CN108913604A - A kind of screening technique of the effectively hydrolyzing bacterial strain of spirit distiller grain - Google Patents
A kind of screening technique of the effectively hydrolyzing bacterial strain of spirit distiller grain Download PDFInfo
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Abstract
The present invention discloses a kind of screening technique of the effectively hydrolyzing bacterial strain of spirit distiller grain, it is characterised in that：It comprises the steps of：The first, related hydrolysis spirit distiller grain may hydrolyze effective fungi of spirit distiller grain, stack to be directly separated in environment from spirit distiller grain and cultivate fungi in strain library；The second, it by the fungi screened using the single polymers for being difficult to hydrolyze in spirit distiller grain as sole carbon source, passes sequentially through the Congo red culture medium of the Congo red culture medium of sodium carboxymethylcellulose-, PDA-aniline blue culture medium, xylan-and carries out preliminary screening；Third cultivates bacterial strain by losing in poor liquid for sole nutrition substance of spirit distiller grain, judges bacterial strain to losing poor hydrolysis ability；4th, compound bacteria group is carried out to enzyme activity test in spirit distiller grain liquid, the bacterium group that screening has synergistic effect, enzyme activity high is high-efficiency composite hydrolysis bacterium group；5th, by high-effective service group with published the enzyme activity of mutant strain with effectively hydrolyzing cellulose and the ability of hydrolyzing biomass is compared.
Description
Technical field
The present invention relates to the optimizing research of hydrolysis spirit distiller grain scheme, in particular to two groups new effectively hydrolyzing spirit distiller grains
Compound bacteria group screening study.
Technical background
Spirit distiller grain is the nutrition barren grain hulls and residual after fermenting and distilling repeatedly and extract alcohol such as cereal
Slag body.It is high to lose poor moisture, lignocellulosic (cellulose, hemicellulose, lignin) content, crude protein, crude fat equal size are low,
Because it is few to lose poor nutritional ingredient, lignocellulosic difficulty hydrolysis, so vinasse cannot effectively be utilized like that as, to make to lose
The conversion of grain is extremely limited.Moreover lose that poor acidity is big, and moisture content is higher, it is easily putrid and deteriorated to cause plant area and surrounding
Environmental pollution.And big and small brewery is distributed in all over China, while these breweries bring income to local resident, also produces
It has given birth to and has largely lost grain, annual emissions have been more than 200,000 tons.How to handle these to lose grain is also local government and brewery administrator
Member's pain in the neck.
In recent years, become hot spot using microbiological treatment biological waste, because with required for physics and chemical treatments
Expensive equipment and reagent is compared, and Biological Pretreatment is under mild conditions by utilizing microbial solid fermentation hydrolytic lignin
Cellulose family biology, equipment requirement is without special, and the microorganism of avirulence hydrolysis of lignocellulose is deposited extensively in the environment
Have many advantages, such as cheap, easily acquisition, environmental protection.
Summary of the invention
The technical problem to be solved in the present invention：For losing, poor acidity is big, nutrition is low, use value is low, pollutes environment
Situation provides one kind with microorganism high-effective service group and loses poor hydrolysis rate to improve, reduce waste residue parking space, reduces waste residue
The technical solution of processing cost promotes to maintain good ecological environment, reaches sustainable and recycle spirit distiller grain, be zero-emission without
The government of pollution advocates business goal and lays the foundation.
Technical solution of the present invention：A kind of screening technique of the effectively hydrolyzing bacterial strain of spirit distiller grain, comprises the steps of：The
One, needed for hydrolysis spirit distiller grain related in Dutch Westerdijk Fungal Biodiversity institute strain library
The key enzyme information wanted may hydrolyze effective fungi of spirit distiller grain to screen, be directly separated training from spirit distiller grain stacking environment
Raise the fungi that can be grown under extremely acid and nutritional ingredient lean conditions；The second, by the fungi screened in spirit distiller grain
Being difficult to the single polymers hydrolyzed is sole carbon source, passes sequentially through the Congo red culture medium of sodium carboxymethylcellulose-, PDA-aniline
The Congo red culture medium of blue culture medium, xylan-carries out preliminary screening；Diameter in second step and hydrolysis, colour developing are enclosed ratio by third
It is worth biggish bacterial strain, is cultivated by losing in poor liquid for sole nutrition substance of spirit distiller grain, test enzyme activity, judges bacterial strain to losing
Poor hydrolysis ability size, as further screening；4th, by the way that the high bacterial strain of enzyme activity value is formed compound bacteria group, by compound bacteria
Group carries out enzyme activity test in spirit distiller grain liquid, and the bacterium group that screening has synergistic effect, enzyme activity high is high efficiency composition water
Bacterium group is solved, and optimizes high-effective service group enzymatic productivity using producing enzyme Optimal Medium；5th, by high-effective service group and openly
The enzyme activity of mutant strain with effectively hydrolyzing cellulose and the ability of hydrolyzing biomass delivered are compared.
The effectively hydrolyzing bacterial strain is 113.65 Oidiodendron echinulatum of CBS, CBS
246.84Phanerochaete chrysosporium、CBS 383.78 Trichoderma reesei、CBS
554.65Aspergillus niger, 110.42 CBS Aspergillus niger and GYM-37 Bjerkandera
One of adusta or more than one combinations.
Preferably, effectively hydrolyzing bacterial strain is CBS 383.78+CBS 554.65 and CBS 383.78+CBS554.65+GYM-
37 two compound bacteria groups.
Being directly separated culture in the spirit distiller grain stacking environment includes：
| No. | Fungi |
| GYM-22 | Monascus sp. |
| GYM-26 | Monascus purpureus |
| GYM-27 | Galactomyces sp. |
| GYM-29 | Strobilurus trullisatus |
| GYM-30 | Moniliella sp. |
| GYM-31 | Bjerkandera adusta |
| GYM-32 | Galactomyces sp. |
| GYM-33 | Aspergillus fumigatus |
| GYM-34 | Byssochlamys sp. |
| GYM-35 | Aspergillus sp. |
| GYM-36 | Bjerkandera adusta |
| GYM-37(bn2(2)) | Bjerkandera adusta |
| GYM-38 | Geotrichum sp. |
The slave Westerdijk is filtered out altogether：
The preliminary screening obtains：
| No. | Fungi |
| CBS 113.65 | Oidiodendron echinulatum |
| CBS 116.64 | Thermomyces verrucosus |
| CBS 138.65 | Penicillium simplicissimum |
| CBS 246.84 | Phanerochaete chrysosporium |
| CBS 613.91 | Pleurotus eryngii |
| CBS 882.68 | Doratomyces nanus |
| CBS 878.68 | Stachybotrys cylindrospora |
| CBS 383.78 | Trichoderma reesei |
| CBS 439.92 | Trichoderma reesei |
| CBS 101526 | Trichoderma viride |
| CBS 554.65 | Aspergillus niger |
| CBS 110.42 | Aspergillus niger |
| GYM-30 | Moniliella sp. |
| GYM-36 | Bjerkandera adusta |
| GYM-37(bn2(2)) | Bjerkandera adusta |
| GYM-38 | Geotrichum sp. |
Most preferably investment mass ratio is respectively 1/2 CBS 383.78+1/2 CBS554.65 when for hydrolysis spirit distiller grain
With 1/3 CBS 383.78+1/3 CBS 554.65+1/3 GYM-37.
Beneficial effects of the present invention
1. research using effectively hydrolyzing bacterial strain is first screened, recycles the composition compound bacteria group hydrolysis of fungi Collaborative experiment to lose
Grain, two degree improve bacterial strain to the hydrolysis ability for losing grain.New high-effective service group is screened and established, the hydrolysis for losing grain is optimized
Efficiency (383.78 cellulose of CBS 554.65+CBS, hemicellulose enzyme activity improve 11.7%-135% compared with single bacterial strain,
CBS 554.65+CBS 383.78+GYM-37 improves hemicellulose 41.8% and cellulase activity compared with single bacterial strain
74%), high hydrolysis efficiency, which more promotes, loses grain and from high molecular polymer is converted into the monosaccharide such as glucose, xylan, solves
Methane-producing bacteria of having determined cannot produce this problem of bio-natural gas using high molecular polymer, lose this all one's life of grain to realize
The transformation of object waste material to bio-natural gas develops circulation, green economy etc. with important to solve the in short supply of current fossil fuel
Effect.
2. research also makes the cellulose of high-effective service group, the enzyme activity of hemicellulose, lignin using culture medium
Power than use merely lose it is poor as nutriment in the case where (383.78 enzyme activity of CBS 554.65+CBS increases 45%-
268%, CBS 554.65+CBS 383.78+GYM-37 enzyme activity increase 55%-758%, and the lignin of two combinations is crucial
Enzyme is also all improved.)
It is mentioned 3. screening and establishing new high-effective service group for excavating ability that fungi hydrolyzes spirit distiller grain and separation
Ectoenzyme caused by pure compound bacteria group promotes the industrialization of ectoenzyme to convert, and reduces market enzyme preparation price etc. with important
Effect.
4. research establish compound bacteria group (wild strain) and SCI cellulose mutant strain reported in the literature to lose grain and
Quite (the content difference for generating reduced sugar is smaller, is shown in Table four) for the hydrolysis ability of other eight kinds of biomass.Promote wild strain
The development and utilization of resource.
Each bacterial strain is in carboxymethyl cellulose element sodium-Congo red culture medium, PDA-aniline blue culture medium, the xylan-the Congo
The ability of preliminary test hydrolysis of lignocellulose is shown in Table one on red culture medium, the results showed that CBS 113.65, CBS 246.84,
The 16 fungal strain diameter such as CBS 383.78, CBS 554.65, CBS 439.92, CBS 110.42, GYM-37 and colour developing, hydrolysis are enclosed
Ratio it is larger.Screening bacterium hydrolysis, colour developing biggish bacterial strain of loop diameter ratio on above-mentioned plate are losing poor liquid culture the 3rd, 7 day
Hydrolysis of lignocellulose ability see Figure 1, the results showed that CBS 113.65, CBS 246.84, CBS 383.78, CBS
554.65, CBS 110.42, GYM-37 have stronger cellulose, hydrolysis of hemicellulose ability, and CBS 113.65 has stronger wood
Quality laccase activity, GYM-37 have stronger lignin peroxidase activity.Fungi Collaborative experiment shows CBS 554.65,
CBS383.78 has synergistic effect, and several celluloses, the key enzyme of hemicellulose are all high compared with single bacterial strain after combination, and enzyme activity
Power is higher in each combination, and CBS 554.65+CBS 383.78+GYM-37 combination and 383.78 phase of CBS554.65+CBS
Than they are suitable or slightly lower to the crucial enzyme activity of cellulose, hemicellulose, and (Cellulase reaches peak on day 3, is 99 μ
Zero) mol/L is gradually decreased down later, and CBS 554.65+CBS 383.78+GYM-37 combination culture first three days also have portion
Divide lignin peroxidase activity, is shown in Table two with 16 groups of compound bacteria groups that six plant heights imitate hydrolysis strain construction.High-effective service
Group CBS 554.65+CBS383.78, CBS 554.65+CBS 383.78+GYM-37 and cellulose mutant bacterium group enzyme activity are tested
Comparison is shown in Table three, is shown in Table four to the hydrolysis ability of eight kinds of biomass, table three, four efficient high-effective service group and fiber as the result is shown
Plain mutant strain is suitable to the hydrolysis ability for losing grain and biomass.
Table one：Bacterium is cultivated 5 days on containing carboxymethyl cellulose element sodium, aniline blue, xylan culture medium and is hydrolyzed
Circle/bacterium diameter ratio table
Annotation：Table one indicates to hydrolyze circle/bacterium diameter after culture 5 days, wherein 3 are expressed as that ratio is big, and 2 are expressed as comparing
It is worth larger, 1 to be expressed as ratio small.Each bacterial strain is repeated 2 times experiment.
Two compound bacteria group of table is to lose the crucial enzyme activity test lost in poor liquid that grain is sole nutrition substance
Three, high-effective service group of table and cellulose mutant bacterium group enzyme enzyme activity test comparison
Four, high-effective service group of table is compared with cellulose mutant bacterium group is to the hydrolysis ability of biomass
Detailed description of the invention
Fig. 1 is the enzyme activity testing research of fungi.Experimental study is to lose grain losing for sole nutrition substance containing 6%
3 days, 7 days are cultivated in poor liquid, and supernatant is taken to carry out enzyme activity determination.A is expressed as β-glucosyl enzym enzyme activity, and b is expressed as cellulose
Enzyme enzyme activity, c are expressed as xylobiase enzyme activity, and d is expressed as xylanase activity power, and e is expressed as laccase activity, and f is indicated
For lignin peroxidase enzyme activity.
Fig. 2 is indicated using standard curve HPLC measurement reduced sugar (glucose, fructose, galactolipin etc.) content and formulated, a
Rhamnose standard curve, b indicate that galactolipin standard curve, c indicate that arabinose standard curve, d indicate glucose standard curve,
E indicates that fructose standard curve, f indicate that galacturonic acid standard curve, g indicate ribose standard curve.
Specific embodiment
The first, the fungi of the corresponding hydrolysis spirit distiller grain of the research center Westerdijk purchase, from losing in poor acidic environment point
From fungal bacterial strain；
1. sampling (acquires spirit distiller grain compost object, fresh spirit distiller grain, wine in brewery in Liquor-making Enterprises & acidic environment
Poor compost object is used to separate the fungi that may have hydrolysis spirit distiller grain, and fresh spirit distiller grain is for being added in culture medium), claim
It takes compost object sample 10g in 250mL triangular flask, 100mL sterile water is added, shakes 3~4h in 30 DEG C of constant temperature oscillation boxes.
1.1 strain isolation：Uniform bacterium solution will be shaken and stand 30min, appropriate supernatant is drawn and carry out 10 times of gradient dilutions,
10 are drawn respectively-1~10-5Each 0.5mL of the bacterium solution of gradient is inoculated in YPD culture medium, and (yeast extract peptone glucose agar medium, contains
Chloramphenicol), 24-48h is cultivated in 28 DEG C of inversions；
1.2 bacterial strain transferred speciess and passage：It is inoculated in YPD solid medium tablets (containing chloramphenicol), method of scoring separates single colonie,
25 DEG C, 24-48h is cultivated, fresh bacterium colony is used as trying bacterium；
1.3 observation colonial morphologies, size, color；
1.4 take for trying bacterium culture smear, oil mirror observation form, arrangement and dyeability after Grain stain；
1.5 DNA are extracted：CTAB method extracts DNA；
1.5.1 strain inoculated 28 DEG C, is cultivated into 72h in PDA culture medium plate；
1.5.2 culture dish is taken out out of incubator and is put on sterilized superclean bench；
1.5.3 it opens water bath and is heated to 60 DEG C；
1.5.4 400 μ l CTAB-buffer 2x and 6-10 sour bulb glass pearl (1.5- are added in 2ml centrifuge tube
2mm), picking colony volume (1-10)) mm is in the PVP that 100 μ l 10% in the centrifuge tube, are then added, on rotation nest vortex mixer
Mix 10min.It is put into water bath, 60 DEG C of water-bath 60min；
1.5.5 after bacterium colony dissolution, 500 μ l SEVAG are added, after shaking 2min, form emulsion.Centrifugation：14000g
10min；
1.5.6 supernatant liquor is collected to be added in another sterile EP tube, it is quantitative；
1.5.7 frost isopropanol (amount is 2/3 volume of supernatant, is mixed) is added, turn upside down centrifuge tube, -20 DEG C of mistakes
Night culture；
1.5.8 taking out, centrifugation：14000g 10min；
1.5.9 supernatant is abandoned, 70% frost alcohol 1ml is added, mixes gently, is centrifuged：14000g 2min. dries；
1.5.10 DNA is precipitated, and TE-buffer 50ul is added and obtains DNA purification liquid；
1.5.11 the DNA extracted, absorbance method detect DNA concentration, and it is spare to be diluted to 1ug/ul freezing.
1.6 PCR amplification
1.6.1 PCR primer：According to document ITS specific primer.PCR amplification the primer is had by the raw work biotechnology in Shanghai
The synthesis of limit company.
Gene primer
ITS ITS5 5’-GGAAGTAAAAGTCGTAACAAGG-3’
ITS4 5'-TCCTCCGCTTATTGATATGC-3'；
PCR reaction system (26 μ L)：
1.6.2 PCR response procedures：
1.6.3 the mentioned DNA of detected through gel electrophoresis
1.6.3.1 1.0g agar Icing Sugar is weighed in 100ml 1.0 × TAE working solution, and it is complete to be heated to agarose with micro-wave oven
It after full thawing, takes out room temperature and is cooled to 60 DEG C or so, 1 μ l green such as blue (DNAGREEN) is added, shakes and mixes, 1.0% will be prepared
Ago-Gel about 60ml pour on the gel casting platform sealed, be inserted into sample comb；After being gelled admittedly, comb is removed
Tooth is put into the horizontal gel electrophoresis instrument added with enough TAE electrophoretic buffers, buffer should be made to be higher by gel 1cm；
1.6.3.2 after adding 3 μ l DNA extracts to mix 1 μ 10 × loading of l buffer, sample solution is prepared, with shifting
Sample solution is added in sample well liquid device；
1.6.3.3 it is powered, 100V electrophoresis about 20min has found that indicator is migrated to being sufficiently separated DNA piece in sample loading buffer
The distance of section stops electrophoresis；It is observed in ultraviolet projectoscope through the green gel such as blue (DNAGREEN) dyeing, observes result；
1.7 sequencing：The raw work sequencing in sea is served after agarose gel electrophoresis detection.
The comparison of 1.8 sequence similarities：Homologous sequence search (BLAST is carried out in GenBank GenBank
Search) to compare the similarity degree of strains tested Yu known bacterial strain corresponding sequence；It determines kind and understands its growth characteristics.
Monascus sp., Bjerkandera adusta., Geotrichum sp. are isolated from brewery,
13 fungal strain such as Aspergillus sp..
| No. | Fungi |
| GYM-22 | Monascus sp. |
| GYM-26 | Monascus purpureus |
| GYM-27 | Galactomyces sp. |
| GYM-29 | Strobilurus trullisatus |
| GYM-30 | Moniliella sp. |
| GYM-31 | Bjerkandera adusta |
| GYM-32 | Galactomyces sp. |
| GYM-33 | Aspergillus fumigatus |
| GYM-34 | Byssochlamys sp. |
| GYM-35 | Aspergillus sp. |
| GYM-36 | Bjerkandera adusta |
| GYM-37(bn2(2)) | Bjerkandera adusta |
| GYM-38 | Geotrichum sp. |
2 in Westerdijk research institute strain library (WWW.FUNG-GROWTH.ORG) needed for related hydrolysis spirit distiller grain
The key enzyme information wanted may hydrolyze effective fungi of spirit distiller grain to screen, thus find respectively hydrolytic lignin, cellulose,
The fungi of hemicellulose.
The first, it is filtered out altogether from Westerdijk
The second, will on sodium carboxymethylcellulose, lignin, hemicellulose plate well-grown bacterial strain, utilize carboxylic first
The Congo red culture medium of base cellulose element sodium-Congo red culture medium, PDA-aniline blue culture medium, xylan-carries out sxemiquantitative sieve
Choosing；
The screening of 2.1 solid mediums
2.1.1 using single polymers in spirit distiller grain as the screening experiment of sole carbon source.
2.1.1.1 culture medium preparation method：
A. carboxymethyl cellulose -- Congo red culture medium prescription：
KH2PO4:0.5g,
MgS04·7H20:0.5g,
Sodium carboxymethylcellulose:10g
Agar:15g,
It is Congo red:0.2g,
Gelatin：2g；
Distilled water 1L, pH 6.8-7.2；
B. potato dextrose agar (PDA) -- aniline blue culture medium
PDA culture medium：Potato：200g, glucose：20g, agar 15g, distilled water 1L
0.01% aniline blue is added in PDA.
C. the Congo red culture medium of xylan
Congo red 0.01g,
MgS04·7H20:0.1g,
Yeast powder:0.5g
KH2PO4:0.1g
Agar:1.5g,
(NH4)2SO4:0.1g
Xylan：0.5g
Nacl:0.5g,ddH2O:100ml；
2.1.1.2 each bacterium is cultivated 3 days on yeast extract peptone glucose agar medium (YPD), it is raw to lawn
After length is good, punched with the punch that diameter is 8mm, pure culture biscuits involvng inoculation is permanent in 25 DEG C in tetra- kinds of culture mediums of above-mentioned a, b, c
Temperature is incubated for, and not to be inoculated with the culture medium of bacterium as control, each bacterial strain is repeated twice experiment, is averaged, PDA-aniline blue culture
Base need to be protected from light incubation)；
2.1.1.3 observation colour developing daily, hydrolysis circle, lawn size, measure its diameter respectively and record.
113.65 246.84 Phanerochaete of Oidiodendron echinulatum, CBS of CBS is filtered out altogether
Chrysosporium, CBS 383.78 Trichoderma reesei, CBS 554.65 Aspergillus niger, CBS
110.42 Aspergillus niger, GYM-37 Bjerkandera adusta, CBS 439.92Trichoderma
16 plants of hydrolysis circles such as reesei and the biggish fungi of bacterium diameter ratio.
2.2 lose poor fluid nutrient medium screening
2.2.1 waste spent grains powder is broken, 80 meshes are crossed, 3g is accurately weighed and 50ml distilled water is added, be made and lose poor liquid.
2.2.2 it will develop the color in tetra- kinds of culture mediums of a, b, c, hydrolyze circle with the big strain inoculated of lawn ratio in YPD liquid
In culture medium, after constant-temperature table is incubated for 3-7 days, it is seeded in and is lost in poor liquid with the amount of 7.5mg/L, 25 DEG C of constant-temperature incubations, not connect
The culture medium of kind bacterium is control, and each bacterial strain is repeated twice experiment, is averaged.
2.2.3 each bacterium growing state in losing poor liquid is observed daily, was taken respectively at the 3rd, 7 day and is lost poor liquid, through 14000rpm
It is centrifuged 10min, supernatant is taken to store 4 DEG C, is used for enzyme activity determination.
2.2.4 enzyme activity determination
2.2.4.1 cellulose key enzymatic determination
(1) sugared (BG) enzyme activity determination of beta-glucosidase
With pNP- β-D-glucopyranose (pNPBGL) for substrate
Reaction system is：1%pNPBGL 50 μ l, 10 μ l (50mM, pH 5.0) NaAc, 40 μ l enzyme supernatants are added to 96
In orifice plate.After reaction system after 25 DEG C are incubated overnight, after 100 μ l of (0.25M) Na2CO3 is added, in ultraviolet specrophotometer,
OD value is measured at 405nm.
(2) β-xylosidase enzyme activity determination
With pNP- β-D-xylopyranose (pNPBXL) for substrate
Reaction system is：1%pNPBXL 50 μ l, 10 μ l (50mM, pH 5.0) NaAc, 40 μ l enzyme supernatants are added to 96
In orifice plate.After reaction system after 25 DEG C are incubated overnight, after 100 μ l of (0.25M) Na2CO3 is added, in ultraviolet specrophotometer,
Absorbance value is measured at 405nm.
(3) Cellulase enzyme activity determination
With microcrystalline cellulose (Avicel) for substrate
Reaction system is：180 μ l of 1%Avicel, 20 μ l supernatants are added in 96 orifice plates, after 25 DEG C are incubated overnight, with
After vortex device mixes, 100 μ l supernatants of transfer to 96 new orifice plates, 150 μ l DNS liquid of addition, 95 DEG C after water-bath 30 minutes, sample
Product cooling hatching in ice bath, and the measurement light absorption value at 540nm.
(4) zytase (xylanase) enzyme activity determination
Using Xylan as substrate
Reaction system is：180 μ l of 1%Xylan, 20 μ l enzyme supernatants are added in 96 orifice plates, after 25 DEG C are incubated overnight,
After being mixed with vortex device, 100 μ l supernatants of transfer to 96 new orifice plates, 150 μ l DNS liquid of addition, 95 DEG C after water-bath 30 minutes,
Sample cooling hatching in ice bath, and the measurement light absorption value at 540nm.
(5) laccase (laccase) enzyme activity determination
With 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) for substrate
Reaction system is：The 140 μ l μ l enzyme supernatants of ddH2O, 20 μ l glycine-HCl (500Mm, pH 3.0), 20,20
After 25 DEG C of μ l ABTS are incubated overnight, light absorption value is measured at 436nm.
(6) lignin peroxidase activity measures
With ABTS and H2O2For substrate
Reaction system is：125μl ddH2O、10μl、10μl H2O2、25μl briffon-Robinson buffer(pH
4.5), 20 μ l enzyme supernatant after 20 25 DEG C of μ l ABTS incubation 1h, measures light absorption value at 436nm.Each sample is repeated 2 times,
It averages and standard error, loses poor supernatant as blank control using be not added bacterium solution.
2.2.4.2 the drafting of standard curve
2.2.4.2.1 the drafting of pNP standard curve
100 μM of pNP (MW=139.11g/mol)
1ml of 100mM:It weighs 0.0139g pNP and presses 1:10 dilutions, then press 1:100 dilutions.
Amount in the above table is added in 96 orifice plates, then (0.25M) Na is added in every hole2CO3100 μ l, at 436nm
Measure light absorption value.Using absorbance value as ordinate, using pNP content as abscissa, standard curve is drawn, standard curve is listed
Equation calculates enzyme activity value.
2.2.4.2.2 the drafting of DNS standard curve
180mg glucose is weighed, is dissolved in 10ml NaAC, dilutes 5 times
| umol/L | Glucose | NaAC | |
| 20 | 100 | 0 | |
| 16 | 80 | 20 | |
| 12 | 60 | 40 | |
| 8 | 40 | 60 | |
| 6 | 30 | 70 | |
| 4 | 20 | 80 | |
| 2 | 10 | 90 | |
| blank | 0 | 0 | 0 |
Amount in the above table is added in 96 orifice plates, then 150 μ l DNS liquid are added in every hole, 95 DEG C water-bath 30 minutes
Afterwards, sample cooling hatching in ice bath, and the measurement light absorption value at 540nm.Using absorbance value as ordinate, with glucose
Content draws standard curve as abscissa, lists calibration curve equation, calculates enzyme activity value.
2.2.4.3 the calculating of enzyme activity force value
PNP and DNS method is calculated by respective standard curvilinear equation, and laccase and lignin peroxidase enzyme activity value press formula：
Δ OD420 × V is always × 106(/ ε × l × V enzyme), ε is the molar extinction coefficient (36000Lmol of ABTS oxidation state in formula-1·
cm-1)。
6 plant heights effect hydrolysis bacterial strain is screened altogether.
CBS 113.65 Oidiodendron echinulatum、CBS 246.84 Phanerochaete
chrysosporium、CBS 383.78 Trichoderma reesei、CBS 554.65 Aspergillus niger、CBS
110.42 Aspergillus niger、GYM-37 Bjerkandera adusta。
2.3 Combined cultures and producing enzyme optimization culture
2.3.1 Combined culture lose poor liquid and bacterium solution prepares according to the above method
The total amount of inoculation bacterium is all 7.5mg/L, enzyme activity determination and the same single bacterial strain of calculation method, loses grain be not inoculated with bacterium
Liquid and single bacterial strain are control.Each compound bacteria group, is repeated 2 times, averages.
It filters out effectively hydrolyzing and loses poor compound bacteria group two：CBS 383.78+CBS 554.65 and CBS 383.78+CBS
554.65+GYM-37。
2.3.2 1%minimal medium (MM) 50ml containing 0.4mM CuSO4 is seeded in by 2.2.3 methods
(Vries RPD et al.2004) and 3g lose in poor fluid nutrient medium, and the 3rd, 5,7 day supernatant of culture is taken to carry out enzyme activity survey
It is fixed.
2.4 high-effective service groups and cellulose mutant strain compare the hydrolysis activity for losing grain
2.4.1 this research cellulose mutant strain used see the table below, bacterial strain be maintained in (Fungal Physiology,
RonaldP.de Vries study group)
2.4.2 it by each mutant strain, combines with this experiment CBS383.78, is inoculated with by 2.3.2 the method, take culture the
3,5,7 days supernatants carry out enzyme activity determination.
2.5 mutation compound strains are compared with two groups of effectively hydrolyzing bacterium groups of this research are to the hydrolysis ability of eight kinds of biomass.
2.5.1 eight kinds of biomass information see the table below
| Middle name of the country | Eeglish name |
| Rice bran | Rice bran(Rb) |
| Wheat bran | Wheat bran(Wb) |
| Corn stover | Corn stover(CS) |
| Birch (tree) powder | Birchwood(biw) |
| Norway spruce powder | Norway sprule(Nan) |
| Megasse | Sugar beet pulp(sugar) |
| Lose grain | waste grain(sg) |
| Wheat stalk | Wheat stover(WS) |
2.5.2 each biomass is added to the Sodium acetate buffer (pH 4.5) of 50mM (containing 0.02%
NaN3 Sodium Afide) be made into 1% concentration biomass.
2.5.3 it takes 500 μ l to be added to the EP pipe of 2ml after each biomass being shaken up, adds the culture supernatant of 100 μ l.
2.5.4 by each sample at 30 DEG C, 100rpm shaking table be incubated for for 24 hours after in 95 DEG C of water-bath 15min, then at 4 DEG C,
14000rpm is centrifuged 10min.
2.5.5 the 100 μ l of supernatant after taking previous step to be centrifuged is surveyed after diluting 10 times with MilliQ water using HPLC
Determine reduced sugar (glucose, xylose, fructose, galactolipin) content, utilizes related reduced sugar (glucose, xylose, fructose, galactolipin)
Standard items do standard curve.
Claims (7)
1. a kind of screening technique of the effectively hydrolyzing bacterial strain of spirit distiller grain, it is characterised in that：It comprises the steps of：The first, from lotus
It is closed required for related hydrolysis spirit distiller grain in blue Westerdijk Fungal Biodiversity Institute strain library
Key enzyme information may hydrolyze effective fungi of spirit distiller grain to screen, stack from spirit distiller grain and be directly separated that turn out can in environment
The fungi grown under extremely acid and nutritional ingredient lean conditions；The second, by the fungi screened to be difficult to water in spirit distiller grain
The single polymers of solution are sole carbon source, pass sequentially through the Congo red culture medium of sodium carboxymethylcellulose-, PDA-aniline blue culture
The Congo red culture medium of base, xylan-carries out preliminary screening；Third, by diameter in second step and hydrolysis, colour developing circle ratio it is larger
Bacterial strain, cultivated by losing in poor liquid for sole nutrition substance of spirit distiller grain, test enzyme activity, judge bacterial strain to losing poor hydrolysis
Capacity of water, as further screening；4th, by the way that the high bacterial strain of enzyme activity value is formed compound bacteria group, by compound bacteria group white
Wine loses progress enzyme activity test in poor liquid, and the bacterium group that screening has synergistic effect, enzyme activity high is high-efficiency composite hydrolysis bacterium group,
And optimize high-effective service group enzymatic productivity using producing enzyme Optimal Medium；5th, it high-effective service group and will publish
The enzyme activity of mutant strain with effectively hydrolyzing cellulose and the ability of hydrolyzing biomass be compared.
2. a kind of screening technique of the effectively hydrolyzing bacterial strain of spirit distiller grain according to claim 1, it is characterised in that：It is described
Effectively hydrolyzing bacterial strain be 113.65 Oidiodendron echinulatum of CBS, 246.84 CBS Phanerochaete
chrysosporium、CBS 383.78 Trichoderma reesei、CBS 554.65 Aspergillus niger、CBS
One of 110.42 Aspergillus niger and GYM-37 Bjerkandera adusta or more than one combinations.
3. a kind of screening technique of the effectively hydrolyzing bacterial strain of spirit distiller grain according to claim 1, it is characterised in that：It is preferred that
, effectively hydrolyzing bacterial strain is that CBS 383.78+CBS 554.65 and CBS 383.78+CBS 554.65+GYM-37 two is compound
Bacterium group.
4. a kind of screening technique of the effectively hydrolyzing bacterial strain of spirit distiller grain according to claim 1, it is characterised in that：It is described
Spirit distiller grain stack and be directly separated culture in environment and include：
5. a kind of screening technique of the effectively hydrolyzing bacterial strain of spirit distiller grain according to claim 1, it is characterised in that：It is described
Slave Westerdijk filter out altogether：
6. a kind of screening technique of the effectively hydrolyzing bacterial strain of spirit distiller grain according to claim 4 or 5, it is characterised in that：
The preliminary screening obtains：
7. according to a kind of screening technique of the effectively hydrolyzing bacterial strain of spirit distiller grain of claim 3, it is characterised in that：It is white for hydrolyzing
Most preferably investment mass ratio is respectively 1/2 CBS 383.78+1/2 CBS 554.65 and 1/3 CBS 383.78+ when wine loses grain
1/3 CBS 554.65+1/3 GYM-37。
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| CN113621674B (en) * | 2021-08-27 | 2022-06-21 | 泸州老窖股份有限公司 | Method for producing L-lactic acid by using liquor distiller grains |
| CN113621527A (en) * | 2021-09-27 | 2021-11-09 | 阜阳师范大学 | Novel smoke tube bacterium SZ-4 and application thereof |
| CN113621527B (en) * | 2021-09-27 | 2022-03-25 | 阜阳师范大学 | Novel smoke tube bacterium SZ-4 and application thereof |
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