CN1696158A - Inverse micelle method in use for degrading cellulose - Google Patents
Inverse micelle method in use for degrading cellulose Download PDFInfo
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Abstract
An anti-micellar method for degradating cellulose includes dissolving cellulase in buffering liquid, preparing the organic solution of surfactant, mixing them together, adding microcrystalline cellulse, phosphoric acid buffering liquid and extractant, strong waving, centrifugal layering, and taking the upper soluiton, laying to obtain glucose solution.
Description
Technical field:
The invention belongs to the field of bioenergy technological development and industrialization, specifically, the present invention relates to a kind of inverse micelle method that is used for degraded cellulose.This method is applied to cellulase catalytic hydrolysis Mierocrystalline cellulose with reverse micelle, and this has the biological enzyme reaction of important application prospect, product glucose then further is processed as products such as feed, grain, chemical feedstocks, microbial proteinous, amino acid, microbiotic, thereby the pollution-free novel process of the biological degradation of developing waste cellulose realizes the comprehensive utilization of cellulose series biomass (as agricultural crop straw etc.).This method overcomes the defective of traditional enzyme hydrolysis method effectively, makes cellulose degradation efficient greatly improve.
Background technology:
Reverse micelle is a tensio-active agent at non-polar organic solvent is aggregate in the solution of body, has polar inner chamber and nonpolar shell.A critical nature of reverse micelle is can a certain amount of water molecules of solubilising (being called core water body or " pond "), but water miscible enzyme molecule again solubilising in " pond " of reverse micelle.Because " pond " in the reverse micelle is the nano level space, is the nano material that reacting field can synthesize 1～100nm with this space, therefore be referred to as nano-reactor again.The enzymology that is fixed in the reverse micelle is that medium engineering has brought new revolution.Utilize reverse micelle to coat enzyme is dissolved in the polarity " pond ", and some water-insoluble compounds then are dissolved in the non-polar organic solvent as cholesterol, alkaloid, lipid etc., transform as enzymatic substrate.Utilize the specificity of enzyme effect, may realize that also the chirality of biocatalysis is synthetic.Reverse micelle system is as the enzyme reaction medium, have form flexibly, Thermodynamically stable, interfacial area are big, can therefore be subjected to people's common concern in recent years by regulating advantages such as realizing the product recovery mutually.Wherein studying maximum is the catalyzed reaction of the synthetic and lipase of peptide.Study the reaction of the synthetic peptide derivant of α 2 chymotrypsin protein enzyme catalysiss in the reverse micelle system of AOT/ octane as Xing etc., obtained 56%～88% productive rate; Surfactant concentration was to the influence of CabdidaRngosa lipase (CRL) hydrolysis vigor when Tsai etc. had studied lipase-catalyzed fat hydrolysis reaction in AOT/ octane-iso/phosphoric acid buffer; Chen etc. have synthesized dipeptides with different proteolytic enzyme in reverse micelle system.Along with deepening continuously of research, the catalytic synthesis that carries out enzyme in reverse micelle system is not only aspect theoretical, and has broad prospects equally in application and development.
Enzyme molecule in the nano-reactor often shows superactivity.On structure, reverse micelle extremely is similar to biomembranous Lipid Bilayer Structure, when using hydrophobic substrate (as Mierocrystalline cellulose), people can study substrate molecule and be distributed in enzyme kinetics in nano-reactor and the organic phase, simultaneously because the huge specific surface of reverse micelle and the very-short-reach between its polarity inner chamber and nonpolar shell, and the collision frequency between the reverse micelle particulate is high, rate of mass transfer therebetween is exceedingly fast, for the effect of enzyme-to-substrate provides best microenvironment, thereby can effectively improve the catalytic efficiency of cellulase and the yield of end products glucose.The reverse micelle technology successfully has been applied to study Tetrahydrofolate dehydrogenase, calcium ion-ATP enzyme, β-lactalase and glucose oxidase etc. recently.
Mierocrystalline cellulose is a recyclability natural resources the abundantest on the earth, and the complete utilization of lignocellulosic material biomass will be to one of of paramount importance biotechnology of the mankind undoubtedly in new thousand.Human to cellulosic utilization, mainly be limited to timber directly as material of construction up to now or be processed into paper, fiber etc., its waste is then as compost or almost use burning disposal.American Chemical Society thinks that unique chemical feedstocks that can replace oil in the future is exactly the annual waste cellulose that surpasses 200,000,000 tons.Simultaneously because China is a large agricultural country; the annual production of agricultural production waste cellulose (as straw, straw etc.) is very huge; therefore actively develop the biological Research on degradation of Mierocrystalline cellulose the development of alleviating human grain and energy dilemma, the natural resource regeneration of acceleration China is significant, in the foundation of the control of environmental pollution and the optimum ecosystem, also can play a significant role simultaneously.
The means that Mierocrystalline cellulose are decomposed into glucose have acid-hydrolysis method and traditional enzyme hydrolysis method.Acid-hydrolysis method needs high temperature, peracid condition and acidproof pressure vessel, though the yield of glucose can reach 50%, serious environment pollution is so abandoned now.Tradition enzymic hydrolysis rule has overcome many deficiencies of acid-hydrolysis method.Natural cellulose is the mechanism of glucose by cellulase system (comprising endoglucanase, exocellobiohydrolase and beta-glucosidase) catalytic hydrolysis in the aqueous solution, still not fully aware of at present, but it is generally acknowledged to be oligose such as cellobiose with the Mierocrystalline cellulose catalytic decomposition at first, by beta-glucosidase these oligose are hydrolyzed to glucose then by the above two.Though cellulosic enzyme hydrolysis method has carried out much quite fruitful research, but Mierocrystalline cellulose is still realized at the end with the pollution-free novel process that technical scale converts glucose to, this mainly is because natural cellulose is a hydrophobic substrate, and the relatively poor and yield that causes glucose in traditional enzyme hydrolysis method of the solubleness in the aqueous solution is less than 50% still.
In the Chinese patent net, the patent that relates to reverse micelle is less, has 10, wherein has 8 to be content with preparation nano level chemical substance.Totally 47 of patents that relate to the biological degraded of Mierocrystalline cellulose, but these patents none relate to this project---the novel method of Mierocrystalline cellulose efficient degradation in the reverse micelle system.China patent CN02289267 " Mierocrystalline cellulose solid phase enzymolysis---liquid fermenting coupling preparation alcoholic acid device ", this device can reduce in the enzymolysis solution carbohydrate to cellulosic restraining effect, thereby help industrial production ethanol; China patent CN97125800 " method for preparing nanometer silicon dioxide particle from alkali-metal silicate " relates to the novel method for preparing nanometer silicon dioxide particle in reverse micelle.It is 200 multinomial that the patent that relates to reverse micelle abroad has, and the preparation of main concentrated chemical material is synthetic, the extracting and separating of biomolecules, and the preparation of dissimilar reverse micelle systems, and some are also arranged is method research to chemical reaction in the reverse micelle.U.S. Pat 2004009117 " Preparation of nanosizedcopper (I) compounds " relates to preparation nano copper compound particulate novel method in the reverse micelle.United States Patent (USP) 03190471 title is " Nanoparticle manganese zinc ferrites synthesized using reverse micelles ", has reported a kind of novel method for preparing ferrous acid zinc-manganese nano-scale particle in reverse micelle.World patent WO03051333 title is " Reverse-micellar delivery system for controlledtransportation and enhanced absorption of agents ", this patent relates to utilizes reverse micelle system as a kind of transmembrane transport system, thereby improves the intestinal absorption function.The degraded of macromolecular substance still belongs to blank field in the reverse micelle system at present.
Summary of the invention:
The objective of the invention is to be to provide a kind of inverse micelle method that is used for degraded cellulose, this method is simple and easy to operate, and the enzyme demand is few, easy to use efficient.The reverse micelle environment that helps cellulase hydrolysis has improved cellulosic degradation efficiency and product pick-up rate, and the reaction efficiency height, production process safety, the degraded with low cost.
In order to realize above-mentioned task, the present invention is by the following technical solutions: the reverse micelle system of the present invention's design can (be the core water body of reverse micelle system in " pond " that comprise, by a certain amount of water molecules of reverse micelle solubilising is formed) in add cellulase (a kind of multi-component prozyme, mainly contain 3 kinds of components: i.e. endo-type beta-glucanase, circumscribed-type beta-glucan cellobiohydrolase and beta-glucosidase), in system, add Mierocrystalline cellulose.Cellulase combines with Mierocrystalline cellulose on the basis of maintenance even enhanced activity and degrades releasing product glucose.Product is dissolved in " pond ", can obtain dispersion liquid from system by method of extraction, then therefrom separates obtaining.Utilization the invention provides reverse micelle system and carries out cellulosic enzymolysis, can be in the short period of time (10 minutes～24 hours) a large amount of degraded celluloses also obtain product glucose, and traditional relatively acid hydrolysis or the enzymolysis isotype in the water solution system are convenient, efficient.
The reverse micelle system of the present invention design by tensio-active agent (as Triton X-100, AOT etc.) be dissolved in spontaneously in the organic solvent (as dimethylbenzene or n-hexyl alcohol or octane-iso etc.) form, thermodynamically stable, optically transparent sphere aggregates (as shown in Figure 1).Its characteristics are that reverse micelle has a shell of being made up of the hydrocarbon chain of surfactant molecule (as Triton X-100, AOT etc.), and hydrophobic tail points to organic solvent (as dimethylbenzene or n-hexyl alcohol or octane-iso etc.), and polar head points to the inner polarity chamber that forms of aggregate.Water molecules forms the pond of nano-scale in the polarity chamber.
Phosphoric acid buffer in the present invention's design is citric acid-Sodium phosphate dibasic damping fluid, SODIUM PHOSPHATE, MONOBASIC-Sodium phosphate dibasic damping fluid or potassium primary phosphate-dipotassium hydrogen phosphate damping fluid.
The invention provides a kind of inverse micelle method of degraded cellulose, it contains the following step:
Under A, the room temperature (20～25 ℃), the buffered soln that contains cellulase (A) of configuration pH5.2～6.4, wherein the concentration of cellulase is 30～900 μ M;
B, outfit contain the organic solution (B) of tensio-active agent, and wherein surfactant concentrations is 0.01～0.20M; Tensio-active agent is polyoxyethylene alkyl phenyl ether (Triton X-100) or 2-ethylhexyl sodium succinate (AOT); Organic solvent is dimethylbenzene or n-hexyl alcohol or octane-iso;
C, with an amount of solution A and solution B uniform mixing, make that the concentration of Cellulase Molecules in mixed solution is 0.15～4.5 μ M, Cellulase Molecules is contained in the pond of reverse micelle system of formation, forms solution C;
D, left standstill 15～25 seconds, treat that solution C forms the transparent system of stable homogeneous after, in the ratio of 0.9～10g/l to wherein adding Microcrystalline Cellulose.Described Microcrystalline Cellulose is fully degraded after 10 minutes～24 hours 30～60 ℃ of reactions;
E, add 10 times to the phosphoric acid buffers of solution A volume and with the isopyknic extraction agent of solution C (chloroform), with the rotating speed high speed centrifugation of 3000～4000rpm 5～10 minutes, make the solution layering behind the thermal agitation.Take out upper solution, be the degraded product glucose solution.
On structure, reverse micelle extremely is similar to biomembranous lipid bilayer, simultaneously because the huge specific surface of reverse micelle and the very-short-reach between its polarity inner chamber and nonpolar shell, and the collision frequency between the reverse micelle particulate is high, rate of mass transfer therebetween is exceedingly fast, this effect for enzyme-to-substrate provides best microenvironment, thereby can effectively improve the efficient of catalytic efficiency and the cellulose degradation and the energy of cellulase.This method is applied to cellulase catalytic hydrolysis Mierocrystalline cellulose with reverse micelle, and this has the biological enzyme reaction of important application prospect, product glucose then further is processed as products such as feed, grain, chemical feedstocks, microbial proteinous, amino acid, microbiotic, thereby the pollution-free novel process of the biological degradation of developing waste cellulose realizes the comprehensive utilization of cellulose series biomass (as agricultural crop straw etc.).This method production process equipment is simple, has the favorable industrial prospect.The experimental result explanation of Fig. 2, with (T=50 ℃ of the same terms, pH=6.0) result of cellulase hydrolysis compares in the following aqueous solution, the cellulase hydrolysis efficient that reverse micelle system especially carries out in the Triton X-100 reverse micelle system has obtained significant raising, and the concentration of product glucose solution has improved more than 30 times.Experiment showed, that the present invention can overcome the defective of traditional enzyme hydrolysis method effectively, makes cellulose degradation efficient greatly improve.In addition, each organic constituent of the reverse micelle system that uses in this method can be reused by the rectifying recovery technology, thereby reduces environmental pollution, and has further reduced cost.
Description of drawings:
Fig. 1 is the reverse micelle system structural representation.A is the pond of the nano-scale that forms in reverse micelle polarity inner chamber of water molecules among the figure; B is organic phase (as dimethylbenzene or n-hexyl alcohol or an octane-iso etc.); C is single surfactant molecule (as Triton X-100, AOT etc.);
Fig. 2 be cellulase hydrolysis after product glucose solution in Triton X-100 and the two kinds of reverse micelle systems of AOT and the aqueous solution concentration ratio, T-shape is designated error among the figure.As seen from Figure 2, with the same terms (T=50 ℃, pH=6.0) down in the aqueous solution result of cellulase hydrolysis compare, the cellulase hydrolysis efficient that reverse micelle system especially carries out in the Triton X-100 reverse micelle system has obtained significant raising.
Embodiment:
Below in conjunction with specific embodiment, further set forth the present invention.Unreceipted concrete experiment condition and method in the following example, usually according to normal condition as: Zhao Yongfang chief editor, press of Wuhan University, 2002, Measurement for Biochemistry principle and application thereof (third edition); Zhou Keyan, the height chief editor that takes the lead, Higher Education Publishing House, 1997, Experiment of Organic Chemistry (third edition); Chen Yuquan, Science Press, 2002, Biochemistry Experiment method and technology.
Embodiment 1: the method for utilizing the reverse micelle degraded Microcrystalline Cellulose of surfactant A OT formation
1. (25 ℃) under the room temperature, the cellulase buffered soln (preparation program of damping fluid is referring to Measurement for Biochemistry principle and application thereof) of preparation 1ml pH6.0, concentration is 380 μ M, is designated as solution A;
2. get 0.15mmol 2-ethylhexyl sodium succinate (AOT) and be dissolved in the 3ml octane-iso, stand for standby use behind the thermal agitation is designated as solution B;
3. from solution A, get 14 μ l and mix, left standstill behind the thermal agitation 20 seconds with solution B, treat its transparent and homogeneous after, be the reverse micelle system that includes Cellulase Molecules;
4. above-mentioned system is added the 10mg Microcrystalline Cellulose in insulation under 50 ℃ of conditions after 10 minutes, and fully reaction 10 minutes in the constant temperature shaking table (50 ℃, 220rpm);
5. after this add 140 μ l phosphoric acid buffers and 3ml chloroform, with the rotating speed high speed centrifugation of 3000rpm 5 minutes, make the solution layering behind the thermal agitation.The upper strata water white transparency is product glucose solution (this step is an extracting operation, referring to Biochemistry Experiment method and technology);
6. measure the decomposed solution of product glucose with sulfuric acid anthrone method (referring to Biochemistry Experiment method and technology), obtain the glucose content in the product solution, simultaneously with the same terms (T=50 ℃, pH=6.0) down in the aqueous solution result of cellulase hydrolysis compare, the result is as shown in Figure 2.The cellulase hydrolysis efficient of carrying out in the AOT reverse micelle system as can be seen from Figure 2, will be much higher than the enzyme digestion reaction efficient of carrying out in the aqueous solution.Detailed data as shown in the table (T=50 ℃, pH=6.0):
| Reaction system | The AOT reverse micelle system | Water solution system |
| Glucose concn (μ g/ml) | ????4.54 | ????0.83 |
Embodiment 2: the method for utilizing the reverse micelle degraded Microcrystalline Cellulose of tensio-active agent Triton X-100 formation
1. (25 ℃) under the room temperature, the cellulase buffered soln (preparation program of damping fluid is referring to Measurement for Biochemistry principle and application thereof) of preparation 1ml pH6.0, concentration is 400 μ M, is designated as solution A;
2. get 0.15mmol polyoxyethylene alkyl phenyl ether (Triton X-100) and be dissolved in the 1ml dimethylbenzene, add the 2ml n-hexyl alcohol again, stand for standby use behind the thermal agitation is designated as solution B;
3. from solution A, get 18 μ l and mix, left standstill behind the thermal agitation about 15 seconds with solution B, treat its transparent and homogeneous after, be the reverse micelle system that includes the enzyme molecule;
4. system is added the 10mg Microcrystalline Cellulose in insulation under 50 ℃ of conditions after 10 minutes, and fully reaction 10 minutes in the constant temperature shaking table (50 ℃, 220rpm);
5. after this add 180 μ l phosphoric acid buffers and 3ml chloroform, with the rotating speed high speed centrifugation of 3500rpm 8 minutes, make the solution layering behind the thermal agitation.The upper strata water white transparency is product glucose solution (this step is an extracting operation, referring to Biochemistry Experiment method and technology);
6. measure the decomposed solution of product glucose with sulfuric acid anthrone method (referring to Biochemistry Experiment method and technology), obtain the glucose content in the decomposed solution, simultaneously with the same terms (T=50 ℃, pH=6.0) down in the aqueous solution result of cellulase hydrolysis compare, the result is as shown in Figure 2.As can be seen from Figure 2, the cellulase hydrolysis efficient of carrying out in reverse micelle system especially Triton X-100 reverse micelle system will be much higher than the enzyme digestion reaction efficient of carrying out in the aqueous solution.Detailed data as shown in the table (T=50 ℃, pH=6.0):
| Reaction system | Triton X-100 reverse micelle system | Water solution system |
| Glucose concn (μ g/ml) | ??28.63 | ????0.83 |
Claims (1)
1, a kind of inverse micelle method that is used for degraded cellulose is characterized in that it contains the following step:
Under A, the room temperature, the buffered soln (A) of cellulase is contained in preparation pH5.2～6.4, and wherein the concentration of cellulase is 30～900 μ M;
B, outfit contain the organic solution (B) of tensio-active agent, and wherein surfactant concentrations is 0.01～0.20M; Tensio-active agent is polyoxyethylene alkyl phenyl ether or 2-ethylhexyl sodium succinate; Organic solvent is dimethylbenzene or n-hexyl alcohol or octane-iso;
C, with solution A and solution B uniform mixing, make that the concentration of Cellulase Molecules in mixed solution is 0.15～4.5 μ M, Cellulase Molecules is contained in the pond of reverse micelle system of formation, forms solution C;
D, left standstill 15～25 seconds, treat that solution C forms transparent system after, to wherein adding Microcrystalline Cellulose, described Microcrystalline Cellulose is fully degraded after 1～24 hour 30～60 ℃ of reactions in the ratio of 0.9～10g/l;
E, 10 times of phosphoric acid buffers to the solution A volume of adding reach and the isopyknic chloroform of solution C, and vibration back centrifugal 5～10 minutes with the rotating speed of 3000～4000rpm makes the solution layering, and the taking-up upper solution is the degraded product glucose solution.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101319244B (en) * | 2008-07-11 | 2011-05-04 | 重庆大学 | Method of preparing glutathione freeze-dried powder |
| CN101695645B (en) * | 2009-11-09 | 2012-03-14 | 湖南大学 | Reverse micelle as well as preparation method and application to cellulose enzymolysis thereof |
| CN109338781A (en) * | 2018-10-12 | 2019-02-15 | 山东金胜粮油集团有限公司 | A kind of steam blasting combines the high-pressure homogeneous method for preparing peanut shell nano-cellulose |
| CN113350381A (en) * | 2021-06-07 | 2021-09-07 | 浙江爱诺生物药业股份有限公司 | Method for improving protein ratio of pearl hydrolysate and treatment solution |
-
2005
- 2005-06-17 CN CN 200510018944 patent/CN1272444C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101319244B (en) * | 2008-07-11 | 2011-05-04 | 重庆大学 | Method of preparing glutathione freeze-dried powder |
| CN101695645B (en) * | 2009-11-09 | 2012-03-14 | 湖南大学 | Reverse micelle as well as preparation method and application to cellulose enzymolysis thereof |
| CN109338781A (en) * | 2018-10-12 | 2019-02-15 | 山东金胜粮油集团有限公司 | A kind of steam blasting combines the high-pressure homogeneous method for preparing peanut shell nano-cellulose |
| CN113350381A (en) * | 2021-06-07 | 2021-09-07 | 浙江爱诺生物药业股份有限公司 | Method for improving protein ratio of pearl hydrolysate and treatment solution |
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| CN1272444C (en) | 2006-08-30 |
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