CN108410742A - The Mi Siming spores aspergillus and its fermentation process of a kind of High Cellulase Production and application - Google Patents
The Mi Siming spores aspergillus and its fermentation process of a kind of High Cellulase Production and application Download PDFInfo
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- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
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Abstract
The invention discloses a kind of Mi Siming spore aspergillus Sartorya Vuill W 10 of High Cellulase Production, and it is CGMCC No.11991 to preserve number.The present invention can obtain the cellulose crude enzyme liquid of high yield by Mi Siming spores aspergillus (Sartorya Vuill W 10) fermentation, and the filter paper enzyme activity of cellulase can reach 1.26U/ml；The application of cellulase that fermentation is obtained can effectively improve stalk residue enzymatic hydrolyzation and ethanol production in bio-ethanol preparation, have certain application value.Not only production cost is low by the present invention, and alcohol getting rate is high, is conducive to commercial applications and the implementation of bio-ethanol, while also providing an approach for the comprehensive utilization of waste biomass.
Description
Technical field
The invention belongs to microbial fermentation engineering technical fields, are related to a kind of Mi Siming that can realize High-Cellulase-Yielding
Spore aspergillus and the method that fermenting and producing cellulase is carried out using the Mi Siming spore aspergillus.
Background technology
The recycling of the lignocellulosics waste such as stalk can not only reduce its negative effect to environment, moreover it is possible to
As renewable, sustainable energy effective source.Second generation bio-ethanol is produced, lignocellulosic has become most viable
The choice of technology (Belal, E.B.Bioethanol production from rice straw residues.Braz J
Microbiol.2013,44(1):225-234).During lignocellulosic dealing with alcohol, using cellulase to lignocellulosic
It is hydrolyzed, and by hydrolysate fermenting and preparing biological ethyl alcohol；Wherein, the production of cellulase directly influences lignocellulosic
Hydrolysis and subsequent fermentations, be dealing with alcohol technology large-scale application major limitation link (Novy V, Longus K,
Nidetzky B.2015)。
Cellulose is polymerized by 1,000 or so glucose molecules, and cellulase energy hydrocellulose generates small point
Son glucose (the flat Han Yun equalitys of Zhou Junqiang Qiu Zhong, the screening of cellulose-degrading bacteria and its Enzymatic characteristic environmental project journals,
2010,(03):705-708).Currently, the strain of production cellulase mainly has fungi and bacterium two major classes, wherein fungi to be produced
Cellulase have higher catalytic efficiency (Xie Zhanling, Wu Run, the progress Practaculture Sciences of cellulase, 2004,21
(4):72-76) and unlike bacterium, most fungi institutes cellulase-producing is all ectoenzyme, this is again so that low cost point
Become possible to that (the easy Chen Hui Wu Qi of Han Xue wait the condition of enzyme production of cellulase-producing bacillus subtilises C-36 to grind from activated liquid enzyme
Study carefully Sichuan Agricultural Universities journal, 2006, (02):178-181).Fermentation is just being widely used in using Cellulase-producing Fungi
(F.Talebnia, D.Karakashev, I.Angelidaki, Production of in the industrial production of production sugar and bio-ethanol
bioethanol from wheat straw:an overview on pretreatment,hydrolysis and
fermentation.Bioresour Technol,2010,101:4744-4753).A variety of cellulase-producings of separated screening
Fungi strain include the mould category of spore, Chaetomium, Trichoderma viride, koning trichoderma, mould, whiterot fungi, sickle-like bacteria and head mold etc.
(Murashima K, Nishimura T, Nakamura Y, et al., 2002Purification and
characterization of new endo-1,4-β-d-glucanases from Rhizopus oryzae.Enzyme
Microb Tech 30:319–326；Happy Chen Xian Li Hui when blue, the selection and breeding of 2003 cellulase-producing t bacteria P1202 and producing enzyme item
Part is studied, 2003,13 (2):12-13；Kovacs K, Marcelli S, Szakacs G et al., 2009Enzymatic
hydrolysis of steam-pretreated lignocellulosic materials with Trichoderma
Atroviride enzymes produced in-house,《Biotechnology for Biofuels》,2009,2(1):
14)。
However the fungi of existing production cellulase is mostly based on composite bacteria agent, sometimes for adding simultaneously in production process
Add cellulase to promote the enzymatic hydrolyzation to lignocellulosic, to improve final alcohol getting rate, this, which will increase, is produced into
This, influences Business Economic Benefit.
Therefore, a kind of single fungal bacterial strain that High-Cellulase-Yielding may be implemented of research is meaningful.
Invention content
The purpose of the present invention is intended to, and for the state of the art for preparing cellulase single bacterial strain is lacked at present, provides one kind
The Mi Siming spore aspergillus Sartorya Vuill W-10 of high-cellulose enzyme high yield may be implemented.
Above-mentioned Mi Siming spores aspergillus submits preservation, depositary institution to be protected for Chinese microorganism strain on January 7th, 2016
Hide administration committee common micro-organisms center (Chnia General Microbiological Culture Collection
Center, CGMCC), preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, is protected
It is CGMCC No.11991 to deposit number, and Classification And Nomenclature is Mi Siming spore aspergillus Sartorya Vuill W-10.
Above-mentioned Mi Siming spores aspergillus can determine its kind with this field conventional means.
It has been investigated that above-mentioned Mi Siming spores aspergillus is raw on seed (PDA, Potato Dextrose Agar) culture medium
Long very fast, 2-3 days colony diameters of constant temperature incubation reach 3-4cm under the conditions of 30 DEG C.Subiculum is relatively thin, and primary hyphae is white, after
Phase bacterium colony is in taupe, and the bacterium colony back side is colourless；Mi Siming spore aspergillus has the conidial head of the double-deck stigma, and conidium is in length
Chain, ascocarp are in sclerotium shape, and ascospore is in oblate spheroid or spherical shape, and ascospore is colourless.
The preparation process for the seed culture medium that the present invention uses for：Peptone 20g, glucose 20g, yeast extract 10g, chlorination
Sodium 0.5g, ferrous sulfate heptahydrate 0.005g, calcium chloride 0.3g, magnesium sulfate 0.5g, dipotassium hydrogen phosphate 2g ratio prepare and planted
Sub- culture medium pre-composition, with deionized water constant volume to 1000mL, and it is molten using the hydrochloric acid of 1mol/L and the sodium hydroxide of 1mol/L
The pH value that liquid adjusts seed culture medium pre-composition is 7, and then sterilize in 121 DEG C 20min, up to seed culture medium after cooling；It obtains
Seed culture medium can be positioned over 4 DEG C store it is spare.
Another object of the present invention is to provide it is a kind of using above-mentioned Mi Siming spores aspergillus ferment cellulase-producing method,
It is as follows：
(1) culture medium is prepared：According to potassium nitrate 4g, lactose or glucose 4g, urea 0.2g, dipotassium hydrogen phosphate 1.5g,
Stalk after 2~6ml of Mandel ' s nutrient solutions, 4~12g of wheat bran, magnesium sulfate 0.5g, calcium chloride 0.3g, NaOH pretreatment
The ratio of powder 30g is prepared to obtain culture medium, is settled to 1000mL with deionized water, and the pH value of culture medium is adjusted using inorganic acid
It is 2.5~3.5；
(2) Mi Siming spore aspergillus seed liquors are inoculated with：According to the 8~15% of culture volume, by Mi Siming spore aspergillus seeds
Liquid is inoculated in the culture medium of step (1) preparation；
(3) cellulase is prepared：The culture for being inoculated with Mi Siming spore aspergillus is based on 45~55 DEG C, is fermented under the conditions of 180r/m
6~8 days up to the crude enzyme liquid containing cellulase.
The method of the above-mentioned cellulase-producing that fermented using Mi Siming spore aspergillus, using potassium nitrate, urea as nitrogen source, with lactose or
Glucose, wheat bran, straw powder are carbon source, using potassium dihydrogen phosphate as phosphorus source, using potassium dihydrogen phosphate, magnesium sulfate, calcium chloride as nothing
Machine salt source.
The method of the above-mentioned cellulase-producing that fermented using Mi Siming spore aspergillus, may be used the routine that this field has disclosed
Means obtain Mandel ' s nutrient solutions, and (bibliography molasses-spirit title page induces the research of koning trichoderma cellulase-producing, Lin Yuan
Mountain, Guangxi University, master thesis, 2004).
The method of the above-mentioned cellulase-producing that fermented using Mi Siming spore aspergillus, may be used the routine that this field has disclosed
Means obtain straw powder (bibliography Y Liu, Z Qiu, the G WangOptimized Alkaline after NaOH pretreatment
Pretreatment Technology of Rice Straw for Ethanol Production[J]Advances in
Engineering Research 2015,1169-1173).
It is above-mentioned using Mi Siming spore aspergillus ferment cellulase-producing method, the inorganic acid be hydrochloric acid, nitric acid or sulfuric acid,
Its a concentration of 1mol/L~5mol/L.
The method of the above-mentioned cellulase-producing that fermented using Mi Siming spore aspergillus, in the step (1), according to 40~60ml/
The bottling amount of 250ml, the culture medium of preparation is dispensed into round.
The method of the above-mentioned cellulase-producing that fermented using Mi Siming spore aspergillus, in the step (2), may be used this field
The conventional means disclosed obtain Mi Siming spore aspergillus seed liquors (bibliography Wang Mei；Shi Jing；Tan Deshui, etc. withered grass gemma
Fujian Journal of Agricultural Sciench property research [J] of bacillus MSJ-5 production 'beta '-mannases, the 03rd phase in 2015)
The method of the above-mentioned cellulase-producing that fermented using Mi Siming spore aspergillus can be further to hair in the step (3)
Ferment products therefrom is centrifuged to remove solid content；3500~4000r/min of centrifugal rotational speed, centrifugation time are 5~10min.
Invention further provides application of the cellulase in preparing bio-ethanol prepared by the above method, one is gone forward side by side
Step illustrates to prepare the preparation process of bio-ethanol using above-mentioned cellulase, respectively by Mi Siming spore aspergillus (Sartorya
Vuill W-10) and aspergillus niger (Aspergillus niger) fermentation respectively obtain containing cellulase and cellobiase
Crude enzyme liquid；Recycle cellulase and cellobiase stalk of the enzymolysis after 2% NaOH pretreatment jointly；It utilizes simultaneously
Saccharomyces cerevisiae synchronizes diastatic fermentation to stalk, you can bio-ethanol is made.Specifically the preparation process of bio-ethanol is：With
For stalk residue as substrate, it is 40 that substrate, the crude enzyme liquid containing cellulase, which are added in round, and obtain concentration of substrate
The mixed liquor of~100g/L, and using the pH value of inorganic acid (with aforementioned) and sodium hydroxide solution adjusting mixed liquor for 5~
6, Tween-80 nonionic surface active agent is added under the conditions of 30~50 DEG C, 120~180r/min, contains cellobiose
The crude enzyme liquid and saccharomyces cerevisiae of enzyme, simultaneous saccharification and fermentation 3~4 days is to get bio-ethanol；Final concentration of the 1 of the Tween-80~
10g/L；The paper filter enzyme activity of every gram of substrate cellulase is 5~15FPU, cellobiase enzyme activity power is 5~15CBU；Institute
State the final concentration of 8g/L of saccharomyces cerevisiae.
In the preparation process of above-mentioned bio-ethanol, the straw powder (namely stalk residue) after the naoh treatment be by
Stalk handles to obtain (bibliography Y Liu, Z Qiu, G WangOptimized by the conventional means that this field has disclosed
Alkaline Pretreatment Technology of Rice Straw for Ethanol Production[J]
Advances in Engineering Research 2015,1169-117).The processing method that the present invention uses for：First by straw
Stalk smash it through 20 mesh sieve, product is air-dried in 60 DEG C to constant weight after sieving, then with a concentration of 2% NaOH solution in 100 DEG C of items
Residue is isolated after pre-processing 2h under part, you can.
In the preparation process of above-mentioned bio-ethanol, the crude enzyme liquid containing cellobiose enzyme is obtained by fermentation of Aspergillus niger
It arrives, conventional means (the bibliography Meng Yong, Wang Zhongyan, Miao Yanfang, Hu Cheng that this field has disclosed may be used in fermentation process
The research of cellobiase fermentation condition is produced about aspergillus niger,《Sichuan University's journal (natural science edition)》, 2002,39 (5)；
938~942).
The stalk that the present invention uses is rice straw or/and wheat stalk.
Compared with prior art, the invention has the advantages that：
1, the present invention can by Mi Siming spores aspergillus (Sartorya Vuill W-10) single bacterial strain fermentation of offer
Cellulase is obtained, the cellulase for promoting lignocellulolyticenzymes yield need not be additionally added, helps to reduce producing
Cost, enterprise economic benefit；
2, cellulose in the crude enzyme liquid that the present invention passes through Mi Siming spores aspergillus (Sartorya Vuill W-10) fermentation acquisition
The filter paper enzyme activity of enzyme can reach 1.26U/ml, have higher producing enzyme efficiency；
3, the application of cellulase that the present invention will be obtained by Mi Siming spores aspergillus (Sartorya Vuill W-10) fermentation
In bio-ethanol preparation, promote enzymolysis process, promote the ratio of glucose in reduced sugar, stalk residue enzymatic hydrolyzation can reach
87%, ethanol production can be up to 20.93DM, not only increase the enzymatic hydrolyzation of stalk residue, and improve bio-ethanol production
Amount has certain application value；
4, not only production cost is low by the present invention, and alcohol getting rate is high, is conducive to commercial applications and the implementation of bio-ethanol, together
When also provide an approach for the comprehensive utilization of waste biomass.
Description of the drawings
Fig. 1 is Mi Siming spore aspergillus pattern schematic diagrames；Wherein (a) is Mi Siming spore aspergillus colony characteristics, is (b) Mi Siming
Form 1 under the mirror of spore aspergillus culture is (c) form 2 under the mirror of Mi Siming spore aspergillus cultures, (d) is Mi Siming spore aspergillus spores.
Specific implementation mode
The present invention is further explained with reference to embodiments.
The preparation process of the Mi Siming spore aspergillus seed liquors used in following example 1~3 for：One ring Mi Siming spores is bent
Mould spore from being inoculated in slant medium on 50ml seed culture mediums, cultivated under the conditions of 30 DEG C, 180r/min 36h to get
Mi Siming spore aspergillus seed liquors, obtained a concentration of OD of seed liquor6002.0 (bibliography Wang Mei；Shi Jing；Tan Deshui, etc. withered
Fujian Journal of Agricultural Sciench property research [J] of careless bacillus MSJ-5 productions 'beta '-mannase, the 03rd phase in 2015).The kind of use
The preparation process of sub- culture medium is：By peptone 20g, glucose 20g, yeast extract 10g, sodium chloride 0.5g, ferrous sulfate heptahydrate
0.005g, calcium chloride 0.3g, magnesium sulfate 0.5g, dipotassium hydrogen phosphate 2g ratio prepare to obtain seed culture medium pre-composition, spend
Ionized water constant volume adjusts seed culture medium using the sodium hydroxide solution of the hydrochloric acid of 1mol/L and 1mol/L and premixes to 1000mL
The pH value of object is 7, and then sterilize in 121 DEG C 20min, up to seed culture medium after cooling；The seed culture medium of acquisition can be put
Be placed in 4 DEG C store it is spare.
Mandel ' the s nutrient solutions used in following example 1~3 be according to《Molasses-spirit title page induces koning trichoderma production
The research of cellulase》Preparation in relation to Mandel ' s nutrient solutions in (the woods Yuanshan mountain, Guangxi University, master thesis, 2004)
What process was prepared.
Straw powder after the NaOH pretreatment used in following example 1~3【Namely substrate in embodiment 4~18
(stalk residue】Preparation process is identical, be according to《Optimized Alkaline Pretreatment Technology of
Rice Straw for Ethanol Production》(Y Liu, Z Qiu, G Wang, [J] Advances in
Engineering Research2015,1169-1173) in the process in relation to NaOH pretreatment straw powder be prepared
, specific preparation process is：20 mesh sieve is crossed after first rice straw is crushed, product is air-dried in 60 DEG C to constant weight after sieving, then is used
A concentration of 2% NaOH solution isolates residue after pre-processing 2h under the conditions of 100 DEG C, you can.
The crude enzyme liquid containing cellobiase used in following example 8~18 be according to《It is produced about aspergillus niger fine
Tie up the research of disaccharidase fermentation condition》(Meng Yong, Wang Zhongyan, Miao Yanfang, Hu Cheng,《Sichuan University's journal (natural science edition)》,
2002,39(5)；938~942) preparation process in relation to cellobiase is prepared in.
The cellulase activity test method of following example 1~3 is：Xinhua's filter paper item of 50mg is added in test tube,
It is added the citrate buffer solution (pH4.8, citric acid concentration is 0.05mol/L in buffer solution) of 1mL, rear that 0.5mL is added is suitably dilute
The crude enzyme liquid released reacts 30min under conditions of 50 DEG C of water-baths, and 2mLDNS reagents are added after reaction, and boiling water bath 5min adds
Distilled water is diluted to mixing after 20mL, colorimetric under the conditions of 520nm, measures absorbance value and calculates reaction knot through glucose mark song
The amount of reduced sugar after beam, deduct enzyme solution and substrate reduced sugar (can by with the control experiment group of enzyme blank and substrate blank ratio
To obtaining) enzyme activity is calculated afterwards.
One filter paper enzyme activity unit of force (FPU) is defined as：The enzyme per minute generated needed for 1 μm of ol glucose in enzymatic reaction
Amount.
In formula, extension rate refers to by the ratio between volume and dilution front volume after gained crude enzyme liquid dilution in Examples 1 to 3.
The present invention provides a kind of Mi Siming spore aspergillus Sartorya Vuill W- that high-cellulose enzyme high yield may be implemented
10.Above-mentioned Mi Siming spores aspergillus, it is Chinese microorganism strain preservation management to submit preservation, depositary institution on January 7th, 2016
Committee's common micro-organisms center (Chnia General Microbiological Culture Collection Center,
CGMCC), preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, preserves number
For CGMCC No.11991, Classification And Nomenclature is Mi Siming spore aspergillus Sartorya Vuill W-10.
As shown in Figure 1, above-mentioned Mi Siming spores aspergillus has the characteristics that：Subiculum is relatively thin, and primary hyphae is white, later stage
Bacterium colony is in taupe, and the bacterium colony back side is colourless；It is in long-chain that Mi Siming spore aspergillus, which has the conidial head of the double-deck stigma, conidium,
Shape, ascocarp are in sclerotium shape, and ascospore is in oblate spheroid or spherical shape, and ascospore is colourless.
Embodiment 1
The specific preparation process that cellulase is prepared using Mi Siming spores aspergillus (Sartorya Vuill W-10) is as follows：
(1) culture medium is prepared：According to potassium nitrate 4g, lactose 4g, urea 0.2g, dipotassium hydrogen phosphate 1.5g, Mandel ' s battalion
The ratio of straw powder 30g after nutrient solution 3ml, wheat bran 6g, magnesium sulfate 0.5g, calcium chloride 0.3g, NaOH pretreatment is with obtained
To culture medium, it is settled to 1000mL with deionized water, and it is 3 to adjust the pH value of culture medium using the hydrochloric acid of 1mol/L；Then it presses
According to the bottling amount of 43mL/250mL, the culture medium of preparation is dispensed into round；
(2) Mi Siming spore aspergillus seed liquors are inoculated with：According to the 10% of culture volume, Mi Siming spore aspergillus seed liquors are connect
Kind in filling in the round of culture medium；
(3) cellulase is prepared：The culture for being inoculated with Mi Siming spore aspergillus is based on 50 DEG C, is fermented 6 days under the conditions of 180r/m,
Then tunning is centrifuged into 5min to get the crude enzyme liquid containing cellulase in 4000r/min.
Cellulase activity test is carried out to obtained crude enzyme liquid, the filter paper enzyme activity for obtaining cellulase is 1.26FPU/
ml。
Embodiment 2
The specific preparation process that cellulase is prepared using Mi Siming spores aspergillus (Sartorya Vuill W-10) is as follows：
(1) culture medium is prepared：According to potassium nitrate 4g, lactose 4g, urea 0.2g, dipotassium hydrogen phosphate 1.5g, Mandel ' s battalion
The ratio of straw powder 30g after nutrient solution 6ml, wheat bran 12g, magnesium sulfate 0.5g, calcium chloride 0.3g, NaOH pretreatment is with obtained
To culture medium, it is settled to 1000mL with deionized water, and it is 2.5 to adjust the pH value of culture medium using the hydrochloric acid of 1mol/L；Then
According to the bottling amount of 60mL/250mL, the culture medium of preparation is dispensed into round；
(2) Mi Siming spore aspergillus seed liquors are inoculated with：According to the 8% of culture volume, Mi Siming spore aspergillus seed liquors are connect
Kind in filling in the round of culture medium；
(3) cellulase is prepared：The culture for being inoculated with Mi Siming spore aspergillus is based on 45 DEG C, is fermented 7 days under the conditions of 180r/m,
Then tunning is centrifuged into 10min to get the crude enzyme liquid containing cellulase in 3500r/min.
Cellulase activity test is carried out to obtained crude enzyme liquid, the filter paper enzyme activity for obtaining cellulase is 0.92U/
ml。
Embodiment 3
The specific preparation process that cellulase is prepared using Mi Siming spores aspergillus (Sartorya Vuill W-10) is as follows：
(1) culture medium is prepared：According to potassium nitrate 4g, lactose 4g, urea 0.2g, dipotassium hydrogen phosphate 1.5g, Mandel ' s battalion
The ratio of straw powder 30g after nutrient solution 2ml, wheat bran 4g, magnesium sulfate 0.5g, calcium chloride 0.3g, NaOH pretreatment is with obtained
To culture medium, it is settled to 1000mL with deionized water, and it is 3.5 to adjust the pH value of culture medium using the hydrochloric acid of 3mol/L；Then
According to the bottling amount of 40mL/250mL, the culture medium of preparation is dispensed into round；
(2) Mi Siming spore aspergillus seed liquors are inoculated with：According to the 15% of culture volume, Mi Siming spore aspergillus seed liquors are connect
Kind in filling in the round of culture medium；
(3) cellulase is prepared：The culture for being inoculated with Mi Siming spore aspergillus is based on 55 DEG C, is fermented 8 days under the conditions of 180r/m,
Then tunning is centrifuged into 5min to get the crude enzyme liquid containing cellulase in 4000r/min.
Cellulase activity test is carried out to obtained crude enzyme liquid, the filter paper enzyme activity for obtaining cellulase is 1.15U/
ml。
Embodiment 4-7
Crude enzyme liquid prepared by embodiment 1 is added to the saccharification that different concentration of substrate are carried out in the container that four fill substrate
Experiment：Under room temperature, digested 72 hours with the rate of 120r/min in shaking table.The concentration of substrate and experimental result of preparation such as table 1
It is shown.
The corresponding saccharification experimental result of 1 embodiment 4-7 difference concentration of substrate of table
From table 1 it follows that with the increase of concentration of substrate, enzymolysis yield is constantly declining；For supervention after ensureing
The produced concentration of alcohol of ferment also requires enzymolysis liquid to have relatively high concentration of reduced sugar, therefore considers enzymolysis yield and go back
Raw sugar concentration, when preparing bio-ethanol, preferably selecting 80g/L concentration of substrate is advisable.
Embodiment 8-12
Crude enzyme liquid prepared by embodiment 1 is added in the container that five fill substrate, it is 80g/L to be configured to concentration of substrate
Mixed liquor, then into mixed liquor be added the crude enzyme liquid (dosage is in terms of the enzyme activity of cellobiase) containing two pool enzyme of fiber and
Tween-80 carries out enzymolysis experiment to substrate respectively：Under room temperature, digested 72 hours with 120r/min in shaking table.Experimental raw is matched
When experimental result is as shown in table 2.
2 embodiment 8-12 enzymolysis experimental raws of table are with when experimental result
Note：Above-mentioned Tween-80 concentration is the densimeter with Tween-80 in container mixed liquor.
From Table 2, it can be seen that substrate enzymatic hydrolyzation is reached as the concentration of Tween-80 increases and rises in Tween-80 concentration
Enzymolysis yield to rice straw 72h when 5g/L reaches maximum value, is 81.8%；But when Tween-80 concentration increases to 10g/
When L, enzymolysis yield is 81%.Therefore, when preparing bio-ethanol, Tween-80 additive amount is preferably advisable with 5g/L.
Embodiment 13-15
Crude enzyme liquid prepared by embodiment 1 is added in the container that three fill substrate, it is 80g/L to be configured to concentration of substrate
Mixed liquor, then be added into mixed liquor containing the mould crude enzyme liquid of cellobiose (dosage is in terms of the enzyme activity of cellobiase) and
Tween-80 carries out enzymolysis experiment to substrate respectively：Under room temperature, digested 72 hours with 120r/min in shaking table.Experimental raw is matched
When experimental result is as shown in table 3.
3 embodiment 13-15 enzymolysis experimental raws of table are with when experimental result
Note：Above-mentioned Tween-80 concentration is the densimeter with Tween-80 in container mixed liquor.
From table 3 it is observed that substrate enzymatic hydrolyzation rises as cellobiose enzyme activity increases, maximum amplification reaches
2.3%, but when cellobiose enzyme activity is more than every gram of substrate of 10CBU/, the promotion for digesting yield is just no longer fairly obvious, because
This, when preparing bio-ethanol, considering cost and enzymolysis yield use enzyme activity for the fiber two of every gram of substrate of 10CBU/
Carbohydrase is advisable.
Embodiment 16-18
Crude enzyme liquid prepared by embodiment 1 is added in the round that three fill substrate, being configured to concentration of substrate is
The mixed liquor of 80g/L, and it is 5-6 to adjust the pH value of mixed liquor using the sodium hydroxide solution of the hydrochloric acid of 1mol/L and 1mol/L,
Tween-80, crude enzyme liquid and saccharomyces cerevisiae containing cellobiose enzyme are added under the conditions of 30~50 DEG C, 120~180r/min,
Diastatic fermentation is synchronized 3-4 days to get bio-ethanol to above-mentioned material.Test raw material is as shown in table 4 with when test result.
4 embodiment 16-18 bio-ethanols of table prepare test raw material with when test result
Note：(1) above-mentioned Tween-80 concentration is the densimeter with Tween-80 in container mixed liquor；
(2) ethanol production %DM refers to the yield of every 100g dry straws production alcohol.
From table 4, it can be seen that the application of cellulase that Mi Siming spores aspergillus provided by the invention is fermented is in biology
In prepared by ethyl alcohol, enzymolysis process can be promoted, promote the ratio of glucose in reduced sugar, and production cost is low, the ethyl alcohol of preparation
Yield is high, to contribute to commercial applications and the implementation of bio-ethanol.
Those of ordinary skill in the art will understand that the embodiments described herein, which is to help reader, understands this hair
Bright principle, it should be understood that protection scope of the present invention is not limited to such specific embodiments and embodiments.This field
Those of ordinary skill can make according to the technical disclosures disclosed by the invention various does not depart from the other each of essence of the invention
The specific variations and combinations of kind, these variations and combinations are still within the scope of the present invention.
Claims (9)
1. a kind of Mi Siming spore aspergillus Sartorya Vuill W-10 of High Cellulase Production, it is CGMCC to preserve number
No.11991。
2. a kind of method for the cellulase-producing that fermented using Mi Siming spores aspergillus described in claim 1, which is characterized in that step
It is as follows：
(1) culture medium is prepared：According to potassium nitrate 4g, lactose or glucose 4g, urea 0.2g, dipotassium hydrogen phosphate 1.5g, Mandel '
Straw powder 30g's after 2~6ml of s nutrient solutions, 4~12g of wheat bran, magnesium sulfate 0.5g, calcium chloride 0.3g, NaOH pretreatment
Ratio is prepared to obtain culture medium, and 1000mL is settled to deionized water, and the pH value for adjusting using inorganic acid culture medium be 2.5~
3.5；
(2) Mi Siming spore aspergillus seed liquors are inoculated with：According to the 8~15% of culture volume, Mi Siming spore aspergillus seed liquors are connect
Kind is in culture medium prepared by step (1)；
(3) cellulase is prepared：The culture of Mi Siming spore aspergillus will be inoculated with based on fermentation 6~8 under the conditions of 45~55 DEG C, 180r/m
It is up to the crude enzyme liquid containing cellulase.
3. the method for using Mi Siming spore aspergillus fermentation cellulase-producing according to claim 2, it is characterised in that the step
Suddenly in (1), inorganic acid is hydrochloric acid, nitric acid or sulfuric acid.
4. the method for using Mi Siming spore aspergillus fermentation cellulase-producing according to claim 2, it is characterised in that the step
Suddenly in (1), according to the bottling amount of 40~60ml/250ml, the culture medium of preparation is dispensed into round.
5. the application of cellulase prepared by claim 2 to 4 any claim the method in preparing bio-ethanol.
6. application of the cellulase in preparing bio-ethanol according to claim 5, it is characterised in that the bio-ethanol
Preparation process be：Using stalk residue as substrate, substrate, the crude enzyme liquid containing cellulase are added in round and obtained
To the mixed liquor of a concentration of 40~100g/L of substrate, and it is 5 to adjust the pH value of mixed liquor using inorganic acid and sodium hydroxide solution
~6, Tween-80 nonionic surface active agent is added under the conditions of 30~50 DEG C, 120~180r/min, contains cellulose two
The crude enzyme liquid and saccharomyces cerevisiae of carbohydrase, simultaneous saccharification and fermentation 3~4 days is to get bio-ethanol.
7. application of the cellulase in preparing bio-ethanol according to claim 6, it is characterised in that fine in every gram of substrate
The paper filter enzyme activity of the plain enzyme of dimension is 5~15FPU, cellobiase enzyme activity power is 5~15CBU.
8. application of the cellulase in preparing bio-ethanol according to claim 6, it is characterised in that the Tween-80
Final concentration of 1~10g/L.
9. application of the cellulase in preparing bio-ethanol according to claim 6, it is characterised in that the saccharomyces cerevisiae
Final concentration of 8g/L.
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| CN112725194B (en) * | 2021-01-21 | 2021-10-22 | 南京林业大学 | Fungus Flavodon sp.x10 for high yield of cellulase and application thereof |
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