CN107012176B

CN107012176B - Method for producing sodium gluconate by using cellulose biomass as raw material through enzymatic conversion

Info

Publication number: CN107012176B
Application number: CN201710264741.4A
Authority: CN
Inventors: 曲音波; 韩小龙
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2017-04-21
Filing date: 2017-04-21
Publication date: 2021-01-15
Anticipated expiration: 2037-04-21
Also published as: CN107012176A

Abstract

The invention discloses a method for producing sodium gluconate by enzymatic conversion of cellulose biomass as raw material, which is to hydrolyze cellulose biomass raw material with cellulase produced in situ or commercial cellulase to prepare high-concentration glucose The cellulose hydrolyzate; then utilize glucose oxidase and catalase to catalyze the oxidation of glucose in the cellulose hydrolyzate to obtain gluconic acid, and neutralize it with sodium hydroxide to obtain sodium gluconate; wherein glucose oxidase and peroxidase can also be used The hydrogenase is immobilized to prepare immobilized enzyme particles to catalyze the oxidation of cellulose hydrolyzate to obtain sodium gluconate. The method is simple in operation, low in production cost, does not need to use culture medium and prepare fermented seeds, shortens the preparation period, and experiments show that the conversion rate of glucose is significantly improved compared with the existing method, and has a large industrial application prospect.

Description

Method for producing sodium gluconate by using cellulose biomass as raw material through enzymatic conversion

Technical Field

The invention relates to a production method of sodium gluconate, in particular to a method for producing sodium gluconate by using cellulose biomass as a raw material and adopting enzymatic conversion (glucose oxidase/catalase or immobilized enzymes of the two enzymes).

Background

Lignocellulose can replace food raw materials used in the fermentation industry, and can be converted into various fermentation products such as bioethanol, biobutanol, 2, 3-butanediol, various organic acids, biopolysaccharides and the like by microorganisms after hydrolysis. The sodium gluconate has wide industrial application, can be used as a chelating agent, a surface cleaning agent for steel and glass, and can be used as a high-efficiency retarder, a high-efficiency water reducing agent and the like in the concrete industry. Aspergillus niger liquid submerged fermentation of starch hydrolysis sugar is a main mode for industrially producing sodium gluconate, because part of carbon source is consumed by thallus growth, and the conversion rate is between 90 and 95 percent. The starch raw material cost accounts for a large proportion of the production cost of the sodium gluconate, and the production of the sodium gluconate by using the lignocellulose can reduce the grain consumption and effectively reduce the raw material cost. Although the production of sodium gluconate from cellulose hydrolysate may not be suitable for the food and pharmaceutical industries, nearly half of the sodium gluconate produced annually in the world is applied to the construction industry, and studies have shown that sodium gluconate fermented from different carbon sources (starch sugar and lignocellulose hydrolysate) does not differ in performance as a cement retarder (ZHANG H, ZHANG J, BAO J. high titanium dioxide acid transfer by Aspergillus niger separated from dry cellulose acid pretreated soil store with out restriction [ J ]. Bioresour technique, 2016,203: 211-. Zhang et al pretreat corn stalk raw material with dry dilute acid, ferment with Aspergillus niger after enzymatic hydrolysis to obtain 76.67g/L gluconic acid, conversion rate 94.91%, furfural, hydroxymethyl furfural and acetic acid existing in non-detoxified corn stalk hydrolysate (corn stalk hydrolysate) have strong inhibition to Aspergillus niger growth, fermentation period is as long as 80-88H (ZHANG H, ZHANG J, BAO J. high titer by Aspergillus niger microorganism by microwave fermentation from dry dilute acid residue [ J ]. BioResource technique, 2016,203:211 + 219.). The corn stalk hydrolysate is biologically detoxified by Amorphotheca resinae and then fermented by Gluconobacter oxydans to obtain 132.46G/L gluconic acid and 38.86G/L xylonic acid (ZHANG H, LIU G, ZHANG J, et al.fermentative production of high glucose and xylonic acids from corn stock fed stock by Gluconobacter oxydans and technique-environmental analysis [ J ] Bioresour technique, 2016,219: 123-131.). However, since the cellulose hydrolysate contains substances that inhibit the growth of microorganisms, the fermentation period is often much longer than when starch or glucose is used as a raw material, and thus detoxification treatment of the hydrolysate is required. Detoxification can reduce the content of inhibitors or alleviate the inhibitory effect, but the detoxification process increases market costs.

Glucose Oxidase (GOD) and Catalase (Catalase, CAT) are used to catalyze the production of gluconic acid from Glucose. Glucose is oxidized into gluconic acid under the action of glucose oxidase and hydrogen peroxide is generated, and hydrogen peroxide can be rapidly decomposed into water and oxygen by adding catalase, so that the toxic effect on the glucose oxidase is avoided, and part of oxygen can be supplemented. And slowly adding alkali liquor with a certain concentration into the reaction system for neutralization in the reaction process so as to maintain the normal pH environment of the system, protect the activity of enzyme and ensure the final generation of sodium gluconate. The enzymatic processes (GOD and CAT) are also capable of catalyzing the oxidation of glucose in the cellulosic hydrolysate to produce gluconic acid and of avoiding the inhibition of microbial growth by inhibitors contained in the cellulosic hydrolysate. The search shows that no example of converting cellulose hydrolysate by using enzyme method (GOD and CAT) to produce sodium gluconate exists so far.

Disclosure of Invention

Aiming at the problems of the prior production method that the fermentation period is prolonged and the like because the growth of microorganisms is inhibited by an inhibitor contained in cellulose hydrolysate, the invention provides a method for producing sodium gluconate by using cellulose biomass as a raw material and adopting an enzymatic method (glucose oxidase/catalase or immobilized enzyme of the two enzymes) for conversion.

The invention relates to a method for producing sodium gluconate by using cellulose biomass as a raw material through enzymatic conversion, which comprises the following steps:

(1) pretreating cellulose biomass by a conventional method to remove hemicellulose and lignin, drying and crushing into particles with the particle size of less than 20 meshes for later use; wherein the cellulosic biomass is suitably pretreated corn stover, wheat straw, xylose residue or waste paper pulp;

(2) hydrolyzing the particles prepared in the step (1) by using cellulase to obtain cellulose hydrolysate with the glucose concentration of not less than 50 g/L;

(3) removing residues from the cellulose hydrolysate prepared in the step (2), adding glucose oxidase and catalase into the hydrolysate, and performing enzymatic conversion to produce sodium gluconate;

the method is characterized in that:

the method for hydrolyzing the particles prepared in the step (1) by using cellulase by taking the particles as a substrate in the step (2) comprises the following steps: measuring the content of glucan in the particles to prepare a hydrolysis system with the mass percent concentration of the particles not lower than 10%, adding cellulase into the hydrolysis system according to the dosage of less than 30FPU/g glucan, respectively supplementing the particles with the mass percent of 3.0-3.5% in the 24h, 36h and 48h of hydrolysis reaction, and correspondingly supplementing the cellulase with 10-14 FPU/g glucan each time for 120h of total hydrolysis;

wherein: the hydrolysis system is prepared by 0.05M citrate buffer solution with pH4.8, the hydrolysis temperature is 50 ℃, and the stirring speed is 200 rpm;

the cellulase is selected from commercial cellulase or crude cellulase liquid produced in situ by trichoderma reesei or penicillium oxalicum;

and (3) adding glucose oxidase and catalase into the hydrolysate, wherein the method for producing sodium gluconate through enzymatic conversion comprises the following steps: determining the content of glucose in the hydrolysate, taking the amount of glucose as a reference, adding glucose oxidase into the hydrolysate according to the amount of 2-20U/g glucose, adding catalase into the hydrolysate according to the amount of 200-2000U/g glucose, reacting under the catalytic reaction conditions of 25-50 ℃, stirring speed of 100-500 r/min and ventilation ratio of 0.4-2 vvm, adding NaOH solution into the reactor during the reaction process to control the pH value to be maintained at 4-6, and finishing the reaction when the concentration of glucose in the reaction solution is less than 5g/L to obtain a reaction solution containing sodium gluconate;

wherein: the glucose oxidase and the catalase are selected from commercial enzyme preparations, or are prepared into immobilized enzymes for repeated use.

In the method for producing sodium gluconate by using cellulose biomass as a raw material through enzymatic conversion: the cellulase in step (2) was initially added in an amount of 26FPU/g glucan.

In the method for producing sodium gluconate by using cellulose biomass as a raw material through enzymatic conversion: each corresponding addition of cellulase in step (2) is preferably carried out at an amount of 12FPU/g glucan.

In the method for producing sodium gluconate by using cellulose biomass as a raw material through enzymatic conversion: the dosage of the glucose oxidase in the step (3) is preferably 16U/g glucose.

In the method for producing sodium gluconate by using cellulose biomass as a raw material through enzymatic conversion: the amount of catalase used in step (3) is preferably 1500U/g glucose.

In the method for producing sodium gluconate by using cellulose biomass as a raw material through enzymatic conversion: the catalytic reaction conditions in the step (3) are preferably that the temperature is controlled at 36 ℃, the pH is controlled at 5.4, the ventilation ratio is controlled at 1.25vvm, and the stirring speed is controlled at 300 r/min.

In the method for producing sodium gluconate by using cellulose biomass as a raw material through enzymatic conversion: the concentration of the NaOH solution in the step (3) is preferably 500 g/L.

The invention discloses a method for producing sodium gluconate by using cellulose biomass as a raw material through enzymatic conversion, which is characterized in that cellulose biomass is hydrolyzed by using cellulase, and glucose contained in cellulose hydrolysate is catalyzed and oxidized by using glucose oxidase and catalase to produce sodium gluconate.

Drawings

FIG. 1: change of glucose concentration in the process of preparing cellulose hydrolysate by batch hydrolysis.

FIG. 2: change of glucose concentration in the process of preparing cellulose hydrolysate by feed hydrolysis.

FIG. 3: when the dosage of the glucose oxidase and the catalase is respectively 8U/g glucose and 500U/g glucose, the glucose concentration and the sodium gluconate concentration in the process of producing the sodium gluconate by catalyzing the cellulose hydrolysate are changed.

FIG. 4: when the dosage of the glucose oxidase and the catalase is respectively 16U/g glucose and 1500U/g glucose, the glucose concentration and the sodium gluconate concentration in the process of producing the sodium gluconate by catalyzing the cellulose hydrolysate are changed.

FIG. 5: the process of producing the sodium gluconate by circularly using the immobilized glucose oxidase-catalase particles.

Wherein a is the change of glucose concentration and sodium gluconate concentration in the production process of sodium gluconate; panel b is the activity retained after repeated use of the immobilized enzyme.

Detailed Description

The present invention will be further described with reference to the following examples. The contents described in the embodiments are only for illustrating the present invention and should not be construed as limiting the scope of the present invention as set forth in the claims.

General description: the construction of Penicillium oxalicum I1-13 is described in "Biotechnology for Biofuels" volume 9 of "Production of a high-efficiency cellulose complex via beta-glucosidase engineering in Penicillium oxalicum" in Novicin, where glucose oxidase is NS28166 and catalase is NS 28196.

Example 1:

crushing corncobs serving as a raw material into particles smaller than 20 meshes, drying the corncobs by using the raw material, and mixing the dried corncobs and prepared cooking liquor (1 per mill acetic acid solution) according to a material-liquid ratio of 1: 8, uniformly mixing the components, putting the mixture into a cooking pot, heating the mixture to 160 ℃, and preserving heat for 1 hour after the temperature reaches the specified temperature. And after the reaction is finished, separating the pretreatment solution from the solid material, washing the solid material with water to be neutral, and drying to obtain the xylose residue. The xylose residue contains 62.6% of cellulose, 2.4% of hemicellulose and 17.7% of lignin.

Mixing the xylose residue with 1% NaOH solution according to a solid-liquid ratio of 1: 8, treating the mixture for 1 hour in a boiling water bath, washing the solid material for 3 to 4 times by using a 2 percent hot sodium hydroxide solution, washing the solid material to be neutral by using distilled water, and drying the solid material to obtain the delignified xylose residues. The delignified xylose residues contain 65.7 percent of cellulose, 1.8 percent of hemicellulose and 3.2 percent of lignin.

Example 2: in-situ production of cellulase from penicillium oxalicum I1-13

The penicillium oxalicum I1-13 is adopted to produce cellulase in situ, and the fermentation method of the cellulase refers to patent 201610976280.9: the preparation method of the cellulase in the method disclosed in the method for producing the cellulase by the ammonium sulfite pulping waste liquor through fed fermentation is in an embodiment 3. The enzyme is produced in a fermentation tank by adopting a fed-batch fermentation method, the volume of the fermentation tank is 7.5L, and the initial liquid loading is 4.5L. Inoculating 0.5L of cultured seeds according to the inoculation amount of 10%, controlling the fermentation temperature at 30 ℃, controlling the ventilation ratio at 0.5 for 0-12 h, controlling the ventilation ratio at 0.75 after 12-24 h, controlling the ventilation ratio at 1 after 24-36 h, and controlling the stirring speed at 300 r/min. After inoculation, the pulping waste liquid is fed at the rate of 0.1mL/L/h for the first 10h, then the feeding rate is doubled every 10h, the feeding rate of the pulping waste liquid reaches 3.2mL/L/h after 50h, the feeding rate of 3.2mL/L/h is kept unchanged, and the feeding is continued to 100 h. After fermentation for 168h, the fermentation broth was collected, centrifuged at 9000rpm at 4 ℃ for 20min to remove the bacterial cells, and then adjusted to pH4.8 with 1M pH4.8 citric acid-sodium citrate buffer solution for saccharification experiments, wherein the crude enzyme solution had FPase, CMCase, pNPCase, and pNPGase at 13.22U/mL, 36.03U/mL, 1.37U/mL, and 108.74U/mL, respectively.

The penicillium oxalicum I1-13 is a cellulase high-yield strain which uses a gene BGL2 promoter to drive BGL1 overexpression on the basis of RE-10(gpdA (p) -clrB-ptra; delta BGL2: hph; delta creA: bar), compared with RE-10, I1-13 greatly up-regulates the expression level of beta-glucosidase, and has good effect on DCCR hydrolysis.

Example 3: hydrolyzing delignified xylose residues in batches to prepare cellulose hydrolysate.

The process for preparing the cellulose hydrolysate by hydrolyzing the delignified xylose residues in batches comprises the following steps: delignified xylose residues with a substrate concentration of 15% and a cellulase dosage of 26FPU/g glucan were hydrolyzed at a stirring speed of 100rpm at a temperature of 50 ℃ with a pH of 4.8 maintained with 0.1M HCl and 0.1M NaOH.

The change in glucose content during hydrolysis is shown in FIG. 1. After 120h of hydrolysis, the concentration of glucose in the hydrolysate can reach 101.17 g/L.

Example 4: preparation of cellulose hydrolysate by hydrolyzing delignified xylose residues through supplement

The process for preparing the cellulose hydrolysate by hydrolyzing the delignified xylose residues with the supplementary materials comprises the following steps: the initial substrate concentration is 15% delignified xylose residue, the cellulase dosage is 26FPU/g glucan, 3.33% delignified xylose residue is supplemented in 24h, 48h and 72h respectively, cellulase corresponding to the supplement dosage is supplemented according to the cellulase dosage of 12FPU/g glucan while supplementing, the stirring speed is 100rpm during hydrolysis, the temperature is 50 ℃, and the pH is maintained at 4.8 by 0.1M HCL and 0.1M NaOH.

The change in glucose content during hydrolysis is shown in FIG. 2. After 120h of hydrolysis, the concentration of glucose in the hydrolysate can reach 145.80 g/L.

Example 5: catalyzing cellulose hydrolysate to produce sodium gluconate when the dosage of glucose oxidase and catalase is respectively 8U/g and 500U/g glucose

And (3) producing sodium gluconate by using the prepared delignified xylose residue hydrolysate as a raw material by using glucose oxidase and catalase. The delignified xylose residue hydrolysate contained 140.17g/L glucose, and the reaction was carried out in an INFORS multiforms fermentation tank with a volume of 1L and a liquid loading of 0.8L. Setting the enzyme reaction temperature at 40 ℃, adding NaOH with the concentration of 500g/L in the reaction process, adjusting and controlling the pH value at 5.5, and adding glucose oxidase and catalase according to the proportion of 8U/g glucose and 500U/g glucose respectively. Stirring speed is 150rpm, ventilation rate is 1VVM, when the glucose concentration in the reaction solution is less than 5g/L, the reaction is ended, samples are taken at intervals of 8h, and after centrifugation at 10000rpm for 10min, the concentrations of glucose and sodium gluconate are measured. The change of glucose concentration and sodium gluconate concentration during the reaction is shown in FIG. 3.

The results show that: when the dosage of the glucose oxidase and the catalase is respectively 8U/g glucose and 500U/g glucose, 161.83g/L sodium gluconate can be obtained after the reaction is carried out for 56 hours, and the conversion rate of the glucose is 95.47%.

Example 6: catalyzing cellulose hydrolysate to produce sodium gluconate when the dosage of glucose oxidase and catalase is 16U/g and 1500U/g glucose respectively

And (3) producing sodium gluconate by using the prepared delignified xylose residue hydrolysate as a raw material by using glucose oxidase and catalase. The delignified xylose residue hydrolysate contained 140.17g/L glucose, and the reaction was carried out in an INFORS multiforms fermentation tank with a volume of 1L and a liquid loading of 0.8L. Setting the enzyme reaction temperature at 40 ℃, adding NaOH with the concentration of 500g/L in the reaction process, adjusting and controlling the pH value at 5.5, and adding glucose oxidase and catalase according to the proportion of 16U/g glucose and 1500U/g glucose respectively. Stirring speed is 150rpm, ventilation rate is 1VVM, when the glucose concentration in the reaction solution is reduced to 5g/L, the reaction is ended, samples are taken at intervals of 8h, and after centrifugation at 10000rpm for 10min, the concentrations of glucose and sodium gluconate are measured. The change of glucose concentration and sodium gluconate concentration during the reaction is shown in FIG. 4.

The results show that: when the dosage of the glucose oxidase and the catalase is respectively 16U/g glucose and 1500U/g glucose, 161.19g/L sodium gluconate can be obtained after 48 hours of reaction, and the conversion rate of the glucose is 95.09%.

Example 7: production of sodium gluconate by recycling immobilized glucose oxidase-catalase particles

Co-immobilizing glucose oxidase and catalase by using two carriers of polyvinyl alcohol and sodium alginate by an embedding method. Preparing a solution containing 8% (w/w) of polyvinyl alcohol and 1.5% (w/w) of sodium alginate by using distilled water, heating and stirring until the polyvinyl alcohol and the sodium alginate are fully dissolved, cooling to 30 ℃, adding a mixed enzyme solution of GOD and CAT prepared according to the ratio of enzyme activity to 1:100, stirring and uniformly mixing by using a magnetic stirrer, standing for 1h, and then dropwise adding a solution containing 1.5% (w/v) of CaCl by using a 50mL syringe₂In the saturated boric acid solution, performing crosslinking reaction in a refrigerator at 4 ℃ for 4 hours, repeatedly washing immobilized enzyme particles for a plurality of times by using distilled water after the crosslinking reaction is finished, absorbing surface moisture by using absorbent paper, and then placing the solution in the refrigerator at 4 ℃ for storage for later use.

And (3) producing sodium gluconate by using the prepared immobilized GOD-CAT particles and the prepared delignified xylose residue hydrolysate as a raw material. The delignified xylose residue hydrolysate contained 140.17g/L glucose, and the reaction was carried out in an INFORS multiforms fermentation tank with a volume of 1L and a liquid loading of 0.8L. The enzyme reaction temperature is set to be 36 ℃, NaOH with the concentration of 500g/L is added in the reaction process to adjust and control the pH value to be 5.4, and the dosage of the immobilized enzyme is added according to 16U/g glucose by taking glucose oxidase as a standard. Stirring speed is 300rpm, ventilation rate is 1.25VVM, when the glucose concentration in the reaction solution is reduced to 5g/L, the reaction is ended, samples are taken at intervals of 8h, and after centrifugation at 10000rpm for 10min, the concentrations of glucose and sodium gluconate are measured. The change of glucose concentration and sodium gluconate concentration during the reaction is shown in FIG. 5.

The results show that: when the immobilized enzyme is added according to 16U/g glucose by taking glucose oxidase as a standard, 166.46g/L sodium gluconate can be obtained after reaction for 48 hours, the conversion rate of glucose is 98.19%, and after the reaction is finished, 86.76% of glucose oxidase activity and 88.74% of catalase activity can still be maintained by the immobilized enzyme granules. After the immobilized enzyme is repeatedly used for 6 times, the immobilized enzyme still can keep more than 60% of activity, the yield of the sodium gluconate is not obviously reduced at 56h, and the immobilized enzyme can still be expected to be continuously used. Therefore, the preparation of liquid glucose oxidase and catalase as immobilized enzyme particles can effectively reduce the dosage of the enzyme preparation, and can also be used for continuous catalytic conversion process.

Claims

1. a method that takes cellulose biomass as a raw material enzymatic conversion to produce sodium gluconate, the steps are:

(1) carry out pretreatment to cellulosic biomass by conventional method, remove hemicellulose and lignin, then dry and pulverize into granules, for subsequent use;

(2) using the particulate matter obtained in step (1) as a substrate, hydrolyzing it with cellulase to obtain a cellulose hydrolyzate with a glucose concentration of not less than 50 g/L; wherein, the cellulase selects commercial cellulose Enzyme or crude cellulase solution produced in situ by Trichoderma reesei or Penicillium oxalate;

(3) removing the residue from the cellulose hydrolyzate obtained in step (2), then adding glucose oxidase and catalase to the hydrolyzate, and enzymatically converting to produce sodium gluconate; wherein, the glucose oxidase and peroxidase are Catalase is selected from commercial enzyme preparations, or prepared as immobilized enzymes for repeated use;

It is characterized by:

In step (1), the cellulosic biomass is appropriately pretreated corn stalk, wheat stalk, xylose residue or waste paper pulp, and the pulverized particle size is less than 20 meshes;

In step (2), the granules obtained in step (1) are used as substrates, and the method for hydrolyzing them with cellulase is as follows: measuring the content of glucan in the granules, and preparing them into hydrolysis with a mass percentage of not less than 10% of the granules. System, add cellulase to the hydrolysis system according to the dosage of less than 30FPU/g glucan, and add 3.0% to 3.5% by mass of particulate matter at the 24th, 36th, and 48th hours of the hydrolysis reaction, and supplement each time accordingly. Add 10～14FPU/g glucan cellulase, hydrolyze for 120h in total;

Wherein: the hydrolysis system is prepared with citrate buffer of 0.05M pH4.8, the hydrolysis temperature is 50°C, and the stirring speed is 200rpm;

Described in step (3), add glucose oxidase and catalase in the hydrolyzate, the method for enzymatic conversion to produce sodium gluconate is: measure the glucose content in the hydrolyzate, take the amount of glucose as a benchmark, press 16U/g glucose The amount of glucose oxidase is added to the hydrolyzed solution, and catalase is added to the hydrolyzed solution by the amount of 1500U/g glucose, at a temperature of 36 ° C, the stirring speed is 300 r/min, and the ventilation ratio is 1.25vvm. The reaction is reacted under the catalytic reaction conditions, During the reaction, a NaOH solution with a concentration of 500 g/L was added to the reactor to control the pH to be maintained at 5.4. When the glucose concentration in the reaction solution was less than 5 g/L, the reaction was terminated to obtain a reaction solution containing sodium gluconate.