CN105462947A - Method for producing cellulose through duckweed - Google Patents
Method for producing cellulose through duckweed Download PDFInfo
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- CN105462947A CN105462947A CN201511029774.8A CN201511029774A CN105462947A CN 105462947 A CN105462947 A CN 105462947A CN 201511029774 A CN201511029774 A CN 201511029774A CN 105462947 A CN105462947 A CN 105462947A
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- C—CHEMISTRY; METALLURGY
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
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Abstract
The invention discloses a method for producing cellulose through duckweed. Cellulose is produced through fermentation with duckweed or processed duckweed residuals as the substrate. Duckweed has the advantages of being easy to obtain, wide in resource, low in price and the like, and has wide market prospects for industrially inducing production of cellulose.
Description
Technical field
The present invention relates to the production technical field of cellulase.More particularly, the present invention relates to a kind of method utilizing duckweed production of cellulose enzyme.
Background technology
Ligno-cellulosic materials be abundant on the earth can in production-goods source, it can be broken down into the monose such as glucose, wood sugar and be utilized bio-based chemical such as producing ethanol further.At present, although the method that the agricultural wastes such as stalk produce ethanol through cellulase degradation extensively admitted, the restriction that stover is several key factor in bio-based chemical process is subject to.Cellulase hydrolysis efficiency is low, production cost height is one of restraining factors.
The production method of cellulase mainly adopts ligno-cellulosic materials to do substrate fermentable, and the Nomenclature Composition and Structure of Complexes of ligno-cellulosic materials affects its hydrolysis efficiency, thus affects the production of cellulase.The lignocellulose substrate of the cellulase induction of report has been had to have corn cob, maize straw, straw, wheat bran etc. in research.They are all containing the xylogen that difference is higher, and compact structure, be difficult to be hydrolyzed utilization.Also have researchist to prepare the inductors such as sophorose, add at fermention medium and carry out the synthesis of cellulase induction, but due to the complicated process of preparation of sophorose, cost is higher, produces be difficult to apply at cellulose fermentation.Therefore a kind of content of lignin is screened less, short texture, can efficient cellulase induction, the substrate that can be efficiently utilized the while of cheap is necessary for the fermentative production of cellulase.
Summary of the invention
An object of the present invention is to solve at least the problems referred to above, and the advantage will illustrated at least is below provided.
A further object of the invention is to provide a kind of method utilizing duckweed production of cellulose enzyme, method of the present invention utilizes duckweed can efficiently induce trichoderma strain to produce Mierocrystalline cellulose, duckweed easily obtains and cheap, can realize low-cost industrial production of cellulose enzyme.
A further object of the invention is to provide a kind of method of duckweed being carried out to pre-treatment, by adopting the pectin composition in sodium carbonate removal duckweed, adopt the starch in amylase and Glucoamylase hydrolysis duckweed, Mierocrystalline cellulose containing high level in duckweed residue and hemicellulose, output and the enzyme that can improve its induction generation cellulase are lived.
In order to realize, according to these objects of the present invention and other advantage, providing a kind of method utilizing duckweed production of cellulose enzyme, duckweed is adopted to be fermenting substrate production of cellulose enzyme.
Preferably, the described method utilizing duckweed production of cellulose enzyme, described duckweed comprises one or more the combination in thready pulse duckweed, little duckweed, blue or green duckweed and duckweed spirodela.
Preferably, the described method utilizing duckweed production of cellulose enzyme, be that substrate prepares seed culture medium and fermention medium with duckweed, wherein in seed culture medium, its addition is 20g/L, and in fermention medium, its addition is 30-50g/L.
Preferably, the described method utilizing duckweed production of cellulose enzyme, before being fermenting substrate production of cellulose enzyme with duckweed, pre-treatment is carried out to duckweed, the concrete mode of described pre-treatment comprises the combination of one or more in physics mode, chemical mode or biological mode, described physics mode comprises pulverizing, grinding or heating, and described chemical mode comprises acid treatment, alkaline purification or free radical process, and described biological mode comprises ferment treatment.
Preferably, the described method utilizing duckweed production of cellulose enzyme, described pre-treatment is specially: duckweed is pulverized to obtain powder, get the citrate buffer solution that 2g duckweed powder is placed in 20mlpH6, add amylase 300u, saccharifying enzyme 150u and 0.106g sodium carbonate, be placed in shaking bath, rotation frequency is 100rpm, after 50 DEG C of concussion hydrolysis 48h, take out after filtering and obtain residue, washing is placed in the air dry oven of 60 DEG C dry.
Preferably, the described method utilizing duckweed production of cellulose enzyme, the bacterial classification that fermentation adopts is that wood is mould.
Preferably, the described method utilizing duckweed production of cellulose enzyme, the bacterial classification that fermentation adopts is Trichodermareesei.
The present invention at least comprises following beneficial effect: the present invention adopts duckweed to be substrate for induction production of cellulose enzyme first, duckweed easily obtains, and it is cheap, the substrate of other induction production of cellulose enzymes is as Microcrystalline Cellulose, sophorose etc., not only complicated process of preparation, use cost is very high, and method of the present invention is that suitability for industrialized production cellulase provides direction; The present invention, by carrying out pre-treatment removing pectin and starch to duckweed, improves it and induces the cellulosic output of generation and enzyme to live, for a large amount of production of cellulose enzyme is laid a good foundation.
Part is embodied by explanation below by other advantage of the present invention, target and feature, part also will by research and practice of the present invention by those skilled in the art is understood.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail, can implement according to this with reference to specification sheets word to make those skilled in the art.
It should be noted that experimental technique described in following embodiment if no special instructions, is ordinary method, described reagent and material, if no special instructions, all can obtain from commercial channels.Following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
Bacterial classification used in the present invention is that Trichodermareesei (trichodermareeseiCCIC13052) ATCC56765 is buied by Chinese microorganism strain preservation center (CICC).
Embodiment 1: undressed duckweed is directly cooked fermenting substrate cellulase-producing
Step one, duckweed through pulverizing after, for preparing seed culture medium and fermention medium, wherein,
Seed culture medium forms: duckweed 20g/L, Dried Corn Steep Liquor Powder 10g/L, glucose 10g/L, pH4.5.
Fermention medium forms: duckweed X, Dried Corn Steep Liquor Powder 17g/L, ammonium sulfate 5g/L, potassium primary phosphate 6g/L, magnesium sulfate 1g/L, tween-80 2ml, glucose 10g/L, pH5.0, wherein the addition X of duckweed be taken as 10 successively, 30,50,70g/L;
Step 2, Trichodermareesei seed is inoculated into PDA inclined-plane, activation culture, cultivates 7 days under 30 DEG C of conditions; Spore suspension is prepared on inclined-plane after activation, and be seeded in seed culture medium according to the ratio of 3% (volume fraction), shaking table 28 DEG C, cultivates 24h under 180rpm condition; Be seeded in fermention medium according to the ratio of 5% (volume fraction) after seed culture, shaking table 26 DEG C, fermentation is stopped after cultivating 120h under 180rpm condition, the International Standards Method adopting IUPAC to recommend measures filter paper enzyme activity, and the protein content (i.e. yield of cellulase) in employing Bradford method mensuration fermented liquid is in table 1.
Embodiment 2: treated duckweed is cooked fermenting substrate cellulase-producing
Step one, get duckweed pulverize after powder, preparation reaction system, according to the concentration of substrate of 10%, namely 2g duckweed is placed in the citrate buffer solution of 20mlpH6, and in citrate buffer solution, add amylase 300u, saccharifying enzyme 150u, 0.106g sodium carbonate (adding 0.05mol sodium carbonate in every 1 liter of citrate buffer solution), obtains mixing solutions;
Step 2, above-mentioned mixing solutions is placed in shaking bath, rotation frequency is 100rpm, after 50 DEG C of concussion hydrolysis 48h, takes out and filters to obtain residue, washing residue is placed in air dry oven (60 DEG C) dry must process after duckweed residue;
Step 3, by above-mentioned duckweed residue through pulverizing after, for preparing seed culture medium and fermention medium,
Wherein, seed culture medium composition: duckweed residue 20g/L, Dried Corn Steep Liquor Powder 10g/L, glucose 10g/L, pH4.5; Fermention medium forms: duckweed residue Y, Dried Corn Steep Liquor Powder 17g/L, ammonium sulfate 5g/L, potassium primary phosphate 6g/L, magnesium sulfate 1g/L, tween-80 2ml, glucose 10g/L, pH5.0, wherein the addition Y of duckweed residue be taken as 10 successively, 30,50,70g/L;
Step 4, Trichodermareesei seed is inoculated into PDA inclined-plane, activation culture, cultivates 7 days under 30 DEG C of conditions; Spore suspension is prepared on inclined-plane after activation, and be seeded in seed culture medium according to the ratio of 3% (volume fraction), shaking table 28 DEG C, cultivates 24h under 180rpm condition; Be seeded in fermention medium according to the ratio of 5% (volume fraction) after seed culture, shaking table 26 DEG C, fermentation is stopped after cultivating 120h under 180rpm condition, the International Standards Method adopting IUPAC to recommend measures filter paper enzyme activity, and the protein content in employing Bradford method mensuration fermented liquid is in table 1.
Embodiment 3: the duckweed in embodiment 1 is replaced with thready pulse duckweed, the method for other induction production of cellulose enzymes is with embodiment 1.
Embodiment 4: the duckweed in embodiment 2 is replaced with little duckweed, the method for other induction production of cellulose enzymes is with embodiment 1.
Embodiment 5: the duckweed in embodiment 3 is replaced with blue or green duckweed, the method for other induction production of cellulose enzymes is with embodiment 1.
Embodiment 6: the duckweed in embodiment 4 is replaced with duckweed spirodela, the method for other induction production of cellulose enzymes is with embodiment 1.
Embodiment 7: the duckweed in embodiment 4 is replaced with duckweed spirodela, the method for other induction production of cellulose enzymes is with embodiment 1.
Embodiment 8: on the basis of embodiment 2, duckweed replaces with the mixture of one or more in duckweed spirodela, blue or green duckweed, little duckweed, thready pulse duckweed, and duckweed is obtained duckweed residue through following pre-treatment, pre-treatment mentioned here comprises: the combination of one or more in physics mode, chemical mode or biological mode, described physics mode comprises pulverizing, grinding or heating, described chemical mode comprises acid treatment, alkaline purification or free radical process, and described biological mode comprises ferment treatment.The present invention carries out pre-treatment to duckweed, one of them object is to remove the material such as starch, pectin contained in duckweed, make the Mierocrystalline cellulose containing high level in the duckweed residue of acquisition and hemicellulose, thus improve the ability of its induction cellulase-producing activity and output, the method of physics mode or chemical mode or other biological mode also can remove starch and pectin composition, therefore the said method of carrying out pre-treatment to duckweed of the present invention is not limited to the method that adopts in embodiment 2.
Reference examples 1:
Step one, preparation seed culture medium and fermention medium, wherein,
Seed culture medium forms: Dried Corn Steep Liquor Powder 10g/L, glucose 10g/L, pH4.5.
Fermention medium forms: Dried Corn Steep Liquor Powder 17g/L, ammonium sulfate 5g/L, potassium primary phosphate 6g/L, magnesium sulfate 1g/L, tween-80 2ml, glucose 10g/L, pH5.0;
Step 2, Trichodermareesei seed is inoculated into PDA inclined-plane, activation culture, cultivates 7 days under 30 DEG C of conditions; Spore suspension is prepared on inclined-plane after activation, and be seeded in seed culture medium according to the ratio of 3% (volume fraction), shaking table 28 DEG C, cultivates 24h under 180rpm condition; Be seeded in fermention medium according to the ratio of 5% (volume fraction) after seed culture, shaking table 26 DEG C, fermentation is stopped after cultivating 120h under 180rpm condition, the International Standards Method adopting IUPAC to recommend measures filter paper enzyme activity, and the protein content in employing Bradford method mensuration fermented liquid is in table 1.
Comparative example 2:
Step one, preparation seed culture medium and fermention medium, wherein,
Seed culture medium forms: Microcrystalline Cellulose 20g/L, Dried Corn Steep Liquor Powder 10g/L, glucose 10g/L, pH4.5.
Fermention medium forms: Microcrystalline Cellulose 50g/L, Dried Corn Steep Liquor Powder 17g/L, ammonium sulfate 5g/L, potassium primary phosphate 6g/L, magnesium sulfate 1g/L, tween-80 2ml, glucose 10g/L, pH5.0;
Step 2, Trichodermareesei seed is inoculated into PDA inclined-plane, activation culture, cultivates 7 days under 30 DEG C of conditions; Spore suspension is prepared on inclined-plane after activation, and be seeded in seed culture medium according to the ratio of 3% (volume fraction), shaking table 28 DEG C, cultivates 24h under 180rpm condition; Be seeded in fermention medium according to the ratio of 5% (volume fraction) after seed culture, shaking table 26 DEG C, fermentation is stopped after cultivating 120h under 180rpm condition, the International Standards Method adopting IUPAC to recommend measures filter paper enzyme activity, and the protein content in employing Bradford method mensuration fermented liquid is in table 1.
Protein content in the cellulase activity measured under table 1 different condition and fermented liquid
As can be seen from Table 1, from embodiment 1 and comparative example 1, directly have significant effect as substrate to induction Trichodermareesei production of cellulose enzyme using duckweed, enzyme work can be increased to 6.8FPU/mL by 0.6FPU/mL, production of enzyme can be increased to 2.94g/L by 0.2g/L, and enzyme work and production of enzyme present the change of first increases and then decreases along with the increase of duckweed consumption in the fermentation medium, simultaneously, when duckweed with the addition of excessive in the fermentation medium, fermented liquid is thickness too;
From embodiment 1 and embodiment 2, the duckweed residue obtained after carrying out pre-treatment removal pectin and starch to duckweed is as substrate for induction Trichodermareesei production of cellulose enzyme, compare and directly use duckweed to carry out induction production of cellulose enzyme, show that the duckweed residue after pre-treatment more contributes to improving the enzyme producing cellulase and lives, as can be seen from protein content, cellulosic output is also significantly increase, and all along with the increase of duckweed residue consumption in the fermentation medium presents the change of first increases and then decreases, so duckweed or duckweed residue content are in the fermentation medium advisable with 30-50g/L.
From embodiment 1 and comparative example 2, although the cellulase that the cellulase that embodiment 1 adopts the duckweed induction of different amount to produce does not use the Microcrystalline Cellulose of weight equivalents to produce is many, but lemna mior raw material easily obtains, cost is also low, X in embodiment 1 is adopted to get three kinds of methods that Y in 50g/L (duckweed), embodiment 2 gets 50g/L (duckweed residue) and comparative example 2 (Microcrystalline Cellulose), preparation 1 × 10
6the cellulase Cost comparisons of FPU is in table 2, as shown in Table 2, produce the cellulase of 1,000,000 FPU, lower than Microcrystalline Cellulose with the cost that duckweed, duckweed residue use, and minimum with the cost of duckweed residue, in addition, Microcrystalline Cellulose raw material comparatively duckweed not easily obtains, the labor capacity of producing cost is also large, and by contrast, duckweed is more suitable for the substrate that industrialization scale operation cellulase uses.
Table 2 duckweed, duckweed residue and Microcrystalline Cellulose do inductor Cost comparisons
Note: calculate (50g/l Microcrystalline Cellulose contrasts for 50g/l duckweed, 50g/l duckweed residue) according to fermention medium composition in embodiment.
Although embodiment of the present invention are open as above, but it is not restricted to listed in specification sheets and embodiment utilization, it can be applied to various applicable the field of the invention completely, for those skilled in the art, can easily realize other amendment, therefore do not deviating under the universal that claim and equivalency range limit, the present invention is not limited to specific details and illustrates here and the embodiment described.
Claims (7)
1. utilize a method for duckweed production of cellulose enzyme, it is characterized in that, adopt duckweed to be fermenting substrate production of cellulose enzyme.
2. utilize the method for duckweed production of cellulose enzyme as claimed in claim 1, it is characterized in that, described duckweed comprises one or more the combination in thready pulse duckweed, little duckweed, blue or green duckweed and duckweed spirodela.
3. utilize the method for duckweed production of cellulose enzyme as claimed in claim 1, it is characterized in that, be that substrate prepares seed culture medium and fermention medium with duckweed, wherein in seed culture medium, its addition is 20g/L, and in fermention medium, its addition is 30-50g/L.
4. as the method utilizing duckweed production of cellulose enzyme as described in arbitrary in claim 1-3, it is characterized in that, before being fermenting substrate production of cellulose enzyme with duckweed, pre-treatment is carried out to duckweed, the concrete mode of described pre-treatment comprises the combination of one or more in physics mode, chemical mode or biological mode, described physics mode comprises pulverizing, grinding or heating, and described chemical mode comprises acid treatment, alkaline purification or free radical process, and described biological mode comprises ferment treatment.
5. utilize the method for duckweed production of cellulose enzyme as claimed in claim 4, it is characterized in that, described pre-treatment is specially: duckweed is pulverized to obtain powder, get the citrate buffer solution that 2g duckweed powder is placed in 20mlpH6, add amylase 300u, saccharifying enzyme 150u and 0.106gl sodium carbonate, be placed in shaking bath, rotation frequency is 100rpm, after 50 DEG C of concussion hydrolysis 48h, take out after filtering and obtain residue, washing is placed in the air dry oven of 60 DEG C dry.
6. utilize the method for duckweed production of cellulose enzyme as claimed in claim 1, it is characterized in that, the bacterial classification that fermentation adopts is that wood is mould.
7. utilize the method for duckweed production of cellulose enzyme as claimed in claim 6, it is characterized in that, the bacterial classification that fermentation adopts is Trichodermareesei.
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Citations (3)
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| CN1607250A (en) * | 2003-10-15 | 2005-04-20 | 中国科学院沈阳应用生态研究所 | Process for preparing high active composite cellulase special for woody fodder |
| CN101223273A (en) * | 2005-06-30 | 2008-07-16 | 诺维信北美公司 | Production of cellulase |
| CN104328056A (en) * | 2014-10-29 | 2015-02-04 | 青岛蔚蓝生物集团有限公司 | Trichoderma reesei capable of producing cellulase in high yield and application of trichoderma reesei |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1607250A (en) * | 2003-10-15 | 2005-04-20 | 中国科学院沈阳应用生态研究所 | Process for preparing high active composite cellulase special for woody fodder |
| CN101223273A (en) * | 2005-06-30 | 2008-07-16 | 诺维信北美公司 | Production of cellulase |
| CN104328056A (en) * | 2014-10-29 | 2015-02-04 | 青岛蔚蓝生物集团有限公司 | Trichoderma reesei capable of producing cellulase in high yield and application of trichoderma reesei |
Non-Patent Citations (4)
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| L LI等: "Relationship between crystallinity index and enzymatic hydrolysis performance of celluloses separated from aquatic and terrestrial plant materials.", 《BIORESOURCES》 * |
| 李莉: "不同植物来源纤维素酶解产糖中构效关系及预处理影响研究", 《中国优秀硕士学位论文全文数据库工程科技I辑》 * |
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