Endoglucanase mutant with acid resistance and application thereof

Abstract

The invention discloses an endoglucanase mutant with acid resistance and application of the endoglucanase mutant, and belongs to the technical field of gene engineering and enzyme engineering. According to the present invention, PCR amplification is adopted to carry out site-specific saturation mutation on endoglucanase tCel5A, and n variants S45D / T55D / T59D and T88H / W255H are constructed and screened; the enzyme activity determination result shows that the enzyme activities of the endoglucanase variants S45D / T55D / T59D and T88H / W255H under the acidic condition are 8828.7 U / mg prot and 5206.7 U / mg prot respectively, and the degradation rates of the endoglucanase variants S45D / T55D / T59D and T88H / W255H to vinasse under the solid-to-liquid ratio of 1: 30 are 53.53% and 50.47% respectively, so that the endoglucanase variants S45D / T55D / T59D and T88H / W255H have high acid resistance and plant cellulose degradation capacity and have great application value in the aspects of agriculture and brewing.

Description

Technical Field

[0001] The invention relates to an acid-resistant endoglucanase mutant and application thereof, belonging to the technical field of gene engineering and enzyme engineering. Background Art

[0002] Endoglucanase is a type of glycoside hydrolase that can catalyze crude fiber. It is widely used in fertilizer decomposition, rice husk degradation, straw recycling and other fields. In the brewing industry, liquor production produces a large amount of vinasse, which as a solid waste will cause serious pollution to the environment. Endoglucanase can hydrolyze crude fiber in vinasse, help vinasse degradation and decomposition, thereby accelerating the recycling of vinasse and promoting environmental protection. However, the high moisture content and strong acidity of vinasse limit the catalytic activity of endoglucanase, thereby affecting its catalytic activity and degradation effect.

[0003] There are existing reports on the modification of endoglucanase expression. For example, Su Shaofeng et al. obtained a high-copy recombinant strain by codon optimization and erythromycin screening (reference: Su Shaofeng, Sa Chula, Liu Hongkui, et al. Optimization and expression of codons of glucan exonuclease and glucan endonuclease genes [J]. Animal Husbandry and Feed Science); Liao Junhua optimized the endoglucanase gene Ends through bioinformatics analysis, and increased the expression by 1.2 times while retaining the same enzymatic properties (reference: Liao Junhua. Study on the expression of the optimized endoglucanase gene Ends in Pichia pastoris [D]. Sichuan Agricultural University, 2010.). However, combined with the application needs of distiller's grains environment, there is currently a lack of endoglucanases with high catalytic activity for acidic substrates, and the poor acid resistance of endoglucanases will limit their application in degrading distiller's grains, making it unable to efficiently hydrolyze crude fiber in distiller's grains. Therefore, modifying the acid resistance of endoglucanase and improving its catalytic activity on acidic substrates are the keys to enhancing its application capabilities. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides an acid-resistant endoglucanase mutant and its application, aiming to solve the technical problem that the existing endoglucanase has low catalytic activity on acidic substrates, and the poor acid resistance of endoglucanase limits its application in degrading vinasse, and the high acidity of the substrate makes it unable to efficiently hydrolyze vinasse crude fiber.

[0005] The first technical solution provided by the present invention is an endoglucanase mutant, wherein the mutant is an endoglucanase parent with an amino acid sequence as shown in SEQ ID NO.1 subjected to any of the following mutations:

[0006] (1) The amino acid at position 45, 55, 59, 61 and / or 260 is mutated to aspartic acid;

[0007] (2) the amino acid at position 87, 298, or 371 is mutated to histidine;

[0008] (3) The amino acid at position 85, 88 and / or 255 is mutated to histidine.

[0009] In certain embodiments, the endoglucanase mutant is obtained by mutating the 45th serine S, the 55th threonine T and the 59th threonine T of the endoglucanase parent as shown in the amino acid sequence of SEQ ID NO.1 to aspartic acid D, or by mutating the 88th threonine T and the 255th tryptophan W of the endoglucanase parent as shown in the amino acid sequence of SEQ ID NO.1 to histidine H.

[0010] In certain embodiments, the nucleotide sequence encoding the parent enzyme endoglucanase is shown as SEQ ID NO.2.

[0011] The second technical solution provided by the present invention is a gene encoding the mutant described in the first technical solution.

[0012] The third technical solution provided by the present invention is a recombinant vector carrying the gene described in the second technical solution.

[0013] In certain embodiments, the recombinant vector uses plasmid pPICZA (alpha) as an expression vector.

[0014] Furthermore, the signal peptide of the expression vector is replaced with SUC2 to form an expression vector.

[0015] The fourth technical solution provided by the present invention is a recombinant microbial cell expressing the mutant described in the first technical solution, or containing the gene described in the second technical solution, or transformed with the recombinant vector described in the third technical solution.

[0016] In certain embodiments, the recombinant microbial cell uses bacteria or fungi as host cells.

[0017] In certain embodiments, the host cell is Pichia pastoris X-33.

[0018] The fifth technical solution provided by the present invention is a method for improving the degradation activity of endoglucanase on acidic substrates, wherein the endoglucanase parent with an amino acid sequence as shown in SEQ ID NO.1 is subjected to any one of the mutations shown in (a) to (j):

[0019] (a) mutating the threonine T at position 55 to aspartic acid D;

[0020] (b) mutating the threonine T at position 59 to aspartic acid D;

[0021] (c) mutating alanine A at position 26 to aspartic acid D;

[0022] (d) Serine S at position 45 and Threonine T at position 55 were both mutated to aspartic acid D;

[0023] (e) Serine S at position 45 and Threonine T at position 59 were both mutated to aspartic acid D;

[0024] (f) mutating both the threonine T at position 55 and the threonine T at position 59 to aspartic acid D;

[0025] (g) mutating the serine at position 45, the threonine at position 55, and the threonine at position 59 to aspartic acid D;

[0026] (h) mutating the threonine T at position 88 to histidine H;

[0027] (i) glycine G at position 85 and threonine T at position 88 were mutated to histidine H;

[0028] (j) The threonine T at position 88 and the tryptophan W at position 255 were both mutated to histidine H.

[0029] The present invention also provides an acid-resistant endoglucanase product, which is a composting agent or a microbial fermentation agent, and the crude fiber degradation product contains the endoglucanase mutant or the recombinant cell.

[0030] In certain embodiments, the industrial degradation product comprises a crude fiber degradation product.

[0031] In certain embodiments, the keratin-degrading products include, but are not limited to, composting agents and microbial fermentation agents.

[0032] The sixth technical solution provided by the present invention is a method for degrading cellulose, wherein the method utilizes the endoglucanase variant described in the first technical solution or the recombinant microbial cell described in the fourth technical solution to degrade cellulose.

[0033] In certain embodiments, the cellulose-containing material includes wine grains, tea leaves, tobacco, straw, rice husks, and the like.

[0034] The seventh technical solution provided by the present invention is the application of the endoglucanase mutant described in the first technical solution, the gene described in the second technical solution, the recombinant vector described in the third technical solution, the recombinant microbial cell described in the fourth technical solution, the method described in the fifth technical solution or the method described in the sixth technical solution in the fields of medicine, animal husbandry, feed and brewing.

[0035] The eighth technical solution provided by the present invention is the use of the endoglucanase mutant described in the first technical solution, the gene described in the second technical solution, the recombinant vector described in the third technical solution, the recombinant microbial cell described in the fourth technical solution, the method described in the fifth technical solution or the method described in the sixth technical solution in degrading crude fiber of wine grains, tea leaves, tobacco, straw, rice husks and other plant raw materials.

[0036] Beneficial Effects

[0037] The present invention obtains two optimal endoglucanase variants S45D / T55D / T59D and T88H / W255H through screening. The results of collagen hydrolysis activity assay show that the keratinase mutants S45D / T55D / T59D and T88H / W255H are tolerant to acidic environments, and their catalytic activities to acidic substrates are 8828.68U / mg prot and 5206.67U / mg prot, respectively. Therefore, this mutant can greatly improve the application value of endoglucanase in acidic high-fiber substrates such as wine lees. The variant can still maintain the relative stability of the protein catalytic center structure in an acidic environment, so it will not lose its catalytic effect on acidic substrates, and it is more efficient during application. Therefore, the acid-resistant endoglucanase variants S45D / T55D / T59D and T88H / W255H provided by the present invention are more valuable and potential for application. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Structural changes of tCel5A before and after Rosetta Supercharging design and specific activities of surface mutants. A) Charge of original protein conformation before tCel5A mutation; B) Charge of protein conformation after Rosetta Supercharging mutation design; C) Specific enzyme activity of single point mutants under optimal reaction conditions and D) Specific enzyme activity of single point mutants under pH 3.0; E) Specific enzyme activity of combined mutants under optimal reaction conditions and F) Specific enzyme activity of combined mutants under pH 3.0.

[0039] Figure 2 Selection of mutation sites in the catalytic center of tCel5A and the relative enzyme activity of single-point or double-point mutants; A) The relative position of cellotetraose and the catalytic active center of tCel5A; B) The relative position of cellotetraose and the catalytic site Relative positions of residues in the range; C) specific enzymatic activity of single-point mutants under optimal reaction conditions; D) specific enzymatic activity of single-point mutants at pH 3.0; E) specific enzymatic activity of double-point mutants under optimal reaction conditions; F) specific enzymatic activity of double-point mutants at pH 3.0.

[0040] Figure 3 The degradation effects of S45D / T55D / T59D and T88H / W255H on liquor lees: A) enzyme activity of fermentation supernatant of tCel5A parent enzyme WT and mutant strain at pH 3.0 and optimal reaction conditions; B) degradation rate of liquor lees by the two mutants at different solid-liquid ratios. DETAILED DESCRIPTION

[0041] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0042] The culture medium involved in the following examples is as follows:

[0043] LB liquid medium (low salt): yeast powder 5g L -1 , Tryptone 10 g·L -1 、NaCl 5g·L -1 .

[0044] LB solid medium (low salt): yeast powder 5g L -1 , Tryptone 10 g·L -1 、NaCl 5g·L -1 , agar powder 20g·L -1 .

[0045] YPD liquid medium: yeast powder 10g·L -1 , Tryptone 20 g·L -1 , D-glucose 20g·L -1 .

[0046] YPDS solid medium: yeast powder 10g·L -1 , Tryptone 20 g·L -1 , D-glucose 20g·L -1 , D-sorbitol 1 mol·L -1 , agar powder 20g·L -1 .

[0047] The detection methods involved in the following embodiments are as follows:

[0048] Endoglucanase activity assay method:

[0049] The protein concentration was determined using a modified Bradford protein assay kit (purchased from Shanghai Shenggong Biotechnology Co., Ltd.), and a standard curve was constructed using bovine serum albumin (BSA). The activity of endoglucanase was determined using the DNS method. The CMC-Na (purchased from McLean Biotechnology Co., Ltd. (Shanghai)) substrate was prepared into a 1% (w / v) uniform solution using acetic acid-sodium acetate buffer (pH 3.0 and pH 4.5). 100 μL of the purified endoglucanase sample solution was prepared into a reaction system with 500 μL of 1% (w / v) CMC-Na solution, and the reaction was shaken at 900 rpm for 60 min at 50 °C. After the reaction was completed, the sample solution was immediately transferred to a boiling water bath to terminate the reaction, and DNS colorimetric solution (purchased from Krypton Road (Guangzhou) Biological Co., Ltd.) was added in a ratio of 1:1 between DNS and the reaction sample, and the solution was allowed to stand in a boiling water bath for 5 min for color development, and then taken out and cooled to room temperature. The reaction solution was diluted with ddH2O, the absorbance was measured at 540 nm, and the results were compared with the standard curve of the glucose solution. There were three parallel experiments. The blank control was inactivated by boiling the sample before adding the substrate. The rest of the operations were the same as above.

[0050] Enzyme activity unit definition: the amount of enzyme (U) required to release 1 μg reducing sugar from the substrate per minute under the corresponding reaction conditions (50°C, pH 3.0 or pH 4.5).

[0051] Detection method of endoglucanase degradation rate of liquor lees:

[0052] Liquor lees were obtained from Anhui Gujing Group Co., Ltd. (Bozhou, Anhui, China). Fresh liquor lees were dried in an oven at 70°C to constant weight and then crushed with a pulverizer. The lees were added to each fermentation supernatant (crude enzyme solution) at a solid-liquid ratio of 1:15 and 1:30. The mixture was degraded at 50°C and 900 rpm for 48 h. The degraded samples were centrifuged (2 min, 10,000 rpm, 20°C). The supernatant and precipitate were separated to calculate the lees degradation rate, and the precipitate was dried at 70°C to constant weight. The degradation rate was calculated as follows:

[0053] Degradation rate = (m0-m1) / m0×100%

[0054] Where m0 is the dry mass of lees added to the system, and m1 is the dry mass of the precipitate after degradation. Three parallel experiments were performed on a single sample.

[0055] The strains and plasmids involved in the following examples are:

[0056] 1. Pichia pastoris X-33: laboratory preserved, used for target protein expression.

[0057] 2. Plasmid pPICZA (alpha): laboratory preserved, used for target protein expression.

[0058] 3. Escherichia coli DH5α: laboratory preserved, used for obtaining multiple copies of plasmids.

[0059] Example 1 Construction of endoglucanase variants

[0060] 1. Construction of mutants

[0061] (1) The construction method of the pAOX1-sSUC2-tCel5A recombinant vector is as follows:

[0062] Based on the plasmid pPICZA (alpha), the endoglucanase gene with the nucleotide sequence of SEQ ID NO.2 was connected to the Kex2 site by chemical synthesis (Genwizhi Co., Ltd.), and the plasmid signal peptide was replaced with SUC2 (nucleotide sequence of SEQ ID NO.3) by whole plasmid PCR. The gene was then transferred into Escherichia coli DH5α to complete the construction and sequence verification.

[0063] (2) Construction of mutants

[0064] In this study, the endoglucanase with the nucleotide sequence of SEQ ID NO.2 was used as the parent enzyme, and seven amino acid sites designed by Rosetta Supercharging on its surface and near the catalytic active center were selected. The 14 amino acid sites within the range were subjected to saturation mutation and the forward mutation sites were combined.

[0065] According to the sequence of endoglucanase (the nucleotide sequence is shown in SEQ ID NO.2, and the amino acid sequence is shown in SEQ ID NO.1), mutation primers (as shown in Table 1) were designed respectively to perform site-directed saturation mutagenesis on the endoglucanase gene on the pAOX1-sSUC2-tCel5A plasmid.

[0066] Table 1: Primers

[0067]

[0068]

[0069] The PCR reaction system was as follows: PrimeSTAR Max Premix (2×) 25 μL, 10 μM forward primer 1 μL, 10 μM reverse primer 1 μL, template DNA 1 μL, and double distilled water was added to 50 μL;

[0070] The amplification conditions of PCR products were as follows: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s, annealing at 55°C for 5 s, extension at 72°C for 2 min, 30 cycles; and finally incubation at 72°C for 10 min.

[0071] The PCR amplification product was detected by 1% agarose gel electrophoresis. After confirming that the amplification product was of the correct size, 0.5 μL of methylated template digestion enzyme (DpnI) was added to 10 μL of the amplification product, mixed, and reacted at 37°C for 1.5 h. The amplification product treated with DpnI was transformed into Escherichia coli DH5α competent cells, and the transformation product was coated on a plate with Zeocin (final concentration 50 μg·L -1 ) in LB solid medium (low salt) at 37°C for 10-12 h, and the single colony grown on the plate was inoculated into a plate supplemented with Zeocin (final concentration 50 μg·L -1 ) in LB liquid medium (low salt), cultured at 37°C and 220 rpm for 8-10 h, and then the plasmid was extracted to obtain recombinant plasmids containing different combinations of expression elements: tCel5A-mutants, including W5D (tryptophan at position 5 mutated to aspartic acid), S45D (serine at position 45 mutated to aspartic acid), S50D (serine at position 50 mutated to aspartic acid), T55D (threonine at position 55 mutated to aspartic acid), T59 D (mutation of threonine at position 59 to aspartic acid), T61D (mutation of threonine at position 61 to aspartic acid), A260D (mutation of alanine at position 260 to aspartic acid), S45D / T55D, S45D / T59D, T55D / T59D, S45D / T55D / T59D, G81H (mutation of glycine at position 81 to histidine), D83H (mutation of aspartic acid at position 83 to histidine), F84H (mutation of Phenylalanine mutated to histidine), G85H (glycine at position 85 mutated to histidine), C86H (cysteine ​​at position 86 mutated to histidine), T87H (threonine at position 87 mutated to histidine), T88H (threonine at position 88 mutated to histidine), Y176H (tyrosine at position 176 mutated to histidine), W255H (tryptophan at position 255 mutated to histidine), S296H (serine at position 296 mutated to histidine ), G297H (glycine at position 297 mutated to histidine), T298H (threonine at position 298 mutated to histidine), F367H (phenylalanine at position 367 mutated to histidine), Y371H (tyrosine at position 371 mutated to histidine), G85H / T88H, G85H / W255H, G85H / G297H, T88H / W255H, T88H / G297H, W255H / G297H.

[0072] Example 2: Construction of recombinant bacteria and expression of keratinase

[0073] The specific steps are as follows:

[0074] (1) The recombinant plasmid tCel5A-mutant obtained in Example 1 was transformed into Pichia pastoris X-33 competent cells, and the transformation products were spread on a plate containing Zeocin (final concentration 100 μg / L -1 ) on YPDS solid medium at 30°C for 2.5-3.0 days, and then the single colony grown on the plate medium was inoculated into a medium containing Zeocin (final concentration 100 μg·L -1 ) in YPD liquid medium at 30°C and 220 rpm for 24 h to prepare seed solutions;

[0075] (2) The seed solution obtained in step (1) was transferred to YPD liquid medium at a ratio of 1% (v / v), and cultured at 30°C and 220 rpm for 24 hours. Methanol was added at a final concentration of 1% (w / v) to induce the expression of tCel5A. The culture process lasted for 72 hours, and methanol was added every 24 hours.

[0076] (3) The fermentation broth was centrifuged at 8000 rpm for 10 min at 4°C to obtain the fermentation supernatant (crude enzyme solution).

[0077] (4) Filter through 0.22 μm PES filter membrane respectively. Pass the filtrate through Ni-NTA gravity column (purchased from Chuanshi Biotechnology Co., Ltd.) to obtain purified endoglucanase.

[0078] (5) SDS-PAGE (Thermo XCell Surelock Mini-Cell, NuPAGE 4-12%, Bis-Tris protein gel) was used to analyze protein expression.

[0079] (6) The catalytic activity of the endoglucanase in the purified solution of step (4) under the optimal conditions and acidic conditions and on CMC-Na was detected respectively. The results are shown in Tables 2, 3 and Figure 1 , Figure 2 , Figure 3 shown.

[0080] Table 2: Cellulose hydrolysis activity of surface-mutated endoglucanases under optimal and acidic conditions

[0081]

[0082]

[0083] Table 3: Cellulose hydrolysis activity of endoglucanases with catalytic center mutations under optimal conditions and acidic conditions

[0084]

[0085]

[0086] The results showed that in the surface charge design strategy, the specific enzyme activity of S45D, T55D, T59D, T61D and A260D mutants increased significantly under the optimal reaction conditions. Under acidic conditions (pH 3.0), the reaction activities of S45D, T55D and T59D were still higher than those of the control, indicating that these three sites were most related to the acid resistance of tCel5A. S45D, T55D and T59D were further combined for mutation. Under acidic and optimal reaction conditions, the enzyme activity of the double-point mutant was significantly improved compared with that of WT, and the enzyme activity of the S45D / T55D / T59D mutant was about 3 times higher than that of WT, indicating that the acid resistance of endoglucanase was significantly improved through surface charge design. In the catalytic center design strategy, only the three single-point mutants G85H, T88H and W255H showed significantly improved enzyme activities in the reaction under acidic conditions (pH 3.0), while the activity of the G297H mutant was close to that of WT under optimal and acidic conditions. Although the T87H, T298H and Y371H mutants showed high catalytic activity under optimal conditions, their activities under acidic conditions were very low. Therefore, four sites, G85, T88, W255 and G297, were selected for double-point His mutation. In the double-point mutation experiment, only the T88H / W255H mutant had a significantly improved specific enzyme activity under optimal conditions, which was about 6345.3U / mgprot; when reacting with acidic substrates (pH 3.0), it was about 5206.7U / mgprot, which was about 2400U / mgprot higher than WT, indicating that it has a better hydrolysis effect on acidic substrates. The fermentation supernatants of S45D / T55D / T59D and T88H / W255H mutants were used for the degradation of vinasse, and the degradation rates were 53.53% and 50.47% respectively at a solid-liquid ratio of 1:30. The degradation effect was higher than the average level (30-40%) and could meet the needs of industrial production.

[0087] The present invention adopts the whole plasmid PCR technology to carry out site-directed saturation mutation on endoglucanase tCel5A, and the obtained endoglucanase variants S45D / T55D / T59D and T88H / W255H have good acid resistance and great application potential in agriculture and brewing industries. The obtained mutants S45D / T55D / T59D and T88H / W255H retain the high activity of the original enzyme under optimal conditions, and the degradation rates of the fermentation supernatants on the lees are 53.53% and 50.47% respectively under a certain solid-liquid ratio, indicating that the mutants are better than the parent enzymes in terms of acid resistance and industrial application.

[0088] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An endoglucanase mutant, characterized in that The mutant is obtained by subjecting the endoglucanase parent with the amino acid sequence shown in SEQ ID NO.1 to any of the following mutations: (1) The amino acid at position 45, 55, 59, 61 and / or 260 is mutated to aspartic acid; (2) the amino acid at position 87, 298, or 371 is mutated to histidine; (3) The amino acid at position 85, 88 and / or 255 is mutated to histidine.

2. The mutant according to claim 1, characterized in that The endoglucanase mutant is obtained by mutating the 45th serine S, the 55th threonine T and the 59th threonine T of the endoglucanase parent as shown in the amino acid sequence of SEQ ID NO.1 to aspartic acid D, or by mutating the 88th threonine T and the 255th tryptophan W of the endoglucanase parent as shown in the amino acid sequence of SEQ ID NO.1 to histidine H.

3. A gene encoding the mutant according to claim 1 or 2.

4. A recombinant vector carrying the gene according to claim 3.

5. The recombinant vector according to claim 4, characterized in that The recombinant vector is an expression vector using plasmid pPICZA (alpha). Optionally, the signal peptide of the expression vector is replaced by SUC2.

6. A recombinant microbial cell expressing the mutant according to claim 1 or 2, or containing the gene according to claim 3, or transformed with the recombinant vector according to claim 4 or 5.

7. The recombinant microbial cell according to claim 6, characterized in that The recombinant microbial cell uses bacteria or fungi as host cells; optionally, the host cell is Pichia pastoris X-33.

8. A method for improving the degradation activity of endoglucanase on acidic substrates, characterized in that: The method comprises subjecting the endoglucanase parent with the amino acid sequence shown in SEQ ID NO.1 to any one of the mutations shown in (a) to (j): (a) mutating the threonine T at position 55 to aspartic acid D; (b) mutating the threonine T at position 59 to aspartic acid D; (c) mutating alanine A at position 26 to aspartic acid D; (d) Serine S at position 45 and Threonine T at position 55 were both mutated to aspartic acid D; (e) Serine S at position 45 and Threonine T at position 59 were both mutated to aspartic acid D; (f) mutating both the threonine T at position 55 and the threonine T at position 59 to aspartic acid D; (g) mutating the serine at position 45, the threonine at position 55, and the threonine at position 59 to aspartic acid D; (h) mutating the threonine T at position 88 to histidine H; (i) glycine G at position 85 and threonine T at position 88 were mutated to histidine H; (j) The threonine T at position 88 and the tryptophan W at position 255 were both mutated to histidine H.

9. A method for degrading cellulose, characterized in that: The method utilizes the endoglucanase variant of claim 1 or 2 or the recombinant microbial cell of claim 6 or 7 to degrade cellulose.

10. Use of the endoglucanase mutant according to claim 1 or 2, the gene according to claim 3, the recombinant vector according to claim 4 or 5, the recombinant microbial cell according to claim 6 or 7, the method according to claim 8 or the method according to claim 9 in degrading crude fiber of vinasse, tea, tobacco, straw, rice husk and other plant raw materials in the fields of medicine, animal husbandry, feed and brewing.

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