was adjusted to 5.2 using acetic acid. The above mixture and steam exploded yellow grass (treated at 185°C for 12 minutes) were mixed at a fresh and steam exploded yellow grass dry weight ratio of 3 / 1, and acetic acid buffer (100 mM, pH 5.2) was added to adjust the substrate concentration to 100 mg / ml.

[0099] Since no swelling-like protein was added, this comparative example omitted the pretreatment stage and directly entered the enzymatic hydrolysis stage. Commercial cellulase was added at 8 FPU / g substrate for enzymatic hydrolysis at a temperature of 46°C for 72 hours. The analysis results showed that the glucose yield was 4.8 mg / ml, and the cellulose conversion rate was 47.0%.

[0100] In the simultaneous saccharification and fermentation ethanol experiment, cellulase was added at 12 FPU / g substrate, and activated yeast bacteria were added (inoculation amount was 0.5 g / L), and the temperature was 30°C for 96 hours. The final ethanol yield was 2.30 mg / ml.

[0101] Compared with Example 5, the glucose yield and ethanol yield of yellow grass treated only with fresh straw cell wall protein were significantly reduced. The glucose yield (6.8 mg / ml) of Example 5 (cooperatively using fresh straw cell wall protein and swelling-like protein) was 41.7% higher than that of Comparative Example 4, and the ethanol yield (3.25 mg / ml) was 41.3% higher. This fully proves that fresh straw cell wall protein and swelling-like protein have a significant synergistic effect in degrading yellow grass.

[0102] Comparative Example 5: Enzymatic hydrolysis and fermentation of yellow grass using only swelling-like protein

[0103] The preparation method of this comparative example is basically the same as that of Example 5, except that no fresh straw cell wall protein is used, and only swelling-like protein is used to promote the enzymatic hydrolysis and fermentation of yellow grass.

[0104] Specifically, the engineered Trichoderma expressing swollenin was inoculated in a medium containing 28 g / L sucrose, 4.5 g / L yeast extract, 1.3 g / L KH2PO4, 0.7 g / L MgSO4·7H2O at an inoculation amount of 1.8 x 10 6 Spores / mL, 190 rpm at 29°C for 58 hours, the fermentation broth was collected, precipitated with 60% saturated ammonium sulfate, and centrifuged at 9500 g for 18 minutes after standing at 4°C for 9 hours. The precipitate was dissolved in 20 mM phosphate buffer (pH 5.0) and dialyzed at 4°C for 28 hours before use. The protein content was determined to be 1.3 mg / mL by the Bradford method.

[0105] The steam-exploded Chinese grass (treated by steam explosion at 185°C for 12 minutes) was directly used, and the prepared swollenin-like protein solution (0.5 mg / g substrate) was added thereto, and the substrate concentration was adjusted to 100 mg / ml by adding acetic acid buffer (100 mM, pH 5.2). The mixture was pretreated at 30°C for 12 hours to allow the swollenin-like protein to fully act on the Chinese grass.

[0106] After the pretreatment, the enzymatic hydrolysis was performed by adding commercial cellulase at 8 FPU / g substrate, at a temperature of 46°C for 72 hours. The analysis results showed that the glucose yield was 5.5 mg / ml, and the cellulose conversion rate was 53.8%.

[0107] In the simultaneous saccharification and fermentation ethanol experiment, cellulase was added at 12 FPU / g substrate, and activated yeast bacteria were added (inoculation amount of 0.5 g / L), at a temperature of 30°C for 96 hours. The final ethanol yield was 2.65 mg / ml.

[0108] Compared with Example 5, the glucose yield and ethanol yield of the Chinese grass treated only with the swollenin-like protein were also significantly lower than those of the synergistic treatment. The glucose yield (6.8 mg / ml) of Example 5 was 23.6% higher than that of Comparative Example 5, and the ethanol yield (3.25 mg / ml) was 22.6% higher. This indicates that although the swollenin-like protein can promote the enzymatic hydrolysis of the Chinese grass by its non-hydrolytic relaxation effect, the effect will be more significant if it is used synergistically with the fresh straw cell wall protein.

[0109] The surface structure of the Chinese grass samples treated in Comparative Example 4 and Comparative Example 5 changed little, and the cellulose exposure degree was much lower than that of the synergistic treatment of Example 5. The sample treated in Comparative Example 4 mainly showed that part of the hemicellulose was degraded, but the cellulose microfibrils were still tightly wrapped. Although some cracks and loosening appeared on the surface of the sample treated in Comparative Example 5, the connection structure between the fiber bundles was not fully destroyed. This further confirms the significant effect of the synergistic action of the fresh straw cell wall protein and the swollenin-like protein on the structural destruction of the Chinese grass.

[0110] The experimental results and analysis are as follows:

[0111] Table 1 lists the glucose yield after enzymatic hydrolysis and ethanol yield after fermentation in different examples and comparative examples.

[0112]

[0113]

[0114] As can be seen from Table 1, the glucose yield and ethanol yield of all substrates treated with fresh straw cell wall protein and expansin-like protein synergistically (Examples 1-5) are significantly higher than those of the comparative examples. The specific analysis is as follows:

[0115] 1. Universality of synergistic effect: For different types of straw substrates, the synergistic use of fresh straw cell wall protein and expansin-like protein shows obvious synergistic effect. Taking corn straw as an example (Example 1), the glucose yield (8.2 mg / ml) when used synergistically is increased by 41.4% compared with the use of fresh straw cell wall protein alone (Comparative Example 1, 5.8 mg / ml), by 26.2% compared with the use of expansin-like protein alone (Comparative Example 2, 6.5 mg / ml), and by 95.2% compared with the use of no synergistic protein (Comparative Example 3, 4.2 mg / ml).

[0116] 2. Effect of substrate difference: The response degree of different types of straw is different, which is related to the structural complexity and component characteristics. The saccharification rate and ethanol yield of corn straw and sorghum straw are relatively high, while those of rice straw and yellow bamboo grass are relatively low. This may be due to the more complex lignin structure or higher lignin content of the latter, which increases the difficulty of enzymatic hydrolysis. However, even for yellow bamboo grass with the most complex structure, synergistic treatment (Example 5) still shows significant synergistic effect compared with the use of fresh straw cell wall protein alone (Comparative Example 4) or expansin-like protein alone (Comparative Example 5), with glucose yield increased by 41.7% and 23.6%, respectively.

[0117] 3. Improvement of cellulose conversion rate: Synergistic treatment significantly improves the cellulose conversion rate of various substrates. The cellulose conversion rates of Examples 1-5 range from 66.5% to 78.3%, while the conversion rates of the comparative groups range from 40.2% to 62.1%. This indicates that the synergistic use of the two proteins can more effectively destroy the structural barriers of straw and improve the accessibility of cellulose.

[0118] The mechanism of the synergistic effect of fresh straw cell wall protein and expansin-like protein is analyzed as follows:

[0119] 1. Structural disruption synergy: The surface of the straw sample treated by the method of Example 1 showed obvious cracking, peeling and porous structure, the connection between fibers was greatly damaged, and the exposure degree of cellulose microfibrils was significantly increased. In contrast, the samples treated by Comparative Example 1 and Comparative Example 2 also had some degree of structural change, but far less obvious than the synergistic treatment. The sample of Comparative Example 3 basically maintained the original dense structure. This shows that the non-hydrolytic swelling effect of the expansin-like protein can destroy the hydrogen bond network in the cellulose crystal structure, and the fresh straw cell wall protein may act together by degrading hemicellulose and part of lignin to make cellulose more susceptible to enzymatic hydrolysis.

[0120] 2. Crystallinity reduction effect: The synergistic treatment (Example 1) significantly reduced the crystallinity index (CrI) of cellulose, from 65.8% of the original straw to 45.2%, while the CrI of the samples treated by Comparative Example 1 and Comparative Example 2 was 52.3% and 50.8%, respectively. The reduction of crystallinity indicates that more amorphous regions are exposed, which is beneficial to the contact and action of cellulase.

[0121] 3. Change of enzyme adsorption behavior: The synergistic treatment significantly changed the interaction mode of cellulase and substrate. The non-productive adsorption (mainly the binding with lignin) of the sample treated by Example 1 to cellulase was reduced by about 40%, while the productive adsorption (binding with cellulose) was increased by about 35%. This shows that the synergistic treatment may optimize the interaction of enzyme and substrate by modifying the surface properties of lignin and / or increasing the accessibility of cellulose.

[0122] 4. Synergistic effect of chemical bond rupture: The synergistic treatment leads to the rupture of specific chemical bonds in the straw, especially the bonds related to the lignin-carbohydrate complex, which cannot be achieved by using either protein alone.

[0123] 5. Removal of hemicellulose barrier: After the treatment of Example 1, the hemicellulose content in the straw was reduced by 58.6%, which was significantly higher than that of Comparative Example 1 (45.2%) and Comparative Example 2 (32.5%). As a barrier to cellulose microfibrils, the effective removal of hemicellulose can greatly increase the accessibility of cellulose.

[0124] 6. Synergistic enhancement of enzyme system: Fresh straw cell wall protein contains various enzyme activities, including endoglucanase, exoglucanase, β-glucosidase, xylanase, etc., which form a synergistic network with the non-hydrolytic relaxation effect of the expansin-like protein. The expansin-like protein first destroys the cellulose crystal structure, increasing the enzyme accessible surface area, and then the endogenous enzyme system more effectively hydrolyzes the exposed polysaccharide chains, further promoting the enzymatic hydrolysis process.

[0125] In summary, the synergistic mechanism of fresh straw cell wall protein and swollen-like protein can be summarized as a structure relaxation-enzymolysis synergistic mode, that is, swollen-like protein mainly destroys the structure of cellulose crystal by non-hydrolytic action, reduces crystallinity, and fresh straw cell wall protein provides various enzyme activities, both of which synergistically act to significantly improve the accessibility of cellulose and the efficiency of enzymolysis. This mechanism not only explains the significant increase in saccharification rate and ethanol yield under synergistic treatment, but also provides a theoretical basis for developing more efficient biomass conversion technology.

[0126] This scheme can significantly improve the efficiency of straw enzymolysis, reduce the amount of enzyme, save the cost of pretreatment, realize the efficient utilization of straw resources, and has important theoretical significance and application value.

[0127] The method for promoting straw cellulose enzymolysis and fermentation provided by the present application has the following industrial application prospects:

[0128] 1. Biofuel production: can be applied to the production of bioethanol, biodiesel and other biofuels, reduces production cost and improves product competitiveness.

[0129] 2. Biochemical production: can be applied to the production of lactic acid, acetone butanol, organic acid and other bio-based chemicals, realizing high-value utilization of biomass resources.

[0130] 3. Agricultural waste treatment: provides a new technical route for the resource utilization of agricultural waste (such as straw, straw, etc.), and solves the environmental problems caused by straw burning.

[0131] 4. Enzyme preparation industry: the synergistic principle of the present application can be applied to the development of new lignocellulose degrading enzyme preparation, improving the performance and market competitiveness of enzyme preparation.

[0132] 5. Papermaking industry: can be applied to the biological pulping process of papermaking industry, reducing energy consumption and chemical consumption, and reducing environmental pollution.

[0133] In summary, the present application has broad industrial application prospects and good economic, social and environmental benefits.

[0134] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for promoting enzymatic hydrolysis and fermentation of straw cellulose by fresh straw wall protein, characterized in that The method comprises the following steps: A. Preparation of fresh straw cell wall protein and expansin-like protein: fresh straw crushed to 40-100 mesh is placed in a cell wall protein extraction solution, the cell wall protein extraction solution is added according to a solid-liquid ratio of 1 / 4-1 / 10, and cell wall protein extraction is carried out by standing at 4°C for 12-24 hours; meanwhile, an engineered bacterium expressing an expansin-like protein is cultured, and the fermentation liquor is collected and the expansin-like protein is enriched by ammonium sulfate precipitation; B. Straw pretreatment and mixing: the pH of the cell wall protein solution is adjusted to 4.2-5.2, the adjusted cell wall protein solution is mixed with steam explosion treated straw according to a fresh steam explosion straw dry weight ratio of 1 / 3-3 / 1, the mixture is added with an expansin-like protein solution, the addition amount is 0.5-2.0 mg / g substrate, and a vinegar acid buffer solution with a pH of 4.2-5.2 is added to adjust the substrate concentration to 20-100 mg / ml; C. Pretreatment stage: the mixture obtained in step B is pretreated at 30-40°C for 4-12 hours; D. Enzymatic hydrolysis stage: cellulase is added according to 8-25 FPU / g substrate for enzymatic hydrolysis, the temperature is 45-50°C, and the time is 48-72 hours; E. Simultaneous saccharification and fermentation of ethanol: cellulase is added according to 8-35 FPU / g substrate, activated yeast is added, the temperature is 30-37°C, and the time is 72-96 hours.

2. The method of claim 1, wherein: The cell wall protein extraction solution is 20-35 mM phosphate buffer, 0.6-1.8 M NaCl, 0.6-2.5 mM benzylsulfonyl fluoride, and pH 4.8-7.

2.

3. The method of claim 1, wherein: The engineered bacterium expressing the expansin-like protein is an Aspergillus or Trichoderma microorganism, which is cultured in a culture medium containing a carbon source and a nitrogen source at 28-30°C for 48-72 hours.

4. The method of claim 3, wherein: The enrichment of the expansin-like protein is carried out by ammonium sulfate precipitation at a saturation degree of 60%-80%, centrifugal collection of the precipitate after standing at 4°C for 6-12 hours, dissolution in 20 mM phosphate buffer, pH 5.0, and desalting by dialysis.

5. The method of claim 1, wherein: In step B, the pH adjustment is carried out using vinegar acid, and the concentration of the vinegar acid buffer solution is 20-100 mM.

6. The method of claim 1, wherein: In step C, the pretreatment aims to make the expansin-like protein fully act on the straw, destroy the crystal structure of cellulose, and increase the accessibility of cellulose.

7. The method of claim 1, wherein: The fresh straw is corn straw, wheat straw, rice straw, sorghum straw or yellow bamboo grass.

8. The method of claim 1, wherein: The steam explosion treatment is carried out at 180-220°C for 3-15 minutes.

9. The method of claim 1, wherein: In step D, the cellulase is a commercial cellulase preparation, which contains endoglucanase, exoglucanase and beta-glucosidase.

10. The method of claim 1, wherein: In step E, the activated yeast is Saccharomyces cerevisiae, and the inoculation amount is 0.5-2.0 g / L. The yeast converts glucose to ethanol during the saccharification and fermentation process.

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