阅读说明：本技术 一种提高纤维素酶解效率的酶解促进剂及其应用 (Enzymolysis accelerant for improving enzymolysis efficiency of cellulose and application thereof ) 是由 张红丹 张贤滨 谢君 李晓垠 于 2021-11-01 设计创作，主要内容包括：本发明公开了一种提高纤维素酶解效率的酶解促进剂及其应用,酶解促进剂包括吐温、茶皂素、蛋白、木聚糖酶。本发明的数据表明,与单独添加吐温、茶皂素、蛋白可以显著提高纤维素酶解效率不同的是,单纯添加木聚糖酶并不能提高纤维素酶的酶解效率,反而会导致纤维素酶的酶解效率略微下降。而将木聚糖酶和吐温、茶皂素、蛋白复配后,出人意料地提高了纤维素酶后期的酶解效率,延长纤维素酶的持续作用时间,具有意料之外的效果；本发明的一些实例酶解24h和48h的葡萄糖得率分别可达89.55%和96.46%,可有效将纤维素酶解的时间从72h缩短至24 h。本发明的一些实例,在缩短酶解时间的同时,延长了纤维素酶的作用时间。(The invention discloses an enzymolysis accelerant for improving the enzymolysis efficiency of cellulose and application thereof, wherein the enzymolysis accelerant comprises tween, tea saponin, protein and xylanase. The data of the invention show that the enzymolysis efficiency of the cellulase cannot be improved by only adding xylanase, but the enzymolysis efficiency of the cellulase is slightly reduced. After the xylanase, the tween, the tea saponin and the protein are compounded, the later enzymolysis efficiency of the cellulase is unexpectedly improved, the continuous action time of the cellulase is prolonged, and an unexpected effect is achieved; according to some embodiments of the invention, the glucose yield of 24h and 48h after enzymolysis can reach 89.55% and 96.46%, respectively, and the enzymolysis time of the cellulase can be effectively shortened from 72h to 24 h. Some embodiments of the invention provide for a reduction in enzymatic time while extending the duration of action of the cellulase enzyme.)

1. An enzymolysis accelerant for improving the enzymolysis efficiency of cellulose is characterized in that: the mass components of the composition are 10-50 parts of tween, 20-60 parts of tea saponin, 20-60 parts of protein and 0.4-1.2 parts of xylanase.

2. The enzymatic hydrolysis enhancer of claim 1, wherein: the mass components of the composite are 10-30 parts of tween, 20-60 parts of tea saponin, 20-60 parts of BSA protein and 0.4-1.2 parts of xylanase.

3. The enzymatic hydrolysis enhancer of claim 1, wherein: the composition comprises 30 parts of tween, 40 parts of tea saponin, 60 parts of BSA protein and 1.2 parts of xylanase by mass.

4. An enzymatic hydrolysis accelerator as defined in any one of claims 1 to 3, wherein: the protein is BSA.

5. An enzymatic hydrolysis accelerator as defined in any one of claims 1 to 3, wherein: the enzyme activity of the xylanase is not less than 10000U/mg.

6. A method for enzymolysis of cellulose comprises mixing pretreatment residue containing cellulose, cellulase, buffer solution and enzymolysis accelerant together for enzymolysis, and is characterized in that: the enzymatic hydrolysis promoter is as defined in any one of claims 1 to 5.

7. The method of claim 6, wherein: the dosage of the enzymolysis accelerant is 50-150 mg/g of oven-dried pretreatment residues.

8. The method of claim 6, wherein: the dosage of the cellulase is not more than 15 FPU/g of oven-dried pretreatment residues.

9. The method according to any one of claims 6 to 8, wherein: the enzymolysis time is not more than 48 h.

10. The application of the enzymolysis accelerant in promoting the enzymolysis of the cellulose is characterized in that: the enzymatic hydrolysis promoter is as defined in any one of claims 1 to 5.

Technical Field

The invention relates to the technical field of efficient conversion and utilization of biomass, in particular to an enzymolysis accelerant for improving the enzymolysis efficiency of cellulose and a method for performing cellulose enzymolysis by using the enzymolysis accelerant.

Background

With the reduction of fossil energy, environmental pollution and the enhancement of environmental awareness, people are forced to find a new clean energy. Biomass has attracted attention as one of them. One of the celluloses is a common biomass, can be enzymolyzed into glucose by cellulase, has wide application and is more and more concerned by people.

The dosage of the cellulase and the enzymolysis efficiency have a crucial influence on the cost of the cellulase. The smaller the amount of cellulase used, the faster the rate of enzymatic hydrolysis (the shorter the time used), the more advantageous it is for cellulase hydrolysis. Generally speaking, the time of the cellulose enzymolysis is generally not less than 72 hours, and the dosage is also larger.

The inventor discloses in CN109457000A that the addition of Tween 80 can shorten the enzymolysis time to a certain extent, reduce the enzyme dosage and improve the glucose yield. But in the later stage of enzymolysis, the activity of the cellulase is obviously reduced, and the effect still needs to be further improved. Other researches show that the dosage of the cellulase can be reduced by some proteins and the like, but the problems of high use cost and poor synergistic effect exist. There is a need for a novel enzymatic hydrolysis promoter that improves the efficiency of enzymatic hydrolysis of cellulose.

Disclosure of Invention

The invention aims to overcome at least one defect of the prior art and provides an enzymolysis accelerant for improving the enzymolysis efficiency of cellulose and a method for performing cellulose enzymolysis by using the enzymolysis accelerant.

The technical scheme adopted by the invention is as follows:

in a first aspect of the present invention, there is provided:

an enzymolysis accelerant for improving the enzymolysis efficiency of cellulose comprises, by mass, 10-50 parts of tween, 20-60 parts of tea saponin, 20-60 parts of protein and 0.4-1.2 parts of xylanase.

In some examples, the enzymatic hydrolysis promoter comprises, by mass, 10-30 parts of tween, 20-60 parts of tea saponin, 20-60 parts of BSA protein, and 0.4-1.2 parts of xylanase.

In some examples, the enzymatic hydrolysis promoter comprises 30 parts of tween, 40 parts of tea saponin, 60 parts of BSA protein and 1.2 parts of xylanase.

In some examples, the enzymatic hydrolysis promoter comprises 10-30 mg of tween, 20-60 mg of tea saponin, 20-60 mg of BSA protein, and not less than 4000U of xylanase.

In some examples, the tween is tween 80.

In some examples, the protein is BSA.

In some examples, the xylanase has an enzymatic activity of not less than 10000U/mg.

In a second aspect of the present invention, there is provided:

a method for enzymatic hydrolysis of cellulose, comprising mixing together a pretreatment residue containing cellulose, cellulase, a buffer, and an enzymatic hydrolysis enhancer as described in the first aspect of the present invention to carry out enzymatic hydrolysis.

In some examples, the amount of the enzymatic hydrolysis enhancer is 50-150 mg/g of oven-dried pretreatment residue.

In some examples, the cellulase enzyme is used in an amount of no more than 15 FPU/g oven dried pretreated residue.

In some examples, the time for enzymatic hydrolysis does not exceed 48 hours.

In some examples, the xylanase is added in an amount of no less than 4000U/g oven dried pretreatment residue.

In a third aspect of the present invention, there is provided:

the use of an enzymatic hydrolysis enhancer of the first aspect of the invention for enhancing enzymatic hydrolysis of a cellulose.

The invention has the beneficial effects that:

some examples of the invention show that, different from the method for obviously improving the enzymolysis speed of the cellulase by independently adding the Tween, the tea saponin and the protein (BSA), the method for improving the enzymolysis efficiency of the cellulase by simply adding the xylanase cannot improve the enzymolysis efficiency of the cellulase, but can cause the enzymolysis efficiency of the cellulase to be slightly reduced. After the xylanase, the Tween, the tea saponin and the protein are compounded, the later-stage enzymolysis efficiency of the cellulase is unexpectedly improved, the continuous action time of the cellulase is prolonged, and the unexpected effect is achieved.

In some embodiments of the present invention, the enzymatic hydrolysis promoter with a specific composition can not only improve the efficiency of the whole enzymatic hydrolysis process, but also improve the final glucose yield, with unexpected effects.

According to some embodiments of the invention, the glucose yield after 24 hours of enzymolysis can reach 89.55%, and the glucose yield after 48 hours of enzymolysis can reach 9.46%. Compared with the prior art, the time for cellulose enzymolysis can be effectively shortened from 72h to 24 h.

Some embodiments of the invention can shorten the enzymolysis time, prolong the action time of the cellulase and facilitate the reduction of the enzyme dosage for the cellulase.

Detailed Description

The present invention will be further described with reference to specific embodiments, but the present invention is not limited to the examples in any way. The starting reagents employed in the examples of the present invention are, unless otherwise specified, those that are conventionally purchased.

Some of the materials and methods in the following examples:

for convenience of comparison, in the following examples or comparative examples, unless otherwise specified:

the bagasse raw material is air-dried, thread-rolled and crushed to obtain the grain diameter<1 mm, the content of the components is 40.2 percent of cellulose, 21.5 percent of hemicellulose and 25.2 percent of lignin. For convenience of proportion, the cellulose used is prepared as follows: adding the raw materials into bagasse according to the absolute dry mass volume ratio of 1 g: 10mL of ethanol aqueous solution of 60% (v/v) was added, and 0.025mol/L of ethanol aqueous solution was added_{Ethanol aqueous solution}CuCl of₂Reacting at 160 ℃ in a high-pressure reaction kettle, stopping heating when the reaction time reaches 10 minutes, immediately condensing with water to reduce the reaction temperature to room temperature, and separating out the pretreatment residues by adopting a vacuum filtration method. The pretreated residue was used for subsequent cellulase hydrolysis tests.

And measuring and analyzing the glucose content in the enzymolysis liquid by using a high performance liquid chromatography.

The enzyme activity of the xylanase is 10000U/mg.

The amount of addition is in mg/g (in absolute terms) of the pretreated residue.

Example 1

2 g (in absolute dry basis) of the pretreatment residue was taken, and 10FPU/g of cellulase (Central second generation), 30mg/g of Tween 80 additive and 100mL of acetic acid-sodium acetate buffer solution with pH 4.8 were added to carry out enzymolysis. The temperature is controlled at 50 ℃ in the enzymolysis process, and the rotating speed is 150 r/min. And (3) taking 1mL of sample after 24 hours, 48 hours and 72 hours of enzymolysis, inactivating, measuring the glucose concentration in the enzymolysis solution by using a high performance liquid phase, and calculating to obtain the glucose yields of 84.06%, 92.17% and 92.32% respectively.

Example 2

The method of example 1 is different in that the additive added during the enzymolysis process is 40mg/g tea saponin, 1mL of samples are taken after 24 hours, 48 hours and 72 hours of enzymolysis and are subjected to inactivation treatment, the glucose concentration in the enzymolysis solution is measured by using a high performance liquid, and the calculated glucose yields are 87.88%, 92.18% and 93.53% respectively.

Example 3

The method of example 1 was identical except that 40mg/g BSA was added as an additive during the enzymatic hydrolysis, 1mL of the sample was taken after 24 hours, 48 hours and 72 hours of the enzymatic hydrolysis and subjected to inactivation treatment, and the glucose concentration in the enzymatic hydrolysate was measured by high performance liquid chromatography, and the glucose yields were calculated to be 88.15%, 92.16% and 93.09%, respectively.

Example 4

The method of example 1 was different in that 1.2mg/g of xylanase was added as an additive during the enzymatic hydrolysis, 1mL of the sample was taken and inactivated after 24 hours, 48 hours and 72 hours of enzymatic hydrolysis, and glucose concentrations in the enzymatic hydrolysate were measured by high performance liquid chromatography, and glucose yields were calculated to be 65.35%, 82.02% and 86.51%, respectively.

Example 5

The method is the same as the method of example 1, except that the additives added in the enzymolysis process are 10mg/g of Tween, 40mg/g of tea saponin, 40mg/g of BSA and 0.8mg/g of xylanase, 1mL of samples are taken out and subjected to inactivation treatment after the enzymolysis is carried out for 24 hours and 48 hours, the glucose concentration in the enzymolysis liquid is measured by using a high performance liquid, and the calculated glucose yields are 84.41% and 92.58% respectively.

Example 6

The method is the same as the method of example 1, except that the additives added in the enzymolysis process are 10mg/g of Tween, 60mg/g of tea saponin, 60mg/g of BSA and 1.2mg/g of xylanase, 1mL of samples are taken out and subjected to inactivation treatment after the enzymolysis is carried out for 24 hours and 48 hours, the glucose concentration in the enzymolysis liquid is measured by using a high performance liquid, and the calculated glucose yields are 85.27% and 93.58% respectively.

Example 7

The method is the same as the method of example 1, except that 30mg/g of tween, 20mg/g of tea saponin, 40mg/g of BSA and 1.2mg/g of xylanase are added during the enzymolysis, 1mL of sample is taken out and subjected to inactivation treatment after the enzymolysis is carried out for 24 hours and 48 hours, the glucose concentration in the enzymolysis solution is measured by using a high performance liquid, and the calculated glucose yield is 85.72% and 93.23% respectively.

Example 8

The method is the same as the method of example 1, except that 30mg/g of tween, 40mg/g of tea saponin, 60mg/g of BSA and 0.4mg/g of xylanase are added during the enzymolysis, 1mL of sample is taken out and subjected to inactivation treatment after the enzymolysis is carried out for 24 hours and 48 hours, the glucose concentration in the enzymolysis solution is measured by using a high performance liquid, and the calculated glucose yield is 85.52% and 94.08% respectively.

Example 9

The method is the same as the method of example 1, except that 30mg/g of tween, 60mg/g of tea saponin, 20mg/g of BSA and 0.8mg/g of xylanase are added during the enzymolysis, 1mL of sample is taken out and subjected to inactivation treatment after the enzymolysis is carried out for 24 hours and 48 hours, the glucose concentration in the enzymolysis solution is measured by using a high performance liquid, and the calculated glucose yield is 85.13% and 93.96% respectively.

Example 10

The method is the same as the method of example 1, except that 30mg/g of tween, 20mg/g of tea saponin, 60mg/g of BSA and 0.8mg/g of xylanase are added during the enzymolysis, 1mL of sample is taken out and subjected to inactivation treatment after the enzymolysis is carried out for 24 hours and 48 hours, the glucose concentration in the enzymolysis solution is measured by using a high performance liquid, and the calculated glucose yield is 87.32% and 93.32% respectively.

Example 11

The method is the same as the method of example 1, except that the additives added in the enzymolysis process are 50mg/g of Tween, 40mg/g of tea saponin, 20mg/g of BSA and 1.2mg/g of xylanase, 1mL of samples are taken out and subjected to inactivation treatment after the enzymolysis is carried out for 24 hours and 48 hours, the glucose concentration in the enzymolysis liquid is measured by using a high performance liquid, and the calculated glucose yields are 86.91% and 93.83% respectively.

Example 12

The method is the same as the method of example 1, except that the additives added in the enzymolysis process are 50mg/g of Tween, 60mg/g of tea saponin, 40mg/g of BSA and 0.4mg/g of xylanase, 1mL of samples are taken out and subjected to inactivation treatment after the enzymolysis is carried out for 24 hours and 48 hours, the glucose concentration in the enzymolysis liquid is measured by using a high performance liquid, and the calculated glucose yields are 86.61% and 93.27% respectively.

Example 13

The method is the same as the method of example 1, except that the additives added in the enzymolysis process are 10mg/g of Tween, 20mg/g of tea saponin, 20mg/g of BSA and 0.4mg/g of xylanase, 1mL of samples are taken out and subjected to inactivation treatment after the enzymolysis is carried out for 24 hours and 48 hours, the glucose concentration in the enzymolysis liquid is measured by using a high performance liquid, and the calculated glucose yields are 80.05% and 92.31% respectively.

Example 14

The method is the same as the method of example 1, except that 30mg/g of tween, 40mg/g of tea saponin, 60mg/g of BSA and 1.2mg/g of xylanase are added during the enzymolysis, 1mL of sample is taken out and subjected to inactivation treatment after the enzymolysis is carried out for 24 hours and 48 hours, the glucose concentration in the enzymolysis solution is measured by using a high performance liquid, and the calculated glucose yield is 89.55% and 96.46% respectively.

Comparative example 1

The method is the same as the method of example 1, except that 10FPU/g cellulase (Cetiric II) is added in the enzymolysis process for enzymolysis, 1mL of sample is taken out and inactivated after 24 hours, 48 hours and 72 hours of enzymolysis, the glucose concentration in the enzymolysis liquid is measured by high performance liquid, and the calculated glucose yield is 65.46%, 85.03% and 89.53% respectively.

And determining the glucose concentration by adopting high performance liquid chromatography and calculating the glucose yield for the enzymolysis solutions in the cases of 24 hours, 48 hours and 72 hours in the cases of the examples 1 to 4 and the comparative example 1 and the enzymolysis solutions in the cases of 24 hours and 48 hours in the cases of 5 to 14. Specific results are shown in table 1:

by comparing the examples with the comparative examples, it can be seen that:

1) as can be seen by comparing examples 1-4 with comparative example 1, under the same conditions, the yield of glucose in the enzymolysis stage can be greatly improved by independently adding Tween 80, tea saponin and BSA in the pretreatment stage, and the yield of glucose in the enzymolysis stage cannot be improved by independently adding xylanase, so that the enzymolysis effect is slightly reduced.

2) Compared with the comparative examples 1-4, in the examples 5-14, the tween 80, the tea saponin, the BSA and the xylanase are added in a compounding way according to a certain proportion, and when the tween 80, the tea saponin and the BSA are added separately in the front stage (24 hours) of enzymolysis, the yield of the enzymolysis glucose is not obviously different; but in the later stage of enzymolysis, cellulose is further consumed in a reaction system, and under the condition that the enzymolysis reaction is inhibited to a certain extent by glucose generated by enzymolysis, the yield of glucose is further improved, and the yield of 48h glucose subjected to enzymolysis in examples 5-14 is equivalent to 72h glucose subjected to enzymolysis in examples 1-4, and is even higher. This shows that the addition of xylanase can further improve the enzymolysis efficiency and the yield of glucose. Possibly related to the unexpected protection effect of xylanase on cellulase, and provides possibility for the enzymatic hydrolysate to be used for cellulase hydrolysis again.

3) Comparing examples 5-14, it can be seen that the composition of the compound enzymolysis accelerant has a certain influence on the enzymolysis rate in the early stage (24 h) of enzymolysis, and the effect of example 13 is the worst, but the enzymolysis effect in the later stage has no significant difference. Example 14 has the best enzymolysis efficiency in the whole enzymolysis process, and the glucose yield of 24h and 48h of enzymolysis is respectively as high as 89.55 percent and 96.46 percent, and the unexpected effect is achieved.

The foregoing is a more detailed description of the invention and is not to be taken in a limiting sense. It will be apparent to those skilled in the art that simple deductions or substitutions without departing from the spirit of the invention are within the scope of the invention.