CN101880637A - Bacillus subtilis and application thereof in sisal hemp degumming - Google Patents

Abstract

The invention discloses Bacillus subtilis and application thereof in sisal hemp degumming. The conservation number of the Bacillus subtilis B2 is CCTCCM208115. When in application, the Bacillus subtilis is used as a single strain which is inoculated in seed culture medium, is placed in a gas bath shaker or water bath shaker with temperature of 37 DEG C and is cultured for 8-12h, 2-20 percent (volume percent) of inoculation quantity is trans-inoculated in degumming culture medium when 0D600 reaches 0.6-1, fresh sisal hemp leaves are added in the degumming culture medium, culture temperature is 25-42 DEG C, the revolution of the shaker is 150-250rpm and the sisal hemp degumming can be completed in 35-55h. The invention has the advantages that the culture conditions are extensive, the growth and the reproduction are rapid, the sisal hemp degumming can be completed in 35h, the fiber is not damaged, the environment is not polluted, and the Bacillus subtilis can substitute for mechanical green removal and can be used for the pretreatment of sisal hemp before sisal hemp degumming and pulping technology.

Description

A kind of subtilis and the application in sisal hemp degumming thereof

Technical field

The present invention relates to microorganism and microbial fermentation field, specifically, the present invention relates to a kind of subtilis that can be used for sisal hemp biological degumming or slurrying with product polygalacturonase and zytase ability, and the application of this bacterial strain in sisal hemp degumming.

Background technology

Sisal hemp is the natural fiber raw material of the characteristic advantage of China, that its fiber has is pure white, strong but pliable in texture, springiness, characteristic such as wear-resisting, the fiber long-width ratio is big, be interweaved often between the fiber in the unit surface when becoming paper, Fiber Distribution is fine and closely woven, and the paper strength height is good paper making raw material, particularly its output height, characteristics such as growth is fast, cutting age is short, for the timber paper making raw material can't be obtained.Although modern science and technology constantly makes progress, that but sisal fibers had was strong but pliable in texture, springiness, pulling force are strong, anti tear, characteristic such as wear-resisting, anticorrosion are that synthon are irreplaceable, the development of new technology, particularly sisal fibers and plastics mixing manufacture degradable plastics and matrix material of being used for automobile light industry etc. are succeeded in developing, the use range of sisal fibers is greatly enlarged, sisal fibers has been used to make fine paper, speciality paper, as coinage paper, aviation, navigation drawing etc.

The research of sisal hemp slurrying focuses mostly in traditional chemical method or mechanical process, and sisal hemp except that cost height, pollution weight, yield are hanged down, has more been destroyed the distinctive tough characteristic of sisal hemp through chemical pulping, has reduced former pulp brightness.The machinery rule has energy consumption height, drawback that slurrying intensity is low.In addition, sisal hemp also need utilize machinery " to remove blue or green " to the sisal hemp peeling before chemical method, mechanical feedback, the sisal hemp scurf is difficult to remove on the sisal fibers bundle but machinery is except that being trapped in behind the green grass or young crops, caustic soda consumption (chemical method) and power consumption (steam explosion) in the follow-up pulping process have been increased on the one hand, on the other hand, influenced the free and pulp brightness of fiber dispersion in the pulping process, ultimate fibre.

Bio-pulping is meant and utilizes microorganism or capacity of decomposition that its zymin had, remove other composition that is unfavorable for slurrying (as pectin, hemicellulose, xylogen, lipid etc.) in pulping raw material or the slurry, make plant tissue and the fiber process of pulp of making separated from one another, it not only can reduce the consumption of mechanical process equal energy source, reduce the environmental pollution that chemical method brought, and also be better than chemical method on the paper performance in every respect, all increase to some extent as pulp strength, tensile index, tear index, burst index and whiteness.At present, domestic and international bio-pulping to crudefiber crop concentrates on the biological degumming slurrying aspect as phloem fiber crudefiber crops such as ramie, bluish dogbane, flax, hemps, and to biological degumming, the slurrying of the sisal hemp in the leaf fibres class fiber crops, rarely has relevant bacterial strain and zymin report, it is relevant that this and sisal hemp growth region, the densification of sisal hemp epidermis quality, pectin content height, common micro-organisms are difficult to reason such as effect.The solution sisal hemp degumming helps the exposure of sisal fibers, for slurrying provides good raw material.Research contriver about sisal hemp degumming takes the lead in having obtained some progress at home and abroad, has certain sisal hemp degumming ability (Chen Qing proposition B2 bacterium, Han Shuanyan etc., the screening of sisal hemp bio-pulping or degumming strain and evaluation and Preliminary Applications thereof, Agricultural engineering institute, 2008,24 (6): 277-281), become and be applied to produce actual bottleneck but reach processing cycle of coming unstuck of 72h hour.Therefore, seek the more suitable condition of coming unstuck, shortening the treatment time of coming unstuck greatly becomes the task of top priority.

Summary of the invention

The purpose of this invention is to provide a kind of have make the effect of sisal hemp biological degumming bacillus subtilis strain Bacillussubtilis B2, and the application of subtilis in sisal hemp degumming is provided, use this bacterial strain and make sisal hemp can realize biological degumming at 35～55 hours.

Purpose of the present invention is achieved through the following technical solutions:

The invention provides that to be used for sisal hemp degumming be the polygalacturonase produced of purpose and the bacterial strain of zytase, this bacterium separates from the soil of sisal hemp planting base for many years and obtains, and grows on nutrient agar.According to " uncle's Jie Shi Bacteria Identification handbook (the 9th edition) and " common bacteria system identification handbook " identify that its physiological and biochemical property conforms to subtilis (Bacillus subtilis) most.(submitted GeneBank to, No.EF639849) carry out the homology comparison in GenBank, the result shows that bacterial strain B2 and Bacillus subtilis have higher homology to its 16S rDNA sequence, and homology reaches 99% simultaneously.Biological characteristics is: thalline is shaft-like, be cotton-shaped growth in Gram-positive, the liquid medium within, gemma is arranged, gemma is cylindricality or ellipse, aerobic, hydrogen peroxide enzyme positive, Gram-positive, to energy fermentation and acids such as glucose, pectinose, seminose, wood sugar, to lactose, semi-lactosi do not produce acid, can hydrolyzed starch, utilize citric acid, well-grown in the meat soup of 7%NaCl.

Subtilis provided by the invention (Bacillus subtilis B2) is submitted Chinese typical culture collection center preservation on August 5th, 2008, and deposit number is CCTCC M 208115.The preservation address is Wuhan City, Hubei Province Wuhan University (430072).

The cultural characteristic of this bacterium, physiological and biochemical property, 16S rDNA sequence are as follows:

Cultural characteristic and morphologic observation

Thalline is cotton-shaped growth in the B2 liquid medium within, and it is shaft-like that cell is, motion, and Gram-positive has gemma, and gemma is cylindricality or ellipse, as shown in Figure 2.

Physiological and biochemical property is identified

According to " uncle Jie Shi Bacteria Identification handbook and " common bacteria system identification handbook ", B2 is carried out Physiology and biochemistry to be identified, its physiological and biochemical property conforms to subtilis (Bacillus subtilis) most, the result is as shown in table 1 for its Physiology and biochemistry, show this bacterium Gram-positive, the hydrogen peroxide enzyme positive, the energy glucose fermentation, pectinose, seminose, wood sugar produces acid and produces acid, to lactose, semi-lactosi does not produce acid, the energy hydrolyzed starch, utilize citric acid, well-grown in the meat soup of 7%NaCl is cultivated, in addition through observing as can be known, under 50 ℃, do not grow, but its colonial morphology is different from common subtilis colonial morphology.

Table 1

(annotate :+expression is positive, and-expression is negative)

16S rDNA complete sequence

Extract the 16S rDNA sequence of B2 bacterium, order-checking is shown as 1446bp, shown in SEQ.ID.NOl.

16S rDNA according to the B2 bacterium sets up phylogenetic tree.The sibship of finding B2 bacterium and bacillus subtilis Baillus subtili subsp.NBRC 101245 and Bacillus subtilis subsp.natto is nearest, proves that further the B2 bacterial strain is subordinate to subtilis.

The application of subtilis in sisal hemp degumming: with subtilis (Bacillus subtilis B2) is that bacterial classification list bacterium is inoculated in seed culture medium, places 37 ℃ of gas baths or shaking bath, and 150rpm～250rpm cultivates 8～12h, to OD ₆₀₀Reach at 0.6～1 o'clock, be transferred in the substratum that comes unstuck with inoculum size 2%～20% (volume ratio), add bright sisal leaves at the substratum that comes unstuck, bright sisal leaves is 0.05～0.2kg/l with the mass volume ratio of the substratum that comes unstuck, 25 ℃～42 ℃ of culture temperature, shaking speed is 150～250rpm, finishes sisal hemp degumming in 35h～55h.

The described medium component that comes unstuck is: 0.02～0.05g/ml carbon source, 0.01～0.03g/ml nitrogenous source, pH5.0～8.0;

Described carbon source is one or more in sisal hemp, xylan, wheat bran, corn cob, pectin, glucose and the wood sugar;

Described nitrogenous source is one or more in ammonium nitrate, ammonium sulfate, urea, analysis for soybean powder, extractum carnis, the peptone.

The composition of described seed culture medium is a nutrient agar.Be to contain extractum carnis 3g in the 1000mL substratum, peptone 10g, NaCl 5g,, agar 20g, pH7.2～7.4,

Described liquid seed culture medium is a nutrient broth medium.Be to contain extractum carnis 3g in the 1000mL substratum, peptone 10g, NaCl 5g, pH7.2～7.4.

The auxiliary slurrying of the sisal hemp biological degumming that the present invention relates to has following advantage:

1. can be exactly to the decomposition of fiber Symbiont, its principle of work is exactly to utilize the specificity of enzyme, thoroughly removes impurity and does not damage fiber itself.The bright leaf of 0.1 kilogram of sisal hemp of prior art report places 1 liter substratum, and (substratum concentration is the analysis for soybean powder of 0.01g/ml, nature pH), as for 37 ℃, the shaking table of 200r/min, 72h can realize coming unstuck fully (screening of sisal hemp bio-pulping or degumming strain and evaluation and Preliminary Applications thereof, Agricultural engineering institute, 2008,24 (6): 277-281), utilize the condition of coming unstuck among the present invention, usually time can shorten 1 times than prior art, and 35h can realize coming unstuck fully.

2. has the biological degumming penetrability.Till decomposing zero exactly and can not occur exceeding the proper limits in righting a wrong.This characteristic is guaranteed the quality control that sisal fibers comes unstuck in industrialized production process.The row yielding of fiber and slurry yield obtain corresponding raising.

3. Wen He condition, simple and direct technology is come unstuck even also can finish under very simple and crude condition.Degumming bacterium is that nutrition can be finished self-reproduction with the sisal hemp epidermis under the pH of gentleness value and temperature, as long as the process time enough just can be finished the degraded to sisal hemp epidermis non-cellulose composition.

4. production cost is low, and degumming quality is stable, on the one hand, has reduced the input of industrial chemicals such as caustic soda significantly, when reducing cost, has reduced inorganic pollutant; On the other hand, the magma intensity of acquisition is big, and whiteness is good, and the yield height is that simple chemical method and mechanical feedback can not possess jointly.

Description of drawings

Fig. 1 a is that the present invention who is observed visually utilizes the design sketch of Bacillus subtilis B2 to sisal hemp degumming;

Fig. 1 b is the prior art that the is observed visually design sketch to sisal hemp degumming;

Fig. 2 a is the Electronic Speculum figure without the sisal hemp of the effect of coming unstuck;

Fig. 2 b is the Electronic Speculum figure of the prior art sisal hemp of coming unstuck;

Fig. 2 c is the Electronic Speculum figure of the embodiment of the invention 4 sisal hemp of coming unstuck.

Embodiment

Below in conjunction with embodiment, the present invention is further detailed explanation.Following examples are the unrestricted technical scheme of the present invention in order to explanation only.Any modification or partial replacement that does not break away from spirit and scope of the invention all should be encompassed in the middle of the claim scope of the present invention.

Embodiment 1: enrichment of sisal hemp degumming bacterium and screening

9 kinds of sisal hemp grown place soil samples are collected on Red Star farm, Zhanjiang City, Guangdong Province, respectively take by weighing 1.00g, join in the triangular flask that 99mL sterilized water and an amount of sterilization granulated glass sphere are housed, the even bacteria suspension that gets vibrates, get the 0.5mL bacteria suspension and insert in the beef peptone enrichment medium, cultivate and from enrichment medium, connect bacterium liquid after 1 day again to the substratum that comes unstuck, at 30 ℃, after cultivating 3d under the 150r/min condition, observation has or not degumming phenomenon.

For making the stable performance of coming unstuck, mixed bacterial with the effect of coming unstuck is tamed, i.e. circulation is come unstuck, concrete grammar is as follows: the come unglued liquid that degumming effect is arranged after coming unstuck 3 days, cultivate 1d in the switching enrichment medium, after switching is come unstuck and cultivated 3d in the substratum again, still have being forwarded to again of the effect of coming unstuck to cultivate 1d in the enrichment medium, so come unstuck and realize domestication by circulation.By domestication, removed the flora of the unstable properties of coming unstuck.Circulation the continuing after three times of coming unstuck has the fermented liquid of degumming effect to coat on the nutrient agar, isolates different single bacterium.Isolating single bacterium is transferred and comes unstuck in the substratum, 30 ℃, cultivate 4d under the 150r/min condition after, find to come unstuck the strongest strain bacterium of ability, called after B2.

Embodiment 2: the classification of bacterial strain is identified

According to " uncle's Jie Shi Bacteria Identification handbook (the 9th edition) and " common bacteria system identification handbook " are identified, thalline is cotton-shaped growth in the B2 liquid medium within, it is shaft-like that cell is, motion, Gram-positive has gemma, gemma is cylindricality or ellipse, its physiological and biochemical property conforms to subtilis (Bacillus subtilis) most, do not grow under 50 ℃, but its colonial morphology is different from common subtilis colonial morphology.(submitted GeneBank to, No.EF639849) carry out the homology comparison in GenBank, the result shows that bacterial strain B2 and Bacillus subtilis have higher homology to its 16S rDNA sequence, and homology reaches 99% simultaneously.Biological characteristics is: thalline is shaft-like, be cotton-shaped growth in Gram-positive, the liquid medium within, gemma is arranged, gemma is cylindricality or ellipse, aerobic, hydrogen peroxide enzyme positive, Gram-positive, to energy fermentation and acids such as glucose, pectinose, seminose, wood sugar, to lactose, semi-lactosi do not produce acid, can hydrolyzed starch, utilize citric acid, well-grown in the meat soup of 7%NaCl, this bacterium submits to the China typical case to cultivate no preservation center preservation, deposit number CCTCC M 208115 on August 5th, 2008.

Embodiment 3: zytase enzyme, polygalacturonase enzyme activity determination in coming unstuck

Because the glycosidic link of polygalacturonase, hemicellulase (being mainly zytase) and cellulose degraded substrate, generation contains the reducing end based products, so adopt reducing sugar test method-DNS method commonly used.

Polygalacturonase (zytase) hydrolyzable substrate pectin (xylan), generate galacturonic acid aldoses such as (wood sugars), aldose and 3,5-dinitrosalicylic acid is the henna aminocompound of thermogenesis altogether, the amount of its reducing sugar and the reaction solution shade that contains the colour generation aminocompound are directly proportional, under 540nm, survey its absorbancy, can calculate polygalacturonase (zytase) enzyme and live.

(1) DNS reagent

With 400mL dissolved in distilled water 6.3g 3,5-dinitrosalicylic acid, progressively add 21g sodium hydroxide, add 185g Rochelle salt, 5.0g phenol, 5.0g sodium sulphite anhydrous 99.3 again, warm water bath (being no more than 48 ℃) is constantly stirred, and is as clear as crystal until solution.Be settled to 1000mL with distilled water, be kept in the brown bottle, isolated with carbonic acid gas, to leave standstill after 5～7 days and use, be 6 months storage period.

(2) phosphoric acid-citric acid solution of pH 7.0

Get Na ₂HPO ₄12H ₂O 71.62g, C ₆H ₈O ₇H ₂O 21.01g, constant volume is pressed 16.47:3.53 and is mixed to 1000mL respectively, gets final product.

(3) preparation of substrate

Pectin substrate: take by weighing 1.00g pectin, with phosphoric acid-citric acid solution dissolving of pH 7.0, constant speed stirs 0.5h on magnetic stirring apparatus, is settled to 100mL with buffered soln.Leave in 0～4 ℃ of refrigerator.

Xylan substrate: take by weighing the 1.00g xylan, add phosphoric acid-citric acid solution of the pH 7.0 of 20mL, be heated to after it dissolves fully, be settled to 100mL with buffered soln.Leave in 0～4 ℃ of refrigerator.

(4) enzyme activity determination

Bacillus subtilis B2 is inoculated in 50ml contains 2% xylan, 1% analysis for soybean powder is cultivated in the liquid nutrient medium of natural pH, takes out the 2mL fermented liquid during fermentation 30h, and 4 ℃, the centrifugal 5min of 10000rpm get supernatant, are crude enzyme liquid.The enzyme activity determination method is: 1. get the 25mL colorimetric cylinder, add crude enzyme liquid 1.0mL, 50 ℃ of insulation 5min add the substrate 1.0mL of constant temperature to 50 in advance ℃, and the tool plug shakes up, accurate response 30min; 2. add 1.5mLDNS reagent and shake up, put the 5min that develops the color in the boiling water immediately, take out the flowing water cooling, adding distil water is diluted to scale, shakes up; 3. compare with inactivator liquid (100 ℃ of deactivation 10min), use the spectrophotometric determination enzymic activity, survey absorbancy, the reducing sugar amount that the reference standard curve calculation generates at wavelength 540nm place.Under the said determination condition, generate the required enzyme amount of 1 μ g reducing sugar with the per minute hydrolysis substrate and be defined as a unit of enzyme activity, unit is U/mL.As calculated, xylanase activity is 92U/mL, polygalacturonase enzyme 178U/mL alive.

Embodiment 4: utilize Bacillus subtilis B2 to sisal hemp degumming

The Bacillus subtilis B2 that is stored in nutrient agar slant medium lines on the nutrient agar plate, after postvaccinal flat board places 37 ℃ of incubators to cultivate 24h, picking list colony inoculation is in the test tube that contains the 5mL nutrient broth medium from flat board, test tube is put in 37 ℃ of gas bath shaking tables, 200rpm, incubated overnight is to OD ₆₀₀=0.6 as seed liquor.(1) get the 1.5mL seed liquor and be inoculated in and 30mL is housed contains wheat bran 0.6g, extractum carnis 0.9g in the 150mL culturing bottle of pH 6.0, adds the bright sisal leaves of sterilization of 3g simultaneously in the culturing bottle, places 30 ℃, the shaking table of 200r/min to come unstuck.(2) with the prior art contrast.With reference to bibliographical informations such as Chen Qing (screening of sisal hemp bio-pulping or degumming strain and evaluation and Preliminary Applications thereof, Agricultural engineering institute, 2008,24 (6): 277-281), getting the 2mL seed liquor is inoculated in simultaneously and 20mL is housed contains the bright leaf of 2g sisal hemp, 0.2g analysis for soybean powder, in the 100mL culturing bottle of natural pH, culturing bottle comes unstuck as in 37 ℃, the shaking table of 200r/min.

Behind the two shake-flask culture 35h, sisal leaves is taken out from shaking in the bottle, use the distilled water wash surface gently, the visual inspection sisal leaves surface situation of coming unstuck.As can be seen, present embodiment sisal hemp epidermis is all removed from Fig. 1 a, exposes white fiber, and comes unstuck under the prior art condition, and during 35h, shown in Fig. 1 b, the sisal fibers major part is also surrounded by green colloid epidermis.Observe under the scanning electron microscope, Fig. 2 a is the Electronic Speculum figure without the sisal hemp of the effect of coming unstuck.Present embodiment comes unstuck and acts on 35h under the condition, and shown in Fig. 2 c, the colloid on fibrous bundle surface is removed fully, and the vertical surface of fiber becomes smooth, does not have colloid between the fibrous bundle, and is separated from one another.Shown in Fig. 2 b, degumming effect is compared greatly and is reduced under the prior art condition, the still residual colloid tectum in sisal fibers surface, and fibrous bundle only exposes slightly.This shows that usually time has shortened 1 times than the 72h of prior art under the sisal hemp degumming condition provided by the invention, can shorten the processing cycle that sisal fibers is used for slurrying, accelerate the technology progress, shorten the flow process time of whole pulping and paper-making and reduce processing cost.

Embodiment 5: to sisal hemp degumming

The Bacillus subtilis B2 that is stored in nutrient agar slant medium lines on the nutrient agar plate, after postvaccinal flat board places 37 ℃ of incubators to cultivate 20h, picking list colony inoculation is in the test tube that contains the 5mL nutrient broth medium from flat board, test tube is put in 37 ℃ of gas bath shaking tables, 250rpm, incubated overnight is to OD ₆₀₀=0.6 o'clock, get 1mL and be inoculated in and be equipped with in the 100mL culturing bottle that 20mL contains nutrient broth medium, be cultured to OD ₆₀₀=1 o'clock as seed liquor.The 20mL seed liquor is inoculated in 200mL and contains 8g pectin, and 4g ammonium sulfate in the 1L culturing bottle of pH7.0, adds the bright sisal leaves of sterilization of 40g simultaneously in the culturing bottle, places 37 ℃, the shaking table of 250r/min to come unstuck, and 40h can come unstuck fully.72h than prior art has shortened 32h.

Embodiment 6: to sisal hemp degumming

The Bacillus subtilis B2 that is stored in nutrient agar slant medium lines on the nutrient agar plate, after postvaccinal flat board places 37 ℃ of incubators to cultivate 36h, picking list colony inoculation is in the test tube that contains the 5mL nutrient broth medium from flat board, test tube is put in 37 ℃ of gas bath shaking tables, 150rpm, incubated overnight is to OD ₆₀₀=0.8 o'clock, to get 2mL and be inoculated in and 40mL is housed contains the 2g corn cob, 1.2g ammonium nitrate in the 250mL culturing bottle of pH8.0 substratum, adds the bright sisal leaves of sterilization of 6g simultaneously in the culturing bottle, place 42 ℃, the shaking table of 250r/min to come unstuck, and 50h can come unstuck fully.72h than prior art has shortened 22h.

Embodiment 7: to sisal hemp degumming

The Bacillus subtilis B2 that is stored in nutrient agar slant medium lines on the nutrient agar plate, after postvaccinal flat board places 37 ℃ of incubators to cultivate 28h, picking list colony inoculation is in the test tube that contains the 5mL nutrient broth medium from flat board, test tube is put in 37 ℃ of gas bath shaking tables, 250rpm, incubated overnight is to OD ₆₀₀=18 o'clock, to get 2mL and be inoculated in and 50mL is housed contains 1g glucose, the 0.5g peptone in the 250mL culturing bottle of pH5.0 substratum, adds the bright sisal leaves of sterilization of 4g simultaneously in the culturing bottle, place 30 ℃, the shaking table of 200r/min to come unstuck, and 55h comes unstuck fully.72h than prior art has shortened 17h.

SEQ.ID.NO1：

TCACCCCAATCATCTGTCCCACCTTCGGCGGCTGGCTCCTAAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCT

CGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTC

CAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTC

GCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCAC

CTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCG

TTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGG

ACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGC

TCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAAT

GCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTAT

CTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTC

CTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAA

TGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTC

CGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCA

AGGTACCGCCCTATTCGAACGGTACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACG

CGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCT

CAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTTGCCTTGGTGAGCCGTTACCTCACCAACTAGC

TAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTATGTTTGAACCATGCGGTTCAAACAACCATC

CGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCT

AACATCAGGGAGCAAGCTCCCATCTGTCCGCTCGACTTGCATGT

Claims (5)

1. a subtilis (Bacillus subtilis B2) is characterized in that: its deposit number CCTCC M 208115.

2. the application of the described subtilis of claim 1 in sisal hemp degumming: it is characterized in that: with subtilis (Bacillus subtilis B2) is that bacterial classification list bacterium is inoculated in seed culture medium, place 37 ℃ of gas baths or shaking bath, 150rpm～250rpm cultivates 8～12h, to OD ₆₀₀Reach at 0.6～1 o'clock, be transferred in the substratum that comes unstuck with inoculum size 2%～20% (volume ratio), add bright sisal leaves at the substratum that comes unstuck, bright sisal leaves is 0.05～0.2kg/l with the mass volume ratio of the substratum that comes unstuck, 25 ℃～42 ℃ of culture temperature, shaking speed is 150～250rpm, finishes sisal hemp degumming in 35h～55h.

3. the application of subtilis according to claim 2 in sisal hemp degumming is characterized in that: the described medium component that comes unstuck is: 0.02～0.05g/ml carbon source, 0.01～0.03g/ml nitrogenous source, pH5.0～8.0;

Described carbon source is one or more in sisal hemp, xylan, wheat bran, corn cob, pectin, glucose and the wood sugar;

Described nitrogenous source is one or more in ammonium nitrate, ammonium sulfate, urea, analysis for soybean powder, extractum carnis, the peptone.

4. the application of subtilis according to claim 2 in sisal hemp degumming is characterized in that: the composition of described seed culture medium is a nutrient agar.

5. the application of subtilis according to claim 2 in sisal hemp degumming is characterized in that: described liquid seed culture medium is a nutrient broth medium.

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