CN105567592A - Bacillus subtilis capable of simultaneously producing pectase and hemicellulase, screening method and applications thereof - Google Patents
Bacillus subtilis capable of simultaneously producing pectase and hemicellulase, screening method and applications thereof Download PDFInfo
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Abstract
The invention relates to a strain of bacillus subtilis capable of simultaneously producing pectase and hemicellulase, a screening method and applications thereof. The strain is named as 16#, and belongs to bacillus subtilis. The preservation number is CGMCC No.11898, the preservation date is 22nd December, 2015, and the preservation organization is China General Microbiological Culture Collection Center (CGMCC), whose address is Beichenxi Road No.1 Yard No.3, Chaoyang District, Beijing. The provided bacillus subtilis is capable of simultaneously producing pectase and hemicellulase, barely produces cellulase, and can be applied to degumming of hemp. The bacillus subtilis is applied to degumming at a room temperature for 96 hours or so, the rate of residual gum can reach 13% or so, the processed hemp can almost reach the requirements of textile industry, and the provided bacillus subtilis can also be applied to degumming of other hemps.
Description
Technical field
The invention belongs to industrial microbial technology field, especially one can produce the subtilis of polygalacturonase and hemicellulase and screening method and application simultaneously.
Background technology
Hemp is annual Moraceae, Cannabis plant, highly about 3 meters.Formal name used at school is Cannabissatival, also referred to as Chinese fiber crops, cold fiber crops, hemp, the male plant of hemp etc.Hemp plantation is very low to the requirement of weather and soil, and without the need to the fertilising in agricultural chemicals and the external world in process of growth, insect pest ability is strong, growth rapidly, plantation is short etc. for harvesting time, the most important thing is that hemp fibre has many distinctive excellent properties, as antistatic, hypo-allergenic, good heat retaining property, thermal diffusivity, fungi-proofing, anticorrosion, uvioresistant etc.Hemp fibre is important textile fibres crop.
Compared with other crudefiber crops (ramie, flax etc.), the biological degumming research of hemp fibre is relatively less, and difficulty of coming unstuck also is higher than other crudefiber crops.Its gum level up to 40 ~ 50%, wherein based on polysaccharide; And its fibrous texture is more special, ultimate fibre difference in length is comparatively large, and fiber separation difference etc., these reasons all considerably increase the difficulty of coming unstuck of hemp.Hemp fibre is had can textile, must remove most colloid, and its ultimate fibre is separated from each other and becomes soft, this process is just called comes unstuck.At present, coming unstuck of flaxen fiber industrially adopts chemical degumming law more, and namely adopt strong acid, highly basic, tensio-active agent, sequestrant etc. process raw ramie at high temperature under high pressure.Although the method that this kind the comes unstuck time used is shorter, within several hours, just can obtain degummed ramie, the trade effluent after coming unstuck can bring great pollution to environment; And required energy consumption is high under High Temperature High Pressure, to coming unstuck, industry increases cost; The most important thing is, the chemical reagent major injury that the uses structure of fiber, reduces the quality of fiber.Along with the enhancing of environmental consciousness, this kind of method no longer advocate by people.
In order to overcome these shortcomings, improve industrial degumming technology, the domestic and international microbial degumming to flaxen fiber or the enzyme with Institute of Micro-biology's product came unstuck and have carried out studying widely in the last few years, achieved many achievements in research simultaneously.Biological degumming is as a kind of Degumming method of novel flaxen fiber, and its wide DEVELOPMENT PROSPECT is apparent.Gum components separated by the polygalacturonase that biological degumming mainly utilizes microorganism to produce and hemicellulase etc. point, but do not damage fibrous texture; Have production cost low, reaction conditions is gentle, pollutes few, the advantage such as to save water and energy.
At present, focus mostly on ramie to the research of the biological degumming of crudefiber crop both at home and abroad, flax, the biological degumming aspects such as bluish dogbane, and relatively less to the biological degumming research of hemp fibre, in the middle of research in the past, hemp fibre treatment cycle is long becomes the Main Bottleneck being applied to and producing reality.Therefore, find more suitable degumming strain and de-alkali additives, the shortening degumming process time becomes the task of top priority.
By retrieval, find patent publication us as relevant to patent application of the present invention in next chapter:
The preparation method of hemp degluing industrial zymin and application (CN1374398) thereof, the preparation method of zymin comprises slant culture and preservation, shake-flask culture, seed tank culture and the fermentor tank production of live high-enzyme kind, select subtilis No.13 bacterial strain, preserving number is CCTCCNO.M200038.The method of hemp degumming comprises fermenting enzyme liquid and crosses filter residue, and dilution enzyme liquid 3 ~ 6 times, puts into glue kettle, and the pH of dilution enzyme liquid is adjusted to 7.5 ~ 9.0, raw ramie pre-treatment, come unglued liquid and raw ramie bath raio (10 ~ 20): 1, usually time 2 ~ 10 hours.The inventive method is pollution-free, and hemp ramie stripes mean length, fineness, the breaking tenacity made, be all better than chemical method.
By contrast, there are the different of essence in patent application of the present invention and above-mentioned patent publication us.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art part, provide a strain can produce the bacterial strain of polygalacturonase and hemicellulase, this bacterial strain can be applied in the biological degumming of hemp fibre under mild conditions simultaneously.
To achieve these goals, the technical solution adopted in the present invention is as follows:
A kind of subtilis that simultaneously can produce polygalacturonase and hemicellulase, name is called 16#, specific name is: Bacillus subtilis, deposit number is: CGMCCNo.11898, preservation date: on December 22nd, 2015, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center.
And step is as follows:
Bacterial strain screening be separated:
Screen from the bacterial strain in the soil of process domestic refuse, and be lyophilized into bacterium powder;
1. enrichment culture: take the bacterium powder collecting also freeze-drying and be placed in hemp enriched liquid substratum, the ratio g:mL of described bacterium powder and hemp enriched liquid substratum is 1:150, at 37 DEG C, 180 ~ 220r/min enrichment culture 24 hours;
2. primary dcreening operation: the bacterium liquid after enrichment culture is according to 10 times of gradient dilutions: the bacterium liquid of enrichment is diluted to 10 with sterilized physiological saline
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7different dilution sample suspension, draws 10 after vibration mixing respectively
-5, 10
-6, 10
-7bacteria suspension 200 μ L, coat in pectin screening solid medium or xylan screening solid medium, at 37 DEG C, be inverted cultivation 24 hours, by Congo red colour developing, measure transparent circle diameter R and colony diameter r, tentatively judge the enzymatic productivity of bacterial strain with the ratio of R/r;
Then picking growing way is better, single bacterium colony that the ratio of R/r is larger, point is received in pectin screening solid medium or xylan screening solid medium, is inverted cultivation 24 hours, selects single bacterium colony separation and purification in LB solid medium that the ratio of R/r is larger at 37 DEG C;
After such separation and purification, the bacterium of gained can produce polygalacturonase and can produce zytase again, and then these bacteriums being preserved in whole mass concentration is in the glycerine pipe of 15%, frozen for subsequent use in-80 DEG C;
3. sieve again: first activated in LB solid medium by the bacterium of primary dcreening operation institute preservation, be inoculated in the substratum of the triangle shaking flask of the 250mL of the liquid LB that 50mL is housed with the inoculum size of a ring, at 37 DEG C, shake-flask culture 16 ~ 24 hours is as seed liquor, cell concentration OD
600reach 2 ~ 2.6;
Take 4-10g raw ramie, load in the triangle shaking flask of 250mL after first boiling water bath process 20 ~ 30min, inoculation 5 ~ 10mL, the tap water containing 0.2% nitrogenous source is added with bath raio 1:20 ~ 25, described nitrogenous source is the mixture of one or more in ammonium sulfate, ammonium nitrate, urea, ammonium oxalate, volatile salt, at 37 DEG C, 180 ~ 220r/min come unstuck 10 ~ 12 hours after take out, detect residual gum content, choose the low bacterial strain of residual gum content as degumming bacterium, called after 16#;
(2) the qualification of bacterial strain:
The individual morphology qualification of bacterial strain: to the bacterium of coming unstuck of sieving first carry out Morphological Identification, find that 16# bacterium bacterium colony is creamy white, bacterium colony is comparatively large, thickness, smooth surface are opaque, neat in edge, and older phage surface plays fold; All show positive through gramstaining, bar is short and small, and forms gemma in thalline central authorities, tentatively predicates bacillus;
16SrDNA gene sequencing and analysis: carry out pcr amplification 16SrDNA with the genomic dna of 16# bacterial strain for template and check order;
NCBI carries out Blast analysis show: the 16SrDNA sequence of 16# bacterial strain and the consistence of subtilis 16SrDNA sequence, up to 99%, obtain the subtilis that simultaneously can produce polygalacturonase and hemicellulase.
And, described step (1) 1. in hemp enriched liquid substratum be:
Hemp powder 2.0g, ammonium nitrate 0.2g, dipotassium hydrogen phosphate 0.05g, sodium sulfate 0.05g, distilled water 100mL, pH7.0.
And, described step (1) 2. in pectin screening solid medium be: pectin 0.5g, ammonium nitrate 0.2g, dipotassium hydrogen phosphate 0.1g, sodium sulfate 0.05g, magnesium sulfate 0.06g, agar powder 2.0g, Congo red 0.02g, distilled water 100mL, pH7.0 ~ 7.5; Or, described step (1) 2. in xylan screening solid medium be: xylan 1.0g, saltpetre 0.1g, magnesium sulfate 0.05g, sodium-chlor 0.05g, dipotassium hydrogen phosphate 0.05g, Congo red 0.02g, agar powder 2.0g, distilled water 100mL, pH7.0 ~ 7.5.
And, described step (1) 2. in LB solid medium be:
Tryptones 1.0g, yeast extract 0.5g, sodium-chlor 1.0g, agar powder 2.0g, distilled water 100mL, pH7.0.
And, described step (1) 3. in liquid LB be;
Tryptones 1.0g, yeast extract 0.5g, sodium-chlor 1.0g, distilled water 100mL, pH7.0.
A kind of application of subtilis in hemp degumming simultaneously producing polygalacturonase and hemicellulase as above.
And, with described subtilis for degumming bacterium is inoculated in liquid seed culture medium, be placed in 37 DEG C of shaking tables, 180 ~ 220rpm is cultured to 16 ~ 24h, is transferred to comes unstuck in substratum with volume ratio 5% ~ 10% inoculum size, come unstuck in substratum containing crudefiber crop raw ramie fiber, crudefiber crop is 1:20 ~ 30 with the ratio g:mL of the substratum that comes unstuck, and temperature of coming unstuck is 25 ~ 45 DEG C, and shaking speed is 180 ~ 220rpm, come unstuck 24 ~ 96h, the hemp after must coming unstuck.
And described liquid seed culture medium is LB liquid nutrient medium, broth culture or minimal medium; Or described crudefiber crop is hemp, ramie or flax; Or, described in the substratum that comes unstuck be retting liquid, this retting liquid is water or is the aqueous solution containing mass percent 0.05% ~ 1% nitrogenous source, or, be 0.05g/mL hemp, 0.002g/mL nitrogenous source, pH7.0.
And described nitrogenous source is the mixture of one or more in ammonium sulfate, ammonium nitrate, urea, ammonium oxalate, volatile salt; Or described liquid seed culture medium is LB liquid nutrient medium: containing NaCl10g, Tryptones 10g in 1000mL substratum, yeast extract 5g, pH7.0.
Accompanying drawing explanation
Fig. 1 is that the present invention sieves to obtain Bacillussubtilis colonial morphology figure;
Fig. 2 is that the present invention sieves to obtain Bacillussubtilis individual morphology figure;
Fig. 3 is the hemp design sketch that the present invention is come unstuck; Wherein, Fig. 3-1 is that hemp sieves to obtain Bacillussubtilis degumming effect figure for hemp through the present invention without degumming effect figure, Fig. 3-2;
Fig. 4 is the Electronic Speculum figure of hemp of the present invention; Wherein, Fig. 4-1 is the Electronic Speculum figure of hemp without the effect of coming unstuck, Fig. 4-2 is the Electronic Speculum figure of hemp through the Bacillussubtilis16# effect of coming unstuck.
Embodiment
Below in conjunction with embodiment, the present invention is further described; Following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
Unless stated otherwise, technique means used in the present invention is method known in those skilled in the art.To those skilled in the art, under the prerequisite not deviating from essence of the present invention and scope, the various change carry out the material component in these embodiments and consumption or change also belong to protection scope of the present invention.
Used medium of the present invention is as follows:
Hemp enrichment medium: hemp powder 2.0g, ammonium nitrate 0.2g, dipotassium hydrogen phosphate 0.05g, sodium sulfate 0.05g, distilled water 100mL, pH7.0.
Pectin screening culture medium: pectin 0.5g, ammonium nitrate 0.2g, dipotassium hydrogen phosphate 0.1g, sodium sulfate 0.05g, magnesium sulfate 0.06g, agar powder 2.0g, Congo red 0.02g, distilled water 100mL, pH7.0 ~ 7.5.
Wood poly-screening sugar culture-medium: xylan 1.0g, saltpetre 0.1g, magnesium sulfate 0.05g, sodium-chlor 0.05g, dipotassium hydrogen phosphate 0.05g, Congo red 0.02g, agar powder 2.0g, distilled water 100mL, pH7.0 ~ 7.5.
LB solid medium: Tryptones 1.0g, yeast extract 0.5g, sodium-chlor 1.0g, agar powder 2.0g, distilled water 100mL, pH7.0.
The composition of described seed culture medium is LB nutrient agar, namely contains NaCl10g, Tryptones 10g, yeast extract 5g in 1000mL substratum, agar 18 ~ 20g, pH7.0.
The composition of described seed liquid nutrient medium is LB substratum, namely contains NaCl10g, Tryptones 10g in 1000mL substratum, yeast extract 5g, pH7.0.
A kind of subtilis that simultaneously can produce polygalacturonase and hemicellulase, name is called 16#, specific name is: Bacillus subtilis, deposit number is: CGMCCNo.11898, preservation date: on December 22nd, 2015, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center.
Can produce a screening method for the subtilis of polygalacturonase and hemicellulase as above, step is as follows simultaneously:
(1) bacterial strain screening be separated:
In the soil of bacterium of the present invention soil sample used from process domestic refuse.
1. enrichment culture: enrichment culture: take the bacterium powder collecting also freeze-drying and be placed in hemp enriched liquid substratum, the ratio g:mL of described bacterium powder and hemp enriched liquid substratum is 1:150, at 37 DEG C, 180 ~ 220r/min enrichment culture 24 hours.
2. primary dcreening operation: the bacterium liquid after enrichment culture is according to 10 times of gradient dilutions.With sterilized physiological saline, the bacterium liquid of enrichment is diluted to 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7etc. different dilution sample suspension, after vibration mixing, draw 10 respectively
-5, 10
-6, 10
-7bacteria suspension 200 μ L, coating pectin (xylan) screens in solid medium, cultivation is inverted 24 hours at 37 DEG C, by Congo red colour developing, measure transparent circle diameter (R) and colony diameter (r), tentatively judge the enzymatic productivity of bacterial strain with the ratio of R/r.Then picking growing way is better, single bacterium colony that the ratio of R/r is larger, and point is received in xylan (pectin) solid medium, is inverted cultivation 24 hours, selects single bacterium colony separation and purification in LB solid medium that the ratio of R/r is larger at 37 DEG C.After such separation and purification, the bacterium of gained can produce polygalacturonase and can produce zytase again, and then these bacteriums being preserved in whole mass concentration is in the glycerine pipe of 15%, frozen for subsequent use in-80 DEG C.
3. sieve again: first activated in LB solid medium by the bacterium of primary dcreening operation institute preservation, be inoculated in the substratum of the triangle shaking flask of the 250mL of the liquid LB that 50mL is housed with the inoculum size of a ring, at 37 DEG C, shake-flask culture 16 hours is as seed liquor.Take 4 ~ 10g raw ramie, load in the triangle shaking flask of 250mL after first boiling water bath process 30min and add tap water 95mL, access seed liquor 5mL (bath raio 1:20), at 37 DEG C, (180 ~ 220r/min) takes out after coming unstuck 10 ~ 12 hours, detect residual gum content, choose the degumming bacterium of the low bacterial strain of residual gum content as lower step.
(2) qualification of bacterial strain:
The individual morphology qualification of bacterial strain: to sieve first bacterium of coming unstuck carries out Morphological Identification, find that bacterium colony is creamy white, bacterium colony is comparatively large, thickness, smooth surface are opaque, and neat in edge, older phage surface plays fold, as shown in Figure 1.All show positive through gramstaining, bar is short and small, and forms gemma in thalline central authorities, tentatively concludes that 2 strain bacterium are all bacillus, as shown in Figure 2.
16SrDNA gene sequencing and analysis: carry out pcr amplification 16SrDNA with the genomic dna of 16# bacterial strain for template and check order.NCBI carries out Blast analysis show: the consistence of the 16SrDNA sequence of 16# bacterial strain and subtilis (Bacillussubtilis) 16SrDNA sequence is up to 99%.
By being separated above, screening and identification of strains result, confirm that this bacterial strain is subtilis (Bacillussubtilis).
Related test results of the present invention:
The present invention can produce the degumming strain polygalacturonase of the subtilis of polygalacturonase and hemicellulase and the mensuration of xylanase activity simultaneously:
1, DNS method mensuration relevant enzyme is lived
(1) configuration of DNS reagent
Accurately take 3,5-dinitrosalicylic acid 10.0g (chemical pure), add redistilled water 600mL, stirring in water bath at 45 DEG C, then 8.0g sodium hydroxide is slowly added, and constantly stir, until solution is as clear as crystal, more progressively add Rochelle salt 312.0g, phenol 2.50g and sodium sulphite anhydrous 99.3 2.50g, constantly stir, until dissolve completely, after being cooled to room temperature, be settled to 1000mL with distilled water, be stored in brown bottle and keep in Dark Place, use after depositing 15d under room temperature, the quality guaranteed period is 6 months.
(2) configuration of the glycine-sodium hydroxide buffer solution of pH9.0
The glycine of 50mL0.2mol/L mixes with the sodium hydroxide of 16.8mL0.2mol/L, and adding distil water is settled to 200mL.
(3) making of typical curve
Accurately take 0.100g galacturonic acid (wood sugar), distilled water dissolves and is settled to 100mL, is the galacturonic standard acid solution of 1mg/mL.Get 16 25mL graduated tube numberings, add the reference liquid of 0,0.1,0.3,0.5,0.7,0.9mL successively, the volume of corresponding interpolation distilled water is 3.0,2.9,2.7,2.5,2.3,2.1mL, and often group 3 is parallel, and the control group 1 not with reference liquid is parallel.Then respectively add the DNS reagent of 2.0mL, vibration mixing after in boiling water bath accurate response 7min, after taking-up immediately with flowing water cooling, be then settled to 25mL with distilled water, 560nm place mensuration light absorption value (OD).With the mole number of galacturonic acid (wood sugar) (μm ol) for X-coordinate, OD value is ordinate zou, drawing standard curve.Obtain galacturonic acid typical curve: Y
1=0.175X
1-0.135, R
2=0.998 directrix curve: Y
2=1.268X
2-0.073, R
2=0.998.
(4) configuration of substrate solution
Pectin substrate: accurately take 1.0000g pectin, by the damping fluid heating for dissolving of pH9.0, and constantly stirs, is settled to 100mL after being cooled to room temperature.Save backup under low temperature, preservation period is 3 days.
Xylan substrate: accurately take 1.0000g xylan, by the damping fluid heating for dissolving of pH9.0, and constantly stirs, is settled to 100mL after being cooled to room temperature.Save backup under low temperature, preservation period is 3 days.
(5) preparation of crude enzyme liquid
After being activated in LB solid medium by degumming strain, picking list colony inoculation is in LB liquid nutrient medium, and at 37 DEG C, 200r/min enrichment culture 16h, at 4 DEG C, the centrifugal 10min of 8000r/min, gained supernatant liquor is crude enzyme liquid.
(6) mensuration that enzyme is alive
The key step of enzyme activity determination:
(1) get the graduated tube of 25mL, add the crude enzyme liquid 1mL through suitable extension rate, substrate 2mL, fully mixes, using the enzyme liquid of deactivation as blank;
(2) in 50 DEG C of water-baths, react 30min, take out;
(3) add 2mLDNS reagent and shake up, immediately boiling water bath accurate color 5min, after taking out, flowing water cooling, is settled to 25mL, measures OD value at 540nm place.An enzyme unit definition alive is that under the conditions described above, the per minute hydrolysis substrate enzyme amount generated needed for 1 μ g reducing sugar is 1 Ge Meihuo unit.Enzyme activity (U/mL)=reducing sugar amount Y × N/30; N is the extension rate of crude enzyme liquid, and 30 is time of enzymatic reacting (min).Recording 16# pectinase activity is 198U/mL, and Xylanase activity is 124U/mL.
Can find out after testing, this subtilis can produce polygalacturonase and hemicellulase simultaneously, cellulase-producing hardly; The residual gum content of hemp fibre can be made in 96h to be reduced to about 13%.
The present invention can produce the come unstuck application of subtilis at hemp fibre of polygalacturonase and hemicellulase simultaneously:
First the degumming strain be stored in glycerine pipe is activated in LB solid medium, be then inoculated in LB liquid seed culture medium, 37 DEG C, 200rpm cultivates 16 ~ 24h cell concentration (OD
600) reach 2-2.6, seed liquor is transferred in the retting liquid of hemp with 5% ~ 10% (volume ratio) inoculum size, retting liquid can be water, also can be the aqueous solution containing 0.05% ~ 1% nitrogenous source, nitrogenous source can be one in ammonium sulfate, ammonium nitrate, urea, ammonium oxalate, volatile salt etc., several or whole, the mass volume ratio of hemp and retting liquid is 1:20 ~ 1:30, temperature of coming unstuck is 25 ~ 45 DEG C, usually time 24 ~ 96h, leave standstill or rotate, shaking speed is 180 ~ 220rpm.
The composition of described liquid seed culture medium is LB liquid nutrient medium.
More preferably, the medium component that comes unstuck described in is: 0.05g/mL hemp, 0.002g/mL nitrogenous source, pH7.0;
More preferably, described nitrogenous source is ammonium nitrate or ammonium sulfate;
After hemp degumming, flaxen fiber is dispersed completely, soft, bright in color pure white (Fig. 3-2), forms sharp contrast with the control group (Fig. 3-1) not adding bacterium liquid.Pass through sem observation, in hempen original ramie, inner some fibrous bundles are closely wrapped up by colloid, almost can't see single fiber structure (Fig. 4-1), after degumming bacterium process about 96h, fiber surface is smooth, does not almost damage, and the colloid on ultimate fibre bundle removes (Fig. 4-2) substantially.Residual gum content after hemp degumming is as table 1, and come unstuck 4 days, hemp residual gum content drops to 8% ~ 15%, is more preferably that residual gum content reaches 13%, almost arrives the requirement of textile industry.
The residual gum content table of table 1. hemp after subtilis is come unstuck
。
Claims (10)
1. one kind can be produced the subtilis of polygalacturonase and hemicellulase simultaneously, it is characterized in that: name is called 16#, specific name is: Bacillus subtilis, deposit number is: CGMCCNo.11898, preservation date: on December 22nd, 2015, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center.
2. can produce a screening method for the subtilis of polygalacturonase and hemicellulase as claimed in claim 1 simultaneously, it is characterized in that: step is as follows:
Bacterial strain screening be separated:
Screen from the bacterial strain in the soil of process domestic refuse, and be lyophilized into bacterium powder;
1. enrichment culture: take the bacterium powder collecting also freeze-drying and be placed in hemp enriched liquid substratum, the ratio g:mL of described bacterium powder and hemp enriched liquid substratum is 1:150, at 37 DEG C, 180 ~ 220r/min enrichment culture 24 hours;
2. primary dcreening operation: the bacterium liquid after enrichment culture is according to 10 times of gradient dilutions: the bacterium liquid of enrichment is diluted to 10 with sterilized physiological saline
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7different dilution sample suspension, draws 10 after vibration mixing respectively
-5, 10
-6, 10
-7bacteria suspension 200 μ L, coat in pectin screening solid medium or xylan screening solid medium, at 37 DEG C, be inverted cultivation 24 hours, by Congo red colour developing, measure transparent circle diameter R and colony diameter r, tentatively judge the enzymatic productivity of bacterial strain with the ratio of R/r;
Then picking growing way is better, single bacterium colony that the ratio of R/r is larger, point is received in pectin screening solid medium or xylan screening solid medium, is inverted cultivation 24 hours, selects single bacterium colony separation and purification in LB solid medium that the ratio of R/r is larger at 37 DEG C;
After such separation and purification, the bacterium of gained can produce polygalacturonase and can produce zytase again, and then these bacteriums being preserved in whole mass concentration is in the glycerine pipe of 15%, frozen for subsequent use in-80 DEG C;
3. sieve again: first activated in LB solid medium by the bacterium of primary dcreening operation institute preservation, be inoculated in the substratum of the triangle shaking flask of the 250mL of the liquid LB that 50mL is housed with the inoculum size of a ring, at 37 DEG C, shake-flask culture 16 ~ 24 hours is as seed liquor, cell concentration OD
600reach 2 ~ 2.6;
Take 4-10g raw ramie, load in the triangle shaking flask of 250mL after first boiling water bath process 20 ~ 30min, inoculation 5 ~ 10mL, the tap water containing 0.2% nitrogenous source is added with bath raio 1:20 ~ 25, described nitrogenous source is the mixture of one or more in ammonium sulfate, ammonium nitrate, urea, ammonium oxalate, volatile salt, at 37 DEG C, 180 ~ 220r/min come unstuck 10 ~ 12 hours after take out, detect residual gum content, choose the low bacterial strain of residual gum content as degumming bacterium, called after 16#;
(2) the qualification of bacterial strain:
The individual morphology qualification of bacterial strain: to the bacterium of coming unstuck of sieving first carry out Morphological Identification, find that 16# bacterium bacterium colony is creamy white, bacterium colony is comparatively large, thickness, smooth surface are opaque, neat in edge, and older phage surface plays fold; All show positive through gramstaining, bar is short and small, and forms gemma in thalline central authorities, tentatively predicates bacillus;
16SrDNA gene sequencing and analysis: carry out pcr amplification 16SrDNA with the genomic dna of 16# bacterial strain for template and check order;
NCBI carries out Blast analysis show: the 16SrDNA sequence of 16# bacterial strain and the consistence of subtilis 16SrDNA sequence, up to 99%, obtain the subtilis that simultaneously can produce polygalacturonase and hemicellulase.
3. the screening method that simultaneously can produce the subtilis of polygalacturonase and hemicellulase according to claim 2, is characterized in that: described step (1) 1. in hemp enriched liquid substratum be:
Hemp powder 2.0g, ammonium nitrate 0.2g, dipotassium hydrogen phosphate 0.05g, sodium sulfate 0.05g, distilled water 100mL, pH7.0.
4. the screening method that simultaneously can produce the subtilis of polygalacturonase and hemicellulase according to claim 2, it is characterized in that: described step (1) 2. in pectin screening solid medium be: pectin 0.5g, ammonium nitrate 0.2g, dipotassium hydrogen phosphate 0.1g, sodium sulfate 0.05g, magnesium sulfate 0.06g, agar powder 2.0g, Congo red 0.02g, distilled water 100mL, pH7.0 ~ 7.5; Or, described step (1) 2. in xylan screening solid medium be: xylan 1.0g, saltpetre 0.1g, magnesium sulfate 0.05g, sodium-chlor 0.05g, dipotassium hydrogen phosphate 0.05g, Congo red 0.02g, agar powder 2.0g, distilled water 100mL, pH7.0 ~ 7.5.
5. the screening method that simultaneously can produce the subtilis of polygalacturonase and hemicellulase according to claim 2, is characterized in that: described step (1) 2. in LB solid medium be:
Tryptones 1.0g, yeast extract 0.5g, sodium-chlor 1.0g, agar powder 2.0g, distilled water 100mL, pH7.0.
6. the screening method that simultaneously can produce the subtilis of polygalacturonase and hemicellulase according to claim 2, is characterized in that: described step (1) 3. in liquid LB be;
Tryptones 1.0g, yeast extract 0.5g, sodium-chlor 1.0g, distilled water 100mL, pH7.0.
7. one kind can be produced the application of subtilis in hemp degumming of polygalacturonase and hemicellulase as claimed in claim 1 simultaneously.
8. the application of subtilis in crudefiber crop is come unstuck simultaneously producing polygalacturonase and hemicellulase according to claim 7, is characterized in that:
With described subtilis for degumming bacterium is inoculated in liquid seed culture medium, be placed in 37 DEG C of shaking tables, 180 ~ 220rpm is cultured to 16 ~ 24h, being transferred to volume ratio 5% ~ 10% inoculum size comes unstuck in substratum, and containing crudefiber crop raw ramie fiber in the substratum that comes unstuck, crudefiber crop is 1:20 ~ 30 with the ratio g:mL of the substratum that comes unstuck, temperature of coming unstuck is 25 ~ 45 DEG C, shaking speed is 180 ~ 220rpm, and come unstuck 24 ~ 96h, the hemp after must coming unstuck.
9. the application of subtilis in crudefiber crop is come unstuck simultaneously producing polygalacturonase and hemicellulase according to claim 8, is characterized in that: described liquid seed culture medium is LB liquid nutrient medium, broth culture or minimal medium; Or described crudefiber crop is hemp, ramie or flax; Or, described in the substratum that comes unstuck be retting liquid, this retting liquid is water or is the aqueous solution containing mass percent 0.05% ~ 1% nitrogenous source, or, be 0.05g/mL hemp, 0.002g/mL nitrogenous source, pH7.0.
10. the application of subtilis in crudefiber crop is come unstuck simultaneously producing polygalacturonase and hemicellulase according to claim 8, is characterized in that: described nitrogenous source is the mixture of one or more in ammonium sulfate, ammonium nitrate, urea, ammonium oxalate, volatile salt; Or described liquid seed culture medium is LB liquid nutrient medium: containing NaCl10g, Tryptones 10g in 1000mL substratum, yeast extract 5g, pH7.0.
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