CN107937430A - A kind of not cellulase-producing bacillus subtilis and its construction method and application - Google Patents
A kind of not cellulase-producing bacillus subtilis and its construction method and application Download PDFInfo
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Abstract
The present invention relates to genetic engineering technology field, in particular to a kind of not cellulase-producing bacillus subtilis and its construction method and application.The construction method includes：The cellulase Endoglucanase encoding gene eglS of 168 bacterial strains of Bacillus subtilis strain are knocked out, obtain the not cellulase-producing bacillus subtilis.The mutant strain that the construction method is built can produce a variety of degummases such as pectase and hemicellulose enzyme; the gum components in ramie phloem can directly be decomposed; the bacterial strain can be applied to ramie microbial degumming; have the advantages that not damage ramee completely, to safety of human and livestock, be conducive to preserve the ecological environment.
Description
Technical field
The present invention relates to genetic engineering technology field, in particular to a kind of not cellulase-producing bacillus subtilis
And its construction method and application.
Background technology
Ramie (Boehmeria nivea) is Urticaceae Section of Genus Boehmeria crop, the single fiber of its bast is long, intensity is big, moisture absorption
With dissipate it is wet it is rapid, heat-conductive characteristic is excellent, be the important source material of textile industry.China is the main place of production of ramie, and yield accounts for
Global more than 90%, there is larger economic benefit and international influence.However, in ramie raw ramie containing 30% or so by
The bonding type non-cellulosic material (being commonly called as " colloid ") of pectin and hemicellulose composition, the presence of these colloids can influence ramie fibre
The quality of dimension, it is impossible to be directly used in weaving.Degumming is that ramee is used for the necessary committed step before weaving, and ramie is de- at present
Gluing method has traditional Process of retting flax with, chemical degumming law and biological degumming method.Traditional Process of retting flax with pollution is big, time-consuming, has been not suitable for
Industrialized production.Industrial common chemical degumming law highly energy-consuming, high pollution, COD contents are high in waste water, and in this method
Damage of the bronsted lowry acids and bases bronsted lowry to ramee is larger, seriously constrains the development of ramee processing industry, the life of high-efficiency environment friendly energy-conservation
Thing degumming mode then changes this present situation.
Strain is the key restriction factors for promoting biological degumming of ramie industrialization, for a long time, in order to can be suitably used for
The biological degumming of ramie bacterial strain of factorial praluction, researcher has screened more plants during China grass degumming has the function of degumming
Strain.At present, the up to tens of strains of the purebred bacterial strain obtained are screened from nature, wherein bacillus proportion is up to
More than 60%.In the bacillus identified, in addition to not identifying the bacterial strain of kind, most of bacterial strain belongs to bacillus subtilis
Bacterium.In publication number CN1157475 C, withered grass gemma is used in the patent of invention in publication date on July 14th, 2004 from Rong by such as Liu
Bacillus carries out China grass degumming；Pan Zukai etc. is adopted in publication number CN1055118 C, publication date in August, 2000 patent of invention of 2 days
China grass degumming is carried out with bacillus subtilis A-5；Li Mingfeng etc. is in publication number CN1055118 C, publication date August in 2000 2 days
Patent of invention in using bacillus subtilis CGMCC No.0213 carry out China grass degumming.It is to utilize gemma in above-mentioned patent
Supernatant after bacillus fermentation carries out Enzymatic Degumming of Ramie.He Lianfang etc. is in publication number CN101575739 B, publication date 2011
Ramie microbial solid degumming is carried out using bacillus subtilis in the patent of invention on December 28, can complete to take off in 1~4 day
Glue.Existing bacterial strain belongs to wild-type microorganisms strain above, and not whether strain used in the angle confirmation from molecular biology
The cellulase of outer secretion type is produced, whether has damage to ramee in scouring processes, and do not assess the strain to ecological environment
Influence.
Bacillus subtilis strain 168 are the type culture (model orgnaism) of bacillus subtilis,
It is widely used as the production host of Food enzyme and important nutrient chemistry product, its product is " generally by FDA certifications
Regarded as safe " (GRAS) security level, is food-grade probiotics.The strain can secrete pectase, zytase, sweet
It is a variety of suitable for pectin and the ectoenzyme of hemicellulose degradation to reveal dextranase, arabinofuranosidase glycosylhydrolase etc., there is ramie life
The potential of thing degumming.Biological degumming of ramie bacterial strain in existing publication belongs to wild-type microorganisms strain, not from point
Whether strain used in the angle confirmation of sub- biology produces the cellulase of outer secretion type, and whether ramee is damaged in scouring processes
Wound, and influence of the strain to ecological environment is not assessed.B.subtilis 168 can also express cellulase, it means that
168 bacterial strains of B.subtilis directly cannot be used for biological degumming of ramie, it is therefore necessary to by biological engineering means to the bacterium
Transformed to expand its application.
In view of this, it is special to propose the present invention.
The content of the invention
Answering the invention discloses a kind of bacillus subtilis of not cellulase-producing and its in ramie microbial degumming
With belonging to field of genetic engineering.The present invention is with Physiology and biochemistry information is clear, genetic background understands and completed genome sequencing
168 bacterial strain of food-grade microorganisms bacillus subtilis as starting strain, pass through homologous recombination double crossing over method knock out it is fine
The plain enzyme gene (eglS) of dimension, makes cellulase no longer express, has blocked the biological approach of degraded cellulose in the bacterial strain, to utilize
Genetic engineering means transform other degumming strains and lay a good foundation.It is more that the mutant strain can produce pectase and hemicellulose enzyme etc.
Kind degummase, can directly decompose the gum components in ramie phloem, the bacterial strain can be applied to ramie microbial degumming, had
Do not damage ramee entirely, to safety of human and livestock the advantages of, be conducive to preserve the ecological environment.
In order to realize the above-mentioned purpose of the present invention, spy uses following technical scheme：
The present invention relates to a kind of construction method of not cellulase-producing bacillus subtilis, including：Knock out Bacillus
The cellulase Endoglucanase encoding gene eglS of 168 bacterial strains of subtilis strain, obtain described not producing cellulose
Enzyme bacillus subtilis.
Containing cellulose enzyme gene eglS in 168 genomes of B.subtilis, the present invention show through bioinformatic analysis
The cellulase EglS (EC 3.2.1.4) of the gene code is the biology enzyme of exocytosis.It is, therefore, intended that cannot be direct
168 bacterial strains of B.subtilis are used for biological degumming of ramie, reapplied after need to being blocked to the eglS genes in the bacterial strain in
China grass degumming.The present invention knocks out cellulase Endoglucanase encoding genes by the technology of homologous recombination double crossing over
(eglS), the approach of 168 strains for degrading celluloses of B.subtilis is blocked, the engineered strain Δ eglS obtained can be used for ramie
Numb biological degumming, and it is environmentally safe pollution-free, it can be achieved that the clean manufacturing of China grass degumming.
The invention further relates to the not cellulase-producing bacillus subtilis that the method is built.
According to an aspect of the present invention, given birth to the invention further relates to the not cellulase-producing bacillus subtilis in ramie
Application in thing degumming.
Compared with prior art, beneficial effects of the present invention are：
1) the not cellulase-producing Endoglucanase of engineered strain bacillus subtilis Δ eglS described in, biological degumming mistake
Ramee will not be damaged in journey completely.
2) engineered strain bacillus subtilis Δ eglS is food-grade microorganisms described in, and safety nuisance free, will not cause ring
Pollute in border.
3) construction method of engineered strains of the present invention can be that the structure of other China grass degumming high efficiency engineering bacterial strains is established
Theoretical foundation and offer practice reference.
Brief description of the drawings
, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution of the prior art
Embodiment or attached drawing needed to be used in the description of the prior art are briefly described, it should be apparent that, in describing below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
Put, other attached drawings can also be obtained according to these attached drawings.
Fig. 1 is homologous recombination double crossing over technical principle schematic diagram of the present invention；
Fig. 2 is the agarose gel electrophoresis detection figure of homology arm in one embodiment of the invention；
Fig. 3 is addition Δ eglS bacterial strains and the biological degumming of ramie ratio for being not added with the bacterial strain in one embodiment of the invention
Compared with figure.
Embodiment
The present invention relates to a kind of construction method of not cellulase-producing bacillus subtilis, including：Knock out Bacillus
The cellulase Endoglucanase encoding gene eglS of 168 bacterial strains of subtilis strain, obtain described not producing cellulose
Enzyme bacillus subtilis.
Preferably, the construction method of cellulase-producing bacillus subtilis, the method for the knockout are not as described above
Homologous recombination method.
Preferably, the construction method of cellulase-producing bacillus subtilis, the method do not specifically include as described above：
Homology arm of the structure containing antibiotics resistance gene is simultaneously transformed into competence Bacillus subtilis strain
In 168 cells, the antibiotics resistance gene is inserted into by cellulase Endoglucanase encoding genes by homologous recombination
In the reading frame of eglS, the engineered strain bacillus subtilis Δ eglS with antibiotic resistance is obtained.
Preferably, the not construction method of cellulase-producing bacillus subtilis as described above, the antibiotic resistance base
Because block that resistance aph genes.
Preferably, the not construction method of cellulase-producing bacillus subtilis as described above, it is described to resist containing antibiotic
The nucleotide sequence such as SEQ ID NO of the homology arm of property gene:Shown in 1.
Preferably, the not construction method of cellulase-producing bacillus subtilis as described above, the competence
The preparation method of 168 cells of Bacillus subtilis strain is CaCl2Method；
Preferably, the preparation method of 168 cells of competence Bacillus subtilis strain includes：
168 strains of Bacillus subtilis strain are cultivated in GM I culture mediums and GM II culture mediums successively
To obtain the final product；
The component of the GM I culture mediums includes：Dipotassium hydrogen phosphate 13g/L~13.8g/L, potassium dihydrogen phosphate 5.3g/L~
6.1g/L, ammonium sulfate 1.7g/L~2.1g/L, sodium citrate 0.8g/L~1.2g/L, epsom salt 0.15g/L~0.25g/
L, glucose 0.4g/L~0.6g/L, casein 0.15g/L~0.25g/L, yeast extract 0.8g/L~1.2g/L；
It is furthermore preferred that the component of the GM I culture mediums includes：Dipotassium hydrogen phosphate 13.4g/L, potassium dihydrogen phosphate 5.7g/L,
Ammonium sulfate 1.9g/L, sodium citrate 1g/L, epsom salt 0.2g/L, glucose 0.5g/L, casein 0.2g/L, yeast extraction
Thing 1g/L；
The component of the GM II culture mediums includes：Dipotassium hydrogen phosphate 13g/L~13.8g/L, potassium dihydrogen phosphate 5.3g/L~
6.1g/L, ammonium sulfate 1.7g/L~2.1g/L, sodium citrate 0.8g/L~1.2g/L, epsom salt 0.15g/L~0.25g/
L, glucose 0.4g/L~0.6g/L, casein 0.03g/L~0.05g/L, yeast extract 0.03g/L~0.05g/L, chlorination
Magnesium 0.48g/L~0.54g/L, calcium chloride 0.34g/L~0.4g/L；
It is furthermore preferred that the component of the GM II culture mediums includes：The formula of the GM II culture mediums is：Dipotassium hydrogen phosphate
13.4g/L, potassium dihydrogen phosphate 5.7g/L, ammonium sulfate 1.9g/L, sodium citrate 1g/L, epsom salt 0.2g/L, glucose
0.5g/L, casein 0.04g/L, yeast extract 0.04g/L, magnesium chloride 0.51g/L, calcium chloride 0.37g/L.
Preferably, the not construction method of cellulase-producing bacillus subtilis as described above, the competence
The preparation method of 168 cells of Bacillus subtilis strain specifically includes：
168 strains of Bacillus subtilis strain are inoculated on LB tablets and cultivate 1~3 in 35 DEG C~39 DEG C
My god, culture is inoculated into the GM I culture mediums of 4~6 parts by volume, is vibrated in 28 DEG C~32 DEG C, shaking table 130rpm~170rpm
8h~16h is cultivated, obtains the first bacterium solution；
First bacterium solution of 1.5~2.5 parts by volume is taken to be transferred in the GM I culture mediums of 16~20 parts by volume, 35 DEG C~
39 DEG C, shaking table 230rpm~270rpm shaken cultivation 3h~4h, obtain the second bacterium solution；
Second bacterium solution of 4~6 parts by volume is taken to be transferred in the GM II culture mediums of 40~50 parts by volume again, 35 DEG C~
39 DEG C, 120rpm~130rpm shaken cultivation 80min~100min, are resuspended up to competent cell after thalline is collected by centrifugation.
Preferably, the construction method of cellulase-producing bacillus subtilis, the thermal shock that is converted into do not turn as described above
Change；
Preferably, the condition of the thermal shock conversion is：43 DEG C~47 DEG C thermal shock 80s~100s；With after 28 DEG C~32 DEG C
Slowly vibrating 680rpm~720rpm, keeps the temperature 50min~70min；
It is furthermore preferred that the condition of the thermal shock conversion is：45 DEG C of thermal shock 90s；With after 30 DEG C of slowly vibrating 700rpm, protect
Warm 60min.
The invention further relates to the not cellulase-producing bacillus subtilis that the method is built.
According to an aspect of the present invention, given birth to the invention further relates to the not cellulase-producing bacillus subtilis in ramie
Application in thing degumming.
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person, the condition suggested according to normal condition or manufacturer carry out.Reagents or instruments used without specified manufacturer, is
Can be with conventional products that are commercially available.
Embodiment 1 is used for the structure of the homology arm of homologous recombination double crossing over
The engineered strain Δ eglS is by the cellulase Endoglucanase in B.subtilis strain 168
Encoding gene (eglS) is knocked out, and structure is unable to the recombined bacillus subtilis of degradation of ramie fiber.Its genome manipulation is former
Reason is as shown in Figure 1, mainly include the following steps that：
(1) in apparent B.subtilis strain 168 degraded cellulose metabolic pathway
Bioinformatic analysis shows, 168 bacterial strains of B.subtilis strain (http on KEGG websites://
www.kegg.jp/kegg-bin/show_pathwayBsu00500 cellulose metabolic pathway shown in) is Starch and
Sucrose Metabolism, its cellulase Endoglucanase (EC：3.2.1.4) for cellulose degradation first step institute
The enzyme needed.
(2) cellulase Endoglucanase encoding genes (eglS) information is obtained
The cellulase Endoglucanase encoding genes eglS (https on NCBI websites://
www.ncbi.nlm.nih.gov/nucleotide/Z29076Report=genbank＆log $=nucl align＆
Blast_rank=29＆RID=2VUNBUNZ015 such as SEQ ID NO of log-on message):Shown in 2.
(3) it is designed for the homology arm of homologous recombination double crossing over
Using the screening of card that resistance progress engineered strain, the homology arm information such as SEQ ID NO according to designed by Fig. 1:1
It is shown.
The agarose gel electrophoresis detection of homology arm is as shown in Figure 2.
Embodiment 2
The present embodiment is related to a kind of 168 cellulase Endoglucanase of B.subtilis strain that knock out and encodes
The method of gene (eglS).
168 strains of B.subtilis strain are seeded in LB tablets (Tryptone 10g/L, NaCl 10g/L, ferment
Female extract 5g/L) upper 37 DEG C cultivate 1 day, culture is accessed into 6mL GM I culture mediums, is vibrated in 30 DEG C of slow shaking table 130rpm
16h is cultivated, obtains the first bacterium solution；The first bacterium solutions of 1.5mL are taken to be transferred in 16mL GM I culture mediums, 37 DEG C of fast shaking tables
(230rpm) shaken cultivation 4h, obtains the second bacterium solution；Second bacterium solution of 4mL is taken to be transferred to 40mL GM II culture mediums again
In, thalline is collected by centrifugation in 5000g, 10min after 37 DEG C of slow shaking table (120rpm) culture 80min.It is light with 5mL original fluid supernatants
Light suspension thalline, the thalline after suspension are competent cell.
Wherein, the formula of the GM I culture mediums is：Dipotassium hydrogen phosphate 13g/L, potassium dihydrogen phosphate 6.1g/L, ammonium sulfate
1.7g/L, sodium citrate 1.2g/L, epsom salt 0.15g/L, glucose 0.6g/L, casein 0.15g/L, yeast extract
1.2g/L；
The formula of the GM II culture mediums is：Dipotassium hydrogen phosphate 13g/L, potassium dihydrogen phosphate 6.1g/L, ammonium sulfate 1.7g/
L, sodium citrate 1.2g/L, epsom salt 0.15g/L, glucose 0.6g/L, casein 0.03g/L, yeast extract
0.05g/L, magnesium chloride 0.48g/L, calcium chloride 0.4g/L.
Appropriate SEQ ID NO are added in 500uL competent cells:DNA (~1ug/ml) shown in 1,43 DEG C of thermal shocks
100s, with after 28 DEG C of slowly vibrating 720rpm, keeps the temperature 70min, it is 50ug/mL's to take 100ul to be coated on kanamycins concentration
On LB tablets, 37 DEG C of overnight incubations, secondary daily inspection and verification transformant.Positive transformant is sent to raw work bioengineering (Shanghai) stock
After part Co., Ltd carries out sequence verification, that is, think to obtain purpose recombination engineering Δ eglS.
Embodiment 3
The present embodiment is related to a kind of 168 cellulase Endoglucanase of B.subtilis strain that knock out and encodes
The method of gene (eglS).
168 strains of B.subtilis strain are seeded in LB tablets (Tryptone 10g/L, NaCl 10g/L, ferment
Female extract 5g/L) upper 37 DEG C cultivate 3 days, culture is accessed into 4mL GM I culture mediums, is vibrated in 30 DEG C of slow shaking table 170rpm
8h is cultivated, obtains the first bacterium solution；The first bacterium solutions of 2.5mL are taken to be transferred in 20mL GM I culture mediums, 37 DEG C of fast shaking tables (270rpm)
Shaken cultivation 3h, obtains the second bacterium solution；Second bacterium solution of 6mL is taken to be transferred in 50mL GM II culture mediums again, 37 DEG C slow
Thalline is collected by centrifugation in 5000g, 10min after shaking table (130rpm) culture 100min.With 5mL original fluids supernatant gently suspended bacteria
Body, the thalline after suspension are competent cell.
Wherein, the formula of the GM I culture mediums is：Dipotassium hydrogen phosphate 13.8g/L, potassium dihydrogen phosphate 5.3g/L, ammonium sulfate
2.1g/L, sodium citrate 0.8g/L, epsom salt 0.25g/L, glucose 0.4g/L, casein 0.25g/L, yeast extract
0.8g/L；
The formula of the GM II culture mediums is：Dipotassium hydrogen phosphate 13.8g/L, potassium dihydrogen phosphate 5.3g/L, ammonium sulfate
2.1g/L, sodium citrate 0.8g/L, epsom salt 0.25g/L, glucose 0.4g/L, casein 0.05g/L, yeast extract
0.03g/L, magnesium chloride 0.54g/L, calcium chloride 0.34g/L.
Appropriate SEQ ID NO are added in 500uL competent cells:DNA (~1ug/ml) shown in 1,47 DEG C of thermal shocks
80s, with after 32 DEG C of slowly vibrating 680rpm, keeps the temperature 50min, takes 100ul to be coated on the LB that kanamycins concentration is 50ug/mL
On tablet, 37 DEG C of overnight incubations, secondary daily inspection and verification transformant.Positive transformant is sent to raw work bioengineering (Shanghai) share
After Co., Ltd carries out sequence verification, that is, think to obtain purpose recombination engineering Δ eglS.
Embodiment 4
The present embodiment is related to a kind of 168 cellulase Endoglucanase of B.subtilis strain that knock out and encodes
The method of gene (eglS).
168 strains of B.subtilis strain are seeded in LB tablets (Tryptone 10g/L, NaCl 10g/L, ferment
Female extract 5g/L) upper 37 DEG C cultivate 2 days, culture is accessed into 5mL GM I culture mediums, is vibrated in 30 DEG C of slow shaking table 150rpm
12h is cultivated, obtains the first bacterium solution；The first bacterium solutions of 2mL are taken to be transferred in 18mL GM I culture mediums, 37 DEG C of fast shaking tables (250rpm)
Shaken cultivation 3.5h, obtains the second bacterium solution；Second bacterium solution of 5mL is taken to be transferred in 45mL GM II culture mediums again, 37 DEG C
Thalline is collected by centrifugation in 5000g, 10min after slow shaking table (125rpm) culture 90min.Gently suspended with 5mL original fluids supernatant
Thalline, the thalline after suspension are competent cell.
Wherein, the formula of the GM I culture mediums is：Dipotassium hydrogen phosphate 13.4g/L, potassium dihydrogen phosphate 5.7g/L, ammonium sulfate
1.9g/L, sodium citrate 1g/L, epsom salt 0.2g/L, glucose 0.5g/L, casein 0.2g/L, yeast extract 1g/
L；
The formula of the GM II culture mediums is：Dipotassium hydrogen phosphate 13.4g/L, potassium dihydrogen phosphate 5.7g/L, ammonium sulfate
1.9g/L, sodium citrate 1g/L, epsom salt 0.2g/L, glucose 0.5g/L, casein 0.04g/L, yeast extract
0.04g/L, magnesium chloride 0.51g/L, calcium chloride 0.37g/L.
Appropriate SEQ ID NO are added in 500uL competent cells:DNA (~1ug/ml) shown in 1,45 DEG C of thermal shocks
90s, with after 30 DEG C of slowly vibrating 700rpm, keeps the temperature 60min, takes 100ul to be coated on the LB that kanamycins concentration is 50ug/mL
On tablet, 37 DEG C of overnight incubations, secondary daily inspection and verification transformant.Positive transformant is sent to raw work bioengineering (Shanghai) share
After Co., Ltd carries out sequence verification, that is, think to obtain purpose recombination engineering Δ eglS.
Embodiment 5
The present embodiment is related to the application in engineered strain bacillus subtilis Δ eglS biological degumming of ramie.
1. cultivate bacterium solution
The engineered strain bacillus subtilis Δ eglS single bacterium colonies that embodiment 4 is prepared are inoculated in containing 20mL LB
In the 150mL conical flasks of fluid nutrient medium, at a temperature of 37 DEG C, the rotating speed of holding 180rpm, in cultivating 18h on shaking table, obtain
Bacterium solution A.
2. biological degumming of ramie shake flask test
Load ramie raw ramie 10g, water 100mL in 300mL conical flasks, bottleneck is covered with sealed membrane.In each conical flask
6mL bacterium solution A, are mixed evenly.Control adds 6mL tap water.At a temperature of 37 DEG C, the rotating speed of holding 100rpm, on shaking table
Culture about 2 days, terminates scouring processes, obtains degummed ramie.
By 60 DEG C after the flushing of the degummed ramie clear water of experimental group and control group, drying to constant weight, and weighing results are respectively
7.04g and 8.82g, the ramie raw ramie weight-loss ratio handled through engineering bacteria are 29.6%, and the ramie raw ramie of undressed journey bacterium processing subtracts
Rate is 11.8% again, and degumming effect is as shown in Figure 3.
Wherein, the computational methods of weight-loss ratio are：
The dry weight of ramie raw ramie before processing is set as M1, and the ramie raw ramie dry weight after degumming is set as M2, then weight-loss ratio
Calculation formula be
Weight-loss ratio=(M1-M2)/M1 × 100%
Finally it should be noted that：The above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe is described in detail the present invention with reference to foregoing embodiments, but it will be understood by those of ordinary skill in the art that：Its
It can still modify to the technical solution described in foregoing embodiments, either to which part or all technical characteristic
Carry out equivalent substitution；And these modifications or replacement, the essence of appropriate technical solution is departed from various embodiments of the present invention skill
The scope of art scheme.
SEQUENCE LISTING
<110>Hemp Inst., China Academy of Agricultural Sciences
<120>A kind of not cellulase-producing bacillus subtilis and its construction method and application
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 2788
<212> DNA
<213>Artificial sequence
<400> 1
atgatgcgaa ggaggaaaag atcagatatg aaacggtcaa tctctatttt tattacgtgt 60
ttattgatta cgttattgac aatgggcggc atgatagctt cgccggcatc agcagcaggg 120
acaaaaacgc cagtagccaa gaatggccag cttagcataa aaggtacaca gctcgttaac 180
cgagacggta aagcggtaca gctgaagggg atcagttcac acggattgca atggtatgga 240
gaatatgtca ataaagacag cttaaaatgg ctgagagatg attggggtat caccgttttc 300
cgtgcagcga tgtatacggc agatggcggt tatattgaca acccgtccgt gaaaaataaa 360
gtaaaagaag cggttgaagc ggcaaaagag cttgggatat atgtcatcat tgactggcat 420
atcttaaatg acggtaatcc aaaccaaaat aaagagaagg caaaagaatt cttcaaggaa 480
atgtcaagcc tttacggaaa cacgccaaac gtcatttatg aaattgcaaa cgaaccaaac 540
ggtgatgtga actggaagcg tgatattaaa ccatatgcgg aagaagtgat ttcagttatc 600
cgcaaaaatg atccagacaa catcatcatt gtcggaaccg gtacatggag ccaggatgtg 660
aatgatgctg ccgatgacca gctaaaagat gcaaacgtta tgtacgcact tcatttttat 720
gccggcacgc acggccaatt tttacgggat aaagcaaact atgcactcag caaaggagca 780
cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 840
agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 900
gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca 960
gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 1020
tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 1080
acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 1140
cagtggaacg aaaactcacg ttaagggatt ttggtcatga acaataaaac tgtctgctta 1200
cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cttgctctag 1260
gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa 1320
tgtcgggcaa tcaggtgcga caatctatcg attgtatggg aagcccgatg cgccagagtt 1380
gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact 1440
aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga 1500
tgatgcatgg ttactcacca ctgcgatccc cgggaaaaca gcattccagg tattagaaga 1560
atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca 1620
ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc 1680
gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg 1740
ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc 1800
agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat 1860
aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct 1920
atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg 1980
tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta 2040
acctattttt gtgacagagt ggggaacaag cgacgcgtct ggcaatggcg gtgtattcct 2100
tgatcaatcg agggaatggc tgaaatatct cgacagcaag accattagct gggtgaactg 2160
gaatctttct gataagcagg aatcatcctc agctttaaag ccgggggcat ctaaaacagg 2220
cggctggcgg ttgtcagatt tatctgcttc aggaacattc gttagagaaa acattctcgg 2280
caccaaagat tcgacgaagg acattcctga aacgccatca aaagataaac ccacacagga 2340
aaatggtatt tctgtacagt acagagcagg ggatgggagt atgaacagca accaaatccg 2400
tccgcagctt caaataaaaa ataacggcaa taccacggtt gatttaaaag atgtcactgc 2460
ccgttactgg tataaagcga aaaacaaagg ccaaaacttt gactgtgact acgcgcagat 2520
tggatgcggc aatgtgacac acaagtttgt gacgttgcat aaaccaaagc aaggtgcaga 2580
tacctatctg gaacttggat ttaaaaacgg aacgttggca ccgggagcaa gcacagggaa 2640
tattcagctc cgtcttcaca atgatgactg gagcaattat gcacaaagcg gcgattattc 2700
ctttttcaaa tcaaatacgt ttaaaacaac gaaaaaaatc acattatatg atcaaggaaa 2760
actgatttgg ggaacagaac caaattag 2788
<210> 2
<211> 1527
<212> DNA
<213> Bacillus subtilis
<400> 2
atgatgcgaa ggaggaaaag atcagatatg aaacggtcaa tctctatttt tattacgtgt 60
ttattgatta cgttattgac aatgggcggc atgatagctt cgccggcatc agcagcaggg 120
acaaaaacgc cagtagccaa gaatggccag cttagcataa aaggtacaca gctcgttaac 180
cgagacggta aagcggtaca gctgaagggg atcagttcac acggattgca atggtatgga 240
gaatatgtca ataaagacag cttaaaatgg ctgagagatg attggggtat caccgttttc 300
cgtgcagcga tgtatacggc agatggcggt tatattgaca acccgtccgt gaaaaataaa 360
gtaaaagaag cggttgaagc ggcaaaagag cttgggatat atgtcatcat tgactggcat 420
atcttaaatg acggtaatcc aaaccaaaat aaagagaagg caaaagaatt cttcaaggaa 480
atgtcaagcc tttacggaaa cacgccaaac gtcatttatg aaattgcaaa cgaaccaaac 540
ggtgatgtga actggaagcg tgatattaaa ccatatgcgg aagaagtgat ttcagttatc 600
cgcaaaaatg atccagacaa catcatcatt gtcggaaccg gtacatggag ccaggatgtg 660
aatgatgctg ccgatgacca gctaaaagat gcaaacgtta tgtacgcact tcatttttat 720
gccggcacgc acggccaatt tttacgggat aaagcaaact atgcactcag caaaggagca 780
cctatttttg tgacagagtg gggaacaagc gacgcgtctg gcaatggcgg tgtattcctt 840
gatcaatcga gggaatggct gaaatatctc gacagcaaga ccattagctg ggtgaactgg 900
aatctttctg ataagcagga atcatcctca gctttaaagc cgggggcatc taaaacaggc 960
ggctggcggt tgtcagattt atctgcttca ggaacattcg ttagagaaaa cattctcggc 1020
accaaagatt cgacgaagga cattcctgaa acgccatcaa aagataaacc cacacaggaa 1080
aatggtattt ctgtacagta cagagcaggg gatgggagta tgaacagcaa ccaaatccgt 1140
ccgcagcttc aaataaaaaa taacggcaat accacggttg atttaaaaga tgtcactgcc 1200
cgttactggt ataaagcgaa aaacaaaggc caaaactttg actgtgacta cgcgcagatt 1260
ggatgcggca atgtgacaca caagtttgtg acgttgcata aaccaaagca aggtgcagat 1320
acctatctgg aacttggatt taaaaacgga acgttggcac cgggagcaag cacagggaat 1380
attcagctcc gtcttcacaa tgatgactgg agcaattatg cacaaagcgg cgattattcc 1440
tttttcaaat caaatacgtt taaaacaacg aaaaaaatca cattatatga tcaaggaaaa 1500
ctgatttggg gaacagaacc aaattag 1527
Claims (10)
1. the not construction method of cellulase-producing bacillus subtilis, it is characterised in that including：Knock out Bacillus
The cellulase Endoglucanase encoding gene eglS of 168 bacterial strains of subtilis strain, obtain described not producing cellulose
Enzyme bacillus subtilis.
2. the construction method of not cellulase-producing bacillus subtilis according to claim 1, it is characterised in that described to strike
The method removed is homologous recombination method.
3. the construction method of the not cellulase-producing bacillus subtilis described in claim 2, it is characterised in that the method has
Body includes：
Homology arm of the structure containing antibiotics resistance gene is simultaneously transformed into competence Bacillus subtilis strain 168
In cell, the antibiotics resistance gene is inserted into by cellulase Endoglucanase encoding genes eglS by homologous recombination
Reading frame in, obtain the engineered strain bacillus subtilis Δ eglS with antibiotic resistance.
4. the construction method of not cellulase-producing bacillus subtilis according to claim 3, it is characterised in that described anti-
Raw element resistant gene is card that resistance aph genes.
5. the construction method of not cellulase-producing bacillus subtilis according to claim 4, it is characterised in that described to contain
There are the nucleotide sequence such as SEQ ID NO of the homology arm of antibiotics resistance gene:Shown in 1.
6. the construction method of not cellulase-producing bacillus subtilis according to claim 3, it is characterised in that the sense
Preparation method by 168 cells of state Bacillus subtilis strain is CaCl2Method；
Preferably, the preparation method of 168 cells of competence Bacillus subtilis strain includes：
By 168 strains of Bacillus subtilis strain, culture is in GM I culture mediums and GM II culture mediums successively
；
The component of the GM I culture mediums includes：Dipotassium hydrogen phosphate 13g/L~13.8g/L, potassium dihydrogen phosphate 5.3g/L~6.1g/
L, ammonium sulfate 1.7g/L~2.1g/L, sodium citrate 0.8g/L~1.2g/L, epsom salt 0.15g/L~0.25g/L, grape
Sugared 0.4g/L~0.6g/L, casein 0.15g/L~0.25g/L, yeast extract 0.8g/L~1.2g/L；
The component of the GM II culture mediums includes：
Dipotassium hydrogen phosphate 13g/L~13.8g/L, potassium dihydrogen phosphate 5.3g/L~6.1g/L, ammonium sulfate 1.7g/L~2.1g/L, lemon
Lemon acid sodium 0.8g/L~1.2g/L, epsom salt 0.15g/L~0.25g/L, glucose 0.4g/L~0.6g/L, casein
0.03g/L~0.05g/L, yeast extract 0.03g/L~0.05g/L, magnesium chloride 0.48g/L~0.54g/L, calcium chloride
0.34g/L~0.4g/L.
7. the construction method of not cellulase-producing bacillus subtilis according to claim 6, it is characterised in that the sense
Specifically included by the preparation method of 168 cells of state Bacillus subtilis strain：
168 strains of Bacillus subtilis strain are inoculated on LB tablets and are cultivated 1~3 day in 35 DEG C~39 DEG C, will
Culture is inoculated into the GM I culture mediums of 4~6 parts by volume, in 28 DEG C~32 DEG C, shaking table 130rpm~170rpm shaken cultivations
8h~16h, obtains the first bacterium solution；
First bacterium solution of 1.5~2.5 parts by volume is taken to be transferred in the GM I culture mediums of 16~20 parts by volume, 35 DEG C~39
DEG C, shaking table 230rpm~270rpm shaken cultivation 3h~4h, obtain the second bacterium solution；
Second bacterium solution of 4~6 parts by volume is taken to be transferred in the GM II culture mediums of 40~50 parts by volume again, 35 DEG C~39 DEG C,
120rpm~130rpm shaken cultivation 80min~100min, are resuspended up to competent cell after thalline is collected by centrifugation.
8. the construction method of not cellulase-producing bacillus subtilis according to claim 3, it is characterised in that described turn
Turn to thermal shock conversion；
Preferably, the condition of the thermal shock conversion is：43 DEG C~47 DEG C thermal shock 80s~100s；With slow after 28 DEG C~32 DEG C
680rpm~720rpm is vibrated, keeps the temperature 50min~70min.
9. the not cellulase-producing bacillus subtilis that any one of claim 1~8 the method is built.
10. application of the not cellulase-producing bacillus subtilis in biological degumming of ramie described in claim 9.
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| CN113881618A (en) * | 2021-10-25 | 2022-01-04 | 长江大学 | Recombinant bacillus subtilis for secreting lactocasein, and construction method and application thereof |
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| CN114304387A (en) * | 2021-12-30 | 2022-04-12 | 上海农乐生物制品股份有限公司 | Protein mulberry leavening agent and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113956336A (en) * | 2021-10-20 | 2022-01-21 | 中国农业科学院麻类研究所 | Application of CitT protein or coding gene thereof in regulating and controlling ramie degumming efficiency of microorganism |
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| CN113881618B (en) * | 2021-10-25 | 2023-10-03 | 长江大学 | Recombinant bacillus subtilis secreting milk casein, and construction method and application thereof |
| CN114304387A (en) * | 2021-12-30 | 2022-04-12 | 上海农乐生物制品股份有限公司 | Protein mulberry leavening agent and preparation method and application thereof |
| CN114304387B (en) * | 2021-12-30 | 2023-08-04 | 上海农乐生物制品股份有限公司 | Protein mulberry starter and preparation method and application thereof |
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