CN113234772B - A kind of method of poplar enzymolysis to produce glucose - Google Patents

Abstract

The invention provides a method for producing glucose by enzymatic hydrolysis _of poplar. First, metal chloride AlCl3 or _CrCl3 is used to pretreat poplar under specific conditions, and then cellulase and surface are added to the pretreated residue. Enzymatic hydrolysis was carried out with the active agent, and the enzymatic hydrolysis efficiency and glucose yield of poplar were improved by adding surfactant to assist metal chloride pretreatment, reducing the amount of enzyme and shortening the time of enzymatic hydrolysis. Under the treatment of the residue, the glucose yield of enzymatic hydrolysis for 24 hours can be as high as 86.94%, which effectively improves the economic benefit.

Description

A kind of method of poplar enzymolysis to produce glucose

technical field

The invention belongs to the technical field of biomass pretreatment, and more particularly relates to a method for producing glucose by enzymatic hydrolysis of poplar.

Background technique

With the development of the times, energy and environmental problems have gradually become prominent. As a new renewable resource, biomass energy has attracted more and more attention. Among them, lignocellulose is considered to be a good source of energy, rich in reserves and inedible. The raw material for preparing biofuel, the method for preparing liquid fuel from lignocellulosic biomass is to hydrolyze hemicellulose and cellulose in the raw material into monosaccharides such as xylose and glucose, and produce ethanol through fermentation.

Poplar has fast growth rate, short growth cycle, strong environmental adaptability, wide planting area and abundant resources. It is an important raw material for the production of bioenergy and biomass chemicals. Biotransformation of poplar to produce bioenergy and chemicals has great potential. . Due to the high anti-lignin content of poplar, during the utilization of lignocellulose, due to the complex structural characteristics of lignocellulose, poplar has a strong degradation barrier, and lignocellulose is difficult to be effectively enzymatically hydrolyzed. Treatment is a key step to improve the enzymatic hydrolysis efficiency of poplar.

Pretreatment removes lignin and degrades hemicellulose in a certain way, causing changes in the chemical composition of lignocellulose, increasing the contact area between enzymes and cellulose, and thus improving the efficiency of enzymatic hydrolysis. The treatment methods include acid pretreatment, alkali pretreatment, ionic liquid pretreatment and organic solvent pretreatment. For example, patent CN201910212819.7 provides a method for improving the enzymatic hydrolysis efficiency of poplar after pretreatment with acetic acid hydrogen peroxide by adding β-glucosidase, but the method still has a long enzymatic hydrolysis time, and the enzymatic hydrolysis time is 72 hours. The amount of enzyme added to the dry matter is 20FPU cellulase and an additional 500nkat to 1000nkat of β-glucosidase. The enzyme dosage is large and the cost is high. Therefore, it is urgent to develop a method that can reduce economic costs and improve the efficiency of poplar enzymolysis. .

SUMMARY OF THE INVENTION

Aiming at the problems of long enzymatic hydrolysis time and large amount of cellulase in the current enzymatic hydrolysis process of poplar to prepare bio-energy, the present invention aims to provide a method for improving poplar enzymatic hydrolysis efficiency through surfactant-assisted metal chloride pretreatment , through a specific poplar pretreatment method, and then adding a specific surfactant to assist the enzymatic hydrolysis of poplar, on the premise of reducing the amount of enzyme and shortening the time of enzymatic hydrolysis, it can also effectively improve the enzymatic hydrolysis efficiency and improve the yield of glucose.

The primary purpose of the present invention is to provide a method for producing glucose by enzymatic hydrolysis of poplar.

Above-mentioned purpose of the present invention is achieved through the following technical solutions:

The invention provides a method for producing glucose by enzymolysis of poplar, comprising the following steps:

S1. Poplar pretreatment: Mix poplar with water at a solid-to-liquid ratio of 1:8 to 15, add metal chloride in an amount of 0.03 to 0.15 mol/L _water , and carry out the pretreatment reaction at 160 °C to 180 °C , the reaction product was washed to neutrality, and separated to obtain poplar pretreatment residue;

S2. Enzymatic hydrolysis: take poplar pretreatment residue, add pH=4.5-5.0 buffer solution, cellulase and surfactant to carry out enzymatic hydrolysis;

Wherein, the metal chloride includes AlCl ₃ or CrCl ₃ ; the mass ratio of poplar pretreatment residue, buffer solution, and surfactant in step S2 is 1 g: 50 mL: 0.1-0.4 g.

In the above method of the present invention, metal chlorides AlCl ₃ and CrCl ₃ are used for pretreatment of poplar, and Lewis acid pretreatment is used to degrade hemicellulose into xylose, remove lignin, and then promote the subsequent enzymatic hydrolysis process; the buffer solution can Maintain the normal pH value required by enzymes, greatly reduce pH value changes, and prevent the decline or loss of enzyme activity caused by large changes in pH value; the addition of surfactants can increase the accessible surface area of cellulose and affect the chemical composition of lignin. The effect is small and can be adsorbed into lignin, preventing the non-productive adsorption of enzymes, thereby improving the yield of glucose.

In some preferred embodiments, the solid-liquid ratio of poplar to water in step S1 is 1:10, see Examples 1-12.

In some preferred embodiments, the addition amount of the cellulase is 17.5-20 FPU/g _{poplar pretreatment residue} , see Examples 1-12.

In some preferred embodiments, the pretreatment reaction in step S1 is a reaction at a speed of 200-300 rpm for 10-30 min, see Examples 1-12.

Preferably, the water in step S1 is ultrapure water.

Preferably, the poplar described in step S1 is obtained by air-drying and pulverizing the poplar; the poplar and the pretreatment residue of the poplar in the present invention are both measured in absolute dry amount.

Preferably, the buffer solution is an acetic acid-sodium acetate buffer solution.

In some preferred embodiments, the surfactant includes one or more of whey protein, calcium lignosulfonate, Tween80, PEG8000, and Triton X-100, see Examples 1-5.

When the metal chloride is AlCl ₃ , the surfactant is most preferably PEG 8000, see Example 1; when the metal chloride is CrCl ₃ , the surfactant is most preferably lignosulfonic acid Calcium, see Example 6.

In some preferred embodiments, the temperature of the enzymatic hydrolysis in step S2 is 40-55° C., and the rotation speed is 100-250 rpm, see Examples 1-12.

Most preferably, the temperature of the enzymatic hydrolysis in step S2 is 50° C., and the rotation speed is 150 rpm, see Example 1.

In some preferred embodiments, the separation described in step S1 is a method of vacuum filtration.

In some of the preferred embodiments, the reaction in step S1 is performed in a reaction kettle.

In addition, the present invention also claims to protect the application of the above method in the production of glucose by enzymatic hydrolysis of poplar.

Compared with the prior art, the beneficial effects of the present invention are:

The method for enzymatic hydrolysis of poplar wood provided by the present invention adopts specific metal chlorides to pretreat poplar wood under specific conditions, and then adds cellulase and surfactant to the pretreated residue for enzymatic hydrolysis. The active agent-assisted metal chloride pretreatment improves the enzymatic hydrolysis efficiency and glucose yield of poplar, reduces the enzyme dosage, and shortens the enzymatic hydrolysis time. Under the enzyme dosage of 17.5-20 FPU/g _{poplar pretreatment residue} , enzymatic hydrolysis of glucose for 24 hours The yield can be as high as 86.94%, which effectively improves the economic benefits.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the raw material reagents used in the examples of the present invention are conventionally purchased raw material reagents.

The poplar used in the examples is taken from a wood factory, and is obtained by air-drying and pulverizing, and its component content is 45.56% of cellulose, 14.98% of hemicellulose, and 22.86% of lignin.

Cellulase (Second Generation) was purchased from Novozymes (China) Biotechnology Co., Ltd.

The glucose content in the enzymatic hydrolyzate was analyzed by high performance liquid phase method, and the calculation method of its yield was as follows:

In the formula, Y represents the glucose yield (%) in the enzymatic hydrolysis solution; C represents the glucose concentration in the enzymatic hydrolysis solution (g/L); V represents the volume of the enzymatic hydrolysis solution (L); M represents the mass of cellulose in the poplar raw material ( g).

Example 1 A method for improving the efficiency of poplar enzymolysis

S1. Poplar pretreatment: 12g (absolute dry amount) poplar and 0.80g AlCl _were added to 120mL ultrapure water, reacted in a reaction kettle at 180°C and 300rpm for 20min, and washed with deionized water until neutral. The poplar pretreatment residue was obtained by vacuum filtration;

S2. Enzymatic hydrolysis: take 2 g of poplar pretreatment residue (absolute dry amount), add 100 mL of acetic acid-sodium acetate buffer solution with pH=4.8, 40 FPU cellulase (Seili II) and 0.3 g of surfactant PEG 8000, Enzymatic hydrolysis was carried out at 50°C and 150rpm.

Example 2 A method for improving the efficiency of poplar enzymolysis

The difference from Example 1 is that the surfactant was replaced with 0.8 g of whey protein, and 100 mL of acetic acid-sodium acetate buffer solution with pH=4.5 was added.

Embodiment 3 A kind of method that improves poplar enzymolysis efficiency

The difference from Example 1 is that the surfactant was replaced with 0.2 g calcium lignosulfonate, and 100 mL of pH=5 acetic acid-sodium acetate buffer solution was added.

Embodiment 4 A kind of method that improves poplar enzymolysis efficiency

The difference from Example 1 is that the surfactant was replaced with Tween 80, and the pretreatment was performed to react at 200 rpm for 30 min.

Example 5 A method for improving the efficiency of poplar enzymolysis

The difference from Example 1 is that the surfactant was replaced with Triton X-100, and the pretreatment was performed to react at 300 rpm for 10 min.

Example 6 A method for improving the efficiency of poplar enzymolysis

S1. Poplar pretreatment: add 12g (absolute dry amount) poplar and 0.95g CrCl ₃ to 120mL ultrapure water, react in a reaction kettle at 160°C and 300rpm for 20min, add deionized water to wash to neutrality, The poplar pretreatment residue was obtained by vacuum filtration;

S2. Enzymatic hydrolysis: take 2 g of poplar pretreatment residue (absolute dry amount), add 100 mL of acetic acid-sodium acetate buffer solution with pH=4.8, 40 FPU cellulase (Seili II) and 0.3 g of surfactant lignosulfonic acid Calcium acid hydrolyzed at 50°C and 150rpm.

Example 7 A method for improving the efficiency of poplar enzymolysis

The difference from Example 6 is that in step S2, the surfactant was replaced with whey protein; enzymatic hydrolysis was performed at 40° C. with a rotation speed of 250 rpm.

Example 8 A method for improving the efficiency of poplar enzymolysis

The difference from Example 6 is that the surfactant in step S2 was replaced with PEG 8000; enzymatic hydrolysis was performed at 55° C. and the rotation speed was 100 rpm.

Example 9 A method for improving the efficiency of poplar enzymolysis

The difference from Example 6 is that the surfactant was replaced with Tween 80.

Embodiment 10 A kind of method that improves poplar enzymolysis efficiency

The difference from Example 6 is that the surfactant was replaced with Triton X-100.

Example 11 A method for improving the efficiency of poplar enzymolysis

The difference from Example 1 is that 0.4g of surfactant PEG 8000 is added in step S2.

Example 12 A method for improving the efficiency of poplar enzymolysis

The difference from Example 1 is that in step S2, 35FPU cellulase (Second generation of Saili) is added.

Comparative Example 1

The same as the method in Example 6, the difference is that the metal chloride is replaced by 0.82 g of ZnCl ₂ , and no surfactant is added to S2.

Comparative Example 2

The same as the method in Example 6, the difference is that the metal chloride is replaced by 0.57 g of MgCl ₂ , and no surfactant is added to S2.

Comparative Example 3

With the method of Example 6, the difference is that the metal chloride is replaced by 0.76g of MnCl ₂ , and no surfactant is added to S2.

Comparative Example 4

The same as the method in Example 6, the difference is that the metal chloride is replaced by 0.97 g of FeCl ₃ , and no surfactant is added to S2.

Comparative Example 5

The same as the method in Example 6, the difference is that the metal chloride is replaced by 0.81 g of CuCl ₂ , and no surfactant is added to S2.

Comparative Example 6

The same as the method in Example 6, the difference is that the metal chloride is replaced by 0.80 g of AlCl ₃ , and no surfactant is added to S2.

Comparative Example 7

With the method of Example 6, the difference is that no surfactant is added in S2.

Comparative Example 8

With the method of Example 1, the difference is that no surfactant is added in S2.

Comparative Example 9

The same as the method in Example 1, the difference is that 0.40 g of AlCl ₃ was added, 120 mL of ultrapure water was added, and no surfactant was added in S2 at 160° C. in the reaction kettle.

Comparative Example 10

The same as the method in Example 1, the difference is that 0.56g of AlCl ₃ was added, 120 mL of ultrapure water was added, and no surfactant was added in S2 at 160° C. in the reaction kettle.

Comparative Example 11

It is the same as the method in Example 1, except that the reaction temperature in step S1 is 150° C., and no surfactant is added in S2.

Comparative Example 12

It is the same as the method in Example 1, except that the reaction temperature in step S1 is 190° C., and no surfactant is added in S2.

Comparative Example 13 A method for improving the efficiency of poplar enzymolysis

The difference from Example 1 is that the reaction temperature of step S1 is 170° C., and no surfactant is added in S2.

Comparative example 14 A kind of method that improves poplar enzymolysis efficiency

The difference from Example 6 is that the reaction temperature of step S1 is 170° C., and no surfactant is added in S2.

Comparative Example 15 A method for improving the efficiency of poplar enzymolysis

The difference from Example 6 is that the reaction temperature of step S1 is 180° C., and no surfactant is added in S2.

Experimental example

Example 1-12 and Comparative Example 1-15 in the enzymatic hydrolysis for 24 hours and 72 hours, respectively take 1 mL of enzymatic hydrolysis solution and carry out 10min inactivation treatment, measure the glucose concentration in the enzymatic hydrolysis solution with high performance liquid phase, and calculate 24 hours respectively. and 72-hour glucose yields. The results are shown in Table 1:

Table 1

By comparing Examples 1 to 5 with Comparative Example 8, it can be seen that the AlCl ₃ pretreatment with added surfactants has a higher yield of glucose, and all five surfactants have a promoting effect on the yield _of glucose. The preprocessing boosts the best. The 24h glucose yield _of AlCl3 pretreatment with PEG 8000 was 86.94%, and the 72h glucose yield was 88.45%.

It can be seen from the comparison between Example 1 and Comparative Example 8 that after adding PEG 8000, the yield of 86.94% in 24 hours after AlCl ₃ pretreatment exceeds that of 81.98% in 72 hours without adding surfactant, which greatly shortens the enzymatic hydrolysis time and improves the Enzymatic yield.

Comparing Examples 6 to 10 with Comparative Example 7, it can be seen that the CrCl ₃ pretreatment with added surfactant has a higher yield of glucose, and all five surfactants have a promoting effect on the yield of glucose. Among them, calcium lignosulfonate had the best effect on CrCl ₃ pretreatment. The CrCl ₃ pretreatment with calcium lignosulfonate was pretreated with 69.69% glucose for 24h and 85.53% for 72h.

It can be seen from the comparison between Example 6 and Comparative Example 7 that after adding calcium lignosulfonate, the yield of 69.69% in 24h can reach 65.94% in 72h without adding surfactant, and the highest glucose yield in 72h reaches 85.53%. The enzymatic hydrolysis time is shortened and the enzymatic hydrolysis yield is improved.

Through the comparison of Comparative Examples 1 to 7, it can be seen that different metal chlorides have different effects on poplar pretreatment at 160 °C, among which AlCl ₃ and CrCl ₃ have relatively good effects.

From the comparison of Comparative Example 6 and Comparative Examples 9 to 10, it can be seen that different ion concentrations have an effect on the AlCl ₃ pretreatment, and when the ion concentration is reduced, the glucose yield will decrease.

From the comparison of Comparative Example 8 and Comparative Examples 11 to 13, it can be seen that different temperatures have a great influence on AlCl ₃ pretreatment, and the glucose yield is poor at too high or too low temperature, and the glucose yield reaches the highest at 180 °C .

Obviously, the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those of ordinary skill in the art, changes or modifications in other different forms can also be made on the basis of the above description. There is no need and cannot be exhaustive of all implementations here. Any modifications, equivalent replacements and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the claims of the present invention.

Claims (9)

1. a method for improving the speed of poplar enzymolysis to produce glucose, is characterized in that, described method is to add surfactant in poplar enzymolysis to produce glucose process, and it comprises the following steps: S1. Poplar pretreatment: Mix poplar with water at a solid-to-liquid ratio of 1:8 to 15, add metal chloride in an amount of 0.03 to 0.05 mol/L water, and carry out pretreatment reaction at 160 °C to 180 °C , the reaction product was washed to neutrality, and separated to obtain poplar pretreatment residue; S2. Enzymatic hydrolysis: take poplar pretreatment residue, add pH=4.5-5.0 buffer solution, cellulase and surfactant to carry out enzymatic hydrolysis; Wherein, the metal chloride is AlCl ₃ or CrCl ₃ ; the mass ratio of poplar pretreatment residue, buffer solution, and surfactant in step S2 is 1 g: 50 mL: 0.1-0.4 g; the addition of the cellulase The amount is 17.5～20FPU/g _{poplar pretreatment residue} . 2 . The method according to claim 1 , wherein the pretreatment reaction in step S1 is a reaction at a speed of 200-300 rpm for 10-30 min. 3 . 3. method according to claim 1, is characterized in that, described buffer solution is acetic acid-sodium acetate buffer solution. 4. The method according to claim 1, wherein the surfactant comprises any one of whey protein, calcium lignosulfonate, Tween 80, PEG 8000, and Triton X-100. 5. The method according to claim ₁ , wherein when the metal chloride is AlCl , the surfactant is PEG 8000; when the metal chloride is _CrCl , the surfactant It is calcium lignosulfonate. 6 . The method according to claim 1 , wherein the temperature of the enzymatic hydrolysis in step S2 is 40-55° C., and the rotation speed is 100-250 rpm. 7 . 7 . The method according to claim 1 , wherein the separation in step S1 is a method of vacuum filtration. 8 . 8. The method according to claim 1, wherein the poplar is obtained by air-drying and pulverizing the poplar. 9 . The method according to claim 1 , wherein the reaction in step S1 is to carry out the reaction in a reaction kettle. 10 .