A method for extracting polyhydroxy fatty acid esters from bamboo powder via enzymatic hydrolysis

Abstract

This invention discloses a method for extracting polyhydroxy fatty acid esters from bamboo powder via enzymatic hydrolysis, belonging to the field of extraction technology. The method includes the following steps: processing whole bamboo to obtain bamboo powder; pretreating the bamboo powder with ozone, followed by enzymatic hydrolysis to obtain enzymatic residue; separating lignin and hemicellulose from the enzymatic residue to obtain lignin; grafting epichlorohydrin onto the lignin to obtain modified lignin; grafting cyclodextrin onto the modified lignin to obtain cyclodextrin-grafted lignin; using cyclodextrin-grafted lignin to encapsulate a dioxapentane polymer to obtain a composite carbon source; culturing microorganisms using the composite carbon source, extracting and purifying the polyhydroxy fatty acid esters; and using high-purity lignin-grafted dioxapentane polymer as a composite carbon source to increase the yield of polyhydroxy fatty acid esters produced by the microorganisms.

Description

Technical Field

[0001] This invention relates to the field of extraction technology, and specifically to a method for extracting polyhydroxy fatty acid esters by enzymatic hydrolysis of bamboo powder. Background Technology

[0002] Polyhydroxyalkanoates (PHAs) are biopolymers synthesized by microorganisms through fermentation under specific nutrient conditions, such as abundant carbon sources and relatively scarce other nutrients like nitrogen and phosphorus. These materials have attracted considerable attention due to their similar physical and chemical properties to traditional plastics, including good mechanical and processing performance. Furthermore, PHAs possess excellent biocompatibility and biodegradability, making them environmentally friendly materials that can be broken down by microorganisms in the natural environment without causing long-term pollution. Therefore, research on PHAs not only helps address the increasingly serious problem of plastic pollution but also has profound significance for alleviating the global energy crisis and promoting the sustainable use of resources.

[0003] With the rapid development of synthetic biology technology and strong national policy support, significant progress has been made in the production process of polyhydroxyalkanoates (PHA). Despite these advancements in production processes, research on PHA extraction technologies remains relatively limited, which to some extent restricts its large-scale production and industrial application. Therefore, future research needs to focus more on optimizing extraction and purification processes to achieve the commercialization and widespread application of PHA, thereby better serving the goals of environmental protection and sustainable resource development.

[0004] Bamboo is rich in lignocellulose biomass, mainly composed of cellulose, hemicellulose, and lignin. Lignin is a three-dimensional network polymer with a three-dimensional structure, widely present in plant cells. It combines with hemicellulose, and the two together fill the spaces between the fine fibers of the cell wall, providing compression resistance and support. As a byproduct of the pulp and paper, fuel ethanol, and biomass refining industries, lignin is typically used for combustion to supply heat energy or directly landfilled, leading to resource waste and environmental pollution. Therefore, the high-value bioconversion of lignin is of great significance for environmental protection and energy security. Summary of the Invention

[0005] The purpose of this invention is to provide a method for extracting polyhydroxy fatty acid esters from bamboo powder by enzymatic hydrolysis, which can improve the bioconversion rate of lignin in bamboo and promote the bioconversion of lignin into high-yield polyhydroxy fatty acid esters.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] A method for extracting polyhydroxy fatty acid esters from bamboo powder by enzymatic hydrolysis includes the following steps:

[0008] S1. Take whole bamboo, process it, crush it, grind it to obtain bamboo powder;

[0009] S2. The bamboo powder is pretreated in an ozone atmosphere to obtain pretreated bamboo powder.

[0010] S3. Take the pretreated bamboo powder and enzymatically hydrolyze it to obtain the enzymatic hydrolysis residue;

[0011] S4. Take the enzymatic hydrolysis residue and perform lignin and hemicellulose separation treatment to obtain lignin;

[0012] S5. Graft the lignin onto epichlorohydrin to obtain modified lignin;

[0013] S6. Take the modified lignin grafted with cyclodextrin to obtain cyclodextrin grafted lignin;

[0014] S7. In a nitrogen atmosphere, 1,3-dioxopentane, initiator and catalyst are added to the polymerization reactor and mixed evenly. The mixture is heated and stirred. Ammonia is added to terminate the reaction. The product is precipitated at low temperature, filtered, washed and vacuum dried to obtain the polymer material.

[0015] S8. Take the cyclodextrin-grafted lignin and deionized water, mix them, add the polymer material, stir ultrasonically, let stand, filter, take the solid phase, wash and dry to obtain the composite carbon source.

[0016] S9. Select a bacterial strain and streak it onto a solid culture medium for activation. Select the activated strain and inoculate it into a liquid culture medium to obtain a liquid seed. Inoculate the liquid seed into a fermentation medium to obtain a fermentation broth. After the fermentation is completed, extract and purify the fermentation broth to obtain the polyhydroxy fatty acid ester.

[0017] The carbon source in the fermentation medium includes glucose and a complex carbon source.

[0018] As a further embodiment of the present invention, step S1 specifically includes: taking whole bamboo, cleaning to remove surface impurities, splitting the whole bamboo and flattening it, peeling it, cutting the peeled bamboo into bamboo strips along the length direction, then cutting it into bamboo blocks, drying the bamboo blocks after centrifuging to remove moisture, crushing them into 2-4mm particles, and grinding them to obtain bamboo powder with a particle size of 300-500 mesh.

[0019] As a further aspect of the present invention, whole bamboo refers to any species of the Bambusoideae subfamily, such as, but not limited to, purple bamboo, yellow-striped bamboo, white bamboo, cinnamon bamboo, and phoenix-tail bamboo.

[0020] As a further aspect of the present invention, step S2, the method for preparing the pretreated bamboo powder specifically includes the following steps:

[0021] Take the bamboo powder and deionized water, mix them, disperse them ultrasonically, adjust the pH to 1.8-3.0, heat and stir, transfer them to a reaction vessel, treat them in an ozone atmosphere, vacuum filter and wash them to obtain pretreated bamboo powder;

[0022] Cellulase adsorption onto the cellulose surface is the first step in enzymatic hydrolysis. However, lignin can irreversibly and nonproductively adsorb onto cellulase through hydrophobic, hydrogen bond, and electrostatic interactions, and it also acts as a physical barrier on the cellulose surface, forming steric hindrance to cellulase. Cellulase is more likely to adsorb onto the lignin surface in the enzymatic hydrolysis system, which has an adverse effect on the enzymatic hydrolysis of cellulose. However, ozone pretreatment reduces the physical barrier of the substrate cellulose, lowers the enzymatic hydrolysis resistance, increases the accessibility of cellulase hydrolysis, and improves the removal rate of lignin.

[0023] Preferably, the mass ratio of bamboo powder to deionized water is 1.8:100. Under this ratio, cellulose is more likely to absorb water and swell, which reduces the cohesive force inside the fiber, makes it easier for ozone molecules to penetrate into the fiber, and improves the delignification effect, reduces the dissolution of hemicellulose and cellulose, and improves the selectivity of delignification.

[0024] Preferably, anhydrous acetic acid is used to adjust the pH value to 2.2, which can reduce the generation of reactive oxygen free radicals, protect carbohydrates from being destroyed, and thus facilitate the removal of lignin.

[0025] As a further aspect of the present invention, in step S3, the method for preparing the enzymatic hydrolysis residue specifically includes the following steps:

[0026] Take the pretreated bamboo powder, deionized water, cellulase and additives, mix them, adjust the pH to 5-6, enzymatically hydrolyze, filter and separate to obtain lignin-rich enzymatic residue;

[0027] The additives include Tween 80, polyethylene glycol 4000, bovine serum albumin, and dodecylbenzenesulfonic acid;

[0028] The mass ratio of Tween 80, polyethylene glycol 4000, bovine serum albumin, and dodecylbenzenesulfonic acid is 0.08-0.25:0.08-0.18:0.06-0.12:0.02-0.05.

[0029] Preferably, the ratio of the pretreated bamboo powder, deionized water, cellulase, and auxiliary agent is 25g:500mL:1100FPU:0.45, and the pH is adjusted to 5.5 with citrate buffer.

[0030] Preferably, the cellulase is Cellulase RS cellulase, CAS number 9012-54-8 (purchased from Beijing Bio-Lab Technology Co., Ltd.).

[0031] By adding nonionic surfactants Tween 80 and polyethylene glycol 4000, lignin can be bound to them through hydrophobic interactions and hydrogen bonding, occupying adsorption sites on the lignin surface and reducing the adsorption between cellulase and lignin. Adding bovine serum albumin (BSA) allows its hydrophobic structure to hydrophobically adsorb onto lignin and occupy its hydrophobic adsorption sites, further reducing the adsorption of cellulase by lignin. Adding anionic surfactant dodecylbenzenesulfonic acid adsorbs onto the lignin surface, forming a hydration film and steric hindrance, further reducing cellulase adsorption. The synergistic effect of Tween 80, polyethylene glycol 4000, BSA, and dodecylbenzenesulfonic acid enhances the dissolution of lignin.

[0032] As a further aspect of the present invention, step S4, the method for preparing lignin specifically includes the following steps:

[0033] Take the enzymatic hydrolysis residue, zinc chloride, and methanol, add hydrochloric acid aqueous solution and formic acid aqueous solution, heat and stir in an oil bath, and then cool in an ice-water bath to separate lignin.

[0034] Through the above extraction steps, the synergistic effect of the zinc chloride-methanol-hydrochloric acid-formic acid mixed system is utilized to sever the connection between lignin and hemicellulose, thereby dissolving lignin relatively uniformly.

[0035] As a further aspect of the present invention, in step S7, the initiator includes at least one of perchloric acid, perchlorate, and perchlorate hydrate.

[0036] As a further aspect of the present invention, in step S7, the catalyst includes at least one of boron trifluoride, boron trifluoride diethyl ether complex, boron trifluoride n-butyl ether complex, tin tetrachloride, titanium tetrachloride, aluminum trichloride, zinc chloride, vanadium trichloride, antimony trichloride, phosphorus pentafluoride, and antimony pentafluoride.

[0037] As a further embodiment of the present invention, in step S9, the solid culture medium is prepared as follows: 5g peptone, 1g yeast extract, 27.5g sodium chloride, 0.7g potassium chloride, 5.4g magnesium chloride hexahydrate, 6.8g magnesium sulfate heptahydrate, 1.4g calcium chloride dihydrate, 0.2g sodium bicarbonate, 0.07g potassium bromide, 20mg boric acid, 5mg sodium silicate, 3mg sodium fluoride, 2mg ammonium nitrate, 1.5g potassium dihydrogen phosphate, and 15g agar. Distilled water is added to bring the volume to 1L, the pH is 7.5, and the mixture is sterilized at 121℃ for 20min.

[0038] As a further embodiment of the present invention, in step S9, the liquid culture medium is prepared as follows: 5g peptone, 1g yeast extract, 90g sodium chloride, 0.7g potassium chloride, 5.4g magnesium chloride hexahydrate, 6.8g magnesium sulfate heptahydrate, 1.4g calcium chloride dihydrate, 0.2g sodium bicarbonate, 0.07g potassium bromide, 20mg boric acid, 5mg sodium silicate, 3mg sodium fluoride, 2mg ammonium nitrate, and 1.5g potassium dihydrogen phosphate. Distilled water is added to bring the volume to 1L, the pH is 7.5, and the mixture is sterilized at 121℃ for 20min.

[0039] As a further embodiment of the present invention, in step S9, the fermentation culture medium is prepared as follows: 5g peptone, 1g yeast extract, 27.5g sodium chloride, 0.7g potassium chloride, 5.4g magnesium chloride hexahydrate, 6.8g magnesium sulfate heptahydrate, 1.4g calcium chloride dihydrate, 0.2g sodium bicarbonate, 0.07g potassium bromide, 20mg boric acid, 5mg sodium silicate, 3mg sodium fluoride, 2mg ammonium nitrate, 1.5g potassium dihydrogen phosphate, 10g glucose, and 10-15g composite carbon source. Distilled water is added to bring the volume to 1L, the pH is 7.5, and the mixture is sterilized at 121℃ for 20min.

[0040] As a further aspect of the present invention, the bacterial strain is *Pseudomonas putida* KT2440, purchased from Shanghai Jiachu Bioengineering Co., Ltd., with strain number SMHCC(SHBCC)D25212.

[0041] As a further aspect of the present invention, the extracted polyhydroxy fatty acid ester is specifically poly(3-hydroxybutyrate).

[0042] The beneficial effects of this invention are:

[0043] The present invention discloses a method for extracting polyhydroxy fatty acid esters by enzymatic hydrolysis of bamboo powder. This method utilizes the high-purity lignin extracted from bamboo powder by enzymatic hydrolysis as a carbon source for the strain to synthesize polyhydroxy fatty acid esters, thereby increasing the yield of polyhydroxy fatty acid esters by the bioconversion of lignin.

[0044] Furthermore, in order to obtain high-purity lignin, ozone pretreatment of bamboo powder reduces the physical barrier of the substrate cellulose, lowers the enzymatic hydrolysis resistance, increases the accessibility of cellulase hydrolysis, and improves the lignin removal rate; the synergistic effect of Tween 80, polyethylene glycol 4000, bovine serum albumin and dodecylbenzene sulfonic acid synergistically improves the dissolution of lignin.

[0045] Furthermore, by grafting lignin with cyclodextrin and encapsulating dioxapentane polymers, the affinity of the ether bonds of the dioxapentane polymers for Pseudomonas putida strains is utilized to allow the strains to attach to the surface of the cyclodextrin-grafted lignin. This avoids the severe adhesion growth and squeezing death caused by strain reproduction, or the uneven growth caused by settling or stratification, thereby increasing the yield of polyhydroxy fatty acid esters. Attached Figure Description

[0046] The invention will now be further described with reference to the accompanying drawings.

[0047] Figure 1 This is the infrared spectrum analysis diagram of the polyhydroxy fatty acid ester extracted in Example 2 of the present invention;

[0048] Figure 2 This is the carbon NMR spectrum of the polyhydroxy fatty acid ester extracted in Example 2 of this invention;

[0049] Figure 3 This is the colony morphology of *Pseudomonas putida* KT2440 cultured in Example 2 of this invention. Detailed Implementation

[0050] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0051] Example 1

[0052] A method for extracting polyhydroxy fatty acid esters from bamboo powder by enzymatic hydrolysis includes the following steps:

[0053] S1. Take whole purple bamboo, clean it to remove surface impurities, split the whole bamboo, flatten it, peel it, cut the peeled bamboo into bamboo strips along the length, cut it into bamboo blocks, dry the bamboo blocks after centrifuging to remove water, crush them into 2mm particles, and grind them to obtain bamboo powder with a particle size of 300 mesh.

[0054] S2. Take the bamboo powder and deionized water, mix them, ultrasonically disperse for 20 min, add anhydrous acetic acid to adjust the pH to 1.8, stir at 60℃ for 1 h, transfer to a reaction vessel, treat in an ozone atmosphere for 1.5 h, vacuum filter and wash to obtain pretreated bamboo powder; the mass ratio of bamboo powder to deionized water is 1.2:100; the ozone concentration in the ozone atmosphere is 90 mg / L, and the ozone flow rate is 3 L / min;

[0055] S3. Take the pretreated bamboo powder, deionized water, cellulase, and auxiliary agent, mix them, add citrate buffer to adjust the pH to 5, and enzymatically hydrolyze them in a water bath shaker at 50°C and 200 rpm for 60 h. Filter and separate to obtain lignin-rich enzymatic hydrolysis residue. The ratio of the pretreated bamboo powder, deionized water, cellulase, and auxiliary agent is 20 g: 500 mL: 800 FPU: 0.16 g.

[0056] The cellulase used is Cellulase RS cellulase.

[0057] The additives include Tween 80, polyethylene glycol 4000, bovine serum albumin, and dodecylbenzenesulfonic acid, wherein the mass ratio of Tween 80, polyethylene glycol 4000, bovine serum albumin, and dodecylbenzenesulfonic acid is 0.08:0.08:0.06:0.02.

[0058] S4. Take the enzymatic hydrolysis residue, zinc chloride, and methanol, add hydrochloric acid aqueous solution and formic acid aqueous solution, heat and stir in an oil bath at 45℃ for 12 minutes, then cool in an ice-water bath, transfer the mixture to a separatory funnel with deionized water, add ethyl acetate to extract lignin, collect the ethyl acetate phase, filter, wash the filter residue, and dry at 80℃ for 10 hours to obtain lignin; the ratio of the enzymatic hydrolysis residue, zinc chloride, hydrochloric acid aqueous solution, formic acid aqueous solution, and methanol is 2g:22g:2mL:2mL:30mL; the concentration of the hydrochloric acid aqueous solution is 2.5mol / L; the concentration of the formic acid aqueous solution is 3mol / L;

[0059] S5. Take the lignin and sodium hydroxide aqueous solution, mix them, stir for 5 min, add epichlorohydrin, stir at 80℃ for 2 h, filter the product under vacuum until dry, wash, and vacuum dry to obtain modified lignin; the ratio of lignin, sodium hydroxide aqueous solution and epichlorohydrin is 1 g: 6 mL: 12 g; the concentration of sodium hydroxide aqueous solution is 1 mol / L.

[0060] S6. Take the modified lignin, β-cyclodextrin, and sodium hydroxide aqueous solution, mix and stir evenly, stir at 35℃ for 2h, filter, take the solid phase for washing, and vacuum dry to obtain cyclodextrin-grafted lignin; the ratio of the modified lignin, β-cyclodextrin, and sodium hydroxide aqueous solution is 2g:4g:30mL; the concentration of the sodium hydroxide aqueous solution is 1mol / L.

[0061] S7. Under a nitrogen atmosphere, 1,3-dioxapentane, perchloric acid, and boron trifluoride diethyl ether complex (purchased from Shandong Yinglang Chemical Co., Ltd.) were added to a polymerization reactor and mixed thoroughly. The mixture was stirred at 40°C for 15 minutes, and ammonia was added to terminate the reaction. The product was precipitated in methanol at 0°C, filtered, washed, and vacuum dried to obtain the polymer material. The ratio of 1,3-dioxapentane, perchloric acid, boron trifluoride diethyl ether complex, and ammonia was 3g:0.0014g:0.0004g:10mL; the concentration of ammonia was 1mol / L.

[0062] S8. Take the cyclodextrin-grafted lignin and deionized water, add the polymer material, ultrasonically stir for 10 min, let stand for 24 h, filter, take the solid phase, wash, and dry to obtain the composite carbon source; the mass ratio of the cyclodextrin-grafted lignin, deionized water and polymer material is 1:50:4.

[0063] S9. Specifically, it includes the following processes:

[0064] S91. Strain activation: Take the KT2440 strain of Pseudomonas putida that was frozen at -80℃, scrape off ice chips with a sterile pipette tip, pick up the strain and streak it onto a solid culture medium, and incubate it in a constant temperature incubator at 30℃ for 20h.

[0065] The solid culture medium is prepared as follows: 5g peptone, 1g yeast extract, 27.5g sodium chloride, 0.7g potassium chloride, 5.4g magnesium chloride hexahydrate, 6.8g magnesium sulfate heptahydrate, 1.4g calcium chloride dihydrate, 0.2g sodium bicarbonate, 0.07g potassium bromide, 20mg boric acid, 5mg sodium silicate, 3mg sodium fluoride, 2mg ammonium nitrate, 1.5g potassium dihydrogen phosphate, and 15g agar. Distilled water is added to bring the volume to 1L, the pH is 7.5, and the medium is sterilized at 121℃ for 20 minutes.

[0066] S92. Preparation of seed liquid: Select activated strains and inoculate them into liquid culture medium, and culture them at 30℃ and 200rpm for 12h to obtain liquid seeds;

[0067] The liquid culture medium is prepared as follows: 5g peptone, 1g yeast extract, 90g sodium chloride, 0.7g potassium chloride, 5.4g magnesium chloride hexahydrate, 6.8g magnesium sulfate heptahydrate, 1.4g calcium chloride dihydrate, 0.2g sodium bicarbonate, 0.07g potassium bromide, 20mg boric acid, 5mg sodium silicate, 3mg sodium fluoride, 2mg ammonium nitrate, and 1.5g potassium dihydrogen phosphate. Distilled water is added to bring the volume to 1L, the pH is 7.5, and the medium is sterilized at 121℃ for 20 minutes.

[0068] S93, Liquid fermentation: Fermentation broth was obtained by inoculating liquid seed and fermentation medium at a volume ratio of 5:100 and shaking culture at 30℃ and 150rpm for 72h.

[0069] The fermentation medium was prepared as follows: 5g peptone, 1g yeast extract, 27.5g sodium chloride, 0.7g potassium chloride, 5.4g magnesium chloride hexahydrate, 6.8g magnesium sulfate heptahydrate, 1.4g calcium chloride dihydrate, 0.2g sodium bicarbonate, 0.07g potassium bromide, 20mg boric acid, 5mg sodium silicate, 3mg sodium fluoride, 2mg ammonium nitrate, 1.5g potassium dihydrogen phosphate, 10g glucose, and 10g composite carbon source. Distilled water was added to bring the volume to 1L, the pH was set to 7.5, and the medium was sterilized at 121℃ for 20min.

[0070] S94. Extraction and purification: After fermentation, the fermentation broth was centrifuged at 12000 rpm for 15 min to collect the bacterial cells. The bacterial cells were resuspended in deionized water and washed three times before being freeze-dried. The freeze-dried bacterial cells were thoroughly ground in a mortar. 10 mL of chloroform was added to each gram of bacterial powder, and the mixture was extracted at 60 °C for 10 h. The mixture was filtered, and the chloroform phase was collected and concentrated in a rotary evaporator. Five times the volume of anhydrous ethanol was added, and the mixture was allowed to stand at 4 °C for 20 h before being centrifuged to collect the solid precipitate. The solid precipitate was then dried under vacuum to obtain polyhydroxy fatty acid ester.

[0071] Example 2

[0072] A method for extracting polyhydroxy fatty acid esters from bamboo powder by enzymatic hydrolysis includes the following steps:

[0073] S1. Take whole purple bamboo, clean it to remove surface impurities, split the whole bamboo, flatten it, peel it, cut the peeled bamboo into bamboo strips along the length, cut it into bamboo blocks, dry the bamboo blocks after centrifuging to remove water, crush them into 3mm particles, and grind them to obtain bamboo powder with a particle size of 400 mesh.

[0074] S2. Take the bamboo powder and deionized water, mix them, ultrasonically disperse for 25 min, add anhydrous acetic acid to adjust the pH to 2.2, stir at 60℃ for 1.5 h, transfer to a reaction vessel, treat in an ozone atmosphere for 2 h, vacuum filter and wash to obtain pretreated bamboo powder; the mass ratio of bamboo powder to deionized water is 1.8:100; the ozone concentration in the ozone atmosphere is 90 mg / L, and the ozone flow rate is 3 L / min;

[0075] S3. Take the pretreated bamboo powder, deionized water, Cellulase RS cellulase, and auxiliary agent, mix them, add citrate buffer to adjust the pH to 5.5, and enzymatically hydrolyze for 72 hours at 50°C and 200 rpm in a water bath shaker. Filter and separate to obtain lignin-rich enzymatic hydrolysis residue. The ratio of the pretreated bamboo powder, deionized water, Cellulase RS cellulase, and auxiliary agent is 25g:500mL:1100FPU:0.45g.

[0076] The cellulase used is Cellulase RS cellulase.

[0077] The mass ratio of Tween 80, polyethylene glycol 4000, bovine serum albumin, and dodecylbenzenesulfonic acid in the additive is 0.16:0.13:0.09:0.035.

[0078] S4. Take the enzymatic hydrolysis residue, zinc chloride, and methanol, add hydrochloric acid aqueous solution and formic acid aqueous solution, heat and stir in an oil bath at 52℃ for 15 min, then cool in an ice-water bath, transfer the mixture to a separatory funnel with deionized water, add ethyl acetate to extract lignin, collect the ethyl acetate phase, filter, wash the filter residue, and dry at 80℃ for 12 h to obtain lignin; the ratio of the enzymatic hydrolysis residue, zinc chloride, hydrochloric acid aqueous solution, formic acid aqueous solution, and methanol is 2.5g:26g:3mL:3mL:30mL; the concentration of the hydrochloric acid aqueous solution is 3mol / L; the concentration of the formic acid aqueous solution is 4mol / L.

[0079] S5. Take the lignin and sodium hydroxide aqueous solution, mix them, stir for 6 min, add epichlorohydrin, stir at 80℃ for 3 h, filter the product under vacuum until dry, wash, and vacuum dry to obtain modified lignin; the ratio of lignin, sodium hydroxide aqueous solution and epichlorohydrin is 1.1 g: 7 mL: 14 g; the concentration of sodium hydroxide aqueous solution is 1 mol / L;

[0080] S6. Mix the modified lignin, β-cyclodextrin, and sodium hydroxide aqueous solution evenly, stir at 40℃ for 3 hours, filter, wash the solid phase, and vacuum dry to obtain cyclodextrin-grafted lignin; the ratio of the modified lignin, β-cyclodextrin, and sodium hydroxide aqueous solution is 2.5g:4.5g:30mL; the concentration of the sodium hydroxide aqueous solution is 1mol / L.

[0081] S7. Under a nitrogen atmosphere, 1,3-dioxapentane, perchloric acid, and boron trifluoride diethyl ether complex were added to a polymerization reactor and mixed thoroughly. The mixture was stirred at 50°C for 20 min, and ammonia was added to terminate the reaction. The product was precipitated in methanol at 0°C, filtered, washed, and vacuum dried to obtain the polymer material. The ratio of 1,3-dioxapentane, perchloric acid, boron trifluoride diethyl ether complex, and ammonia was 4 g: 0.0016 g: 0.0005 g: 11 mL; the concentration of ammonia was 1 mol / L.

[0082] S8. Take the cyclodextrin-grafted lignin and deionized water, add the polymer material, ultrasonically stir for 15 min, let stand for 24 h, filter, take the solid phase, wash, and dry to obtain the composite carbon source; the mass ratio of the cyclodextrin-grafted lignin, deionized water and polymer material is 2:50:6.

[0083] S9. Specifically, it includes the following processes:

[0084] S91. Strain activation: Take the KT2440 strain of Pseudomonas putida that was frozen at -80℃, scrape off ice chips with a sterile pipette tip, pick up the strain and streak it onto a solid culture medium, and incubate it in a constant temperature incubator at 30℃ for 30h.

[0085] The solid culture medium is prepared as follows: 5g peptone, 1g yeast extract, 27.5g sodium chloride, 0.7g potassium chloride, 5.4g magnesium chloride hexahydrate, 6.8g magnesium sulfate heptahydrate, 1.4g calcium chloride dihydrate, 0.2g sodium bicarbonate, 0.07g potassium bromide, 20mg boric acid, 5mg sodium silicate, 3mg sodium fluoride, 2mg ammonium nitrate, 1.5g potassium dihydrogen phosphate, and 15g agar. Distilled water is added to bring the volume to 1L, the pH is 7.5, and the medium is sterilized at 121℃ for 20 minutes.

[0086] S92. Preparation of seed liquid: Select activated strains and inoculate them into liquid culture medium, and culture them at 30℃ and 200rpm for 15h to obtain liquid seeds;

[0087] The liquid culture medium is prepared as follows: 5g peptone, 1g yeast extract, 90g sodium chloride, 0.7g potassium chloride, 5.4g magnesium chloride hexahydrate, 6.8g magnesium sulfate heptahydrate, 1.4g calcium chloride dihydrate, 0.2g sodium bicarbonate, 0.07g potassium bromide, 20mg boric acid, 5mg sodium silicate, 3mg sodium fluoride, 2mg ammonium nitrate, and 1.5g potassium dihydrogen phosphate. Distilled water is added to bring the volume to 1L, the pH is 7.5, and the medium is sterilized at 121℃ for 20 minutes.

[0088] S93, Liquid fermentation: Fermentation broth was obtained by inoculating liquid seed and fermentation medium at a volume ratio of 8:100 and shaking culture at 30℃ and 150rpm for 72h.

[0089] The fermentation medium was prepared as follows: 5g peptone, 1g yeast extract, 27.5g sodium chloride, 0.7g potassium chloride, 5.4g magnesium chloride hexahydrate, 6.8g magnesium sulfate heptahydrate, 1.4g calcium chloride dihydrate, 0.2g sodium bicarbonate, 0.07g potassium bromide, 20mg boric acid, 5mg sodium silicate, 3mg sodium fluoride, 2mg ammonium nitrate, 1.5g potassium dihydrogen phosphate, 10g glucose, and 13g composite carbon source. Distilled water was added to bring the volume to 1L, the pH was set to 7.5, and the medium was sterilized at 121℃ for 20min.

[0090] S94. Extraction and purification: After fermentation, the fermentation broth was centrifuged at 12000 rpm for 15 min to collect the bacterial cells. The bacterial cells were resuspended in deionized water and washed three times before being freeze-dried. The freeze-dried bacterial cells were thoroughly ground in a mortar. 10 mL of chloroform was added to each gram of bacterial powder, and the mixture was extracted at 60 °C for 10 h. The mixture was filtered, and the chloroform phase was collected and concentrated in a rotary evaporator. Five times the volume of anhydrous ethanol was added, and the mixture was allowed to stand at 4 °C for 20 h before being centrifuged to collect the solid precipitate. The solid precipitate was then dried under vacuum to obtain polyhydroxy fatty acid ester.

[0091] like Figure 1 As shown, in the infrared spectrum of the polyhydroxyalkanoate extracted in this embodiment, the 1720 cm⁻¹... -1It is the C=O stretching vibration absorption peak, which is a characteristic absorption peak of polyhydroxy fatty acid esters.

[0092] like Figure 2 As shown, the carbon NMR spectrum of the polyhydroxy fatty acid ester extracted in this embodiment shows peaks at 19.746 ppm, 40.757 ppm, 77.000 ppm and 169.135 ppm corresponding to methyl (-CH3), methylene (-CH2-), methine (-CH-) and carbonyl (C=O) respectively, indicating that its intracellular polymer is specifically poly(3-hydroxybutyrate).

[0093] like Figure 3 As shown, the colony morphology of *Pseudomonas putida* KT2440 grown on fermentation medium.

[0094] Example 3

[0095] A method for extracting polyhydroxy fatty acid esters from bamboo powder by enzymatic hydrolysis includes the following steps:

[0096] S1. Take whole purple bamboo, clean it to remove surface impurities, split the whole bamboo, flatten it, peel it, cut the peeled bamboo into bamboo strips along the length, cut it into bamboo blocks, dry the bamboo blocks after centrifuging to remove water, crush them into 4mm particles, and grind them to obtain bamboo powder with a particle size of 500 mesh.

[0097] S2. Take the bamboo powder and deionized water, mix them, ultrasonically disperse for 30 min, add anhydrous acetic acid to adjust the pH to 3.0, stir at 60℃ for 2 h, transfer to a reaction vessel, treat in an ozone atmosphere for 2.5 h, vacuum filter and wash to obtain pretreated bamboo powder; the mass ratio of bamboo powder to deionized water is 2.5:100; the ozone concentration in the ozone atmosphere is 90 mg / L, and the ozone flow rate is 3 L / min;

[0098] S3. Take the pretreated bamboo powder, deionized water, Cellulase RS cellulase, and auxiliary agent, mix them, add citrate buffer to adjust the pH to 6, and enzymatically hydrolyze them in a water bath shaker at 50°C and 200 rpm for 80 h. Filter and separate to obtain lignin-rich enzymatic hydrolysis residue. The ratio of the pretreated bamboo powder, deionized water, Cellulase RS cellulase, and auxiliary agent is 30 g: 500 mL: 1500 FPU: 0.75 g.

[0099] The mass ratio of Tween 80, polyethylene glycol 4000, bovine serum albumin, and dodecylbenzenesulfonic acid in the additive is 0.25:0.18:0.12:0.05.

[0100] S4. Take the enzymatic hydrolysis residue, zinc chloride, and methanol, add hydrochloric acid aqueous solution and formic acid aqueous solution, heat and stir in an oil bath at 58℃ for 18 minutes, then cool in an ice-water bath, transfer the mixture to a separatory funnel with deionized water, add ethyl acetate to extract lignin, collect the ethyl acetate phase, filter, wash the filter residue, and dry at 80℃ for 15 hours to obtain lignin; the ratio of the enzymatic hydrolysis residue, zinc chloride, hydrochloric acid aqueous solution, formic acid aqueous solution, and methanol is 3g:30g:4mL:4mL:30mL; the concentration of the hydrochloric acid aqueous solution is 3.5mol / L; the concentration of the formic acid aqueous solution is 5mol / L;

[0101] S5. Take the lignin and sodium hydroxide aqueous solution, mix them, stir for 7 min, add epichlorohydrin, stir at 80℃ for 4 h, filter the product under vacuum until dry, wash, and vacuum dry to obtain modified lignin; the ratio of lignin, sodium hydroxide aqueous solution and epichlorohydrin is 1.2 g: 8 mL: 16 g; the concentration of sodium hydroxide aqueous solution is 1 mol / L.

[0102] S6. Take the modified lignin, β-cyclodextrin, and sodium hydroxide aqueous solution, mix and stir evenly, stir at 45℃ for 4 hours, filter, take the solid phase for washing, and vacuum dry to obtain cyclodextrin-grafted lignin; the ratio of the modified lignin, β-cyclodextrin, and sodium hydroxide aqueous solution is 3g:5g:30mL; the concentration of the sodium hydroxide aqueous solution is 1mol / L.

[0103] S7. Under a nitrogen atmosphere, 1,3-dioxapentane, perchloric acid, and boron trifluoride diethyl ether complex were added to a polymerization reactor and mixed thoroughly. The mixture was stirred at 60°C for 25 minutes, and ammonia was added to terminate the reaction. The product was precipitated in methanol at 0°C, filtered, washed, and vacuum dried to obtain the polymer material. The ratio of 1,3-dioxapentane, perchloric acid, boron trifluoride diethyl ether complex, and ammonia was 5 g: 0.0018 g: 0.0006 g: 12 mL; the concentration of ammonia was 1 mol / L.

[0104] S8. Take the cyclodextrin-grafted lignin and deionized water, add the polymer material, ultrasonically stir for 20 min, let stand for 24 h, filter, take the solid phase, wash, and dry to obtain the composite carbon source; the mass ratio of the cyclodextrin-grafted lignin, deionized water and polymer material is 3:50:8.

[0105] S9. Specifically, it includes the following processes:

[0106] S91. Strain activation: Take the KT2440 strain of Pseudomonas putida that was frozen at -80℃, scrape off ice chips with a sterile pipette tip, pick up the strain and streak it onto a solid culture medium, and incubate it in a constant temperature incubator at 30℃ for 40h.

[0107] The solid culture medium is prepared as follows: 5g peptone, 1g yeast extract, 27.5g sodium chloride, 0.7g potassium chloride, 5.4g magnesium chloride hexahydrate, 6.8g magnesium sulfate heptahydrate, 1.4g calcium chloride dihydrate, 0.2g sodium bicarbonate, 0.07g potassium bromide, 20mg boric acid, 5mg sodium silicate, 3mg sodium fluoride, 2mg ammonium nitrate, 1.5g potassium dihydrogen phosphate, and 15g agar. Distilled water is added to bring the volume to 1L, the pH is 7.5, and the medium is sterilized at 121℃ for 20 minutes.

[0108] S92. Preparation of seed liquid: Select activated strains and inoculate them into liquid culture medium, and culture them at 30℃ and 200 rpm for 16 h to obtain liquid seeds;

[0109] The liquid culture medium is prepared as follows: 5g peptone, 1g yeast extract, 90g sodium chloride, 0.7g potassium chloride, 5.4g magnesium chloride hexahydrate, 6.8g magnesium sulfate heptahydrate, 1.4g calcium chloride dihydrate, 0.2g sodium bicarbonate, 0.07g potassium bromide, 20mg boric acid, 5mg sodium silicate, 3mg sodium fluoride, 2mg ammonium nitrate, and 1.5g potassium dihydrogen phosphate. Distilled water is added to bring the volume to 1L, the pH is 7.5, and the medium is sterilized at 121℃ for 20 minutes.

[0110] S93, Liquid fermentation: Fermentation broth was obtained by inoculating liquid seed and fermentation medium at a volume ratio of 5-10:100 and shaking culture at 30℃ and 150rpm for 72h.

[0111] The fermentation medium was prepared as follows: 5g peptone, 1g yeast extract, 27.5g sodium chloride, 0.7g potassium chloride, 5.4g magnesium chloride hexahydrate, 6.8g magnesium sulfate heptahydrate, 1.4g calcium chloride dihydrate, 0.2g sodium bicarbonate, 0.07g potassium bromide, 20mg boric acid, 5mg sodium silicate, 3mg sodium fluoride, 2mg ammonium nitrate, 1.5g potassium dihydrogen phosphate, 10g glucose, and 15g composite carbon source. Distilled water was added to bring the volume to 1L, the pH was set to 7.5, and the medium was sterilized at 121℃ for 20 minutes.

[0112] S94. Extraction and purification: After fermentation, the fermentation broth was centrifuged at 12000 rpm for 15 min to collect the bacterial cells. The bacterial cells were resuspended in deionized water and washed three times before being freeze-dried. The freeze-dried bacterial cells were thoroughly ground in a mortar. 10 mL of chloroform was added to each gram of bacterial powder, and the mixture was extracted at 60 °C for 10 h. The mixture was filtered, and the chloroform phase was collected and concentrated in a rotary evaporator. Five times the volume of anhydrous ethanol was added, and the mixture was allowed to stand at 4 °C for 20 h before being centrifuged to collect the solid precipitate. The solid precipitate was then dried under vacuum to obtain polyhydroxy fatty acid ester.

[0113] Comparative Example 1

[0114] The difference from Example 2 is that the bamboo powder obtained by grinding in step S1 is directly enzymatically hydrolyzed without ozone pretreatment.

[0115] Comparative Example 2

[0116] The difference from Example 2 is that the mass ratio of Tween 80, polyethylene glycol 4000, bovine serum albumin and dodecylbenzenesulfonic acid in the additives described in step S3 is 0:0.29:0.09:0.035.

[0117] Comparative Example 3

[0118] The difference from Example 2 is that the mass ratio of Tween 80, polyethylene glycol 4000, bovine serum albumin and dodecylbenzenesulfonic acid in the additives described in step S3 is 0.29:0:0.09:0.035.

[0119] Comparative Example 4

[0120] The difference from Example 2 is that the mass ratio of Tween 80, polyethylene glycol 4000, bovine serum albumin and dodecylbenzenesulfonic acid in the additives described in step S3 is 0.16:0.22:0:0.035.

[0121] Comparative Example 5

[0122] The difference from Example 2 is that the mass ratio of Tween 80, polyethylene glycol 4000, bovine serum albumin and dodecylbenzenesulfonic acid in the additives described in step S3 is 0.16:0.13:0.125:0.

[0123] Comparative Example 6

[0124] The difference from Example 2 is that the fermentation medium is prepared as follows: 5g peptone, 1g yeast extract, 27.5g sodium chloride, 0.7g potassium chloride, 5.4g magnesium chloride hexahydrate, 6.8g magnesium sulfate heptahydrate, 1.4g calcium chloride dihydrate, 0.2g sodium bicarbonate, 0.07g potassium bromide, 20mg boric acid, 5mg sodium silicate, 3mg sodium fluoride, 2mg ammonium nitrate, 1.5g potassium dihydrogen phosphate, and 23g glucose. Distilled water is added to bring the volume to 1L, the pH is 7.5, and the mixture is sterilized at 121℃ for 20min.

[0125] Comparative Example 7

[0126] The difference from Example 2 is that the cyclodextrin-grafted lignin obtained in step S6 is a composite carbon source.

[0127] Test Example 1

[0128] The lignin obtained from Examples 1-3 and Comparative Examples 1-5 were tested for extraction rate and purity according to the National Renewable Energy Laboratory (NREL) standard method. The data results are shown in Table 1 below.

[0129] Table 1

[0130]

[0131] As shown in Table 1, the enzymatic hydrolysis methods used in Examples 1-3 of this application for extracting lignin from bamboo powder have high extraction rates and purity. In Comparative Example 1, the lack of ozone pretreatment reduced the enzymatic hydrolysis effect of cellulase on cellulose in bamboo powder, resulting in a significant decrease in both the purity and extraction rate of the obtained lignin. In Comparative Example 2, the absence of Tween 80 as an adjuvant reduced the adsorption of cellulase on the lignin surface, leading to a decrease in the enzymatic hydrolysis effect of cellulase on cellulose in bamboo powder, thus reducing the purity and extraction rate of the obtained lignin. In Comparative Example 3, the absence of polyethylene glycol 4000 as an adjuvant reduced the cellulose... Enzyme adsorption on the lignin surface reduces the enzymatic hydrolysis effect of cellulase on cellulose in bamboo powder, resulting in lower purity and extraction rate of the obtained lignin. In Comparative Example 4, bovine serum albumin was not added as an adjuvant, and cellulase adsorption on the lignin surface reduced the enzymatic hydrolysis effect of cellulase on cellulose in bamboo powder, resulting in lower purity and extraction rate of the obtained lignin. In Comparative Example 5, dodecylbenzenesulfonic acid was not added as an adjuvant, and cellulase adsorption on the lignin surface reduced the enzymatic hydrolysis effect of cellulase on cellulose in bamboo powder, resulting in lower purity and extraction rate of the obtained lignin.

[0132] Test Example 2

[0133] The yields of polyhydroxy fatty acid esters obtained after culturing for 72 hours in Examples 1-3 and Comparative Examples 1-7 are shown in Table 2 below.

[0134] Table 2

[0135]

[0136] As shown in Table 2, Examples 1-3 of this application yielded a relatively high amount of polyhydroxyalkanoate. In Comparative Examples 1-5, the low purity of lignin led to a decrease in the yield of polyhydroxyalkanoate; in Comparative Example 6, the absence of a lignin-containing composite carbon source resulted in a significant decrease in the yield of polyhydroxyalkanoate; and in Comparative Example 7, the lack of encapsulation of the dioxapentane polymer led to a decrease in the yield of polyhydroxyalkanoate.

[0137] The foregoing has described some embodiments of the present invention in detail, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. A method for extracting polyhydroxy fatty acid esters from bamboo powder by enzymatic hydrolysis, characterized in that, Includes the following steps: S1. Process whole bamboo to obtain bamboo powder; S2. The bamboo powder is pretreated in an ozone atmosphere to obtain pretreated bamboo powder. S3. Take the pretreated bamboo powder and enzymatically hydrolyze it to obtain the enzymatic hydrolysis residue; S4. Take the enzymatic hydrolysis residue and perform lignin and hemicellulose separation treatment to obtain lignin; S5. Graft the lignin onto epichlorohydrin to obtain modified lignin; S6. Take the modified lignin grafted with cyclodextrin to obtain cyclodextrin grafted lignin; S7. Using 1,3-dioxopentane as raw material, an initiator and a catalyst are added to prepare dioxopentane polymer; S8. A composite carbon source was obtained by grafting lignin with cyclodextrin to encapsulate dioxapentane polymer. S9. Select a bacterial strain and streak it onto a solid culture medium for activation. Select the activated strain and inoculate it into a liquid culture medium to obtain a liquid seed. Inoculate the liquid seed into a fermentation medium to obtain a fermentation broth. After the fermentation is completed, extract and purify the fermentation broth to obtain the polyhydroxy fatty acid ester. The carbon source in the fermentation medium includes glucose and a complex carbon source; In step S3, the enzymatic hydrolysis process includes auxiliary agents; the auxiliary agents include Tween 80, polyethylene glycol 4000, bovine serum albumin, and dodecylbenzenesulfonic acid; In step S7, the initiator is perchloric acid, and the catalyst is boron trifluoride diethyl ether complex; In step S9, the bacterial strain is *Pseudomonas putida* KT2440. The extracted polyhydroxy fatty acid ester is specifically poly(3-hydroxybutyrate).

2. The method for extracting polyhydroxy fatty acid esters from bamboo powder by enzymatic hydrolysis according to claim 1, characterized in that, In step S2, the method for preparing the pretreated bamboo powder specifically includes the following steps: The bamboo powder and deionized water are mixed, ultrasonically dispersed, and the pH is adjusted to 1.8-3.

0. The mixture is heated and stirred, then transferred to a reaction vessel and treated in an ozone atmosphere. The mixture is then vacuum filtered and washed to obtain pretreated bamboo powder.

3. The method for extracting polyhydroxy fatty acid esters from bamboo powder by enzymatic hydrolysis according to claim 1, characterized in that, In step S3, the preparation method of the enzymatic hydrolysis residue specifically includes the following steps: Take the pretreated bamboo powder, deionized water, cellulase and additives, mix them, adjust the pH to 5-6, enzymatically hydrolyze, filter and separate to obtain lignin-rich enzymatic residue; The mass ratio of Tween 80, polyethylene glycol 4000, bovine serum albumin and dodecylbenzenesulfonic acid in the additive is 0.08-0.25: 0.08-0.18: 0.06-0.12: 0.02-0.

05.

4. The method for extracting polyhydroxy fatty acid esters from bamboo powder by enzymatic hydrolysis according to claim 1, characterized in that, In step S4, the method for preparing lignin specifically includes the following steps: The enzyme hydrolysis residue, zinc chloride, and methanol were mixed, and hydrochloric acid aqueous solution and formic acid aqueous solution were added. The mixture was heated and stirred in an oil bath under sealed conditions, and then cooled in an ice-water bath to separate lignin.

5. The method for extracting polyhydroxy fatty acid esters from bamboo powder by enzymatic hydrolysis according to claim 1, characterized in that, In step S9, the solid culture medium is prepared as follows: 5g peptone, 1g yeast extract, 27.5g sodium chloride, 0.7g potassium chloride, 5.4g magnesium chloride hexahydrate, 6.8g magnesium sulfate heptahydrate, 1.4g calcium chloride dihydrate, 0.2g sodium bicarbonate, 0.07g potassium bromide, 20mg boric acid, 5mg sodium silicate, 3mg sodium fluoride, 2mg ammonium nitrate, 1.5g potassium dihydrogen phosphate, and 15g agar. Distilled water is added to bring the volume to 1L, the pH is 7.5, and the medium is sterilized at 121℃ for 20min.

6. The method for extracting polyhydroxy fatty acid esters from bamboo powder by enzymatic hydrolysis according to claim 1, characterized in that, In step S9, the liquid culture medium is prepared as follows: 5g peptone, 1g yeast extract, 90g sodium chloride, 0.7g potassium chloride, 5.4g magnesium chloride hexahydrate, 6.8g magnesium sulfate heptahydrate, 1.4g calcium chloride dihydrate, 0.2g sodium bicarbonate, 0.07g potassium bromide, 20mg boric acid, 5mg sodium silicate, 3mg sodium fluoride, 2mg ammonium nitrate, and 1.5g potassium dihydrogen phosphate. Distilled water is added to bring the volume to 1L, the pH is 7.5, and the mixture is sterilized at 121℃ for 20min.

7. The method for extracting polyhydroxy fatty acid esters from bamboo powder by enzymatic hydrolysis according to claim 1, characterized in that, In step S9, the fermentation medium is prepared as follows: 5g peptone, 1g yeast extract, 27.5g sodium chloride, 0.7g potassium chloride, 5.4g magnesium chloride hexahydrate, 6.8g magnesium sulfate heptahydrate, 1.4g calcium chloride dihydrate, 0.2g sodium bicarbonate, 0.07g potassium bromide, 20mg boric acid, 5mg sodium silicate, 3mg sodium fluoride, 2mg ammonium nitrate, 1.5g potassium dihydrogen phosphate, 10g glucose, and 10-15g composite carbon source. Distilled water is added to bring the volume to 1L, the pH is 7.5, and the medium is sterilized at 121℃ for 20min.

Images