Preparation method of gluconic acid

Abstract

The invention relates to an enzyme catalysis technology, and discloses a method for preparing gluconic acid from lignocellulose. The method comprises the following steps: a lignocellulose raw material and a compound enzyme are mixed for an enzymatic hydrolysis reaction, and the compound enzyme contains cellulase, lysing polysaccharide monooxygenase and glucose oxidase. According to the method, agricultural waste wood fibers which are low in cost and easy to obtain are used as raw materials, and the conversion rate of converting lignocellulose into gluconic acid is effectively increased through the synergistic catalysis effect of the compound enzyme.

Description

Technical Field

[0001] The invention relates to enzyme catalysis technology, and in particular to a method for preparing gluconic acid. Background Art

[0002] Gluconic acid is an important platform compound with a wide range of applications. Gluconic acid can be used as a water reducer and dispersant for cement, as an acidulant in food formulations, or used to prepare household or factory cleaning agents, textile and metal processing additives, leather alum tanning agents, metal rust removers, plasticizers for concrete in the construction industry, biodegradable chelating agents, and anti-precipitation agents for secondary oil recovery.

[0003] The preparation methods of gluconic acid include fermentation, electrochemical catalysis and chemical oxidation. Among them, the biological fermentation method has become the mainstream method for synthesizing gluconic acid due to its green, safe and efficient characteristics; however, the fermentation process of the biological fermentation method must strictly control sterility, the fermentation cycle is long, and the production of gluconic acid is affected by the presence of impurities such as bacteria, which affects the product purity of gluconic acid. Compared with the fermentation method, the enzymatic method for preparing gluconic acid has the advantages of no need to strictly control sterility, simple process, high substrate concentration and good purity, and easy refining. However, whether it is the fermentation method or the enzymatic method, the raw material for preparing gluconic acid is mainly starch-based glucose, and the raw material price is relatively expensive, resulting in excessively high costs. Replacing starch with wood fiber to prepare glucose can reduce raw material costs.

[0004] At present, gluconic acid is prepared from wood fiber by first hydrolyzing wood fiber into glucose through cellulase, and then converting glucose into gluconic acid through glucose oxidase. However, due to the natural anti-biodegradation barrier of wood fiber, the glucose conversion efficiency is low. In addition, a large amount of hydrogen peroxide is produced during the oxidation of glucose catalyzed by glucose oxidase, which has strong oxidizing properties and can lead to enzyme inactivation. Summary of the invention

[0005] The purpose of the present invention is to overcome the problems existing in the prior art and provide a method for preparing gluconic acid, which uses wood fiber as raw material, adopts composite enzyme synergistic catalysis, effectively improves the conversion efficiency of gluconic acid, and realizes efficient conversion of wood fiber.

[0006] In order to achieve the above-mentioned object, the first aspect of the present invention provides a method for preparing gluconic acid, comprising the following steps: mixing a lignocellulose raw material with a composite enzyme for enzymatic hydrolysis, wherein the composite enzyme contains cellulase, a lytic polysaccharide monooxygenase and glucose oxidase.

[0007] Preferably, based on the dry weight of 1 g of the lignocellulose raw material, the amount of the cellulase is 5-20 mg, the amount of the lytic polysaccharide monooxygenase is 0.5-10 mg, and the amount of the glucose oxidase is 0.01-0.1 mg.

[0008] Preferably, the weight ratio of the cellulase, the lytic polysaccharide monooxygenase and the glucose oxidase is 200-500:50-100:1.

[0009] Preferably, the conditions of the enzymatic hydrolysis reaction include at least: a temperature of 30-45° C., a stirring rate of 180-220 rpm, and a time of 60-80 h.

[0010] Preferably, the lignocellulosic raw material is selected from at least one of corn straw, rice straw, rape straw, corn cob, soybean straw, wheat straw and sugarcane bagasse.

[0011] Preferably, the particle size of the lignocellulosic raw material is less than or equal to 0.18 mm.

[0012] Preferably, the lignocellulosic raw material is mixed with the composite enzyme in the form of a buffer solution, and the buffer solution is obtained by mixing the lignocellulosic raw material with a buffer solution having a pH of 5-6.

[0013] Preferably, the solid content of the buffer solution is 5-10 wt %.

[0014] Preferably, the lignocellulosic raw material is pretreated before being mixed with the composite enzyme, and the pretreatment process comprises: mixing the lignocellulosic raw material with a pretreatment agent in a reaction solvent for pre-reaction, wherein the pretreatment agent is a strong base or a strong acid.

[0015] Preferably, the weight ratio of the reaction solvent, the pretreatment agent and the lignocellulosic raw material is 100:0.2-1.2:8-12.

[0016] Preferably, the pre-reaction conditions at least include: a temperature of 110-130° C. and a time of 25-35 min.

[0017] Preferably, the cellulase is obtained by fermentation culture of a cellulase-producing strain.

[0018] Preferably, the cellulase producing strain is Trichoderma reesei and / or Myceliophthora thermophila.

[0019] Preferably, the fermentation culture conditions at least include: an inoculation amount of 2-5% by volume, a temperature of 25-50° C., and a time of 3-7 days.

[0020] Preferably, the fermentation culture medium contains: KH2PO4 1-3g / L, (NH4)2SO4 1-2g / L, MgSO40.1-0.5g / L, FeSO4 0.002-0.008g / L, MnSO4 0.05-0.1g / L, ZnSO40.001-0.002g / L, CoCl20.001-0.003g / L, CaCl2 0.2-0.4g / L, peptone 0.5-1.5g / L, and rice straw 8-12g / L.

[0021] Preferably, the amino acid sequence of the lytic polysaccharide monooxygenase is shown in SEQ ID NO:1.

[0022] Preferably, the nucleotide sequence of the lytic polysaccharide monooxygenase is shown in SEQ ID NO:2.

[0023] Through the above technical solution, the beneficial effects of the present invention are:

[0024] The preparation method of gluconic acid provided by the present invention uses cheap and easily available agricultural waste lignocellulose as a raw material, and directly prepares gluconic acid through the multi-enzyme synergistic catalysis of lytic polysaccharide monooxygenase, cellulase, and glucose oxidase; the synergistic effect between the three composite enzymes provided by the present invention effectively promotes the conversion rate of lignocellulose to generate gluconic acid, improves the production efficiency of gluconic acid, and the method has a simple process flow and low process cost. DETAILED DESCRIPTION

[0025] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0026] The first aspect of the present invention provides a method for preparing gluconic acid, comprising the following steps: mixing a lignocellulose raw material with a composite enzyme for enzymatic hydrolysis, wherein the composite enzyme contains cellulase, a lytic polysaccharide monooxygenase and glucose oxidase.

[0027] The preparation method of gluconic acid provided by the present invention uses cheap and readily available agricultural waste wood cellulose as raw material, and directly produces gluconic acid through the synergistic catalytic action of multiple enzymes including cleavage polysaccharide monooxygenase, cellulase, and glucose oxidase; wherein, cellulase can hydrolyze cellulose in wood fiber to produce glucose, and glucose can be converted into gluconic acid by glucose oxidase, while producing hydrogen peroxide, and hydrogen peroxide can be used as a substrate of cleavage polysaccharide monooxygenase to enhance its hydrolysis of cellulose crystal structure in wood cellulose, thereby improving the conversion rate of cellulase hydrolyzing cellulose to produce glucose. The synergistic action between the three composite enzymes provided by the present invention can promote the conversion efficiency of wood cellulose to produce gluconic acid.

[0028] The preparation method of gluconic acid provided by the invention does not need to control aseptic conditions during the entire preparation process, thus reducing energy consumption, simplifying the process flow, and reducing process costs.

[0029] According to the present invention, preferably, based on the dry weight of 1g of the lignocellulose raw material, the amount of the cellulase is 5-20mg, the amount of the lytic polysaccharide monooxygenase is 0.5-10mg, and the amount of the glucose oxidase is 0.01-0.1mg. The inventors found that under this preferred embodiment, it is beneficial to promote the synergistic effect of the lytic polysaccharide monooxygenase, cellulase and glucose oxidase, and further improve the conversion rate of lignocellulose degradation to form gluconic acid. Among them, the amount of cellulase, lytic polysaccharide monooxygenase and glucose oxidase is controlled by the protein concentration in the feed liquid.

[0030] According to the present invention, the amount of the cellulase, the lytic polysaccharide monooxygenase and the glucose oxidase can be any ratio, preferably, the weight ratio of the cellulase, the lytic polysaccharide monooxygenase and the glucose oxidase is 200-500:50-100:1, more preferably 200-500:90-100:1. The inventors found that under this preferred embodiment, it is beneficial to improve the conversion rate of lignocellulose degradation into gluconic acid.

[0031] According to the present invention, preferably, the conditions of the enzymolysis reaction include at least: the temperature is 30-45°C, which can be 30°C, 35°C, 40°C, 45°C, or any value between the above two values; the stirring rate is 180-220rpm, which can be 180rpm, 190rpm, 200rpm, 210rpm, 22rpm, or any value between the above two values; the time is 60-80h, which can be 60h, 65h, 70h, 75h, 80h, or any value between the above two values. The inventors found that under this preferred embodiment, it is beneficial to improve the enzymolysis efficiency of the composite enzyme on lignocellulose.

[0032] According to the present invention, the lignocellulose raw material can be selected from any raw material containing lignocellulose, for example, agricultural solid waste or food processing solid waste. Preferably, the lignocellulose raw material is selected from at least one of corn stalks, rice stalks, rape stalks, corn cobs, soybean stalks, wheat stalks and bagasse. In this preferred embodiment, the resource utilization of agricultural solid waste can be further improved, and the production cost of gluconic acid can be effectively reduced.

[0033] According to the present invention, preferably, the particle size of the lignocellulosic raw material is less than or equal to 0.18 mm. Exemplarily, the lignocellulosic raw material is crushed by conventional crushing equipment and then passed through an 80-mesh sieve.

[0034] According to the present invention, the lignocellulosic raw material can be directly mixed with the complex enzyme. Preferably, the lignocellulosic raw material is mixed with the complex enzyme in the form of a buffer solution, and the buffer solution is obtained by mixing the lignocellulosic raw material with a buffer solution with a pH of 5-6. Among them, the buffer solution can adopt an acetate-phosphate buffer with a pH of 5-6. In this preferred embodiment, it is beneficial to improve the enzymolysis efficiency of the complex enzyme on the lignocellulosic raw material and improve the conversion rate of gluconic acid. Further preferably, the solid content in the buffer solution is 5-10wt%.

[0035] According to the present invention, preferably, the lignocellulosic raw material is pretreated before being mixed with the complex enzyme, and the pretreatment process includes: in a reaction solvent, the lignocellulosic raw material is mixed with a pretreatment agent for pre-reaction, wherein the pretreatment agent is a strong base or a strong acid. Further preferably, after the lignocellulosic raw material is pretreated, it is mixed with a buffer solution with a pH of 5-6 to obtain a buffer solution. In this preferred embodiment, it is beneficial to improve the enzymatic hydrolysis rate of the lignocellulosic raw material, thereby improving the conversion rate of gluconic acid.

[0036] According to the present invention, preferably, the weight ratio of the reaction solvent, the pretreatment agent and the lignocellulosic raw material is 8-12:0.2-1.2:100. Generally, the pretreatment agent strong acid or strong alkali is dissolved in the reaction solvent to form a dilute acid solution or a dilute alkali solution, and then mixed with the lignocellulosic raw material. The reaction solvent can be water. In this preferred embodiment, it is beneficial to improve the pretreatment effect of the lignocellulosic raw material, thereby improving the enzymatic hydrolysis efficiency of the composite enzyme.

[0037] According to the present invention, preferably, the conditions of the pre-reaction include at least: a temperature of 110-130°C, which can be 110°C, 115°C, 120°C, 125°C, 130°C, or any value between the above two values; a time of 25-35min, which can be 25min, 27min, 29min, 31min, 33min, 35min, or any value between the above two values.

[0038] In the present invention, the cellulase can be commercially available or prepared by itself. Preferably, the cellulase is obtained by fermenting and culturing a cellulase-producing strain. The cellulase-producing strain can be any strain that can ferment and produce cellulase. Preferably, the cellulase-producing strain is Trichoderma reesei and / or Myceliophthora thermophila.

[0039] In the present invention, the cellulase-producing strain can obtain a seed culture solution through seed culture, and then the seed culture solution is fermented and cultured. The seed culture process is beneficial to quickly restore the vitality of bacteria, promote the rapid growth of bacteria, and is also beneficial to the formation of cellulase during the fermentation culture process.

[0040] According to the present invention, the fermentation culture conditions such as temperature and time are selected according to the specific characteristics of the cellulase-producing strain to meet the growth requirements of the cellulase-producing strain; the fermentation culture process can be static fermentation, or stirring can be used to improve the utilization efficiency of the cellulase-producing strain on the sugar substances in the fermentation culture medium. Preferably, the fermentation culture conditions at least include: an inoculation amount of 2-5% by volume, a temperature of 25-50°C, and a time of 3-7 days, wherein the culture temperature of Trichoderma reesei is more preferably 25-35°C, and the culture temperature of Myceliophthora thermophila is more preferably 35-50°C.

[0041] In the present invention, the culture medium used for fermentation culture of the cellulase-producing strain can provide the corresponding strain with the nutrients required for growth. Preferably, the culture medium for fermentation culture contains: KH2PO4 1-3g / L, (NH4)2SO4 1-2g / L, MgSO4 0.1-0.5g / L, FeSO4 0.002-0.008g / L, MnSO4 0.05-0.1g / L, ZnSO4 0.001-0.002g / L, CoCl2 0.001-0.003g / L, CaCl20.2-0.4g / L, peptone 0.5-1.5g / L, rice straw 8-12g / L.

[0042] In the present invention, the fermentation liquid obtained by fermentation and cultivation of the cellulase-producing strain is subjected to solid-liquid separation to remove the bacterial body to obtain a crude cellulase enzyme solution, which is then concentrated by freeze-drying to obtain a cellulase concentrated solution. At this time, the cellulase is mixed with the lignocellulose raw material in the form of the crude enzyme solution.

[0043] According to the present invention, preferably, the lytic polysaccharide monooxygenase is MtLPMO9 (NCBI: XP_003661787), and its amino acid sequence is shown in SEQ ID NO: 1; further preferably, the nucleotide sequence of the lytic polysaccharide monooxygenase is shown in SEQ ID NO: 2. The lytic polysaccharide monooxygenase in the present invention can be commercially available or prepared by itself. Exemplarily, the preparation process of the lytic polysaccharide monooxygenase is as follows: the lytic polysaccharide monooxygenase gene MtLPMO9 (XP_003661787) from Myceliophthora thermophila is cloned, and then the pPICZαC plasmid is used as a vector to construct a recombinant plasmid pPICZαC-MtLPMO9, and the recombinant plasmid pPICZαC-MtLPMO9 is transformed into a Pichia pastoris X33 competent state by a conventional method for expression to obtain a recombinant strain, and the recombinant strain is fermented to obtain a recombinant strain fermentation broth, and the recombinant strain fermentation broth is concentrated by freeze drying to obtain a lytic polysaccharide monooxygenase crude enzyme solution. At this time, the lytic polysaccharide monooxygenase is mixed with the lignocellulosic raw material in the form of the crude enzyme solution.

[0044] MtLPMO9 amino acid sequence (SEQ ID NO: 1): mskasallag ltgaalvaah ghvshivvng vyyrnydpttdwyqpnpptvigwtaadqdngfvepnsfgt pdiichksatpggghatvaagdkinivwtpewpeshigpv idylaacngdcetvdksslrwfkidgagyd kaagrwaada lrangnswlvqipsdlkagnyvlrheiialhgaqspngaq aypqcinlrv tgggsnlpsg vagtslykatdpgilfnpyvsspdytvpgpaliagaassi aqstsvatat gtatvpgggg anptatttaatsaapsttlrttttsaaqttappsgdvqtk ygqcggngwt gptvcapgss csvlnewysqcl;

[0045] MtLPMO9 nucleotide sequence (SEQ ID NO: 2): atgtccaagg cctctgctct cctcgctggcctgacgggcg cggccctcgt cgctgcacatggccacgtca gccacatcgt cgtcaacggcgtctactacaggaactacga ccccacgacagactggtacc agcccaaccc gccaacagtc atcggctggacggcagccgatcaggataatggcttcgttg aacccaacag ctttggcacg ccagatatcatctgccacaagagcgccacccccggcggcg gccacgctac cgttgctgcc ggagacaagatcaacatcgtctggacccccgagtggcccg aatcccacat cggccccgtc attgactacctagccgcctgcaacggtgactgcgagaccg tcgacaagtc gtcgctgcgc tggttcaagattgacggcgccggctacgacaaggccgccg gccgctgggc cgccgacgct ctgcgcgccaacggcaacagctggctcgtccagatcccgt cggatctcaa ggccggcaac tacgtcctccgccacgagatcatcgccctccacggtgctc agagccccaa cggcgcccag gcctacccgcagtgcatcaacctccgcgtcaccggcggcg gcagcaacct gcccagcggc gtcgccggcacctcgctgtacaaggcgaccgacccgggca tcctcttcaa cccctacgtc tcctccccggattacaccgtccccggcccggccctcattg ccggcgccgc cagctcgatc gcccagagcacgtcggtcgccactgccaccggcacggcca ccgttcccgg cggcggcggc gccaaccctaccgccaccaccaccgccgccacctccgccg ccccgagcac caccctgagg acgaccactacctcggccgcgcagactaccgccccgccct ccggcgatgtgcagaccaag tacggccagtgtggtggcaacggatggacgggcccgacgg tgtgcgcccc cggctcgagc tgctccgtcctcaacgagtggtactcccagtgtttgtaa.

[0046] In the present invention, the enzymolysis solution obtained by the enzymolysis reaction is subjected to solid-liquid separation, and the obtained liquid is used as a product containing gluconic acid. The solid-liquid separation can be carried out in a conventional manner in the art, such as filtering or centrifuging.

[0047] As a relatively preferred specific embodiment of the present invention, the preparation method of gluconic acid comprises the following steps:

[0048] (1) culturing Trichoderma reesei and / or Myceliophthora thermophila to obtain a seed culture solution, inoculating the seed culture solution into a fermentation medium at an inoculum amount of 2-5% by volume, fermenting at a temperature of 25-50° C. for 3-7 days to obtain a cellulase fermentation solution, performing solid-liquid separation on the cellulase fermentation solution to remove bacterial cells, obtaining a crude cellulase enzyme solution, and concentrating the solution by freeze drying to obtain a cellulase concentrate;

[0049] (2) crushing the lignocellulosic raw material into particles with a particle size of less than or equal to 0.18 mm, mixing with a dilute acid or a dilute alkali so that the weight ratio of the reaction solvent, the pretreatment agent and the lignocellulosic raw material is 8-12:0.2-1.2:100, reacting at a temperature of 110-130° C. for 25-35 minutes to obtain a pretreated raw material, and mixing the pretreated raw material with a buffer solution with a pH of 5-6 to obtain a buffer solution with a solid content of 5-10 wt%;

[0050] (3) Mixing the buffer solution with the cellulase concentrate, the cleavage polysaccharide monooxygenase crude enzyme solution and the glucose oxidase obtained in step (1) (based on the dry weight of 1 g of the lignocellulose raw material, the amount of the cellulase is 5-20 mg, the amount of the cleavage polysaccharide monooxygenase is 0.5-10 mg, the amount of the glucose oxidase is 0.01-0.1 mg, and the weight ratio of the cellulase, the cleavage polysaccharide monooxygenase and the glucose oxidase is 200-500:50-100:1), performing enzymolysis reaction at a temperature of 30-45° C. and a stirring rate of 180-220 rpm for 60-80 h to obtain an enzymolysis solution, performing solid-liquid separation on the enzymolysis solution, and obtaining a liquid as a product containing gluconic acid.

[0051] The present invention will be described in detail below through examples.

[0052] In the following examples, Trichoderma reesei was purchased from China Industrial Microorganism Culture Collection Center with a number of CICC 13052; Myceliophthora thermophila was purchased from Shanghai Collection Biotechnology Center with a number of SHBCC D82838; Pichia pastoris X33 was purchased from Invitrogen; glucose oxidase was purchased from Shanghai Yingxin Laboratory Co., Ltd. with a product number of TX20017; corn straw, rice straw, wheat straw, and rape straw were all sourced from a place in Henan, and the remaining raw materials and reagents were conventional commercial products.

[0053] In the following examples, protein concentration was determined by Lowry method; gluconic acid was determined by high performance liquid chromatography, using an Aminex HPX 87H column, 5 mmol / L H2SO4 solution as the mobile phase, a flow rate of 0.6 mL / min, and a UV detector for detection.

[0054] In the following examples, the preparation process of the crude enzyme solution of the lytic polysaccharide monooxygenase is as follows: the lytic polysaccharide monooxygenase gene MtLPMO9 (XP_003661787) from Myceliophthora thermophila is cloned, and then the pPICZαC plasmid is used as a vector to construct a recombinant plasmid pPICZαC-MtLPMO9, and the recombinant plasmid pPICZαC-MtLPMO9 is transformed into a Pichia pastoris X33 competent state by a conventional method for expression to obtain a recombinant strain, and the recombinant strain is fermented to obtain a recombinant strain fermentation liquid, and the recombinant strain fermentation liquid is concentrated by freeze drying to obtain a crude enzyme solution of the lytic polysaccharide monooxygenase.

[0055] Example 1

[0056] The formula of Trichoderma reesei fermentation medium is: KH2PO4 2g / L, (NH4)2SO4 1.4g / L, MgSO40.3g / L, FeSO4 0.005g / L, MnSO4 0.075g / L, ZnSO4 0.0015g / L, CoCl2 0.002g / L, CaCl2 0.3g / L, peptone 1g / L, rice straw 10g / L;

[0057] (1) culturing Trichoderma reesei to obtain a seed culture solution, inoculating the seed culture solution into a fermentation medium at an inoculum amount of 3% by volume, fermenting at a temperature of 28° C. for 5 days to obtain a cellulase fermentation solution, performing solid-liquid separation on the cellulase fermentation solution to remove bacterial cells, obtaining a crude cellulase enzyme solution, and concentrating the solution by freeze drying (temperature of −50° C. for 48 h) to obtain a cellulase concentrate;

[0058] (2) crushing corn stalks and passing them through an 80-mesh sieve to obtain particles with a particle size of less than or equal to 0.18 mm, and then mixing them with a 0.05M NaOH dilute alkali solution (water as a solvent) so that the weight ratio of corn stalks, NaOH and water is 10:0.2:100, reacting at a temperature of 120° C. for 30 minutes to obtain a pretreated raw material, and mixing the pretreated raw material with an acetate-phosphate buffer solution with a pH of 5.6 to obtain a buffer solution with a solid content of 8 wt%;

[0059] (3) The buffer solution is mixed with the cellulase concentrate, the crude polysaccharide monooxygenase solution and the glucose oxidase obtained in step (1) (based on the dry weight of 1 g of corn stalk, the amount of cellulase is 20 mg protein / g stalk, the amount of lytic polysaccharide monooxygenase is 10 mg protein / g stalk, and the amount of glucose oxidase is 0.1 mg protein / g stalk), and an enzymatic hydrolysis reaction is carried out at a temperature of 37° C. and a stirring rate of 200 rpm for 72 h to obtain an enzymatic hydrolyzate containing gluconic acid.

[0060] Example 2

[0061] The formula of the fermentation medium of Thermomycete is: KH2PO4 1g / L, (NH4)2SO4 1g / L, MgSO40.1g / L, FeSO4 0.002g / L, MnSO4 0.05g / L, ZnSO4 0.001g / L, CoCl2 0.001g / L, CaCl2 0.2g / L, peptone 0.5g / L, rice straw 12g / L;

[0062] (1) culturing Myceliophthora thermophila to obtain a seed culture solution, inoculating the seed culture solution into a fermentation medium at an inoculum amount of 2% by volume, fermenting at a temperature of 45° C. for 6 days to obtain a cellulase fermentation solution, performing solid-liquid separation on the cellulase fermentation solution to remove bacterial cells, obtaining a crude cellulase enzyme solution, and concentrating the solution by freeze drying to obtain a cellulase concentrate;

[0063] (2) crushing wheat straw and passing it through an 80-mesh sieve to obtain particles with a particle size of less than or equal to 0.18 mm, mixing the particles with dilute hydrochloric acid so that the weight ratio of wheat straw, HCl and water is 8:1:100, reacting at a temperature of 110° C. for 35 minutes to obtain a pretreated raw material, and mixing the pretreated raw material with an acetate-phosphate buffer having a pH of 5.6 to obtain a buffer solution having a solid content of 5 wt %;

[0064] (3) The buffer solution is mixed with the cellulase concentrate, the crude polysaccharide monooxygenase solution and the glucose oxidase obtained in step (1) (based on the dry weight of 1 g of rice straw, the amount of cellulase is 10 mg protein / g straw, the amount of lytic polysaccharide monooxygenase is 2 mg protein / g straw, and the amount of glucose oxidase is 0.02 mg protein / g straw), and an enzymatic hydrolysis reaction is carried out at a temperature of 30° C. and a stirring rate of 220 rpm for 80 h to obtain an enzymatic hydrolyzate containing gluconic acid.

[0065] Example 3

[0066] The formula of the fermentation medium of Thermomycete is: KH2PO4 1g / L, (NH4)2SO4 2g / L, MgSO40.5g / L, FeSO4 0.008g / L, MnSO4 0.1g / L, ZnSO4 0.002g / L, CoCl2 0.003g / L, CaCl2 0.4g / L, peptone 1.5g / L, rice straw 8g / L;

[0067] (1) culturing Myceliophthora thermophila to obtain a seed culture solution, inoculating the seed culture solution into a fermentation medium at an inoculum amount of 5% by volume, fermenting at a temperature of 50° C. for 4 days to obtain a cellulase fermentation solution, performing solid-liquid separation on the cellulase fermentation solution to remove bacterial cells, obtaining a crude cellulase enzyme solution, and concentrating the solution by freeze drying to obtain a cellulase concentrate;

[0068] (2) crushing rice straw and passing it through an 80-mesh sieve to obtain particles with a particle size of less than or equal to 0.18 mm, mixing the particles with dilute sulfuric acid so that the weight ratio of water, H2SO4 and rice straw is 100:1.2:10, reacting the particles at a temperature of 130°C for 25 minutes to obtain a pretreated raw material, and mixing the pretreated raw material with an acetate-phosphate buffer having a pH of 5.6 to obtain a buffer solution having a solid content of 10 wt%;

[0069] (3) The buffer solution is mixed with the cellulase concentrate, the crude polysaccharide monooxygenase solution and the glucose oxidase obtained in step (1) (based on the dry weight of 1 g of rice straw, the amount of cellulase is 16 mg protein / g straw, the amount of lytic polysaccharide monooxygenase is 4 mg protein / g straw, and the amount of glucose oxidase is 0.04 mg protein / g straw), and an enzymatic hydrolysis reaction is carried out at a temperature of 45° C. and a stirring rate of 180 rpm for 60 h to obtain an enzymatic hydrolyzate containing gluconic acid.

[0070] Example 4

[0071] An enzymatic hydrolyzate containing gluconic acid was prepared according to the method of Example 3, except that the amounts of cellulase, cleavage polysaccharide monooxygenase and glucose oxidase in step (3) were replaced by: based on 1 g of dry weight of rice straw, the amount of cellulase was 8 mg protein / g straw, the amount of cleavage polysaccharide monooxygenase was 1.5 mg protein / g straw, and the amount of glucose oxidase was 0.015 mg protein / g straw.

[0072] Example 5

[0073] An enzymatic hydrolyzate containing gluconic acid was prepared according to the method of Example 3, except that the amounts of cellulase, cleavage polysaccharide monooxygenase and glucose oxidase in step (3) were replaced by: based on 1 g of dry weight of rice straw, the amount of cellulase was 5 mg protein / g straw, the amount of cleavage polysaccharide monooxygenase was 0.5 mg protein / g straw, and the amount of glucose oxidase was 0.01 mg protein / g straw.

[0074] Example 6

[0075] An enzymatic hydrolyzate containing gluconic acid was prepared according to the method of Example 3, except that the amounts of cellulase, cleavage polysaccharide monooxygenase and glucose oxidase in step (3) were replaced by: based on 1 g of dry weight of rice straw, the amount of cellulase was 7 mg, the amount of cleavage polysaccharide monooxygenase was 3.5 mg, and the amount of glucose oxidase was 0.04 mg.

[0076] Example 7

[0077] The enzymatic hydrolyzate containing gluconic acid was prepared according to the method of Example 3, except that the temperature of the enzymatic hydrolysis reaction in step (3) was replaced by: the temperature was 28° C. and the stirring rate was 250 rpm.

[0078] Example 8

[0079] (1) crushing corn stalks and passing them through an 80-mesh sieve to obtain particles with a particle size of less than or equal to 0.18 mm, mixing them with a 0.05 M NaOH dilute alkali solution (water as a solvent) so that the weight ratio of water, NaOH and corn stalks is 10:0.2:100, reacting them at a temperature of 120° C. for 30 minutes to obtain a pretreated raw material, and mixing the pretreated raw material with an acetate-phosphate buffer solution with a pH of 5.6 to obtain a buffer solution with a solid content of 8 wt %;

[0080] (2) The buffer solution was mixed with cellulase (purchased from Xiasheng Company, product number FDG-2226), crude enzyme solution of cleavage polysaccharide monooxygenase and glucose oxidase (based on the dry weight of 1 g of corn stover, the amount of cellulase was 12 mg, the amount of cleavage polysaccharide monooxygenase was 5 mg, and the amount of glucose oxidase was 0.05 mg), and the enzymatic hydrolysis reaction was carried out at a temperature of 37°C and a stirring rate of 200 rpm for 72 h to obtain an enzymatic hydrolyzate containing gluconic acid.

[0081] Example 9

[0082] The enzymatic hydrolyzate containing gluconic acid was prepared according to the method of Example 1, except that step (2) was replaced by:

[0083] (2) The corn stalks are crushed and passed through a 30-mesh sieve to obtain particles with a particle size of 0.2-0.6 mm, and then mixed with a 0.05 M NaOH dilute alkali solution (water as a solvent) so that the weight ratio of water, NaOH and corn stalks is 100:0.2:10, and reacted at a temperature of 120° C. for 30 minutes to obtain a pretreated raw material, and the pretreated raw material is mixed with an acetate phosphate buffer solution with a pH of 5.6 to obtain a buffer solution with a solid content of 8 wt%.

[0084] Example 10

[0085] The enzymatic hydrolysate containing gluconic acid was prepared according to the method of Example 1, except that step (2) was replaced by:

[0086] (2) The corn stalks are crushed and passed through an 80-mesh sieve to obtain particles with a particle size less than or equal to 0.18 mm, and then mixed with a 0.05M NaOH dilute alkali solution (water as a solvent) so that the weight ratio of water, NaOH and corn stalks is 100:0.2:10, and reacted at a temperature of 120°C for 30 minutes to obtain a pretreated raw material, and the pretreated raw material is mixed with a PBS buffer solution with a pH of 7.4 to obtain a buffer solution with a solid content of 8wt%.

[0087] Comparative Example 1

[0088] (1) culturing Trichoderma reesei to obtain a seed culture solution, inoculating the seed culture solution into a fermentation medium at an inoculum amount of 3% by volume, fermenting at a temperature of 28° C. for 5 days to obtain a cellulase fermentation solution, performing solid-liquid separation on the cellulase fermentation solution to remove bacterial cells, obtaining a crude cellulase enzyme solution, and concentrating the solution by freeze drying to obtain a cellulase concentrate;

[0089] (2) crushing corn stalks and passing them through an 80-mesh sieve to obtain particles with a particle size of less than or equal to 0.18 mm, and then mixing them with a 0.05M NaOH dilute alkali solution (water as a solvent) so that the weight ratio of water, NaOH and corn stalks is 10:0.2:100, reacting them at a temperature of 120° C. for 30 minutes to obtain a pretreated raw material, and mixing the pretreated raw material with an acetate-phosphate buffer solution with a pH of 5.6 to obtain a buffer solution with a solid content of 8 wt%;

[0090] (3) The buffer solution is mixed with the cellulase concentrate and glucose oxidase obtained in step (1) (based on 1 g of dry weight of corn stalks, the amount of cellulase is 12 mg, and the amount of glucose oxidase is 0.05 mg), and an enzymatic hydrolysis reaction is carried out at a temperature of 37° C. and a stirring rate of 200 rpm for 72 h to obtain an enzymatic hydrolyzate containing gluconic acid.

[0091] Comparative Example 2

[0092] (1) culturing Trichoderma reesei to obtain a seed culture solution, inoculating the seed culture solution into a fermentation medium at an inoculum amount of 3% by volume, fermenting at a temperature of 28° C. for 5 days to obtain a cellulase fermentation solution, performing solid-liquid separation on the cellulase fermentation solution to remove bacterial cells, obtaining a crude cellulase enzyme solution, and concentrating the solution by freeze drying to obtain a cellulase concentrate;

[0093] (2) crushing corn stalks and passing them through an 80-mesh sieve to obtain particles with a particle size of less than or equal to 0.18 mm, and then mixing them with a 0.05M NaOH dilute alkali solution (water as a solvent) so that the weight ratio of water, NaOH and corn stalks is 10:0.2:100, reacting them at a temperature of 120° C. for 30 minutes to obtain a pretreated raw material, and mixing the pretreated raw material with an acetate-phosphate buffer solution with a pH of 5.6 to obtain a buffer solution with a solid content of 8 wt%;

[0094] (3) The buffer solution is mixed with the cellulase concentrate, cellobiose dehydrogenase and glucose oxidase obtained in step (1) (based on the dry weight of 1 g of corn stalk, the amount of cellulase is 12 mg, the amount of cellobiose dehydrogenase is 5 mg, and the amount of glucose oxidase is 0.05 mg), and an enzymatic hydrolysis reaction is carried out at a temperature of 37° C. and a stirring rate of 200 rpm for 72 h to obtain an enzymatic hydrolyzate containing gluconic acid.

[0095] Comparative Example 3

[0096] (1) crushing corn stalks and passing them through an 80-mesh sieve to obtain particles with a particle size of less than or equal to 0.18 mm, mixing them with a 0.05 M NaOH dilute alkali solution (water as a solvent) so that the weight ratio of water, NaOH and corn stalks is 10:0.2:100, reacting them at a temperature of 120° C. for 30 minutes to obtain a pretreated raw material, and mixing the pretreated raw material with an acetate-phosphate buffer solution with a pH of 5.6 to obtain a buffer solution with a solid content of 8 wt %;

[0097] (2) The buffer solution was mixed with cellulase (purchased from Xiasheng Company, product number FDG-2226) and glucose oxidase (based on 1 g of dry weight of corn straw, the amount of cellulase was 12 mg and the amount of glucose oxidase was 0.05 mg), and the enzymatic hydrolysis reaction was carried out at a temperature of 37°C and a stirring rate of 200 rpm for 72 h to obtain an enzymatic hydrolyzate containing gluconic acid.

[0098] Test Example 1

[0099] The concentration and yield of gluconic acid in the enzymatic hydrolysates containing gluconic acid obtained in Examples 1 to 10 and Comparative Examples 1 to 3 were measured respectively, and the enzymatic hydrolysis rate of lignocellulose was calculated. The results are shown in Table 1.

[0100] Among them, the gluconic acid concentration was determined by high performance liquid chromatography, using an Aminex HPX 87H column, 5mmol / L H2SO4 solution as the mobile phase, a flow rate of 0.6mL / min, and a UV detector for detection. The cellulose content in the wood fiber was determined using the NREL / TP-510-42618 standard to calculate the maximum theoretical conversion of gluconic acid. The gluconic acid conversion rate is equal to the amount of gluconic acid generated by enzymatic hydrolysis divided by the maximum theoretical conversion of gluconic acid, and the enzymatic hydrolysis rate is equal to the amount of cellulose hydrolyzed divided by the total amount of cellulose in the original wood cellulose.

[0101] Table 1

[0102]

[0103]

[0104] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A method for preparing gluconic acid, characterized in that, The following steps are involved: The lignocellulose raw material is mixed with a composite enzyme to carry out an enzymatic hydrolysis reaction, wherein the composite enzyme contains cellulase, lytic polysaccharide monooxygenase and glucose oxidase.

2. The method according to claim 1, characterized in that Based on the dry weight of 1 g of the lignocellulose raw material, the dosage of the cellulase is 5-20 mg, the dosage of the lytic polysaccharide monooxygenase is 0.5-10 mg, and the dosage of the glucose oxidase is 0.01-0.1 mg.

3. The method according to claim 2, characterized in that The weight ratio of the cellulase, the lytic polysaccharide monooxygenase and the glucose oxidase is 200-500:50-100:

1.

4. The method according to any one of claims 1 to 3, characterized in that The conditions of the enzymatic hydrolysis reaction at least include: a temperature of 30-45° C., a stirring rate of 180-220 rpm, and a time of 60-80 h.

5. The method according to any one of claims 1 to 3, characterized in that: The lignocellulose raw material is selected from at least one of corn straw, rice straw, rape straw, corn cob, soybean straw, wheat straw and sugarcane bagasse; Preferably, the particle size of the lignocellulosic raw material is less than or equal to 0.18 mm.

6. The method according to any one of claims 1 to 3, characterized in that: The lignocellulosic raw material is mixed with the composite enzyme in the form of a buffer solution, wherein the buffer solution is obtained by mixing the lignocellulosic raw material with a buffer solution having a pH of 5-6; Preferably, the solid content of the buffer solution is 5-10 wt %.

7. The method according to claim 6, characterized in that The lignocellulosic raw material is pretreated before being mixed with the composite enzyme, and the pretreatment process comprises: mixing the lignocellulosic raw material with a pretreatment agent in a reaction solvent for pre-reaction, wherein the pretreatment agent is a strong base or a strong acid; Preferably, the weight ratio of the reaction solvent, the pretreatment agent and the lignocellulosic raw material is 100: 0.2-1.2: 8-12; Preferably, the pre-reaction conditions at least include: a temperature of 110-130° C. and a time of 25-35 min.

8. The method according to any one of claims 1 to 3, characterized in that The cellulase is obtained by fermentation and cultivation of a cellulase-producing strain; Preferably, the cellulase producing strain is Trichoderma reesei and / or Myceliophthora thermophila.

9. The method according to claim 8, characterized in that The fermentation culture conditions at least include: an inoculation amount of 2-5% by volume, a temperature of 25-50° C., and a time of 3-7 days; Preferably, the fermentation culture medium contains: KH2PO4 1-3g / L, (NH4)2SO4 1-2g / L, MgSO4 0.1-0.5g / L, FeSO4 0.002-0.008g / L, MnSO4 0.05-0.1g / L, ZnSO40.001-0.002g / L, CoCl2 0.001-0.003g / L, CaCl2 0.2-0.4g / L, peptone 0.5-1.5g / L, and rice straw 8-12g / L.

10. The method according to any one of claims 1 to 3, characterized in that: The amino acid sequence of the lytic polysaccharide monooxygenase is shown in SEQ ID NO: 1; Preferably, the nucleotide sequence of the lytic polysaccharide monooxygenase is shown in SEQ ID NO:2.

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