CN101955887A - Raw-starch amylase producing penicillium and raw-starch amylase preparation produced thereby - Google Patents

Raw-starch amylase producing penicillium and raw-starch amylase preparation produced thereby Download PDF

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CN101955887A
CN101955887A CN2010102720653A CN201010272065A CN101955887A CN 101955887 A CN101955887 A CN 101955887A CN 2010102720653 A CN2010102720653 A CN 2010102720653A CN 201010272065 A CN201010272065 A CN 201010272065A CN 101955887 A CN101955887 A CN 101955887A
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living
gxu20
mould
diastase
penicillium
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CN101955887B (en
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冯家勋
林海娟
段承杰
刘君梁
罗雪梅
唐纪良
张秋江
冼亮
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Guangxi University
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Abstract

The invention discloses raw-starch amylase producing penicillium and a raw-starch amylase preparation produced thereby. The invention provides penicillium sp. GXU20 CGMCC No.3690. The invention also provides the raw-starch amylase preparation produced by fermentation of penicillium GXU20. The penicillium GXU20 and the raw-starch amylase preparation both can be used in alcohol production for avoiding thermophilic digestion of raw materials and saving a large amount of energy; the addition of alpha-amylase is avoided, and cost is saved; and saccharification and fermentation are performed in the same container, and equipment cost is reduced. The raw-starch amylase producing penicillium and the raw-starch amylase preparation produced thereby are expected to solve the problem of high alcohol production cost caused by the thermophilic digestion of starch.

Description

Produce the living diastase of giving birth to diastatic Penicillium notatum and preparation thereof
Technical field
The present invention relates to a kind of living diastase of giving birth to diastatic Penicillium notatum and preparation thereof that produces, be specifically related to a strain produce give birth to diastatic Penicillium notatum, with the liquid living diastase and the application of said preparation in living tapioca (flour) simultaneous saccharification and fermentation production alcohol of this bacterial strain preparation.
Background technology
Starch is the abundantest polysaccharide material of plant storage on the earth.In foodstuffs industry, it is a kind of raw material of economy, can be used to produce sweeting agents such as glucose, maltose, high fructose syrup, also is used for producing microbiotic, amino acid, alcohol and organic edible acid etc.Become raw spirit behind the dehydration of alcohol; raw spirit becomes fuel alcohol through denaturing treatment; a plurality of countries use fuel alcohol in the world; to extenuating the world energy sources crisis, improve environment, promote significant (Sanchez OJ such as agricultural sustainable development; Cardona CA.Trends in biotechnologicalproduction of fuel ethanol from different feedstocks.Bioresource Technology2008,99:5270-5295).
With starch is in traditional biomass to alcohol conversion process of raw material, the high temperature steaming that the necessary process earlier of raw material is about 90-110 ℃, add high temperature resistant α-Dian Fenmei the long-chain starch in the gelatinization raw material is cut into the short chain dextrin, make mash viscosity reduce, this is the gelatinization and the liquefaction process of starch; The short chain dextrin can't be utilized by yeast, the raw material after the liquefaction need be cooled to about 60 ℃, adds saccharifying enzyme the short chain dextrin is degraded into glucose, and this is a saccharifying; Last glucose produces alcohol through yeast fermentation.Or after raw material carried out gelatinization and liquefaction, reduce to about 30 ℃, add saccharifying enzyme and yeast simultaneously, simultaneous saccharification and fermentation produce alcohol (Liang Yuchao, Li Kaimian, cassava alcohol fermentation research progress, Guangxi light industry, 2007,98:16-18).
When being the raw material production fuel alcohol with tapioca (flour) and W-Gum at present is to use above-mentioned biomass to alcohol conversion process.Because the high temperature steaming (gelatinization and liquefaction) in prices of raw and semifnished materials height and the traditional biomass to alcohol conversion process is a very process of power consumption, probably need to consume 35% (Wang Chenxia of the required energy of whole Alcohol Production process, Du Fengguang, Rigen's moral, starch material raw material fermentation method is produced the alcohol general introduction, grain and grease, 2008,6:11-13).Production of Fuel Ethanol cost height, the no market competitiveness, manufacturing enterprise also needs government subsidy to survive, and therefore, the production cost that reduces fuel alcohol is very necessary.
Give birth to amylase and refer to a kind of prozyme, directly useful effect generally comprises α-Dian Fenmei (α-1,4-D-Glucanohydrolase in the raw material starch granules without the virgin state of boiling or processing; EC 3.2.1.1), beta-amylase (β-amylase; EC 3.2.1.2) and glucoamylase (Glucoamylase; EC 3.2.1.3) etc.α-Dian Fenmei is cut α-1,4 glycosidic link of starch molecule inside at random, generates the short chain dextrin; Beta-amylase is from non reducing end hydrolyzed starch molecule α-1,4 glycosidic link separately of starch molecule, and product is a maltose, can not hydrolyzing alpha-1,6 glycosidic link, and product also has limit dextrin when acting on amylopectin.Glucoamylase is commonly called as saccharifying enzyme, can not only hydrolyzing alpha-1,4 glycosidic link, can also weakly hydrolyse α-1,6 glycosidic links, downcut glucose one by one from the non reducing end of starch molecule, final product is glucose (Wang Jingyan, Zhu Shenggeng, Xu Changfa, Higher Education Publishing House on the biological chemistry, Beijing, 2002:43).
The simultaneous saccharification and fermentation that living amylase is used for raw material starch produces alcohol, (30-40 ℃) directly changes raw material starch into fermentable sugar at low temperatures, simultaneous yeast produces alcohol with the fermentability sugar-fermenting that hydrolysis produces, saccharification and fermentation are carried out in same reactor like this, can greatly simplify traditional biomass to alcohol conversion process, the cost of minimizing equipment aspect improves plant factor.The more important thing is, can save the big energy that the high temperature steaming process is consumed, greatly cut down the consumption of energy.Therefore, living amylase is applied in the Production of Fuel Ethanol aspect tempting development prospect, research and development are given birth to amylase to reducing significant (the Li S-Z of production cost of fuel alcohol, Chan-Halbrendt C.Ethanol production in China:Potential and technologies.Applied Energy 2009,86:162-169).
Can produce living diastatic microorganism has a lot, if diastatic microbial host fungi is given birth in can producing of having reported, such as Aspergillus sp., Rhizopus sp., also have and report that the amylase that derives from bacterium also can act on living starch (Sun H, Ge X, Wang L, et al.Microbial production of raw starchdigesting enzymes.African Journal of Biotechnology 2009,8:1734-1739).
Summary of the invention
The purpose of this invention is to provide a kind of living diastase of giving birth to diastatic Penicillium notatum and preparation thereof that produces.
Penicillium notatum provided by the invention (Peniciilium sp.), name is called GXU20, belong to Penicillium (Penicillium sp.), separation is from the forest soil of Guangxi Shiwan Dashan, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 03 25th, 2010 and (be called for short CGMCC, the address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.3690.Mould (Penicillium sp.) GXU20 CGMCC No.3690 is called for short mould GXU20.It is glucose that the product that discharges behind the Tapioca Starch is given birth in mould GXU20 effect, can improve the utilization ratio of living starchy material effectively.
The present invention also provides the substratum that is used to cultivate mould GXU20, is with wheat bran, soybean cake powder, KH 2PO 4, MgSO 47H 2O, FeSO 47H 2O, CaCl 2Mixing obtains with water; Contain wheat bran 30g in every liter of described substratum, soybean cake powder 25g, KH 2PO 43g, MgSO 47H 2O 0.2g, FeSO 47H 2O 0.025g, CaCl 20.13g.This culture medium raw material cheapness, production cost is low, is easy to preparation.
The present invention also provides a kind of method of producing living diastase, is fermentation mould GXU20, obtains living diastase.The condition of described fermentation can be pH4.0-6.0,26-34 ℃, is preferably pH5.0,28 ℃.
Described fermentation can comprise the steps: the spore liquid of mould GXU20 is seeded in the described substratum, and pH5.0-6.0,26-32 ℃, 180rpm (centrifugal radius is 4.5-5.5cm) were cultivated 10-14 days;
Described fermentation specifically comprises the steps:
(1) with mould GXU20 activation 5-7 days;
(2) spore with the activatory bacterial classification mixes with water, and getting spore concentration is 1 * 10 10The spore liquid of individual/mL;
(3) spore liquid is inserted in the described substratum of claim 2,5.0,28 ℃ of pH, 180rpm (centrifugal radius is 4.5-5.5cm) shaking table was cultivated 11 days.
More than the living diastase for preparing of arbitrary described method also belong to protection scope of the present invention.
Living diastase provided by the invention: at pH3.5-6.5 the active more than 60% of high enzymatic activity arranged all, preferred pH4.0-6.0, optimum pH is 4.5; At 35-60 ℃ the active more than 60% of high enzymatic activity arranged all, preferred 40-60 ℃, optimum temperuture is 50 ℃; The suitableeest action condition is near the fermentation condition (4.0,40 ℃ of pH) of thermotolerant yeast bacterium, gives birth to amylase activities down up to 216U/mL pH4.0,40 ℃.
Mould GXU20 and/or described living diastase can be used for producing alcohol.Can avoid the high temperature steaming of raw material, save big energy, reduce the interpolation of α-Dian Fenmei, saccharification and fermentation are carried out in a container simultaneously, reduce equipment cost.
When using described living diastase and producing alcohol, the pH value can be 3.5-6.5, and temperature can be 35-60 ℃; When using described living diastase, the pH value is preferably 4.5, and temperature is preferably 50 ℃.
The present invention also protects a kind of method of producing alcohol, comprises the steps: that to give birth to Tapioca Starch be substrate, adds described living diastase and Angel yeast saccharomyces cerevisiae, and pH4.0,40 ℃, 150rpm (centrifugal radius is 4.5-5.5cm) fermentation obtain alcohol.
Mould GXU20 provided by the invention and living diastase thereof are mainly used in living starch simultaneous saccharification and fermentation and produce alcohol technology, can avoid the high temperature steaming of raw material, save big energy; Need not add α-Dian Fenmei, save cost; Saccharification and fermentation are carried out in a container simultaneously, reduce equipment cost.The present invention is expected to solve the expensive problem of being brought because of high temperature steaming starch of Alcohol Production.
Description of drawings
Fig. 1 is the colonial morphology of mould GXU20 on separating plate.
Fig. 2 is the starch degradation circle of mould GXU20 on separating plate.
Fig. 3 is the colonial morphology of mould GXU20 on the PDA flat board.
Fig. 4 is the microphotograph of mould GXU20.
Fig. 5 is that pH produces living diastatic influence to mould GXU20.
Fig. 6 is that temperature produces living diastatic influence to mould GXU20.
Fig. 7 is that different carbon sources, nitrogenous source produce living diastatic influence to mould GXU20; A: different carbon sources produce mould GXU20 and give birth to diastatic influence; B: different nitrogen sources produces mould GXU20 and gives birth to diastatic influence.
Fig. 8 gives birth to diastatic wheat bran, soybean cake powder concentration for the suitableeest mould GXU20 produces; A: the most suitable mould GXU20 produces and gives birth to diastatic wheat bran concentration; B: the suitableeest mould GXU20 produces and gives birth to diastatic soybean cake powder concentration.
Fig. 9 is that mould GXU20 produces the comparison of giving birth to the amylase ability before and after the medium optimization.
Figure 10 is the product evaluation that Tapioca Starch is given birth in the liquid living diastase hydrolysis of mould GXU20; A: saccharide, G1 is a glucose, and G2 is a maltose, and G3 is a trisaccharide maltose; B: hydrolysis 5min; C: hydrolysis 10min; D: hydrolysis 30min; E: hydrolysis 4h.
Figure 11 is the suitableeest action pH curve of the liquid living diastase of mould GXU20.
Figure 12 is the liquid living diastase optimum temperature curve of mould GXU20.
Figure 13 is the liquid living diastase of mould GXU20 is given birth to Tapioca Starch to different concns a hydrolytic action.
Figure 14 is the Tapioca Starch particle electromicroscopic photograph after the liquid living diastase of mould GXU20 is handled.
Figure 15 is the measurement result of the liquid living diastase of mould GXU20 in giving birth to Tapioca Starch simultaneous saccharification and fermentation product alcohol.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.% among the following embodiment if no special instructions, is the quality percentage composition.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.Rotating speed in following examples, being at radius is rotating speed under the centrifugal radius of 4.5-5.5cm.
Basic fermention medium: give birth to Tapioca Starch 10g, (NH 4) 2SO 42.5g, Tryptones 2g, K 2HPO 43g, MgSO 47H 2O 0.2g, FeSO 47H 2O 0.0255g, CaCl 20.13g, be settled to 1L with distilled water; The pH value is regulated with the 2M HCl aqueous solution or the 2M NaOH aqueous solution; 121 ℃ of moist heat sterilization 20min.
Sodium phosphate dibasic-citrate buffer solution of pH4.0: with 7.71 milliliters of 0.2mol/L Na 2HPO 4The aqueous solution and 12.29 milliliters of 0.1mol/L aqueous citric acid solutions mix.
Among embodiment 1, embodiment 2, the embodiment 3, give birth to amylase activity and measure employing 3,5-dinitrosalicylic acid (3,5-dinitrosalicylate, DNS) method (Miller GL.Use of dinitrosalicyclic acidreagent for determination of reducing sugar.Analytical Chemistry1959,31:426-428), concrete steps are as follows:
1, glucose is dissolved in the sterile distilled water, makes the glucose reference liquid of different concns; Add 1mL DNS in 500 μ L glucose reference liquids, boiling water bath 5min colour developing is cooled to room temperature, and the 540nm place measures absorbance value; Obtain the typical curve of absorbance value and glucose concn, the functional expression of typical curve is y=2.7489x-0.0168 (R 2=0.9994) (y is an absorbance value, and x is a glucose concn).
2,2g is given birth to Tapioca Starch and be suspended from Sodium phosphate dibasic-citrate buffer solution of 100mL pH4.0, obtain giving birth to the Tapioca Starch suspension; In 2mL EP pipe, add 450 μ L and give birth to the Tapioca Starch suspension, add the liquid living diastase of 50 μ L then, 40 ℃ of reaction 30min down, during constantly vibration, enzyme-to-substrate is fully contacted, reaction finishes the back and adds 1mL DNS, and boiling water bath 5min colour developing is cooled to room temperature, 1, the centrifugal 1min of 000rpm, the 540nm place measures absorbance value, calculates enzyme activity according to typical curve and absorbance value.Enzyme activity definition: pH4.0, Tapioca Starch is given birth in hydrolysis under 40 ℃ of conditions, and it is an enzyme activity unit (1U) that 1h discharges the required enzyme amount of 1mg reducing sugar (glucose that is equivalent to equivalent).
The acquisition of embodiment 1, mould GXU20
One, the acquisition of bacterial strain
1, the collection of pedotheque
Gather the soil of the shallow soil layer of the Guangxi China Fangchenggang City about 8-20cm of Shiwan Dashan forest.
2, the separation screening of bacterial strain
(1) prepares isolation medium with distilled water; Contain in every liter of isolation medium: give birth to Tapioca Starch 10g, NaNO 33g, KH 2PO 41g, FeSO 47H 2O 0.001g, MgSO 47H 2O 0.5g, agar 15g; PH5.5; Other component except that Tapioca Starch is mixed the back at 121 ℃ of moist heat sterilization 20min, give birth to Tapioca Starch through Co 60Aseptic adding when irradiation sterilization, the Tapioca Starch after the sterilization other component after sterilization is cooled to 45 ℃, mixing falls dull and stereotyped.
(2) get the 1g soil sample and put into the 150mL Erlenmeyer flask, add the 19mL sterilized water, on magnetic stirring apparatus, stir 30min, get suspension liquid gradient dilution (10 -2, 10 -3, 10 -4With 10 -5), respectively get 50 μ L and be coated on the isolation medium flat board, 28 ℃ of cultivations.
(3) after 3-5 days, observe the colony growth situation, select an amount of gradient of colony number to be coated on 8 separating plates 28 ℃ of cultivations in a large number.
After (4) 5 days, use I 2/ KI dyeing, periphery of bacterial colonies transparent hydrolysis circle occurs the living amylase of this bacterial strain secretion then are described, the tangible fungi of picking hydrolysis circle is forwarded to new separating plate, and purifying is single bacterium colony.
(5) the basic fermention medium of preparation pH 5.5.
(6) the purpose inoculation that step (4) is obtained is to basic fermention medium (liquid), and 28 ℃, 180rpm were cultivated 5-7 days, gets supernatant liquor and gives birth to amylase activity and measure, and therefrom filters out the highest bacterial strain GXU20 of enzyme activity.
The form of bacterial strain GXU20 on isolation medium seen Fig. 1, and the living starch degradation circle of bacterial strain GXU20 on isolation medium seen Fig. 2.
Two, the evaluation of bacterial strain
The colonial morphology of bacterial strain on the PDA flat board seen Fig. 3, and this bacterial strain is grown on the PDA substratum comparatively fast, and colony diameter can reach 4-5cm in 7 days, the colony growth initial stage mycelia be white, differentiate that colony colour is dark green behind the conidium, substratum back side non-pigment.
The microphotograph of bacterial strain is seen Fig. 4, and it is more that opticmicroscope is observed down visible spore, and conidium be an ellipse or circular, and the conidium stigma is two verticillate, and conidiophore is the broom shape, and mycelia has tabula.
According to the morphological specificity of this bacterial strain,, bacterial strain GXU20 is accredited as Penicillium (Penicillium) with reference to " fungi identification handbook ".Mould GXU20 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preserving number is CGMCC No.3690.
Embodiment 2, mould GXU20 productions are given birth to the optimization of diastatic culture condition
One, the preparation of spore liquid
1, with 121 ℃ of sterilizations of PDA substratum 20min.
2,5-7 days the spore of mould GXU20 of activation that goes down to posterity on the PDA flat board is made spore suspension after with aseptic washing, spore concentration is 1 * 10 10Individual/mL.
Two, the optimization of pH value
1, the basic fermention medium of the different pH values of preparation (4.0,4.5,5.0,5.5 or 6.0).
2, the spore liquid of mould GXU20 is seeded in the substratum of step 1 by 1% inoculum size (volumn concentration), 28 ℃, 180rpm were cultivated 5 days.
3, collect crude enzyme liquid as solution to be measured, give birth to amylase activity and measure.
The results are shown in Figure 5, mould GXU20 produces and gives birth to the suitableeest diastatic cultivation pH value is 5.0.
Three, the optimization of temperature
1, the basic fermention medium of preparation pH5.0.
2, the spore liquid of mould GXU20 is seeded in the substratum of step 1 by 1% inoculum size (volumn concentration), places the shaking table 180rpm of differing temps (26 ℃, 28 ℃, 30 ℃, 32 ℃ or 34 ℃) to cultivate respectively 5 days.
3, collect crude enzyme liquid as solution to be measured, give birth to amylase activity and measure.
The results are shown in Figure 6, mould GXU20 produces and gives birth to the suitableeest diastatic culture temperature is 28 ℃.
Four, the suitableeest carbon source of substratum and nitrogenous source determines
1, prepares various substratum
The substratum (pH5.0) that contains various carbon sources: other carbon source (potato powder, sweet potato powder, Semen Maydis powder, rice meal, buckwheat flour, Zulkovsky starch, sticky rice flour or wheat bran) of quality such as use to replace living Tapioca Starch in the basic fermention medium, obtain containing the substratum of various carbon sources.
The substratum (pH5.0) that contains various nitrogenous sources: give birth to Tapioca Starch 10g, nitrogenous source (urea, SODIUMNITRATE, ammonium sulfate, ammonium chloride, Tryptones, yeast extract paste, extractum carnis or soybean cake powder) 10g, K 2HPO 43g, MgSO 47H 2O 0.2g, FeSO 47H 2O 0.0255g, CaCl 20.13g, be settled to 1L with distilled water; The pH value is regulated with the 2M HCl aqueous solution or the 2M NaOH aqueous solution; 121 ℃ of moist heat sterilization 20min.
2, the spore liquid of mould GXU20 is seeded to respectively in the various substratum of step 1 by 1% inoculum size (volumn concentration), 28 ℃, 180rpm were cultivated 7 days.
3, collect crude enzyme liquid as solution to be measured, give birth to amylase activity and measure.
The results are shown in Figure 7.Mould GXU20 produces and gives birth to the suitableeest diastatic carbon source is wheat bran, and the suitableeest nitrogenous source is a soybean cake powder.
Five, wheat bran and soybean cake powder optimal concentration determines
1, the wheat bran optimal concentration determines
(1) prepares various substratum
The substratum (pH5.0) that contains various concentration wheat bran: wheat bran (5g, 10g, 15g, 20g, 25g, 30g, 35g or 40g), ammonium sulfate 2.5g, Tryptones 2g, K 2HPO 43g, MgSO 47H 2O 0.2g, FeSO 47H 2O 0.0255g, CaCl 20.13g, be settled to 1L with distilled water; The pH value is regulated with the 2M HCl aqueous solution or the 2M NaOH aqueous solution; 121 ℃ of moist heat sterilization 20min.
(2) spore liquid of mould GXU20 is seeded to respectively in the various substratum of step (1) by 1% inoculum size (volumn concentration), 28 ℃, 180rpm were cultivated 7 days.
(3) collect crude enzyme liquid as solution to be measured, give birth to amylase activity and measure.
The results are shown in Figure 8A.Mould GXU20 produces and gives birth to the suitableeest diastatic wheat bran concentration is 3% (g/100mL).
2, the soybean cake powder optimal concentration determines
(1) prepares various substratum
The substratum (pH5.0) that contains various concentration soybean cake powder: give birth to Tapioca Starch 10g, soybean cake powder (10g, 15g, 20g, 25g, 30g or 35g), K 2HPO 43g, MgSO 47H 2O 0.2g, FeSO 47H 2O 0.0255g, CaCl 20.13g, be settled to 1L with distilled water; The pH value is regulated with the 2M HCl aqueous solution or the 2M NaOH aqueous solution; 121 ℃ of moist heat sterilization 20min.
(2) spore liquid of mould GXU20 is seeded to respectively in the various substratum of step (1) by 1% inoculum size (volumn concentration), 28 ℃, 180rpm were cultivated 7 days.
(3) collect crude enzyme liquid as solution to be measured, give birth to amylase activity and measure.
The results are shown in Figure 8B.Mould GXU20 produces and gives birth to the suitableeest diastatic soybean cake powder concentration is 2.5% (g/100mL).
Six, mould GXU20 produces the comparison of enzyme situation in the fermention medium of basic fermention medium and optimization
1, prepares various substratum
(1) the basic fermention medium of preparation pH5.0.
(2) the optimization fermention medium of preparation pH5.0: wheat bran 30g, soybean cake powder 25g, KH 2PO 43g, MgSO 47H 2O0.2g, FeSO 47H 2O 0.025g, CaCl 20.13g be settled to 1L with distilled water; The pH value is regulated with the 2M HCl aqueous solution or the 2M NaOH aqueous solution; 121 ℃ of moist heat sterilization 20min.
2, the spore liquid of mould GXU20 is seeded to respectively in the various substratum of step 1 by 1% inoculum size (volumn concentration), 28 ℃, 180rpm are cultivated.
3, in the culturing process, regularly get crude enzyme liquid every day, give birth to amylase activity and measure as solution to be measured.
The results are shown in Figure 9.Mould GXU20 produces and gives birth to diastatic climax at the 5th day in the basic fermention medium, just begin to descend but produced enzyme activity since the 6th day, and the product enzyme climax of optimizing fermention medium is at the 11st day, and produces enzyme activity in 11-16 days and keep stable.The result shows: optimize the basic fermention medium practicality of fermention medium, at first its raw material wheat bran and soybean cake powder be than Tryptones and ammonium sulfate cheapness, and next produces the enzyme back enzyme that peaks and lives and keep stable and do not lose, and is easy to prepare zymin.After adopting the optimization fermention medium, the living amylase activity of mould GXU20 fermented liquid is brought up to about original 6 times.
The prescription of optimizing fermention medium is as follows: wheat bran 30g, soybean cake powder 25g, KH 2PO 43g, MgSO 47H 2O 0.2g, FeSO 47H 2O 0.025g, CaCl 20.13g be settled to 1L with distilled water; The pH value is regulated with the 2M HCl aqueous solution or the 2M NaOH aqueous solution; 121 ℃ of moist heat sterilization 20min.
The preparation of embodiment 3, liquid living diastase
1, fermention medium is optimized in preparation
The optimization fermention medium of preparation pH5.0.
2, the preparation of spore liquid
(1) with 121 ℃ of sterilizations of PDA substratum 20min.
(2) 5-7 days the spore of mould GXU20 of activation that goes down to posterity on the PDA flat board is made spore suspension after with aseptic washing, spore concentration is 1 * 10 10Individual/mL.
3, the preparation of liquid living diastase
(1) 50mL being optimized fermention medium places 250mL to shake bottle.
(2) spore liquid of mould GXU20 is seeded to by 1% inoculum size (volumn concentration) optimizes in the fermention medium, 28 ℃, 180rpm were cultivated 11 days.
The centrifugal culture of (3) 13,000rpm is removed thalline, collects supernatant liquor as solution to be measured, gives birth to amylase activity and measures.
The living amylase activity of solution to be measured is 216U/mL.
This solution to be measured is liquid living diastase, carries out the experiment of embodiment 4, embodiment 5 and embodiment 6.
The enzymatic property of embodiment 4, liquid living diastase
One, the Tapioca Starch product analysis is given birth in liquid living diastase hydrolysis
1, the Tapioca Starch suspension is given birth in preparation
2g is given birth to Tapioca Starch be suspended from Sodium phosphate dibasic-citrate buffer solution of 100mL pH4.0, obtain giving birth to the Tapioca Starch suspension.
2, product analysis
In the finger-type bottle, add 6mL and give birth to the Tapioca Starch suspension, add the liquid living diastase of 2mL then, places 40 ℃ under, the 150rpm shaking table reacts.Get 500 μ L samples during respectively at 5min, 10min, 30min and 4h, boiling water boils the 5min deactivation and gives birth to amylase, cooling; 12, the centrifugal 5min of 000rpm gets supernatant liquor, and HPLC detects the sugar (standard specimen is glucose, maltose, trisaccharide maltose) in the supernatant liquor.
The results are shown in Figure 10.The result shows: Tapioca Starch is given birth in liquid living diastase effect, even at very short reaction times (in the 5min), the product that discharges also has only glucose, and glucose prolongation in time has the trend that increases.Infer in the liquid living diastase of mould GXU20 it mainly is to give birth to diastatic enzyme to play hydrolytic action thus.
Two, the suitableeest action pH value and the optimum temperature of liquid living diastase
1, the suitableeest action pH value
Detect the difference of liquid living diastase enzyme activity under the condition of different pH: use Sodium phosphate dibasic-citrate buffer solution of Sodium phosphate dibasic-citrate buffer solution (pH 3.0,3.5,4.0,4.5,5.0,5.5,6.0,6.5 or 7.0) replacement pH4.0 of different pH respectively, other is with the living amylase activity measuring method of embodiment 3; The enzyme activity definition: Tapioca Starch is given birth in hydrolysis under 40 ℃ of conditions, and it is an enzyme activity unit (1U) that 1h discharges the required enzyme amount of 1mg reducing sugar (glucose that is equivalent to equivalent).
It is as follows to give birth to the amylase activity measurement result: 85.04U/mL (pH 3.0), 164.30U/mL (pH3.5), 216.00U/mL (pH4.0), 271.78U/mL (pH4.5), 256.44U/mL (pH5.0), 231.66U/mL (pH5.5), 210.86U/mL (pH6.0), 159.83U/mL (pH6.5), 127.07U/mL (pH 7.0).With high enzymatic activity is 100%, and the enzyme activity of other pH value is relative enzyme activity with the ratio of high enzymatic activity, is X-coordinate with the pH value, and enzyme activity is the ordinate zou mapping relatively, sees Figure 11.The result shows that the suitableeest action pH of the liquid living diastase of mould GXU20 in the time of 40 ℃ is about 4.5, and relative enzyme activity is 79% during pH4.0.
2, optimum temperature
Detect the difference of liquid living diastase enzyme activity under the condition of different temperatures: adopt different temperature (20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃ or 60 ℃) respectively, other is with the living amylase activity measuring method of embodiment 3; The enzyme activity definition: Tapioca Starch is given birth in hydrolysis under the pH4.0 condition, and it is an enzyme activity unit (1U) that 1h discharges the required enzyme amount of 1mg reducing sugar (glucose that is equivalent to equivalent).
It is as follows to give birth to the amylase activity measurement result: 71.64U/mL (20 ℃), 81.43U/mL (25 ℃), 126.03U/mL (30 ℃), 168.87U/mL (35 ℃), 216.00U/mL (40 ℃), 235.27U/mL (45 ℃), 248.70U/mL (50 ℃), 232.69U/mL (55 ℃), 212.07U/mL (60 ℃).As 100%, the enzyme activity of other temperature is relative enzyme activity with the ratio of high enzymatic activity, is X-coordinate with the temperature with high enzymatic activity, and enzyme activity is the ordinate zou mapping relatively, sees Figure 12.The result shows that the optimum temperature when pH4.0 of the liquid living diastase of mould GXU20 is about 50 ℃, is 87% at 40 ℃ of relative enzyme activities.
Three, the substrate specificity of liquid living diastase
Detect the difference of liquid living diastase enzyme activity under the different substrate conditions: use the different substrates (give birth to Tapioca Starch, living Semen Maydis powder, raw potato powder, living sweet potato powder, give birth to sticky rice flour, uncooked rice powder, give birth to buckwheat flour or Zulkovsky starch) of equal in quality to replace giving birth to Tapioca Starch respectively, other is with the living amylase activity measuring method of embodiment 3; The enzyme activity definition: it is an enzyme activity unit (1U) that pH4.0, hydrolysis substrate under 40 ℃ of conditions, 1h discharge the required enzyme amount of 1mg reducing sugar (glucose that is equivalent to equivalent).
Use Micro BCA analysis of protein reagent (Pierce, Rockford, IL) protein concentration of the liquid living diastase of detection.
The living amylase activity measurement result of liquid living diastase sees Table 1 under the different substrate conditions.
The substrate specificity of the liquid living diastase of table 1 mould GXU20
Substrate Specific enzymes vigor (U/mg) Relative enzyme activity (%)
Give birth to Tapioca Starch 71.8±1.03? 100±1.4?
Living Semen Maydis powder 151.7±6.9? 211±9.7?
The uncooked rice powder 148.3±3.67? 206±5.2?
The raw potato powder 68.8±0.28? 96±0.4?
Give birth to sweet potato powder 45.5±0.76? 63±1.0?
Give birth to buckwheat flour 39.1±0.75? 54±1.0?
Give birth to sticky rice flour 24.4±0.19? 34±0.3?
Zulkovsky starch 2.8±0.06? 4±0.09?
With to the enzyme activity of giving birth to Tapioca Starch as 100%, be relative enzyme activity to the enzyme activity of other substrate with ratio to the enzyme activity of life Tapioca Starch.The liquid living diastase of mould GXU20 to the enzyme activity of various substrates by to weak order being by force: corn, rice, cassava, potato, Ipomoea batatas, buckwheat, glutinous rice, Zulkovsky starch.The liquid living diastase of mould GXU20 is stronger to the enzyme activity of corn and rice, illustrates that the liquid living diastase of mould GXU20 except using, also has application potential in the processing of living tapioca (flour) in other starch processing industry.
Embodiment 5, utilize liquid living diastase hydrolysis to give birth to Tapioca Starch
1, liquid living diastase is to giving birth to the hydrolysis of Tapioca Starch
5 groups of processing are set, every group of processing is provided with 12 repetitions: add the Sodium phosphate dibasic-citrate buffer solution (add-on of each component sees Table 2 in each group processing) of giving birth to Tapioca Starch, liquid living diastase and pH4.0 in the triangular flask of 150mL, obtain reaction system; The reaction system mixing is placed in 40 ℃, 150rpm shaking table; Every 12h gets 2 bottles for every group, detects the percent hydrolysis of living tapioca (flour), calculating mean value.
The add-on of each component during each group of table 2 is handled
Figure BSA00000257416500111
The method of calculation of living tapioca (flour) percent hydrolysis (Shariffa YN, Karim AA, Fazilah A, ZaidulISM Enzymatic hydrolysis of granular native and mildly heat-treated tapiocaand sweet potato starches at sub-gelatinization temperature.FoodHydrocolloid 2009,23:434-440): the remnants after will acting on give birth to Tapioca Starch distilled water wash 3 times, place 40 ℃ of baking ovens to dry to constant weight, weighing, weight are W 2(g); Take by weighing simultaneously and this group is handled the livings Tapioca Starch of used living Tapioca Starch identical weight, behind the usefulness distilled water wash 3 times, also place 40 ℃ of baking ovens to dry to constant weight, weighing, weight is W 1(g); Living tapioca (flour) percent hydrolysis is calculated as follows: percent hydrolysis (%)=(W 1-W 2) * 100/W 3W 3It is the total content (starch content that every after testing 1g gives birth to Tapioca Starch is 0.76g) that is not hydrolyzed the starch in the Tapioca Starch in previous existence.
With time is X-coordinate, and living tapioca (flour) percent hydrolysis is the ordinate zou mapping, sees Figure 13.The result shows that mould GXU20 gives birth to the amylase living Tapioca Starch of hydrolysis different concns effectively, for the living Tapioca Starch of concentration 15%, reaches 95% with liquid living diastase effect 72h posthydrolysis per-cent.
2, the Tapioca Starch particulate electron microscopic observation after the liquid living diastase of mould GXU20 is handled
Living Tapioca Starch before handling and the above-mentioned living Tapioca Starch of remnants that is numbered the 3rd group of each time period of handling are delivered to fixedly metal spraying of material engineering institute of Guangxi University, the living Tapioca Starch particulate metamorphosis before and after handling by electron microscopic observation.The Electronic Speculum model is S-3400N, and Hitachi company produces.
Living Tapioca Starch particle surface smooth (seeing Figure 14 A) before handling, the living Tapioca Starch particle that the liquid living diastase of process mould GXU20 was handled is surface irregularity then, form many pittings that cause not of uniform size, a simultaneously inner visible big hole (wherein above-mentioned be numbered the 3rd group handle seen Figure 14 B by 12 hours living Tapioca Starch of enzymolysis).After living amylorrhexis Tapioca Starch particulate surface was described, the duck eye of formation was beneficial to the inside that living amylase penetrates into starch granules, and starch is given birth in hydrolysis better then.This shows that the living amylase of the liquid living diastase of mould GXU20 has played the intensive Degradation to giving birth to Tapioca Starch.
Embodiment 6, utilize liquid living diastase simultaneous saccharification and fermentation to give birth to Tapioca Starch (150g/L) to produce alcohol
Angel yeast saccharomyces cerevisiae dry yeast is available from Hubei Angel Yeast Co.,Ltd; Activate according to packing instruction.
12 repetitions are set: the aqueous solution of urea (containing 0.3g urea) of in the 250mL triangular flask, add the liquid living diastase (216U/mL) of 22.5mL, 15g giving birth to Tapioca Starch, 5mL 6%, 5mL activatory Angel yeast saccharomyces cerevisiae (containing dry yeast 0.1g approximately), sterile distilled water 67.5mL; With the 2M HCl aqueous solution pH is transferred to 4.0; With bottleneck sealing, be positioned in 40 ℃, 150rpm shaking table (substrate is fully contacted with enzyme, be beneficial to the hydrolytic action of enzyme) the life Tapioca Starch; Every 12h gets 2 bottles, and measurement volumes is measured reducing sugar amount, remaining amount of starch, alcohol output.
Residual reducing sugar test: the reducing sugar content in the usefulness DNS method mensuration fermented liquid (%, g/100mL).
Remaining starch test: get the fermented liquid that 1mL has measured reducing sugar content, the concentrated hydrochloric acid that adds 1mL 12M, adding 4mL distilled water again, to make cumulative volume be 6mL, the final concentration of hydrochloric acid is 2M in the solution, and boiling water bath 15min is cooled to room temperature, be neutralized to neutrality with the 2M NaOH aqueous solution, the DNS method is measured the reducing sugar amount, and the calculation of starch residual volume (%, g/100mL).
Ethanol concn is measured: in the fermented liquid that has been left after reducing sugar and remaining starch have been surveyed in sampling, every bottle adds 100mL distilled water, heating distills out the 100mL distillate, measures the ethanol content of distillate with Ebullioscope, the ethanol concn when measuring temperature post-equalization to 20 ℃ simultaneously.
Fermentation rate is calculated: the theoretical alcohol (g) that produces of the tapioca (flour) of the corresponding consumption of alcohol (g) * 100/ that fermentation rate (%)=fermentation produces.
The results are shown in Figure 15.The result shows, after having added the liquid living diastase of mould GXU20, give birth to Tapioca Starch and be degraded to glucose, yeast becomes alcohol with glucose fermentation simultaneously, and enzymic hydrolysis Tapioca Starch and yeast fermentation glucose carry out in same container simultaneously, and liquor output can reach 53.3g/L behind 48h, fermentation rate is 92%, reducing sugar and remaining starch content very low (＜0.5%, g/100mL), meet the requirement of residual sugar amount in the starch alcohol industry, illustrating that fermentation carries out fully, thoroughly.These data show that all livings amylase that mould GXU20 produces can be applied to give birth in the technology of simultaneous saccharification and fermentation product alcohol of Tapioca Starch, has broad application prospects.

Claims (10)

1. mould (Penicillium sp.) GXU20, its deposit number is CGMCC No.3690.
2. being used to cultivate the substratum of mould (Penicillium sp.) GXU20 CGMCC No.3690, is with wheat bran 30g, soybean cake powder 25g, KH 2PO 43g, MgSO 47H 2O 0.2g, FeSO 47H 2O 0.025g and CaCl 20.13g being mixed to cumulative volume with water is that 1L obtains.
3. a method of producing living diastase is fermentation mould (Penicillium sp.) GXU20 CGMCCNo.3690, obtains living diastase.
4. method as claimed in claim 3 is characterized in that: the condition of described fermentation is pH4.0-6.0,26-34 ℃, is preferably pH5.0,28 ℃.
5. method as claimed in claim 3, it is characterized in that: described fermentation comprises the steps: the spore liquid of mould (Penicillium sp.) GXU20 CGMCC No.3690 is seeded in the described substratum of claim 2, and pH5.0-6.0,26-32 ℃, 180rpm were cultivated 10-14 days.
6. method as claimed in claim 3 is characterized in that: described fermentation comprises the steps:
(1) with mould (Penicillium sp.) GXU20 CGMCC No.3690 activation 5-7 days;
(2) spore with the activatory bacterial classification mixes with water, and getting spore concentration is 1 * 10 10The spore liquid of individual/mL;
(3) spore liquid is inserted in the described substratum of claim 2,5.0,28 ℃ of pH, 180rpm shaking table were cultivated 11 days.
7. the living diastase that arbitrary described method prepares in the claim 3 to 6.
8. mould (Penicillium sp.) GXU20 CGMCC No.3690 and/or the described living diastase of claim 7 application in producing alcohol.
9. application as claimed in claim 8 is characterized in that: when using described living diastase, the pH value is 3.5-6.5, and temperature is 35-60 ℃; When using described living diastase, the pH value is preferably 4.5, and temperature is preferably 50 ℃.
10. a method of producing alcohol comprises the steps: that to give birth to Tapioca Starch be substrate, adds described living diastase of claim 6 and Angel yeast saccharomyces cerevisiae, and pH4.0,40 ℃, 150rpm fermentation obtain alcohol.
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CN109679937A (en) * 2019-01-07 2019-04-26 安徽大学 A kind of produced amylolysis enzyme with high specific enzyme activity power, its encoding gene and its application

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CN104673861A (en) * 2015-02-05 2015-06-03 江南大学 Method for improving enzymolysis efficiency of raw starch
CN104673861B (en) * 2015-02-05 2018-10-16 江南大学 A method of improving farinose enzymolysis efficiency
CN105925594A (en) * 2016-06-13 2016-09-07 广西大学 Raw starch-digesting glucoamylase, preparation method thereof and application of raw starch-digesting glucoamylase to raw starch hydrolysis and preparation of ethanol by simultaneous saccharification and fermentation of raw starch
CN109679937A (en) * 2019-01-07 2019-04-26 安徽大学 A kind of produced amylolysis enzyme with high specific enzyme activity power, its encoding gene and its application
CN109679937B (en) * 2019-01-07 2022-06-07 安徽大学 Raw starch hydrolase with high specific enzyme activity, encoding gene and application thereof

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