CN113430142B - Bacillus cereus and application thereof - Google Patents
Bacillus cereus and application thereof Download PDFInfo
- Publication number
- CN113430142B CN113430142B CN202110831065.0A CN202110831065A CN113430142B CN 113430142 B CN113430142 B CN 113430142B CN 202110831065 A CN202110831065 A CN 202110831065A CN 113430142 B CN113430142 B CN 113430142B
- Authority
- CN
- China
- Prior art keywords
- bacillus
- cyclodextrin
- bacillus cereus
- beta
- fermentation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
- C12N9/1074—Cyclomaltodextrin glucanotransferase (2.4.1.19)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/20—Preparation of compounds containing saccharide radicals produced by the action of an exo-1,4 alpha-glucosidase, e.g. dextrose
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/01—Hexosyltransferases (2.4.1)
- C12Y204/01019—Cyclomaltodextrin glucanotransferase (2.4.1.19)
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a bacillus creek and application thereof, belonging to the field of microorganisms. To obtain beta-cyclodextrin glucosyltransferase with high purity. The invention provides a rivulet bacillus STB08 and a method for preparing high-purity beta-cyclodextrin glucosyltransferase by rivulet bacillus STB08, which can obtain the high-purity beta-cyclodextrin glucosyltransferase by plate marking, seed culture, fermentation culture and centrifugal collection, solves the problems in research and production, omits the steps of separation and purification and is beneficial to the preparation of cyclodextrin. Meanwhile, the enzyme has good thermal stability and high product specificity, and has potential industrial application value.
Description
Technical Field
The invention relates to bacillus cereus and application thereof, belonging to the technical field of microorganisms.
Background
Cyclodextrin is a cyclic polymer formed by connecting 6 or more glucose units via alpha-1, 4-glycosidic bonds, and has a hollow cylindrical shape with a slight conical shape. The most common are alpha-, beta-and gamma-cyclodextrins, formed by the linkage of 6, 7 and 8 glucose units, respectively, which are also the most industrially applicable, especially beta-cyclodextrins. The cyclodextrin has a hydrophilic surface and a hydrophobic cavity structure because glucose hydroxyl-OH surrounds an outer ring and an ether bond C-O-C inwards surrounds and arranges, is often used for including hydrophobic object molecules so as to improve the physical and chemical properties of the molecules, such as solubility, volatility, chemical performance and the like, and is widely applied to the industries of food, medicine, cosmetics and the like.
Currently, cyclodextrin is produced mainly by enzymatic synthesis by the action of cyclodextrin glycosyltransferase (cgtase) on starch or related derivatives, the main product being a mixture of α -, β -and γ -cyclodextrins. The primary products of the reaction are named alpha-CGTase, beta-CGTase and gamma-CGTase, respectively, according to the difference of the primary products of the reaction, wherein the beta-CGTase is the most studied. After the CGT enzyme is expressed by wild bacteria or genetically engineered bacteria, a large amount of miscellaneous enzymes exist in an enzyme solution, which causes the following problems: (1) many miscellaneous enzymes need to be separated and purified, but the process is complex and complicated, and great difficulty is brought to the research of the enzymology property and the product analysis. (2) In the production process of cyclodextrin, a plurality of byproducts are produced, and certain influence is brought to the production efficiency of cyclodextrin and the separation and purification of products. Rosso et al, screened and separated to obtain a strain Bacillus circulators DF9R of high-yield beta-CGT enzyme, wherein the enzyme activity in the fermentation liquid is 5.8U/mL, and a plurality of hybrid bands exist in an electrophoretogram, but all the hybrid bands have the defects of long fermentation period, special fermentation conditions, less extracellular enzyme production, low purity, difficult separation and purification and poor thermal stability, so that the strain is limited in industrial application. Therefore, the method for screening the high-activity beta-cyclodextrin glucosyltransferase with high purity and strong stability has wide application prospect.
Disclosure of Invention
The invention aims to obtain the beta-cyclodextrin glucosyltransferase with high purity, solves the problems in research and production, omits a separation and purification step, and is beneficial to the preparation of cyclodextrin.
The invention provides a Bacillus cereus (Bacillus xiaoxinensis), which is numbered as STB08 with the preservation number of CGMCC NO.22625 and is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation date of 2021 year, 5 month and 28 days.
The invention also provides a microbial preparation containing the bacillus creeper.
The invention also provides an application of the brook bacillus or the microbial preparation in producing beta-cyclodextrin glucosyltransferase.
The invention also provides a method for preparing beta-cyclodextrin glucosyltransferase, which is to ferment the bacillus creeper at 25-30 ℃ for at least 72 h.
In one embodiment, the medium used for fermentation uses yeast powder as a carbon source.
In one embodiment, the medium used for the fermentation is fish peptone as nitrogen source.
In one embodiment, the fermentation medium for fermentation comprises corn steep liquor dry powder, Na2CO3、MgSO4·7H2O、KH2PO4And tapioca starch.
The invention also provides application of the bacillus creeper in producing cyclodextrin.
The invention also provides a method for producing cyclodextrin, which uses the bacillus creeper to produce beta-cyclodextrin glucosyltransferase, and then uses the beta-cyclodextrin glucosyltransferase according to the proportion of 2U/gMaltodextrin dry baseAdding into a system containing maltodextrin, reacting at 45 deg.C for 24 hr, boiling to inactivate enzyme for 10min, and adding 2U/g(maltodextrin dry basis)Saccharifying the saccharifying enzyme of (1) at 30 ℃ for 1 hour.
The invention also provides application of the bacillus creeper in decomposing maltodextrin in the field of food.
The invention has the beneficial effects that:
(1) the Bacillus rivieri STB08 provided by the invention can secrete beta-cyclodextrin glucosyltransferase with higher purity, has better thermal stability, keeps the temperature at 50 ℃ and below for 2 hours, keeps the enzyme activity at more than 80%, has half-life periods of 42min and 9min at 60 ℃ and 65 ℃, has potential industrial application value, is beneficial to the preparation of cyclodextrin, realizes that beta-cyclodextrin obtained by decomposing maltodextrin reaches 11.6g/L, accounts for 74.3% of the total cyclodextrin, and has higher beta-cyclodextrin product specificity.
(2) According to the invention, the rapid and normal growth of the Bacillus rivieri STB08 is realized through the optimization of the culture conditions of the flat plate and the seeds, and the preservation and the subsequent utilization of the bacteria are facilitated;
(3) the invention realizes the high secretion and high-purity expression of beta-cyclodextrin glucosyltransferase by optimizing fermentation culture conditions.
Biological material preservation
Bacillus cereus (Bacillus xiaoxinensis) STB08, classified and named Bacillus xiaoxinensis, has been preserved in China Committee for culture Collection of microorganisms at 28 months 5 and 2021, with the preservation number of CGMCC NO.22625 and the preservation address of Beijing City Shangyang district No. 1 Beijing Homeh No. 3.
Drawings
FIG. 1 is a SDS-PAGE gel of beta-cyclodextrin glucosyltransferase;
FIG. 2 is a graph of a thermal stability analysis of β -cyclodextrin glycosyltransferase, where the abscissa is time and the ordinate is relative activity;
fig. 3 is a graph of the process of producing cyclodextrin by the action of β -cyclodextrin glucosyltransferase on maltodextrin (DE ═ 4), wherein the abscissa is time and the ordinate is cyclodextrin production.
Detailed Description
The method for measuring the enzyme activity comprises the following steps: 0.1mL of an appropriately diluted enzyme solution was added to a test tube containing 0.9mL of a 1% (w/v) maltodextrin (DE ═ 4) solution prepared in advance with 10mM phosphate buffer (pH 6.5), and after reaction at 50 ℃ for 10min, 3.5mL of 30mM NaOH was added to stop the reaction, and 0.5mL of a 5mM Na-containing solution was added2CO30.02% (w/v) phenolphthalein solution prepared from the solution was developed at room temperature for 20min, and the absorbance was measured at 550 nm. The inactivated enzyme was used as a blank. One unit of enzyme activity is defined as the amount of enzyme required to produce 1. mu. mol of beta-cyclodextrin per minute under the above conditions.
The purity detection method of the beta-cyclodextrin glucosyltransferase comprises the following steps:
the purity of cyclodextrin glucosyltransferase was determined by SDS-PAGE gel electrophoresis: SDS-PAGE gel electrophoresis was performed with reference to the SDS-PAGE gel rapid preparation kit instructions for Biyuntian, using 5% concentration of the concentrate gel and 10% concentration of the isolate gel, and staining was performed with 0.125% Coomassie Brilliant blue G-250. Finally, electrophoretic analysis was performed using a gel imaging analyzer.
The method for measuring the thermal stability of beta-cyclodextrin glucosyltransferase comprises the following steps:
keeping the temperature of the enzyme at 55 ℃, keeping the temperature at different temperatures (40, 45, 50, 55, 60, 65, 70 and 80 ℃), sampling at different time points, rapidly cooling in an ice bath, determining the residual activity of the enzyme according to the cyclization activity, and making a time-relative activity curve by taking the enzyme activity of the sample which is not kept at the temperature of 25 ℃ as 100%.
Example 1 isolation and characterization of Bacillus cereus (Bacillus xiaoxiensis)
1. Separation: soil from the vicinity of a starch factory was collected, filled in a sterile bottle, and 15 strains were isolated in a dish according to a conventional method.
2. And (3) identification: and carrying out strain identification and analysis of physicochemical properties on the separated strains.
(1) Analysis of physicochemical Properties
Inoculating the strain on the plate into a seed culture medium for activation, then inoculating an activation solution into a fermentation culture medium for fermentation, centrifugally collecting fermentation liquor, measuring enzyme activity, and selecting the strain with the highest enzyme activity for strain identification.
(2) The strain identification method comprises the following steps:
and (3) carrying out strain identification by adopting PCR amplification of a 16S rRNA gene sequence. The sequence of the PCR amplification primer of the strain 16S rRNA is as follows: 27F (5'-AGAGTTTGATCCTGGCTCAG-3', shown in SEQ ID NO: 1); 1492R (5'-AAGTCGTAACAAGGTAACC-3', shown in SEQ ID NO: 2).
Before PCR, activated bacterium liquid is firstly centrifuged for 3min at 8000 Xg for enrichment, supernatant is poured off, 1mL of distilled water is added for suspension, centrifugation and enrichment are carried out again, supernatant is poured off, and then 500 mL of solution is addedmu.L of distilled water was suspended for use. PCR reaction (25. mu.L): 2 × Taq Plus MasterMix (Dye)12.5 μ L, ddH2O10.5. mu.L, primer 27F (100. mu.M) 0.5. mu.L, primer 1492R (100. mu.M) 0.5. mu.L, and template (bacterial suspension) 1. mu.L. The PCR reaction parameters were as follows: pre-denaturation at 95 ℃ for 10min, denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 30s, circulation for 34 times, and extension at 72 ℃ for 10 min. And (3) carrying out sample sequencing on the PCR product, wherein the PCR amplified fragment is about 1500bp, and the sequencing work is completed by the Huada gene.
Results and analysis: the 16S rRNA sequences of the tested strains were analyzed by aligning them with the 16S rRNAs of known strains in the database using Blast (https:// Blast. The sequencing result shows that the 16S rRNA sequence of the strain is 1413 bp. The 16S rRNA sequence of Bacillus xiaoxiensis STB08 is shown in SEQ ID NO. 3. the 5 species with the highest homology identified using Blast comparison are listed in Table 1 and can be found: it has the highest sequence homology with 16S rRNA of Bacillus pumilus (Bacillus xiaoxiensis) and only has 6 base differences. Meanwhile, the homology of the strain sequence with a certain model strain sequence or a non-model strain sequence is more than or equal to 99 percent and is higher than that of other strains by 0.8 percent, so that the strain can be identified and inferred to be derived from the Bacillus cereus (Bacillus xiaoxinensis), and is named as Bacillus xiaoxinensis STB 08.
TABLE 1 homology analysis of 16S rRNA sequences of CGT-Com strains
3. Optimum pH for culturing Bacillus rivieri STB08
Bacillus cereus STB08 was cultured as follows:
(1) plate scribing: dipping the bacterial liquid of the Bacillus rivieri STB08 by using an inoculating loop, and adding the bacterial liquid into a prepared plate culture medium (6 g/L yeast powder, 6g/L fish peptone and K)2HPO4·3H2O1 g/L, tapioca starch 12g/L, MgSO4·7H2O0.2 g/L, agar 15g/L, adjusted to pH 10.0 with NaOH), streaked, and cultured in an incubator at 30 ℃ for 12 hours.
(2) Seed culture: single colonies were picked from the plates into seed medium and cultured at 30 ℃ and 200 rpm.
The seed culture medium comprises the following components: 6g/L of yeast powder, 6g/L of fish peptone and K2HPO4·3H2O1 g/L, tapioca starch 12g/L, MgSO4·7H2O0.2 g/L, adjusted to pH 7.0, 8.0, 9.0, 10.0, 11.0, 12.0 with NaOH, respectively.
The seed culture was carried out in seed culture media of different pH values, and the growth of the bacterial liquid is shown in Table 2. The Bacillus rivieri STB08 can be found to be suitable for growth under the condition of pH 10.0-11.0. In the pH 10.0 seed culture medium, the bacterial liquid grows turbid in 12h, which indicates that the optimal growth pH of the bacterial liquid is 10.0.
TABLE 2 Effect of different pH seed media on growth of the strains
Example 2 secretion of beta-Cyclodextrin glucosyltransferase by Bacillus cereus (Bacillus xiaoxiensis)
Bacillus cereus STB08 was cultured as follows:
(1) plate scribing: dipping the bacterial liquid of the Bacillus rivieri STB08 by using an inoculating loop, and adding the bacterial liquid into a prepared plate culture medium (6 g/L yeast powder, 6g/L fish peptone and K)2HPO4·3H2O1 g/L, tapioca starch 12g/L, MgSO4·7H2O0.2 g/L, agar 15g/L, adjusted to pH 10.0 with NaOH), streaked, and cultured in an incubator at 30 ℃ for 12 hours.
(2) Seed culture: picking single colony from plate to seed medium (yeast powder 6g/L, fish peptone 6g/L, K)2HPO4·3H2O1 g/L, tapioca starch 12g/L, MgSO4·7H2O0.2 g/L, adjusted to pH 10.0 with NaOH), and cultured at 30 ℃ and 200rpm for 12 hours.
(3) Fermentation culture: the seed culture was inoculated into a 250mL Erlenmeyer flask containing 50mL of the fermentation medium at an inoculum size of 4% (v/v), and cultured at 200rpm at 25 ℃ for 72 hours.
The formula of the fermentation medium is as follows: 26.6g/L of corn steep liquor dry powder and Na2CO3 4.14g/L，MgSO4·7H2O 0.21g/L，KH2PO41.63g/L and 12g/L of cassava starch, and adjusting the initial pH values to 7.0, 8.0, 9.0, 10.0 and 11.0 by NaOH respectively.
Fermentation in fermentation media at different pH values produced cyclodextrin glycosyltransferase, the results are shown in Table 3. The enzyme activity of the Bacillus rivieri STB08 can reach 10.7U/mL at 25 ℃ and pH of 9.0. This level of secretion is at a higher level in the wild-type strain producing cyclodextrin glycosyltransferase.
TABLE 3 influence of pH on fermentation of Cyclodextrin glucosyltransferase
(4) Collecting enzyme liquid: centrifuging the cultured bacterial liquid obtained in the step (3) at 4 ℃ and 10000 Xg for 15min to obtain supernatant, namely the beta-cyclodextrin glucosyltransferase. The formula of the fermentation medium is as follows: 26.6g/L of corn steep liquor dry powder and Na2CO34.14g/L，MgSO4·7H2O 0.21g/L，KH2PO41.63g/L, tapioca starch 12g/L, pH 9.
(5) And (3) enzyme activity detection: the cyclodextrin glucosyltransferase obtained by centrifugation was subjected to purity detection and property analysis, and the results are shown in FIGS. 1 and 2. As can be seen in FIG. 1, the beta-cyclodextrin glucosyltransferase prepared from Bacillus rivularis STB08 shows a single band and has high purity when analyzed by SDS-PAGE. Meanwhile, the enzyme has good thermal stability, the temperature is kept at 50 ℃ and below for 2h, the figure 2 shows that the enzyme activity is kept above 80%, and the half-life periods at 60 ℃ and 65 ℃ are 42min and 9min respectively. The cyclodextrin glucosyltransferase of the invention is one enzyme protein corresponding to one band, and the single band indicates that the purity of the enzyme is high.
Example 3 beta-Cyclodextrin glucosyltransferase enzyme secreted by Bacillus cereus (Bacillus xiaoxiensis) decomposes maltodextrin
At 5% (dry basis, w/v, 5g/100mL) wheatAdding 2U/g of bud dextrin (DE ═ 4) as substrate(maltodextrin dry basis)The beta-cyclodextrin glycosyltransferase obtained in example 2 was reacted at 45 ℃ and sampled at different time points (0, 1, 2, 3, 6, 9, 12, 24 hours), boiled to inactivate the enzyme for 10 minutes, 2U/g (maltodextrin dry basis) of the glucoamylase was added, saccharification was carried out at 30 ℃ for 1 hour, boiled to inactivate the enzyme for 10 minutes, centrifugation was carried out at 10000r/min for 20 minutes, and then the supernatant was filtered through a 0.45 μm ultrafiltration membrane and subjected to product analysis by High Performance Liquid Chromatography (HPLC).
HPLC determination conditions: waters 600 high performance liquid chromatograph (equipped with differential refractive index detector), chromatographic column Lichrosorb NH2(4.6 mm. times.150 mm), acetonitrile-water (68% -32%) as a mobile phase, a column temperature of 30 ℃ and a flow rate of 1 mL/min.
As a result: after the beta-cyclodextrin glucosyltransferase obtained in example 2 acts on maltodextrin (DE ═ 4) for 24h, FIG. 3 shows that the main product beta-cyclodextrin reaches 11.6g/L, which is 74.3% of total cyclodextrin, and has higher beta-cyclodextrin product specificity.
Example 4 secretion of beta-Cyclodextrin glucosyltransferase by Bacillus cereus (Bacillus xiaoxiensis)
Bacillus cereus STB08 was cultured as follows:
(1) plate scribing: dipping the bacterial liquid of the Bacillus rivieri STB08 by using an inoculating loop, and adding the bacterial liquid into a prepared plate culture medium (6 g/L yeast powder, 6g/L fish peptone and K)2HPO4·3H2O1 g/L, tapioca starch 12g/L, MgSO4·7H2O0.2 g/L, agar 15g/L, adjusted to pH 10.0 with NaOH), streaked, and cultured in an incubator at 30 ℃ for 12 hours.
(2) Seed culture: single colonies were picked from the plates into seed medium and cultured at 30 ℃ and 200 rpm.
The seed culture medium comprises the following components:
seed culture medium A: 6g/L of yeast powder, 6g/L of fish peptone and K2HPO4·3H2O1 g/L, tapioca starch 12g/L, MgSO4·7H2O0.2 g/L, adjusted to pH 10.0 with NaOH.
Seed culture medium B: 6g/L yeast powder and 6g soybean peptoneL，K2HPO4·3H2O1 g/L, tapioca starch 12g/L, MgSO4·7H2O0.2 g/L, adjusted to pH 10.0 with NaOH.
Seed culture medium C: 6g/L yeast powder, 6g/L tryptone and K2HPO4·3H2O1 g/L, tapioca starch 12g/L, MgSO4·7H2O0.2 g/L, adjusted to pH 10.0 with NaOH.
Seed culture medium D: 6g/L of yeast powder, 6g/L of casein peptone and K2HPO4·3H2O1 g/L, tapioca starch 12g/L, MgSO4·7H2O0.2 g/L, adjusted to pH 10.0 with NaOH.
Seed culture medium D: 6g/L of yeast powder, 6g/L of bovine bone peptone and K2HPO4·3H2O1 g/L, tapioca starch 12g/L, MgSO4·7H2O0.2 g/L, adjusted to pH 10.0 with NaOH.
The seed culture was carried out in different seed culture media, and the growth of the bacterial liquid is shown in Table 4. It can be found that fish peptone is essential for the normal growth of bacillus cereus STB 08.
TABLE 4 Effect of different kinds of peptone on growth of the strains
Example 5 secretion of beta-Cyclodextrin glucosyltransferase by Bacillus cereus (Bacillus xiaoxiensis)
Bacillus cereus STB08 was cultured as follows:
(1) plate scribing: dipping the bacterial liquid of the Bacillus rivieri STB08 by using an inoculating loop, and adding the bacterial liquid into a prepared plate culture medium (6 g/L yeast powder, 6g/L fish peptone and K)2HPO4·3H2O1 g/L, tapioca starch 12g/L, MgSO4·7H2O0.2 g/L, agar 15g/L, adjusted to pH 10.0 with NaOH), streaked, and cultured in an incubator at 30 ℃ for 12 hours.
(2) Seed culture: single colonies were picked from the plates into seed medium and cultured at 30 ℃ and 200 rpm.
The seed culture medium comprises the following components:
a: 6g/L of yeast powder, 6g/L of fish peptone and K2HPO4·3H2O1 g/L, tapioca starch 12g/L, MgSO4·7H2O0.2 g/L, adjusted to pH 10.0 with NaOH.
B: 6g/L yeast extract, 6g/L fish peptone and K2HPO4·3H2O1 g/L, tapioca starch 12g/L, MgSO4·7H2O0.2 g/L, adjusted to pH 10.0 with NaOH.
As a result of seed culture in these 2 seed media, it was found that the seed medium containing yeast powder was turbid after 12 hours of culture, whereas the seed medium containing yeast extract was turbid after 24 hours of culture. Therefore, the yeast powder plays an important role in the rapid growth of the strain and is beneficial to the high-efficiency production of the cyclodextrin glucosyltransferase in industry.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> Bacillus rivieri and application thereof
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 1
<210> 2
<211> 19
<212> DNA
<213> Artificial Synthesis
<400> 2
aagtcgtaac aaggtaacc 19
<210> 3
<211> 1413
<212> DNA
<213> Bacillus xiaoxiensis STB08
<400> 3
gctggctcca aaggttacct caccgacttc gggtgttaca aactctcgtg gtgtgacggg 60
cggtgtgtac aagacccggg aacgtattca ccgcggcatg ctgatccgcg attactagca 120
attccggctt catgtaggcg agttgcagcc tacaatccga actgagaatg gctttatggg 180
attggctcca cctcacggct tcgcaaccct ttgtaccatc cattgtagca cgtgtgtagc 240
ccaggtcata aggggcatga tgatttgacg tcgtccccac cttcctccgg tttgtcaccg 300
gcagtcacct tagagtgccc aactaaatgc tggcaactaa gatcaagggt tgcgctcgtt 360
gcgggactta acccaacatc tcacgacacg agctgacgac aaccatgcac cacctgtcac 420
tttgcccccg aaggggaagc tctgtctcca gagtggtcaa aggatgtcaa gacctggtaa 480
ggttcttcgc gttgcttcga attaaaccac atgctccact gcttgtgcgg gtccccgtca 540
attcctttga gtttcagcct tgcggccgta ctccccaggc ggagtgctta atgtgtttac 600
ttcggcacta cgggcatcga aacccctaac acctagcact catcgtttac ggcgtggact 660
accagggtat ctaatcctgt ttgctcccca cgctttcgcg cctcagcgtc agttacagac 720
cagagagtcg ccttcgccac tggtgttcct ccacatatct acgcatttca ccgctacacg 780
tggaattcca ctctcctctt ctgtactcaa gcttcccagt ttccaatggc cgctcggggt 840
tgagccccga gatttcacat cagacttaag aagccgcctg cgcgcgcttt acgcccaata 900
attccggaca acgcttgcca cctacgtatt accgcggctg ctggcacgta gttagccgtg 960
gctttctggt gaggtaccgt cacggtaccg gtagttacgc cggtacttgt tcttccctca 1020
caacagagct ttacgacccg aaggccttcc tcactcacgc ggcattgctc cgtcagactt 1080
tcgtccattg cggaagattc cctactgctg cctcccgtag gagtctgggc cgtgtctcag 1140
tcccagtgtg gccgatcacc ctctcaggtc ggctacgcat cgtcgccttg gtaagccgtt 1200
accttaccaa ctagctaatg cgccgcgggc ccatccctta gtgacagcac aaaggccatc 1260
tttcaacaga gaaccaggag gttccttgta ttattcggta ttagcttcgg tttcccgaag 1320
ttatcccaat ctaaggggca ggttgcccac gtgttactca cccgtccgcc gctgacttcc 1380
gggagcaagc tcccttctgt ccgctcgact tgc 1413
Claims (10)
1. Bacillus cereus (B.parviensis)Bacillus xiaoxiensis) The bacillus cereus is characterized in that the bacillus cereus is preserved in China general microbiological culture Collection center (CGMCC) at 28 th 5 th 2021 with the preservation number of CGMCC NO. 22625.
2. A microbial preparation comprising the Bacillus cereus of claim 1.
3. Use of a bacillus creeper as claimed in claim 1 or a microbial preparation as claimed in claim 2 for the production of β -cyclodextrin glucosyltransferase.
4. A method of producing β -cyclodextrin glucosyltransferase, comprising fermenting bacillus cereus of claim 1 at 25-30 ℃ for at least 72 hours.
5. The method according to claim 4, wherein the medium for fermentation uses yeast powder as a carbon source.
6. The process according to claim 4, wherein the medium used for the fermentation is fish peptone as nitrogen source.
7. The method according to claim 4, wherein the fermentation medium for fermentation comprises corn steep liquor dry powder, Na2CO3、MgSO4·7H2O 、KH2PO4And tapioca starch.
8. Use of Bacillus cereus of claim 1 for the production of cyclodextrins.
9. A process for producing cyclodextrin, characterized in that beta-cyclodextrin glycosyltransferase is produced by Bacillus cereus as claimed in claim 1, and said beta-cyclodextrin glycosyltransferase is added to a system containing maltodextrin in an amount of 2U/g on a dry basis of maltodextrin, reacted at 45 ℃ for 24 hours, boiled to inactivate the enzyme for 10 minutes, and then saccharified at 30 ℃ for 1 hour by adding 2U/g on a dry basis of maltodextrin to the system.
10. Use of Bacillus cereus according to claim 1 for decomposing maltodextrin in the field of food.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110831065.0A CN113430142B (en) | 2021-07-22 | 2021-07-22 | Bacillus cereus and application thereof |
| PCT/CN2021/143099 WO2023000618A1 (en) | 2021-07-22 | 2021-12-30 | Bacillus xiaoxiensis and application thereof |
| JP2022538391A JP2023538160A (en) | 2021-07-22 | 2021-12-30 | Bacillus xiaoxiensis and its use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110831065.0A CN113430142B (en) | 2021-07-22 | 2021-07-22 | Bacillus cereus and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113430142A CN113430142A (en) | 2021-09-24 |
| CN113430142B true CN113430142B (en) | 2021-11-23 |
Family
ID=77761361
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110831065.0A Active CN113430142B (en) | 2021-07-22 | 2021-07-22 | Bacillus cereus and application thereof |
Country Status (3)
| Country | Link |
|---|---|
| JP (1) | JP2023538160A (en) |
| CN (1) | CN113430142B (en) |
| WO (1) | WO2023000618A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113430142B (en) * | 2021-07-22 | 2021-11-23 | 江南大学 | Bacillus cereus and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103981125A (en) * | 2014-04-15 | 2014-08-13 | 福建省农业科学院土壤肥料研究所 | Geobacillus caldoxylosilyticus strain producing cyclodextrin glycosyltransferase |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2004253985A1 (en) * | 2003-07-01 | 2005-01-13 | Novozymes A/S | CGTase variants |
| EP1781779A2 (en) * | 2004-08-02 | 2007-05-09 | Novozymes A/S | Creation of diversity in polypeptides |
| CN101130761A (en) * | 2007-07-13 | 2008-02-27 | 云南师范大学 | Generation bacterium of ring dextrin glucosyl transferase |
| CN101503680B (en) * | 2009-01-06 | 2011-01-05 | 江南大学 | Mutant of cyclodextrin glucosyl transferase having highly beta-cyclodextrin yielding property and mutation method |
| CN102250931B (en) * | 2011-06-23 | 2013-01-09 | 广西大学 | Gene for coding beta-cyclodextrin glucosyltransferase and application thereof |
| CN103667102B (en) * | 2013-09-23 | 2016-05-18 | 江南大学 | A kind of cyclodextrin glycosyltransferase is produced bacterial strain and application thereof |
| CN109706131A (en) * | 2018-12-28 | 2019-05-03 | 合肥工业大学 | A kind of genetic engineering bacterium that expressing high specific beta cyclodextrin glycosyl transferase and its construction method and application |
| CN113430142B (en) * | 2021-07-22 | 2021-11-23 | 江南大学 | Bacillus cereus and application thereof |
-
2021
- 2021-07-22 CN CN202110831065.0A patent/CN113430142B/en active Active
- 2021-12-30 WO PCT/CN2021/143099 patent/WO2023000618A1/en active Application Filing
- 2021-12-30 JP JP2022538391A patent/JP2023538160A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103981125A (en) * | 2014-04-15 | 2014-08-13 | 福建省农业科学院土壤肥料研究所 | Geobacillus caldoxylosilyticus strain producing cyclodextrin glycosyltransferase |
Non-Patent Citations (2)
| Title |
|---|
| Bacillus xiaoxiensis sp.nov., a slightly halophilic bacterium isolated from non-saline forest soil;Yi-Guang Chen et al.;《Int J Syst Evol Microbiol》;20110930;第61卷(第Pt9期);第2095-2100页 * |
| 环状糊精葡萄糖基转移酶的性质及环糊精的转化条件;张心平等;《南开大学学报（自然科学）》;19941231(第4期);第63-67页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2023000618A1 (en) | 2023-01-26 |
| CN113430142A (en) | 2021-09-24 |
| JP2023538160A (en) | 2023-09-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Gungormusler et al. | Continuous production of 1, 3-propanediol using raw glycerol with immobilized Clostridium beijerinckii NRRL B-593 in comparison to suspended culture | |
| CN106754508B (en) | Bacillus subtilis strain and application thereof in soy sauce fermentation and aroma enhancement | |
| CN106701627B (en) | Marine vibrio with high yield of alginate lyase and application thereof | |
| CN113832083B (en) | Bacillus beleisi and application thereof in vinegar brewing | |
| WO2006053480A1 (en) | A acetoin high yield bacillus pumilus strain | |
| CN114250157B (en) | Saccharomycopsis fibuligera cx-3 strain for high yield of aroma substances and application thereof | |
| CN113430142B (en) | Bacillus cereus and application thereof | |
| CN105695340A (en) | Aspergillus oryzae and application thereof | |
| Wittlich et al. | Conversion of glycerol to 1, 3-propanediol by a newly isolated thermophilic strain | |
| CN111826308B (en) | Marine sediment-derived chitin efficient degrading bacterium and application thereof | |
| CN112574920B (en) | Fibro-cellulose microbacterium PX02, method for producing dextranase by using fibro-cellulose microbacterium PX02 and application of method | |
| CN106754486B (en) | Pseudomonas for high-yield trehalose synthase and fermentation enzyme production method thereof | |
| Sun et al. | Simultaneous liquefaction, saccharification, and fermentation of l-lactic acid using aging paddy rice with hull by an isolated thermotolerant Enterococcus faecalis DUT1805 | |
| CN100374555C (en) | Method for preparing beta-cyclodextrin by yeast | |
| CN106754829B (en) | Method for producing chitosanase by using bacillus HS17 fermentation and application thereof | |
| Fang et al. | Optimization of growth medium and fermentation conditions for improved antibiotic activity of Xenorhabdus nematophila TB using a statistical approach | |
| JP5083735B2 (en) | Clostridial genus, cellulase-containing cellulase and method for producing hemicellulase | |
| CN110777096B (en) | Streptomyces capable of producing trypsin with high yield and application thereof | |
| CN108018246B (en) | Bacterial strain for co-production of chitosanase and gamma-polyglutamic acid and application thereof | |
| JP2004344084A (en) | Alcohol fermentative yeast | |
| CN107574132B (en) | Bacillus for degrading insoluble phosphorus and organophosphorus pesticide | |
| Kitcha et al. | Cyclodextrin glycosyltransferase from a newly isolated alkalophilic Bacillus sp. C26. | |
| Ramli et al. | Potential cyclodextrin glycosyltransferase producer from locally isolated bacteria | |
| CN115011507B (en) | High-yield beta-glucosidase lactobacillus and application thereof | |
| JP7197086B2 (en) | Microorganism producing allitol and D-talitol from D-allulose and method for producing allitol and D-talitol using the same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |