CN102757914A - Paenibacillus xylanilyticus strain and method for preparing xyloglucanase by using same - Google Patents
Paenibacillus xylanilyticus strain and method for preparing xyloglucanase by using same Download PDFInfo
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
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Abstract
The invention provides a Paenibacillus xylanilyticus strain and a method for preparing xyloglucanase by using the same. Paenibacillus xylanilyticus B20 CCTCC M 2012194 is used as a fermentation strain to carry out liquid fermentation so as to prepare the xyloglucanase. The preparation method comprises the following steps: activating the strain on a slant culture medium, carrying out seed culture in the liquid seed culture medium, and carrying out liquid state fermentation in the fermentation culture medium to finally obtain the fermentation liquid containing xyloglucanase. Since the Paenibacillus xylanilyticus B20 CCTCC M 2012194 is utilized to prepare the xyloglucanase by fermentation, the invention has the advantages of short fermentation time and high enzyme yield, the production conditions are easy to control, and the products are easy to separate and purify.
Description
Technical field
The present invention relates to mikrobe and system zymotechnic field thereof, relate in particular to a strain and separate xylan series bacillus and system enzyme method thereof.
Background technology
Xyloglucanase enzymes (xyloglucanase, XEG) (numbering EC3.2.1.151) is β-1 in the hydrolysis xyloglucan main chain, one type of xyloglucan specificity restriction endonuclease of 4-glycosidic link; For a kind of extracellular enzyme, at GH5, GH12; GH16 all finds its existence in GH44 and the GH74 family.Xyloglucanase enzymes belongs to a kind of small component enzyme in the cellulase system, and to β-1, the 4-glycosidic link is that the saccharan of backbone structure, especially xyloglucan have the specificity degradation capability, and the xyloglucan of therefore can degrading is produced oligose.The xyloglucanase enzymes particularly xyloglucanase enzymes of bacterial origin generally has thermostability preferably, and under alkaline condition, has greater activity, therefore can be widely used in all trades and professions.1. in plant production, xyloglucanase enzymes is a kind of important enzyme of coordinate plant growth, can be used for the cellulose raw material that directional transformation goes out to be beneficial to enzymolysis; 2. in the bio-ethanol industry, xyloglucanase enzymes can be worked in coordination with the cellulose degraded cellulose raw material, produces bio-ethanol; 3. in food service industry, xyloglucanase enzymes can make the muddiness in the fruit juice become clarification, improves juice quality; 4. in washing composition,, can be used as the enzyme addn in washing powder and the washing liquid because xyloglucanase enzymes shows stable enzyme activity in washing composition.Xyloglucanase enzymes also has application potential at other field, helps drug delivery like zytase, also can be used as novel surfactant.
Utilize the method for microbial fermentation can produce enzyme, still, the mikrobe of the generation xyloglucanase enzymes that prior art is reported is less, and have that fermentation time is long, working condition is wayward, product not easy separation and easy purification, produce shortcomings such as enzyme level is low.
Summary of the invention
Above-mentioned defective to the existence of prior art for preparing xyloglucanase enzymes; The object of the present invention is to provide a strain to separate the xylan series bacillus and prepare the method for xyloglucanase enzymes with it; Prepare xyloglucanase enzymes with said strain fermentation, fermentation time is short, and yield of enzyme is high.
Technical scheme of the present invention is following:
Xylan series bacillus (Paenibacillus xylanilyticus) B20 is separated in one strain, and biological preservation information is: CCTCC No.M 2012149.
A kind of method for preparing xyloglucanase enzymes, with the above-mentioned xylan series bacillus B20 that separates, CCTCC No.M 2012149 carries out liquid fermenting as fermentation strain, and concrete steps are following:
(1) slant strains activation: with the said xylan series bacillus B20 that separates, CCTCC No.M2012149 streak inoculation is cultivated 24～36h in 28～32 ℃ on slant medium; Said slant medium composition is counted by grams per liter: peptone 8～12; Yeast extract paste 4～6, NaCl 8～12, agar 18～22; All the other compositions are water, regulate pH value to 6.5～7.0;
(2) seed culture: the bacterial classification of growing on the said slant medium of step (1) is scraped, be inoculated in the liquid seed culture medium that places shaking table, shaking speed 100～300r/min; In 28～32 ℃ of shaking culture 12～18h, said liquid seed culture medium composition is counted by grams per liter: peptone 8～12, yeast extract paste 4～6; NaCl 8～12; Wood sugar 2～5, all the other compositions are water, regulate pH value to 6.0～7.0;
(3) liquid fermentation and culture: will pass through step (2) and cultivate the gained activated seed liquid inoculum size of per-cent 5～10% by volume, and be inoculated in the fermention medium that places shaking table, shaking speed 150～200r/min; In 28～32 ℃ of shaking culture 30～40h, stop fermentation, obtain fermented liquid; Said fermention medium composition is counted by grams per liter: peptone 8～12, and yeast extract paste 20～24, NaCl 5～7; Wood sugar 1～3.5, all the other compositions are water, regulate pH value to 6.5～8.0.
The said xylan series bacillus B20 that separates obtains through the following steps screening and separating:
(1) avenges unrestrained mountain from China Wuxi City, Jiangsu Province in March, 2010 and descend collection soil sample in the mud of rice field;
(2) from pumpkin, extract thick xyloglucan according to the routine techniques means: with pumpkin chopping oven dry, clay into power, in 65.2 ℃ of water, soak 8h, soak solution is carried out spinning; Collecting precipitation immerses gained deposition oven dry back in the 0.1M NaOH solution, collects extracting solution behind the immersion 4h; And this extracting solution is carried out centrifugal, collect residue, again with in the residue obtained immersion 2.7M NaOH solution; Soak 12.6h, collect extracting solution, regulate its pH value to 5.0 with Glacial acetic acid min. 99.5; Supernatant is collected in centrifugal back, and this supernatant is dialysed, concentrated, and obtains thick xyloglucan through freeze-drying; With the thick xyloglucan preparation of gained primary dcreening operation substratum, said primary dcreening operation medium component percentage ratio is by weight counted: 0.1% thick xyloglucan, 0.2% (NH4) 2SO4 at last; 0.05%MgSO47H2O, 0.1%KH2PO4,0.05%NaCl; 1.5% agar powder, all the other compositions are water (pH7.0).
(3) enrichment culture: get the pedotheque that 1g step (1) gathered and be suspended in the 50mL saline water, add granulated glass sphere and break up mixing 30min, the bacteria suspension after getting 1mL and breaing up adds shaking culture 14h in the 50mL LB liquid nutrient medium; And 106 times of bacteria suspension dilutions after 0.1mL cultivated, be applied in the primary dcreening operation substratum that step (2) prepared, in 30 ℃ of overnight cultures in incubator; Bacterium colony to growing carries out the plate streaking separation and Culture, obtains single bacterium colony, in 4 ℃ of preservations; Said LB liquid culture based component percentage ratio is by weight counted: 1% yeast extract paste; 0.5% peptone, 1%NaCl, all the other compositions are water.
(4) dibbling of picking step (3) gained list bacterium colony is in being on 0.1% the multiple sieve culture medium flat plate through Remazol light blue (Remazol Brilliant Blue) painted xyloglucan (AZO-Xyloglucan) mass percent; In 30 ℃ of overnight cultures in incubator; Picking can produce the bacterial strain of transparent circle, in 4 ℃ of preservations.Said sieve again medium component by weight percentage ratio count: 0.1%AZO-xyloglucan, 0.2% (NH4) 2SO4,0.05%MgSO47H2O, 0.1%KH2PO4,0.05%NaCl, 1.5% agar powder, all the other compositions are water (pH7.0).
The inoculation of (5) step (4) being preserved is in liquid fermentation medium; Measure the xyloglucan enzyme activity after cultivating 40h; The bacterium producing multi enzyme preparation that is that enzyme activity is arranged; From above-mentioned bacterium producing multi enzyme preparation, filter out the highest bacterial strain of a strain xyloglucan production of enzyme, called after is separated xylan series bacillus (Paenibacillus xylanilyticus) B20.Said liquid fermentation medium composition percentage ratio is by weight counted: 0.1%AZO-xyloglucan, and 0.2% (NH4) 2SO4,0.05%MgSO47H2O, 0.1%KH2PO4,0.05%NaCl, 1.5% agar powder, all the other compositions are water (pH7.0).
Xylan series bacillus (Paenibacillus xylanilyticus) B20 that separates provided by the present invention; Chinese typical culture collection center (CCTCC), deposit number: CCTCC M 2012149, address: China have been preserved in on April 27th, 2012; Wuhan, Wuhan University.This bacterial strain hereinafter to be referred as: separate xylan series bacillus B20CCTCC M 2012149.
Said xylan series bacillus B20CCTCC M 2012194 bacterial strains of separating have following microbial characteristic:
1. morphological specificity
Separate the xylan series bacillus B20CCTCC M 2012194 bacterial strains 18h that on the LB plate culture medium, grows, the bacterium colony of formation is bigger, colony diameter 5～8mm; The surface is fold slightly, and the edge is irregular, can spread growth; Be little yellow, opaque, observation is rod-short under oily mirror.
2. physiological and biochemical property
According to " uncle Jie Shi handbook (R.E. Buchanan; N.E. basic this volume such as grade, Science Press, 1984) said authentication method; Carry out the Physiology and biochemistry evaluation to separating xylan series bacillus B20 CCTCC M 2012194 bacterial strains, its major physiological biochemical character is seen table 1.
Table 1
Annotate: "-" expression is negative; "+" expression is positive; The weak positive reaction of " (+) " expression.
3. genetics characteristic
Adopt 16S rDNA order-checking to carry out the taxonomy Molecular Identification.Bacterial classification extracting genome DNA process is following: utilize acetone to destroy bacteria cell wall membrane lipid structure; Abolish bacterial cell membrane to discharge nucleic acid in the born of the same parents with lysate; Remove protein and polysaccharide through phenol/chloroform/primary isoamyl alcohol, and, use TE damping fluid dissolving DNA at last with absolute ethyl alcohol deposit D NA.Utilize the 16S rDNA of this bacterial strain of round pcr amplification, wherein, upstream primer: 5 '-AGAGTTTGATCATGGCTCAG-3 ', downstream primer:
5’-TAGGGTTACCTTGTTACGACTT-3’。Transfer to Shanghai BGI Technology Co., Ltd. after pcr amplification product is purified and carry out sequencing, obtain the base sequence of the 16S rDNA of this bacterial strain through sequencing analysis, shown in SEQ ID NO:1.Submit to GeneBank to carry out sequence alignment and homology analysis this sequence, prove the 16S rDNA base sequence of this bacterial strain and separate xylan series bacillus (Paenibacillus xylanilyticu) type strain (Genbank accession number: have 99% homology AB727983.1).
The foregoing explanation; Bacterial strain of the present invention on morphological specificity and physiological and biochemical property with separate xylan series bacillus (Paenibacillus xylanilyticu) and match; With separate xylan series bacillus (Paenibacillus xylanilyticu) reference culture and compare; Have 99% homology aspect the 16S rDNA base sequence, therefore, can judge that bacterial strain of the present invention is for separating xylan series bacillus (Paenibacillus xylanilyticu).
The measuring method of above-mentioned xyloglucan enzyme activity is following: the preparation mass percent concentration is 0.2% xyloglucan solution, with the citric acid-sodium citrate damping fluid dissolving of pH 5.0, in 50 ℃ of 4h that in the constant temperature water bath vibrator, vibrate; Get 100 μ L fermented liquids, add in the above-mentioned xyloglucan solution of 900 μ L mixing; In 50 ℃ of water-baths, react 5min, in boiling water bath, boil 10min (enzyme reaction of going out) then, to reach the purpose of the enzyme that goes out; Add 1mL DNS solution, in boiling water bath, boil 5min, be settled to 10mL after the cooling fast; Measure its absorbance in the 540nm place with ultraviolet spectrophotometer, contrast solution be 100 μ L in advance through the go out fermented liquid of enzyme reaction of 10min, institute survey absorbance substitution glucose typical curve equation (is set up standard working curve with standard glucose solution; With the light absorption value is X-coordinate; Amount with glucose is an ordinate zou), try to achieve the amount of contained reducing sugar in the enzyme activity determination system, and definition calculates enzyme activity according to enzyme activity unit.Said enzyme activity unit (U) is defined as: at pH 5.0, under the condition that temperature is 50 ℃, PM is that the needed enzyme amount of reducing sugar amount that is equivalent to 1 μ mol glucose amount is an enzyme activity unit with substrate conversion.
Beneficial technical effects of the present invention is:
The present invention relates to a kind of xylan series bacillus B20 CCTCC M 2012149 that separates, carry out liquid fermentation and culture as fermentation strain, not only possess the general advantage that fermentation using bacteria produces enzyme with the said xylan series bacillus of separating; Fermentation time is short, working condition is easy to control, product is easy to separation and purification; In addition, this strain enzyme-producing level is high, under best technological condition for fermentation; Yield of enzyme reaches as high as 10.88U/mL, is fit to large-scale industrial production.
Embodiment
Do further description below in conjunction with the embodiment specific embodiments of the invention, following examples are convenient to understand better the present invention, but do not limit the present invention.
Embodiment 1
The concrete steps that xylan series bacillus B20CCTCC M 2012149 preparation xyloglucanase enzymes are separated in utilization are following:
(1) slant strains activation: separate xylan series bacillus B20CCTCC M 2012149 streak inoculations on slant medium with said, under 28 ℃, cultivate 24h, the slant culture based component is counted by grams per liter: peptone 8; Yeast extract paste 4; NaCl 12, and agar 22, all the other compositions are water; Regulate pH value to 6.5, in 115 ℃ of autoclaving 20min;
(2) seed culture: the bacterial classification of the 1cm2 area of growing on the said slant medium of step (1) is scraped, be inoculated in the liquid activation substratum, in 28 ℃ of shaking culture 12h; Shaking speed 100r/min, the liquid seed culture medium composition is counted by grams per liter: peptone 8, yeast extract paste 4; NaCl 8, and wood sugar 2, all the other compositions are water; Regulate pH value to 6.0, in 115 ℃ of autoclaving 20min;
(3) liquid fermentation and culture: will pass through step (2) and cultivate the gained activated seed liquid inoculum size of per-cent 5% by volume, and be inoculated in the fermention medium, fermention medium is loaded in the triangular flask of 250mL; Loading amount is 50mL, with postvaccinal fermentation culture based on 32 ℃ of shaking culture 40h, shaking speed 150r/min; Fermentation finishes, and obtains fermented liquid, and the fermentation culture based component is counted by grams per liter: peptone 12; Yeast extract paste 20, NaCl 7, wood sugar 1; All the other compositions are water, regulate pH value to 6.5, in 115 ℃ of autoclaving 20min.
Measure the enzyme activity of xyloglucanase enzymes in the gained fermented liquid according to the measuring method of above-mentioned xyloglucan enzyme activity, and to calculate yield of enzyme be 6.08U/mL.
Embodiment 2
The concrete steps that xylan series bacillus B20CCTCC M 2012149 preparation xyloglucanase enzymes are separated in utilization are following:
(1) slant strains activation: separate xylan series bacillus B20CCTCC M 2012149 streak inoculations on slant medium with said, under 29 ℃, cultivate 26h, the slant culture based component is counted by grams per liter: peptone 9; Yeast extract paste 5; NaCl 11, and agar 21, all the other compositions are water; Regulate pH value to 6.6, in 115 ℃ of autoclaving 20min;
(2) seed culture: the bacterial classification of the 1cm2 area of growing on the said slant medium of step (1) is scraped, be inoculated in the liquid activation substratum, in 29 ℃ of shaking culture 13h; Shaking speed 150r/min, the liquid seed culture medium composition is counted by grams per liter: peptone 9, yeast extract paste 5; NaCl 9, and wood sugar 3, all the other compositions are water; Regulate pH value to 6.2, in 115 ℃ of autoclaving 20min;
(3) liquid fermentation and culture: will pass through step (2) and cultivate the gained activated seed liquid inoculum size of per-cent 6% by volume, and be inoculated in the fermention medium, fermention medium is loaded in the triangular flask of 250mL; Loading amount is 50mL, with postvaccinal fermentation culture based on 31 ℃ of shaking culture 38h, shaking speed 160r/min; Fermentation finishes, and obtains fermented liquid, and the fermentation culture based component is counted by grams per liter: peptone 11; Yeast extract paste 21, NaCl 6, wood sugar 1.5; All the other compositions are water, regulate pH value to 7.0, in 115 ℃ of autoclaving 20min.
Measure the enzyme activity of xyloglucanase enzymes in the gained fermented liquid according to the measuring method of above-mentioned xyloglucan enzyme activity, and to calculate yield of enzyme be 8.75U/mL.
Embodiment 3
The concrete steps that xylan series bacillus B20CCTCC M 2012149 preparation xyloglucanase enzymes are separated in utilization are following:
(1) slant strains activation: separate xylan series bacillus B20CCTCC M 2012149 streak inoculations on slant medium with said, under 30 ℃, cultivate 30h, the slant culture based component is counted by grams per liter: peptone 10; Yeast extract paste 6; NaCl 10, and agar 20, all the other compositions are water; Regulate pH value to 6.7, in 115 ℃ of autoclaving 20min;
(2) seed culture: the bacterial classification of the 1cm2 area of growing on the said slant medium of step (1) is scraped, be inoculated in the liquid activation substratum, in 30 ℃ of shaking culture 14h; Shaking speed 200r/min, the liquid seed culture medium composition is counted by grams per liter: peptone 10, yeast extract paste 6; NaCl 10, and wood sugar 4, all the other compositions are water; Regulate pH value to 6.4, in 115 ℃ of autoclaving 20min;
(3) liquid fermentation and culture: will pass through step (2) and cultivate the gained activated seed liquid inoculum size of per-cent 7% by volume, and be inoculated in the fermention medium, fermention medium is loaded in the triangular flask of 250mL; Loading amount is 50mL, with postvaccinal fermentation culture based on 30 ℃ of shaking culture 36h, shaking speed 170r/min; Fermentation finishes, and obtains fermented liquid, and the fermentation culture based component is counted by grams per liter: peptone 10; Yeast extract paste 22, NaCl 5, wood sugar 2.0; All the other compositions are water, regulate pH value to 7.5, in 115 ℃ of autoclaving 20min.
Measure the enzyme activity of xyloglucanase enzymes in the gained fermented liquid according to the measuring method of above-mentioned xyloglucan enzyme activity, and to calculate yield of enzyme be 7.36U/mL.
Embodiment 4
The concrete steps that xylan series bacillus B20CCTCC M 2012149 preparation xyloglucanase enzymes are separated in utilization are following:
(1) slant strains activation: separate xylan series bacillus B20CCTCC M 2012149 streak inoculations on slant medium with said, under 31 ℃, cultivate 32h, the slant culture based component is counted by grams per liter: peptone 11; Yeast extract paste 4; NaCl 9, and agar 19, all the other compositions are water; Regulate pH value to 6.8, in 115 ℃ of autoclaving 20min;
(2) seed culture: the bacterial classification of the 1cm2 area of growing on the said slant medium of step (1) is scraped, be inoculated in the liquid activation substratum, in 31 ℃ of shaking culture 15h; Shaking speed 250r/min, the liquid seed culture medium composition is counted by grams per liter: peptone 11, yeast extract paste 4; NaCl 11, and wood sugar 5, all the other compositions are water; Regulate pH value to 6.6, in 115 ℃ of autoclaving 20min;
(3) liquid fermentation and culture: will pass through step (2) and cultivate the gained activated seed liquid inoculum size of per-cent 8% by volume, and be inoculated in the fermention medium, fermention medium is loaded in the triangular flask of 250mL; Loading amount is 50mL, with postvaccinal fermentation culture based on 29 ℃ of shaking culture 34h, shaking speed 180r/min; Fermentation finishes, and obtains fermented liquid, and the fermentation culture based component is counted by grams per liter: peptone 9; Yeast extract paste 23, NaCl 7, wood sugar 2.5; All the other compositions are water, regulate pH value to 8.0, in 115 ℃ of autoclaving 20min.
Measure the enzyme activity of xyloglucanase enzymes in the gained fermented liquid according to the measuring method of above-mentioned xyloglucan enzyme activity, and to calculate yield of enzyme be 10.88U/mL.
Embodiment 5
The concrete steps that xylan series bacillus B20CCTCC M 2012149 preparation xyloglucanase enzymes are separated in utilization are following:
(1) slant strains activation: separate xylan series bacillus B20CCTCC M 2012149 streak inoculations on slant medium with said, under 32 ℃, cultivate 34h, the slant culture based component is counted by grams per liter: peptone 12; Yeast extract paste 5; NaCl 8, and agar 18, all the other compositions are water; Regulate pH value to 6.9, in 115 ℃ of autoclaving 20min;
(2) seed culture: the bacterial classification of the 1cm2 area of growing on the said slant medium of step (1) is scraped, be inoculated in the liquid activation substratum, in 32 ℃ of shaking culture 16h; Shaking speed 300r/min, the liquid seed culture medium composition is counted by grams per liter: peptone 12, yeast extract paste 5; NaCl 12, and wood sugar 2, all the other compositions are water; Regulate pH value to 6.8, in 115 ℃ of autoclaving 20min;
(3) liquid fermentation and culture: will pass through step (2) and cultivate the gained activated seed liquid inoculum size of per-cent 9% by volume, and be inoculated in the fermention medium, fermention medium is loaded in the triangular flask of 250mL; Loading amount is 50mL, with postvaccinal fermentation culture based on 28 ℃ of shaking culture 32h, shaking speed 190r/min; Fermentation finishes, and obtains fermented liquid, and the fermentation culture based component is counted by grams per liter: peptone 8; Yeast extract paste 24, NaCl 6, wood sugar 3; All the other compositions are water, regulate pH value to 6.5, in 115 ℃ of autoclaving 20min.
Measure the enzyme activity of xyloglucanase enzymes in the gained fermented liquid according to the measuring method of above-mentioned xyloglucan enzyme activity, and to calculate yield of enzyme be 6.26U/mL.
Embodiment 6
The concrete steps that xylan series bacillus B20CCTCC M 2012149 preparation xyloglucanase enzymes are separated in utilization are following:
(1) slant strains activation: separate xylan series bacillus B20CCTCC M 2012149 streak inoculations on slant medium with said, under 32 ℃, cultivate 36h, the slant culture based component is counted by grams per liter: peptone 12; Yeast extract paste 6; NaCl 9, and agar 19, all the other compositions are water; Regulate pH value to 7.0, in 115 ℃ of autoclaving 20min;
(2) seed culture: the bacterial classification of the 1cm2 area of growing on the said slant medium of step (1) is scraped, be inoculated in the liquid activation substratum, in 28 ℃ of shaking culture 18h; Shaking speed 300r/min, the liquid seed culture medium composition is counted by grams per liter: peptone 8, yeast extract paste 6; NaCl 11, and wood sugar 3, all the other compositions are water; Regulate pH value to 7.0, in 115 ℃ of autoclaving 20min;
(3) liquid fermentation and culture: will pass through step (2) and cultivate the gained activated seed liquid inoculum size of per-cent 10% by volume, and be inoculated in the fermention medium, fermention medium is loaded in the triangular flask of 250mL; Loading amount is 50mL, with postvaccinal fermentation culture based on 30 ℃ of shaking culture 30h, shaking speed 200r/min; Fermentation finishes, and obtains fermented liquid, and the fermentation culture based component is counted by grams per liter: peptone 12; Yeast extract paste 20, NaCl 5, wood sugar 3.5; All the other compositions are water, regulate pH value to 8.0, in 115 ℃ of autoclaving 20min.
Measure the enzyme activity of xyloglucanase enzymes in the gained fermented liquid according to the measuring method of above-mentioned xyloglucan enzyme activity, and to calculate yield of enzyme be 9.14U/mL.
Claims (3)
1. xylan series bacillus (Paenibacillus xylanilyticus) B20 is separated in a strain, and biological preservation information is: CCTCC No.M 2012149.
2. a method for preparing xyloglucanase enzymes is characterized in that CCTCC No.M 2012149 carries out liquid fermenting as fermentation strain with the said xylan series bacillus B20 that separates of claim 1, the preparation xyloglucanase enzymes.
3. according to the said method for preparing xyloglucanase enzymes of claim 2, it is characterized in that concrete steps are following:
(1) slant strains activation: with the said xylan series bacillus B20 that separates, CCTCC No.M2012149 streak inoculation is cultivated 24～36h in 28～32 ℃ on slant medium; Said slant medium composition is counted by grams per liter: peptone 8～12; Yeast extract paste 4～6, NaCl 8～12, agar 18～22; All the other compositions are water, regulate pH value to 6.5～7.0;
(2) seed culture: the bacterial classification of growing on the said slant medium of step (1) is scraped, be inoculated in the liquid seed culture medium that places shaking table, shaking speed 100～300r/min; In 28～32 ℃ of shaking culture 12～18h, said liquid seed culture medium composition is counted by grams per liter: peptone 8～12, yeast extract paste 4～6; NaCl 8～12; Wood sugar 2～5, all the other compositions are water, regulate pH value to 6.0～7.0;
(3) liquid fermentation and culture: will pass through step (2) and cultivate the gained activated seed liquid inoculum size of per-cent 5～10% by volume, and be inoculated in the fermention medium that places shaking table, shaking speed 150～200r/min; In 28～32 ℃ of shaking culture 30～40h, stop fermentation, obtain fermented liquid; Said fermention medium composition is counted by grams per liter: peptone 8～12, and yeast extract paste 20～24, NaCl 5～7; Wood sugar 1～3.5, all the other compositions are water, regulate pH value to 6.5～8.0.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108004186A (en) * | 2018-01-09 | 2018-05-08 | 北京工商大学 | A kind of bacterial strain and application produced hydrolyzable and prepare high polymerization degree XOS zytases |
| CN109576188A (en) * | 2018-12-28 | 2019-04-05 | 天津开发区坤禾生物技术有限公司 | A kind of prevention and treatment tuber of pinellia root rot microbial inoculum and its preparation method and application |
| CN113046340A (en) * | 2021-01-28 | 2021-06-29 | 青岛农业大学 | High-efficiency xyloglucanase and application thereof |
| CN117050913A (en) * | 2023-08-24 | 2023-11-14 | 河北省科学院生物研究所 | Paenibacillus CBP-2 and application thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108004186A (en) * | 2018-01-09 | 2018-05-08 | 北京工商大学 | A kind of bacterial strain and application produced hydrolyzable and prepare high polymerization degree XOS zytases |
| CN109576188A (en) * | 2018-12-28 | 2019-04-05 | 天津开发区坤禾生物技术有限公司 | A kind of prevention and treatment tuber of pinellia root rot microbial inoculum and its preparation method and application |
| CN109576188B (en) * | 2018-12-28 | 2021-07-09 | 天津开发区坤禾生物技术有限公司 | Bacterial agent for preventing and treating pinellia ternata root rot and preparation method and application thereof |
| CN113046340A (en) * | 2021-01-28 | 2021-06-29 | 青岛农业大学 | High-efficiency xyloglucanase and application thereof |
| CN113046340B (en) * | 2021-01-28 | 2022-07-01 | 青岛农业大学 | Xyloglucanase and application thereof |
| CN117050913A (en) * | 2023-08-24 | 2023-11-14 | 河北省科学院生物研究所 | Paenibacillus CBP-2 and application thereof |
| CN117050913B (en) * | 2023-08-24 | 2024-01-26 | 河北省科学院生物研究所 | Paenibacillus CBP-2 and application thereof |
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