CN112251390A - Genetically engineered bacterium for synthesizing vanillin by converting lignin-containing biomass and application thereof - Google Patents

Genetically engineered bacterium for synthesizing vanillin by converting lignin-containing biomass and application thereof Download PDF

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CN112251390A
CN112251390A CN202011011447.0A CN202011011447A CN112251390A CN 112251390 A CN112251390 A CN 112251390A CN 202011011447 A CN202011011447 A CN 202011011447A CN 112251390 A CN112251390 A CN 112251390A
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朱道辰
孙建中
朱彬
许令侠
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Abstract

The invention discloses a genetically engineered bacterium for converting lignin-containing biomass to synthesize vanillin and application thereof, belonging to the technical field of biology. The strain is named as Bacillus lignphilus L1-vdh, which is preserved in China center for type culture Collection in 1 month in 2020 with the preservation number: CGMCC No. 19226. The invention relates to a method for knocking out a gene coding vanillic acid dehydrogenase of Bacillus lignophilus L1(Bacillus ligniniphilus L1) to inactivate the gene so as to accumulate vanillin in a large amount. Thereby effecting a conversion from lignin to vanillin. The application of the invention can convert lignin biomass and depolymerized products thereof into vanillin. The invention can convert the agricultural and forestry waste biomass resources such as sawdust and straw, industrial lignin, papermaking black liquor and other cheap substances into aromatic compound vanillin with high added value, solves the bottleneck of high-value utilization of the agricultural and forestry waste, and has important significance for environmental protection.

Description

Genetically engineered bacterium for synthesizing vanillin by converting lignin-containing biomass and application thereof
Technical Field
The invention relates to a method for converting lignin-containing biomass or industrial lignin and depolymerized products thereof into vanillin by using a lignin-containing biomass genetic engineering bacterium (hereinafter referred to as a genetic engineering bacterium) capable of accumulating vanillin in large quantity, which is obtained by reconstructing a metabolic pathway of a lignin-containing bacillus strain by using a molecular biology technology, and belongs to the field of biotechnology.
Background
Vanillin (Vanillin, C)8H8O3) The 3-methoxy-4-hydroxybenzaldehyde is also called vanillin, is a broad-spectrum high-grade spice with the largest yield and the widest application in the world, and is applied to the industries of food, daily chemicals, rubber, plastics, medicines and the like.
The vanillin synthesized by the chemical synthesis method is a main vanillin source in the market, the annual yield is up to 20000 tons, but the chemically synthesized vanillin has low cost and large scale, but easily causes environmental pollution and cannot meet the requirements of people on natural foods.
Natural vanillin is mainly obtained by extracting vanilla beans at present, but artificial pollination is needed in planting of the vanilla beans, so that the labor intensity is high, and large-scale planting is difficult. At present, the planting amount of the vanilla is only about 2000-3000 tons every year, the yield of the extracted natural vanillin is only about 20 tons, and the market price is up to 3200 dollars/kg. In recent years, the demand of consumers for natural vanillin is increasing, and some foreign (for example, European) legislative bodies recommend the use of natural vanillin in food, but the extraction method with plant tissues has low yield and high cost, and causes the shortage of natural vanillin.
The microbial degradation and conversion of lignin has the advantages of simple conversion process, high yield, low energy consumption, safe product and the like. The U.S. food and drug administration states that "natural flavors must be products derived from plant or animal sources, either by physical, enzymatic or microbial processes". Therefore, the microbial method for transforming and synthesizing vanillin is an important trend for future development.
Lignin has not been appreciated and utilized for a long time as the major source of the most abundant natural aromatic compounds worldwide. How to convert the lignin components into high-value products is a feasible way for improving the comprehensive utilization of depolymerized lignin. The lignin depolymerization product contains a large amount of aromatic compounds such as ferulic acid, cinnamic acid, vanillic acid, vanillin and coumaric acid. The aromatic compound in the lignin is converted into the specific single aromatic compound by a biological method, which has important practical significance for realizing high-value utilization of the lignin and is also the requirement for realizing sustainable development of agriculture and forestry. Chemical degradation of lignin, separation and purification of vanillin, such as oxidative depolymerization of lignin under alkaline conditions followed by extraction and separation of vanillin, has been reported to have disadvantages in that the catalysts used are mainly noble metal and transition metal based catalysts, which are expensive and difficult to separate and purify, and are liable to cause pollution, and thus are difficult to be industrially applied. The lignin is converted into vanillin by a biological method under a mild condition, so that the use of a catalyst and the generation of environmental pollutants are avoided, and the method is one of feasible methods for producing vanillin by converting the lignin in the future. It has been reported before that the constructed engineering bacterium Rhodococcus jostii RHA045 can produce vanillin by using wheat straw and glucose as raw materials, and the yield of 96mg/L can be achieved after 6 days of fermentation (Paul D.Sainsbury, et al. ACS chemical biology,2013,8, 2151-. However, the strain can not utilize lignin as a single carbon source, and only obtains 1.0-1.3g/L of vanillin after fermenting for 48-72 hours by using alkaline lignin as a carbon source, and obviously the strain is not suitable for producing vanillin by utilizing lignin. And the production efficiency of vanillin is also influenced by the overlong fermentation time of 6 days, so that the production of vanillin by lignin conversion is difficult to realize.
The Bacillus lignophilus L1 is an extreme microbe which is screened from deep sea sediments and is alkalophilic and salt-tolerant, can survive in the environment with pH 7-11, has strong growth adaptability of L1 (10-50 ℃, pH 6.0-11.0, 0-10% NaCl, and optimal growth pH 9), and is one of the highest bacterial strains for alkali tolerance in the currently known lignin-degrading bacteria. Our earlier studies showed that: vanillin and vanillic acid are common intermediates of the β -aryl ether, biphenyl, diarylpropane and ferulic acid pathways, and represent up to 30-44.2% of the aromatic products of L1 depolymerizing alkaline lignin (Zhu D, et al Biotechnology for Biofuels,2017,10(1): 44). It is shown that vanillin is one of the most desirable target products for the biological depolymerization and conversion of lignin. Vanillin can be converted to vanillic acid by means of a vanillin dehydrogenase. Therefore, the vanillin dehydrogenase gene is knocked out, the degradation of vanillin is blocked, and the large-scale accumulation of vanillin can be realized.
The invention aims to solve the technical problem of providing a method for efficiently converting lignin biomass and depolymerized products thereof into vanillin by constructing a genetic engineering strain.
Disclosure of Invention
The invention aims to disclose a method for converting lignin, including various industrial lignin, papermaking black liquor and other cheap substances, into aromatic compound vanillin with high added value.
The invention discloses a genetically engineered bacterium for synthesizing vanillin by converting lignin-containing biomass, which is named as a lignin-philic Bacillus (Bacillus ligniniphilus) L1-vdh, which is preserved in China general microbiological culture collection management center of institute of microbiology, China academy of sciences, No.1 institute 3, north Chen West Lu, in the south area of the republic of China for 20 days in 2019, and is named as Bacillus ligniniphilus, wherein the accession number is as follows: CGMCC No. 19226.
The method for synthesizing vanillin by converting lignin-containing biomass by using the genetic engineering bacterium for converting lignin-containing biomass comprises the following steps:
(1) preparation of lignin depolymerization mixture: the industrial lignin or pretreated liquor containing the lignin after pretreatment is treated by one of a thermal cracking method, an enzymolysis polymerization method, microwaves, a metal catalyst cracking method, an alkali hydrolysis method and the like to achieve the aim of depolymerizing the lignin, and the pH value of the depolymerized reaction liquor is adjusted to be neutral for later use.
Wherein the thermal cracking method of step (1): thermally cracking the lignin aqueous solution with the temperature of 250-450 ℃ for 5-30 minutes in a reaction kettle. The pressure is controlled within 30 MPa.
The enzymatic polymerization method in the step (1) comprises the following steps: laccase was dissolved in acetate buffer (pH 4) at a concentration of 0.1g/L, and then the laccase solution was mixed uniformly with 10% lignin solution at a ratio of 1:1(v/v), followed by reaction at 30 ℃ for 12 hours.
Wherein the alkaline hydrolysis method of step (1): adding sodium hydroxide solution with concentration not higher than 10% (g/v) into lignin (lignin content is not higher than 10g/L), and treating at 80-100 deg.C for 1-6 hr.
Wherein the metal catalyst cracking method in the step (1): treating with metal catalyst (nickel, titanium, zinc, etc.) as catalyst at 100-300 deg.C in 10% lignin solution system for 0.5-1 h. Dissolving lignin in 2-10% NaCl solution at 1-10% (g/L), reacting at 60-200 deg.C for 1-3 hr, and collecting filtrate.
(2) A gene engineering bacterium L1-vdh (preservation number: CGMCC No.19226) of Bacillus lignophilus (Bacillus ligniniphilus) is frozen and inoculated to a freezing tube containing kanamycin (50 mu g-1) The activation pre-culture is carried out for 24h at 37 ℃ in a 2216E culture medium, then a fermentation culture medium is inoculated into the fermentation culture medium according to the proportion of 1:10(V/V) and cultured for 24h at 37 ℃ in a fermentation tank, the pH is adjusted to be about 9 through NaOH and HCl, glucose is supplemented when the glucose is lower than 5g/L in the culture process in a feeding mode, the fermentation is stopped when the fermentation OD value is above 30, then the fermentation liquid is replaced by MM63 culture medium taking lignin depolymerization products as single carbon sources, the culture is continued for 24h at 37 ℃, the pH is adjusted to be about 9 through NaOH and HCl, and the synthesis amount of vanillin is detected by HPLC.
Wherein the 2216E culture medium in the step (2) comprises the following components: 5g/L of peptone, 1g/L of yeast powder, 0.1g/L of ferric citrate, 19.45g/L of sodium chloride, 5.97g/L of magnesium chloride, 3.24g/L of sodium sulfate, 1.8g/L of calcium chloride, 0.16g/L of sodium carbonate, 0.08g/L of potassium bromide, 0.034g/L of strontium chloride, 0.022g/L of boric acid, 0.004g/L of sodium silicate, 0.0024g/L of sodium fluoride, 0.0016g/L of sodium nitrate and 0.008g/L of sodium dihydrogen phosphate.
Wherein the components of the fermentation medium in the step (2) are as follows: 50g/L of enzymolysis soybean meal, 30g/L of corn pulp powder, 10g/L of glucose and FeSO4 0.1g/L,MgSO40.5g/L, phosphate buffer 50 mM.
Wherein the MM63 medium in the step (2) comprises the following components: 100mM KH2PO4,75mM KOH,15mM(NH4)2SO4,1mM MgSO4,3.9μM FeSO4And lignin depolymerization product 1-30 g/L.
(3) And (4) centrifuging or filtering the fermentation liquor by a ceramic membrane to collect supernatant, and placing the supernatant into a storage tank for later use. And crushing the collected thalli by a high-pressure homogenizer, filtering by a ceramic membrane to collect supernatant, and then combining the supernatant with fermentation liquor to be placed in a storage tank.
(4) Removing other polymers such as heteroprotein, polypeptide and polysaccharide from the supernatant by polysulfone ultrafiltration membrane, and collecting the filtrate.
(5) The filtrate is subjected to organic solvent extraction, concentration, crystallization and other conventional steps to prepare the vanillin.
The invention has the beneficial effects that:
the present invention relates to a new method for synthesizing vanillin from various lignins by means of high-efficiency microbial conversion. Lignin has complex composition, and as many as dozens of compounds are produced after depolymerization, it is difficult to separate, purify and utilize lignin. The invention can transform lignin and depolymerized products into the main product vanillin by the biosynthesis of genetically engineered microorganisms. Provides an effective method for high-value utilization of lignin. And the microbial synthesis of vanillin is considered as one of natural vanillin, and can be used as an additive of natural perfume.
Drawings
FIG. 1 is a schematic diagram of the vanillin dehydrogenase gene knockout;
FIG. 2 shows the content of vanillin measured by HPLC method in the fermentation broth.
Detailed Description
The invention will now be described with reference to the following examples, which are provided for illustrative purposes only, and the invention is not limited to these examples, but encompasses all variations which are significant as a result of the guidance provided herein.
Modes for carrying out the invention
Embodiments of the present invention are described in detail below.
The invention relates to a method for synthesizing vanillin by microbial conversion by using lignin as a substrate, which comprises the steps of taking the lignin as a raw material, or taking the lignin depolymerized by a series of biological, chemical or physical methods as a raw material, depolymerizing and converting the lignin by using genetic engineering bacteria to synthesize vanillin, extracting the vanillin into an organic solvent phase, and further refining and purifying the vanillin.
In the method for producing vanillin by microbial synthesis of the present invention, the lignin biomass as a substrate is not particularly limited as long as it is derived from a plant biomass containing, as one of the main components, lignin containing phenylpropane units. Specific examples thereof include: poplar, willow, pine, wood chips, miscanthus, switchgrass, sorghum straw, corn straw, rice straw, wheat straw, bagasse, rice hull powder, wheat bran, alkaline lignin, sodium lignosulfonate, ground lignin, xylose residue, vinegar residue, black paper liquor and the like.
The microorganism selected by the invention is a typical strain of the Bacillus lignophilus L1.
The vanillin dehydrogenase gene knockout method of the selected Bacillus lignophilus L1 can be a homologous arm recombination method or a CRISP-Cas9 gene editing method or other methods capable of inactivating vanillin dehydrogenase genes.
In the synthetic preparation method of vanillin, the lignin depolymerization method comprises but not limited to thermal cracking, enzyme method, microwave, metal catalyst and alkali catalysis.
The term "depolymerization" as used herein means that the lignin structure is such that monomeric aromatic compounds such as ferulic acid and p-coumaric acid are produced.
The medium and method for culturing the genetically engineered bacterium of the present invention are not particularly limited, and may be appropriately selected as appropriate for the nutrient components and culturing method for growth and transformation of the bacterium. The incubation time is also not particularly limited as long as the lignin depolymerization product can be converted to synthesize the desired target amount of vanillin. The preferable bacteria growth medium is 2216E medium (component g/L, peptone 5, yeast powder 1, ferric citrate 0.1, sodium chloride 19.45, magnesium chloride 5.97, sodium sulfate 3.24, calcium chloride 1.8, sodium carbonate 0.16, potassium bromide 0.08, strontium chloride 0.034, boric acid 0.022, sodium silicate 0.004, sodium fluoride 0.0024, sodium nitrate 0.0016, sodium dihydrogen phosphate 0.008).
The culture time of the genetically engineered bacteria in the present invention is not particularly limited, as long as the lignin depolymerization product can be converted into vanillin with a desired target amount, preferably 24 h. The production amount of vanillin in the above-mentioned case is not particularly limited according to the purpose, and may be, for example, 0.2mg/L or more, preferably 100mg/L or more, more preferably 300mg/L or more in terms of, for example, the content of vanillin in the unit medium.
In the method for synthesizing and converting vanillin of the present invention, when vanillin is extracted from the culture solution, vanillin may be extracted directly from the culture solution of the microorganism, or the bacterial cells may be disrupted as necessary to obtain a disrupted cell product, and then extracted from the disrupted cell product. Extraction and disruption may also be performed simultaneously.
The organic solvent used in the extraction of vanillin in the present invention includes, but is not limited to, one or more of methanol, ethanol, acetone, butanone, cyclohexanone, diethyl ether, petroleum ether, n-hexane, ethyl acetate, butyl acetate, n-propyl acetate, and dimethyl sulfoxide, which are mixed in different proportions.
The method for separating the cells may be a centrifugal method or a ceramic membrane filtration method. The filtrate is further removed of biomacromolecules such as polysaccharide, protein, cell wall fragments and the like through an ultrafiltration membrane.
The purification method can be ion exchange column, macroporous resin or silica gel column elution.
Example 1: example construction of homologous arm Gene knockout engineering bacterium L1-vdh
The upstream and downstream homologous arm primers are designed by using Primer design software (Primer Premier 5.0) according to the upstream and downstream 500bp range of the vanillin dehydrogenase gene vdh sequence (SEQ ID NO.1), and enzyme cutting sites (KpnI, HindIII, NotI and PstI) are respectively added (the primers designed for constructing the gene knockout bacteria are shown in Table 1). Construction of cloning vector: the upstream and downstream homology arms of the target gene (named ch1-ch7) were cloned by PCR, the product was purified and ligated to pMD19-T plasmid and transformed into E.coli BL21 competent cells (purchased from Hongxi Biotech, Suzhou) to identify positive clones (pT-ch-up, pT-ch-down). Construction of the knockout vector: the upstream positive cloning plasmid and pKS1 temperature-sensitive plasmid were digested with endonucleases KpnI and HindIII, respectively, to obtain homologous fragments carrying the same sticky ends, the upstream homologous fragment was cloned into pKS1 and the positive clone (pKS1-ch-up) was verified by double digestion. pT-ch-down and pKS1-ch-up were digested with endonuclease NotI and PstI respectively and purified double digestion products were recovered, and the downstream homologous fragments were cloned into pKS1-ch-up plasmid to verify positive clones (pKS 1-ch-up-down). Transferring the positive clone pKS1-ch-up-down to competent cell transformation of the bacterium L1 by an electrotransformation method, obtaining knockout positive bacteria by a temperature induction knockout method, and verifying the successful knockout of genes by electrophoresis (the flow is shown in figure 1). The engineering bacteria is named as lignin-philic Bacillus (Bacillus ligniniphilus) L1-vdh, which has been preserved in the general microbiological culture collection center of China academy of sciences microbiological research institute No. 3 of West Lu No.1 in the morning area of the Yangxi, Beijing, China for 12 months and 20 days in 2019, the suggested classification is named as Bacillus ligniniphilus, and the preservation number is: CGMCC No. 19226.
TABLE 1 primer information
Figure BDA0002697685290000071
Example 2: preparation of lignin depolymerization mixture
The alkaline lignin is prepared by mixing 1: dissolving 10 g/V water, and performing hydrothermal depolymerization on the lignin mixture in a batch high-pressure reaction kettle at 280 deg.C under 0.5-6MPa for 60 min. And (4) placing the depolymerized product into a storage tank for later use.
Example 3: fermentation conversion of alkali lignin by engineering bacteria
The genetically engineered bacterium L1-vdh was inoculated into 2216E medium (containing 10. mu.g/L kanamycin), test tube expansion was carried out for 24 hours, then inoculated into a 500ml Erlenmeyer flask containing 100ml MM63 medium (containing 10g/L alkaline lignin), then cultured at 37 ℃ for 5 days at 220 revolutions of a shaker, and the vanillin content in the fermentation broth was examined by HPLC. HPLC detection indicated a significant vanillin peak at a retention time of 6.22 minutes (fig. 2). Calculation by peak area reference to standard curve measurements indicated that the yield of vanillin in shake flask culture for 24 hours had reached a maximum of 123mg/L, whereas the wild-type strain (laboratory screening stock) control of L1 was only 6.4 mg/L. The yield of vanillin of the engineering bacteria is 19.2 times of that of the wild bacteria (the wild strain is inoculated in a 2216E culture medium test tube for propagation for 24h and then inoculated in a 500ml triangular flask containing 100ml MM63 culture medium (containing 10g/L of alkaline lignin), and other fermentation conditions and detection methods are the same as those of the genetic engineering bacteria)
Example 4: fermentation conversion of lignin depolymerization mixture by engineering bacteria
Inoculating the genetically engineered bacterium L1-vdh into 2216E culture medium (containing erythromycin 10 mu g/L) according to the ratio of 1:100(V/V), carrying out shake flask propagation for 24 hours, then inoculating into a 50L fermentation tank according to the ratio of 1:50(V/V), wherein the fermentation medium is 2216E culture medium, removing culture solution after fermenting for 24 hours, and replacing with MM63 culture medium containing 10% (V/V) lignin depolymerization mixture solution. The culture was then continued for 24 hours. The acid and alkali are adjusted to pH 9 + -0.2 by 10% NaOH and 10% HCl during fermentation, and the content of vanillin is 0.86g/L at most in 24 hours by HPLC detection.
Example 5: extraction and purification preparation of vanillin
Filtering the fermentation liquid with ceramic membrane at a speed of 8m/h, a pressure range of 0.4-1MPa and a temperature of 40 deg.C, and collecting supernatant. And putting the supernatant into a storage tank for later use. After the cell precipitation is subjected to wall breaking by a homogenizer, the cell precipitation is subjected to high-speed centrifugation at 16000 rpm, and supernatants are collected and combined. Then the supernatant is ultrafiltered by polysulfone membrane with cut-off molecular weight above 2500 nm and 10-100nm, and the filtrate is collected. The filtrate was extracted with petroleum ether at a ratio of 1:1(v/v) continuous extraction, and then carrying out reduced pressure concentration by a double-effect evaporator to recover petroleum ether, thereby obtaining saturated concentrated solution. And pumping the saturated concentrated solution into a first-stage crystallizing tank, and then enabling the saturated feed liquid to enter a second-stage crystallizing tank from the first-stage crystallizing tank. Then enters a third-stage crystallizing tank. Finally, the crystallization rate is more than 80%. And washing the crystallized vanillin with cold water, and drying to obtain a finished product with the purity of 99.5% by HPLC detection.
Example 6: the process for synthesizing vanillin by converting paper-making black liquor as substrate and extracting and separating
And (3) carrying out spray drying on the papermaking black liquor, and collecting the papermaking black liquor dry powder for later use. The genetically engineered bacterium L1-vdh was inoculated into 2216E medium (containing 10. mu.g/L kanamycin), the flask was expanded for 24 hours, and then inoculated into a 50L fermentor, the fermentation medium was 2216E medium, and after 18 hours of fermentation, the medium was replaced with MM63 medium containing 10% of spray-dried powder of black liquor. Then, the culture was continued for 24 to 36 hours. The content of vanillin in the culture solution was 1.23g/L by HPLC. And (3) breaking the wall of the fermentation liquor by a homogenizer, centrifuging at 5000 revolutions, collecting supernatant, extracting the supernatant by using diethyl ether with the volume of 5 times, and then concentrating under reduced pressure to recover the diethyl ether to obtain vanillin concentrated solution. Mixing vanillin concentrate with 10 times weight of styrene nonpolar macroporous resin, stirring, loading into column, adsorbing, and adjusting pH to 6-7. Eluting with 10% ethanol water solution, identifying vanillin eluting component by silica gel thin layer chromatography (n-butanol: glacial acetic acid: water: 5:1:3), mixing the eluates containing vanillin, and concentrating under reduced pressure. Cooling the concentrated solution to separate out crystals, and then recrystallizing to obtain vanillin crystalline powder. Purity by HPLC 98%. The yield thereof was found to be 7.6%.
Figure BDA0002697685290000091
Sequence listing
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Claims (9)

1. A genetically engineered bacterium for synthesizing vanillin by transforming lignin-containing biomass is named as Bacillus lignophilus (Bacillus ligniniphilus) L1-vdh, and the suggested classified name is Bacillus ligniniphilus with the accession number as follows: CGMCC No. 19226.
2. The method for synthesizing vanillin by converting lignin-containing biomass with the genetically engineered bacterium for converting lignin-containing biomass to synthesize vanillin according to claim 1 is characterized by comprising the following steps:
(1) preparation of lignin depolymerization mixture: treating industrial lignin or pretreated lignin-containing pretreatment liquid by one of thermal cracking method, enzymolysis polymerization method, microwave, metal catalyst cracking method, alkali hydrolysis method, etc. to depolymerize lignin, and adjusting pH of depolymerization reaction liquid to neutral;
(2) a gene engineering bacterium L1-vdh (preservation number: CGMCC No.19226) of Bacillus lignophilus (Bacillus ligniniphilus) is frozen and inoculated to a freezing tube containing kanamycin (50 mu g-1) The 2216E culture medium is activated and pre-cultured for 24h at 37 ℃, then the culture medium is inoculated into a fermentation culture medium according to the proportion of 1:10(V/V) and cultured for 24h at 37 ℃ in a fermentation tank, the pH is adjusted to be about 9 through NaOH and HCl, glucose is supplemented when the glucose is lower than 5g/L in the culture process in a feeding mode, the fermentation is stopped when the fermentation OD value is above 30, then the fermentation broth is replaced by MM63 culture medium taking lignin depolymerization products as a single carbon source, the culture is continued for 24h at 37 ℃, the pH is adjusted to be about 9 through NaOH and HCl, and the synthesis amount of vanillin is detected by HPLC;
(3) centrifuging or filtering the fermentation liquor by a ceramic membrane to collect supernatant, and placing the supernatant into a storage tank for later use; crushing the collected thalli by a high-pressure homogenizer, filtering by a ceramic membrane to collect supernatant, and then mixing with the supernatant of the fermentation liquor to put into a storage tank;
(4) removing other polymers such as heteroprotein, polypeptide and polysaccharide from the supernatant by polysulfone ultrafiltration membrane, and collecting the filtrate;
(5) the filtrate is subjected to organic solvent extraction, concentration, crystallization and other conventional steps to prepare the vanillin.
3. The method for synthesizing vanillin by converting lignin-containing biomass with genetically engineered bacteria according to claim 2, wherein the thermal cracking method in step (1) comprises: thermally cracking the lignin aqueous solution with the temperature of 250-450 ℃ for 5-30 minutes in a reaction kettle, wherein the lignin aqueous solution is not higher than 10% (v/v); the pressure is controlled within 30 MPa.
4. The method for synthesizing vanillin by transforming lignin-containing biomass with genetically engineered bacteria according to claim 2, wherein the enzymatic polymerization method in step (1) comprises: laccase was dissolved in acetate buffer (pH 4) at a concentration of 0.1g/L, and then the laccase solution was mixed uniformly with 10% lignin solution at a ratio of 1:1(v/v), followed by reaction at 30 ℃ for 12 hours.
5. The method for synthesizing vanillin by converting lignin-containing biomass with genetically engineered bacteria according to claim 2, wherein the alkaline hydrolysis method in step (1) comprises: adding sodium hydroxide solution with concentration not higher than 10% (g/v) into lignin (lignin content is not higher than 10g/L), and treating at 80-100 deg.C for 1-6 hr.
6. The method for synthesizing vanillin by converting lignin-containing biomass with genetically engineered bacteria according to claim 2, wherein the metal catalyst cracking method in step (1) comprises: treating with metal catalyst (nickel, titanium, zinc, etc.) as catalyst at 100-300 deg.C in 10% lignin solution system for 0.5-1 h; dissolving lignin in 2-10% NaCl solution at 1-10% (g/L), reacting at 60-200 deg.C for 1-3 hr, and collecting filtrate.
7. The method for synthesizing vanillin by transforming lignin-containing biomass with genetically engineered bacteria according to claim 2, wherein the medium components in step (2)2216E are as follows: 5g/L of peptone, 1g/L of yeast powder, 0.1g/L of ferric citrate, 19.45g/L of sodium chloride, 5.97g/L of magnesium chloride, 3.24g/L of sodium sulfate, 1.8g/L of calcium chloride, 0.16g/L of sodium carbonate, 0.08g/L of potassium bromide, 0.034g/L of strontium chloride, 0.022g/L of boric acid, 0.004g/L of sodium silicate, 0.0024g/L of sodium fluoride, 0.0016g/L of sodium nitrate and 0.008g/L of sodium dihydrogen phosphate.
8. The method for synthesizing vanillin by transforming lignin-containing biomass with genetically engineered bacteria according to claim 2, wherein the fermentation medium of step (2) comprises the following components: 50g/L of enzymolysis soybean meal, 30g/L of corn pulp powder, 10g/L of glucose and FeSO4 0.1g/L,MgSO40.5g/L, phosphate buffer 50 mM.
9. The method for synthesizing vanillin by transforming lignin-containing biomass with genetically engineered bacteria according to claim 2, wherein the medium MM63 in step (2) comprises the following components: 100mM KH2PO4,75mM KOH,15mM(NH4)2SO4,1mM MgSO4,3.9μM FeSO4And lignin depolymerization product 1-30 g/L.
CN202011011447.0A 2020-09-23 2020-09-23 Genetically engineered bacterium for synthesizing vanillin by converting lignin-containing biomass and application thereof Pending CN112251390A (en)

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