CN112226423A - Method for improving cellulase recovery rate by successive adsorption of low-solid-content substrate - Google Patents
Method for improving cellulase recovery rate by successive adsorption of low-solid-content substrate Download PDFInfo
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- 108010059892 Cellulase Proteins 0.000 title claims abstract description 60
- 229940106157 cellulase Drugs 0.000 title claims abstract description 60
- 238000001179 sorption measurement Methods 0.000 title claims abstract description 50
- 238000000034 method Methods 0.000 title claims abstract description 30
- 238000011084 recovery Methods 0.000 title claims abstract description 26
- 239000000758 substrate Substances 0.000 title claims abstract description 25
- 239000000243 solution Substances 0.000 claims abstract description 22
- 238000002474 experimental method Methods 0.000 claims abstract description 15
- 239000007853 buffer solution Substances 0.000 claims abstract description 13
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 12
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 12
- 239000001509 sodium citrate Substances 0.000 claims abstract description 12
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims abstract description 12
- 239000011259 mixed solution Substances 0.000 claims abstract description 10
- 238000000926 separation method Methods 0.000 claims abstract description 10
- 239000007788 liquid Substances 0.000 claims abstract description 9
- 239000001913 cellulose Substances 0.000 claims abstract description 7
- 229920002678 cellulose Polymers 0.000 claims abstract description 7
- 238000002156 mixing Methods 0.000 claims abstract description 3
- 239000007787 solid Substances 0.000 claims description 26
- 239000011521 glass Substances 0.000 claims description 20
- 238000011085 pressure filtration Methods 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 229920001410 Microfiber Polymers 0.000 claims description 11
- 239000003658 microfiber Substances 0.000 claims description 11
- 239000007791 liquid phase Substances 0.000 claims description 3
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 claims description 2
- 238000004064 recycling Methods 0.000 claims description 2
- 238000004043 dyeing Methods 0.000 claims 1
- 239000000706 filtrate Substances 0.000 abstract description 14
- 238000006243 chemical reaction Methods 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 5
- 230000005764 inhibitory process Effects 0.000 abstract description 2
- 238000003756 stirring Methods 0.000 abstract description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 10
- 238000001914 filtration Methods 0.000 description 10
- 238000004519 manufacturing process Methods 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 1
- 229920002488 Hemicellulose Polymers 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000036983 biotransformation Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000000446 fuel Substances 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 239000012978 lignocellulosic material Substances 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000002787 reinforcement Effects 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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Abstract
The invention discloses a method for improving the recovery and utilization rate of cellulase by successive adsorption of a low-solid-content substrate, which comprises the following steps: mixing the sodium citrate buffer solution and the cellulase solution to obtain a mixed solution containing cellulase; adding a fresh cellulose substrate, and performing an adsorption experiment to obtain a mixed solution after adsorption is finished; and (4) performing solid-liquid separation, and analyzing the concentration of the non-adsorbed protein in the filtrate obtained by separation. The method is simple to operate, can effectively reduce the stirring cost in the process and the substrate inhibition effect in the subsequent reaction, and greatly improves the recovery efficiency of the cellulase.
Description
Technical Field
The invention belongs to the technical field of lignocellulose enzymolysis and also belongs to the technical field of industrial biology, and particularly relates to a method for improving the cellulase recovery rate by successive adsorption of a low-solid-content substrate.
Background
The production of fuel ethanol from lignocellulosic materials rich in cellulose and hemicellulose is one of the most promising production routes for the development of green, sustainable energy. Cellulase is a catalyst in the process of converting lignocellulose into sugar platform compounds, however, due to the characteristics of low activity, high consumption, high cost and the like, cellulase becomes a main obstacle in the commercialization process of cellulosic ethanol. Most of the cellulase still has activity after the conversion, and can be recovered and recycled through various ways, thereby reducing the cost of enzyme. Therefore, the development and the reinforcement of the high-efficiency recycling process of the cellulase are beneficial to greatly reducing the cost of the cellulase, and become an important way for solving the problem.
The method for recovering the cellulase by the re-adsorption method has simple process and low equipment requirement, is suitable for large-scale operation and has wide industrial prospect. In the method of re-adsorption by using fresh materials, the existing reports almost all add substrates with the same mass as that of the first hydrolysis to recover free enzymes in the hydrolysate, complete re-adsorption, then carry out solid-liquid separation, further supplement buffer solution, and enter the second hydrolysis or synchronous saccharification and fermentation process. Due to the economics of ethanol distillation, substrate solids levels greater than 10% are required in biomass conversion. And a large amount of fresh materials are used for re-adsorbing the free enzymes, the solid-liquid separation process needs longer time and large energy consumption, and more importantly, more product sugar or ethanol can be adsorbed and carried into the second round of reaction, so that the second round of biotransformation is seriously inhibited. Therefore, the cellulase recovered from the low-solid-content substrate is used as a novel convenient enzyme recovery method, the operation convenience is greatly improved, and the method is expected to be applied to the actual industrial process.
Disclosure of Invention
[ problem to be solved ]
Aiming at the defects in the prior art, the invention aims to provide a simpler and more convenient and efficient cellulase recovery method, so as to improve the utilization rate of cellulase and reduce the cost of cellulase in the production process of bioethanol, and is expected to be used in the actual industrial process.
[ technical solution of the present invention ]
A method for improving the recovery and utilization rate of cellulase by successive adsorption of a low-solid-content substrate comprises the following operation steps:
the method comprises the following steps: mixing the sodium citrate buffer solution and the cellulase solution to obtain a mixed solution containing cellulase;
step two: adding a fresh cellulose substrate into the mixed solution obtained in the step one, and performing an adsorption experiment to obtain a mixed solution after adsorption is finished;
step three: and D, performing solid-liquid separation on the mixed liquid obtained in the step two, and using the liquid phase obtained by separation for non-adsorption protein concentration analysis.
Preferably, the preparation of the mixed solution containing cellulase in the first step is carried out as follows: a10-100 mM sodium citrate buffer solution having a pH of 4.8 and a 0.1-1 FPU/mL cellulase solution are added to a conical flask having a glass stopper.
Preferably, the preparation of the mixed solution after the adsorption in the second step is performed as follows: adding a fresh cellulose substrate Avicel PH-101 (the solid content is 1% -15%) into the solution, and carrying out an adsorption experiment in a reciprocating constant-temperature water bath oscillating table at the temperature of 4-25 ℃ and the rotation speed of 0-200 rpm for 30-180 min.
Preferably, in the solid-liquid separation in the third step, the solution obtained in the previous step is filtered by means of reduced pressure filtration through glass ultra-fiber filter paper (Whatman 1821-025), the solid is filtered, and the liquid phase is analyzed for the concentration of non-adsorbed protein by Coomassie brilliant blue staining method.
Preferably, the fresh cellulosic substrate having a solids content of 1% to 15% is added in a single feed or in successive feeds.
[ PROBLEMS ] the present invention
(1) The method for improving the recovery and utilization rate of the cellulase by successive adsorption of the low-solid-content substrate can effectively avoid the waste of the cellulase released into the hydrolysate and greatly improve the utilization efficiency of the cellulase;
(2) the operation of successive adsorption of the low-solid-content fresh substrate is favorable for reducing the stirring cost in the process and the substrate inhibition effect in the subsequent reaction;
(3) the method has the advantages of simple process, convenient operation and low cost, is easy to establish an economical and feasible cellulase recovery process, and has very important application prospect in the industrial production of ethanol.
Drawings
FIG. 1 is a schematic diagram of a process for improving cellulase recovery and utilization rate by successive adsorption of a low-solid-content substrate.
FIG. 2 is a graph showing the effect of cellulase recovery in examples 3, 6 and 7.
Detailed Description
The present invention will be further illustrated by the following specific examples.
The following examples are intended to enable those skilled in the art to better understand the present invention and are not intended to limit the claims in any way.
Example 1
Adding 10 mM sodium citrate buffer solution with the pH value of 4.8 and cellulase solution into a conical flask with a glass plug, wherein the concentration of the cellulase is 1FPU/mL, then adding Avicel PH-101 (the solid content is 10%) into the solution at one time, carrying out an adsorption experiment in a reciprocating constant-temperature water bath oscillating table with the temperature of 4 ℃ and no rotating speed, wherein the adsorption time is 30min, finally carrying out reduced-pressure filtration, filtering out solids through glass microfiber filter paper, reserving filtrate for subsequent non-adsorption protein concentration analysis, and calculating to obtain the cellulase recovery rate of 27.5%.
Example 2
Adding 100 mM sodium citrate buffer solution with the pH value of 4.8 and cellulase solution into a conical flask with a glass plug, wherein the concentration of cellulase is 0.1FPU/mL, then adding Avicel PH-101 (the solid content is 2%) into the solution, carrying out an adsorption experiment in a reciprocating constant temperature water bath oscillating table with the temperature of 25 ℃ and the rotating speed of 200rpm, carrying out reduced pressure filtration after 180min, filtering out solids through glass microfiber filter paper, and keeping filtrate for carrying out a second round of cellulase recovery; and adding Avicel PH-101 (solid content is 2%) into the obtained filtrate again, performing adsorption experiment for 120min in a reciprocating water bath oscillation table with the temperature of 25 ℃ and the rotation speed of 200rpm, then performing reduced pressure filtration, filtering out solids through glass microfiber filter paper, collecting the filtrate, performing subsequent non-adsorption protein concentration analysis, and calculating to obtain the cellulase recovery rate of 60.2%.
Example 3
Adding 50 mM sodium citrate buffer solution with the pH value of 4.8 and cellulase solution into a conical flask with a glass plug, wherein the concentration of the cellulase is 1FPU/mL, then adding Avicel PH-101 (the solid content is 4%) into the solution, carrying out an adsorption experiment in a reciprocating constant-temperature water bath oscillating table with the temperature of 25 ℃ and no rotating speed, carrying out reduced-pressure filtration after 120min, filtering out solids through glass microfiber filter paper, and keeping the filtrate for carrying out a second round of cellulase recovery; and adding Avicel PH-101 (solid content is 4%) into the obtained filtrate again, performing an adsorption experiment in a reciprocating constant-temperature water bath oscillating table with the temperature of 25 ℃ and no rotating speed for 120min, then performing reduced pressure filtration, filtering out solids through glass microfiber filter paper, finally collecting the filtrate for subsequent non-adsorption protein concentration analysis, and calculating to obtain the cellulase recovery rate of 71.58%, wherein the data result is shown in figure 2 (4 + 4).
Example 4
Adding 50 mM sodium citrate buffer solution with the pH value of 4.8 and cellulase solution into a conical flask with a glass plug, wherein the concentration of cellulase is 0.2 FPU/mL, then adding Avicel PH-101 (the solid content is 5%) into the solution at one time, carrying out an adsorption experiment in a reciprocating constant temperature water bath oscillating table with the temperature of 25 ℃ and the rotating speed of 50 rpm, wherein the adsorption time is 120min, finally carrying out reduced pressure filtration, filtering out solids through glass microfiber filter paper, keeping filtrate for subsequent non-adsorption protein concentration analysis, and calculating to obtain the cellulase recovery rate of 23.49%.
Example 5
Adding 50 mM sodium citrate buffer solution with the pH value of 4.8 and cellulase solution into a conical flask with a glass plug, wherein the concentration of the cellulase is 1FPU/mL, then adding Avicel PH-101 (the solid content is 10%) into the solution at one time, carrying out an adsorption experiment in a reciprocating constant-temperature water bath oscillating table with the temperature of 25 ℃ and the rotating speed of 150 rpm, wherein the adsorption time is 120min, finally carrying out reduced-pressure filtration, filtering out solids through glass microfiber filter paper, reserving the filtrate for subsequent non-adsorption protein concentration analysis, and calculating to obtain the cellulase with the recovery rate of 34.92%.
Example 6
Adding 50 mM sodium citrate buffer solution with the pH value of 4.8 and cellulase solution into a conical flask with a glass plug, wherein the concentration of the cellulase is 1FPU/mL, adding Avicel PH-101 (the solid content is 10%) into the solution at one time, carrying out an adsorption experiment in a reciprocating constant-temperature water bath oscillating table with the temperature of 25 ℃ and no rotating speed, wherein the adsorption time is 120min, finally carrying out reduced-pressure filtration, filtering out solids through glass microfiber filter paper, reserving filtrate, carrying out subsequent non-adsorption protein concentration analysis, and calculating to obtain the cellulase recovery rate of 58.84%. The data results are shown in fig. 2 (10).
Example 7
Adding 50 mM sodium citrate buffer solution with the pH value of 4.8 and cellulase solution into a conical flask with a glass plug, wherein the concentration of the cellulase is 1FPU/mL, then adding Avicel PH-101 (the solid content is 2%) into the solution, carrying out an adsorption experiment in a reciprocating constant-temperature water bath oscillating table with the temperature of 25 ℃ and no rotating speed, carrying out reduced-pressure filtration after 120min, filtering out solids through glass microfiber filter paper, and keeping the filtrate for carrying out a second round of cellulase recovery; and adding Avicel PH-101 (solid content is 2%) into the obtained filtrate again, performing adsorption experiment in a reciprocating constant-temperature water bath oscillating table with the temperature of 25 ℃ and no rotating speed for 120min, then performing reduced pressure filtration, filtering out solids through glass microfiber filter paper, collecting the filtrate, performing subsequent non-adsorption protein concentration analysis, and calculating to obtain the cellulase recovery rate of 67.38%. The data results are shown in fig. 2 (2 + 2).
Claims (5)
1. The method for improving the recovery and utilization rate of the cellulase by successive adsorption of the low-solid-content substrate is characterized by comprising the following operation steps of:
the method comprises the following steps: mixing the sodium citrate buffer solution and the cellulase solution to obtain a mixed solution containing cellulase;
step two: adding a fresh cellulose substrate into the mixed solution obtained in the step one, and performing an adsorption experiment to obtain a mixed solution after adsorption is finished;
step three: and D, performing solid-liquid separation on the mixed liquid obtained in the step two, and using the liquid phase obtained by separation for non-adsorption protein concentration analysis.
2. The method for improving the recovery and utilization rate of the cellulase through successive adsorption of the low-solid-content substrate according to claim 1, wherein in the first step, the concentration of the sodium citrate buffer solution is 10-100 mM, the pH value is 4.8, and the dosage of the cellulase is 0.1-1 FPU/mL.
3. The method for improving the cellulase recovery rate through successive adsorption of the low-solid-content substrate according to claim 1, wherein the solid content of the fresh cellulose substrate added in the second step is 1% -15%, the adsorption experiment is carried out in a reciprocating constant-temperature water bath oscillating table with the temperature of 4-25 ℃ and the rotation speed of 0-200 rpm, and the adsorption time is 30-180 min.
4. The method for improving the cellulase recovery and utilization rate by successive adsorption of a low-solid-content substrate according to claim 1, wherein the solid-liquid separation in the third step is to perform non-adsorption protein concentration analysis by passing the solution obtained in the second step through glass microfiber filter paper by means of reduced pressure filtration and adopting a Coomassie brilliant blue dyeing method.
5. The method for improving the cellulase recycling rate through successive adsorption of the low-solid-content substrate according to claim 3, wherein the fresh cellulose substrate with the solid content of 1% -15% is added in a one-time feeding mode or a batch-by-batch feeding mode.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974570A (en) * | 2010-09-09 | 2011-02-16 | 天津大学 | Method for recycling cellulose complete components in fuel ethanol production |
| CN105492615A (en) * | 2012-08-01 | 2016-04-13 | 因比肯公司 | Methods of processing lignocellulosic biomass using single-stage autohydrolysis pretreatment and enzymatic hydrolysis |
| CN107488651A (en) * | 2017-10-13 | 2017-12-19 | 西北农林科技大学 | A kind of method for recycling enzyme hydrolysis solid residue cellulase |
| CN109097406A (en) * | 2018-09-26 | 2018-12-28 | 天津科技大学 | A method of improving the cellulase rate of recovery |
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- 2020-11-08 CN CN202011235187.5A patent/CN112226423A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974570A (en) * | 2010-09-09 | 2011-02-16 | 天津大学 | Method for recycling cellulose complete components in fuel ethanol production |
| CN105492615A (en) * | 2012-08-01 | 2016-04-13 | 因比肯公司 | Methods of processing lignocellulosic biomass using single-stage autohydrolysis pretreatment and enzymatic hydrolysis |
| CN107488651A (en) * | 2017-10-13 | 2017-12-19 | 西北农林科技大学 | A kind of method for recycling enzyme hydrolysis solid residue cellulase |
| CN109097406A (en) * | 2018-09-26 | 2018-12-28 | 天津科技大学 | A method of improving the cellulase rate of recovery |
Non-Patent Citations (2)
| Title |
|---|
| 李强: "重吸附法回收利用纤维素酶的工艺优化", 《化学工程》 * |
| 杨静: "几种纤维素酶制剂水解和吸附性能的研究", 《林产化学与工业》 * |
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