CN106047744B - A kind of bacillus amyloliquefaciens and its application - Google Patents
A kind of bacillus amyloliquefaciens and its application Download PDFInfo
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Abstract
The present invention provides a bacillus amyloliquefaciens WZS01.The bacterial strain is preserved in China typical culture collection center, deposit number CCTCC NO:M2016096, and preservation Time of Day is on March 8th, 2016, depositary institution is China typical culture collection center, preservation address is Wuhan, China, Wuhan University, postcode 430072.The advantages that bacterial strain can be used for being catalyzed cane sugar-6-acetic ester synthesis and sucralose-6-acetic ester in Sucralose preparation and hydrolyze two steps after once cultivating, and have reaction condition mild, and good reaction selectivity, product is single, high conversion rate.The present invention prepares Sucralose for biological method and provides new approach.
Description
Technical field
The invention belongs to biocatalysis technology fields, and in particular to a kind of bacillus amyloliquefaciens and its application.
Background technique
Sucralose, also referred to as 4,1 ', 6 '-sucraloses, be uniquely using sucrose as the functional sweetener of raw material,
Sugariness is 600 times or so of sucrose.Sucralose has the characteristics that noenergy, sugariness are high, sweet taste is pure, safe, is at present most
One of outstanding functional sweetener, oneself is led by more than 30 a state approvals including China as sweetener, and in food
Domain is widely used.
The mode of synthesizing trichloro has one chemical method of chemical method and enzyme.Chemical method such as the U.S. using full radical protection method
Patent US 4617269 uses the United States Patent (USP) US 4889928 of single radical protection method, US 5449772, US 4889928 and US
5449772.Chemical synthesis product yield is higher, but reaction condition is harsher, production cost is higher, reaction selectivity compared with
Difference is easy to produce by-product.Enzyme-chemically method is the stereocpecificity and catalytic activity using enzyme, occurs that group reaction in institute
The position needed, such as by 6 hydroxyls in sucrose on glucose molecule with enzyme process be acylated protect, then through chloro, deacylation base, point
From and etc. synthesizing trichloro.United States Patent (USP) US 4617269 and GB 2145080A is disclosed by dextrose and saccharose biology
The method that method prepares cane sugar-6-acetic ester, molar yield 50% or so.Chinese patent CN 101268090A discloses utilization can
Generate the microorganism of transfructosylase class (such asAureobasidium pullulansDeng) Whole Cell Biocatalysis glucose-
The method that 6- acetic acid esters is converted into cane sugar-6-acetic ester, yield are 45% or so.United States Patent (USP) US 4826962 is with raffinose
Raw material, after chosen property chlorination, with deriving fromMortierella vinacea,Circinella muscaeOrAspergillus nigeAlpha-galactosidase, hydrolysis removal raffinose in lactose base be made Sucralose, molar yield
70% or so.Although this method molar yield is high, a large amount of gossypose raw material is more difficult to get.United States Patent (USP) US 5141860
The method of preparation acyl sucrose derivative is disclosed, some lipase, esterase, amylase, galactosidase, protease are used for
The selective hydrolysis of sucrose acetate, de- ester after further chlorination, available Sucralose, but these reaction process steps
More, enzyme dosage is big, and the reaction time is long, and mixture and isomers are more in product.Chinese patent CN 101886100A is used
Novi letter commercialization immobilized lipase Lipozyme TLIM in the tert-butyl alcohol-dimethyl sulfoxide (volume ratio 4:1) system,
It is catalyzed sucrose and vinyl acetate carries out transesterification reaction, 30 DEG C are reacted 6 hours, and molar yield reaches 89%.However commodity in use
Lipase higher cost.In addition the present invention uses the tert-butyl alcohol-dimethylformamide (volume ratio 4:1) as reaction medium, with
In the case where above-mentioned patent CN 101886100A same substrate concentration, the molar yield of product can achieve 95.6%.Reactant
Dimethylformamide in system is without removal, because of the next step reaction of Sucralose preparation, the i.e. chlorination of cane sugar-6-acetic ester
Step needs carry out in dimethylformamide.
After the completion of chlorinating step, Sucralose acetic acid esters needs deacetylate group to generate Sucralose.About trichlorine
The deprotection reaction of sucrose monoester, patent US5023329, US5128248, US5470969, CN1176094C are molten in methanol
In liquid, catalyst is made using sodium methoxide or sodium hydroxide and is reacted.However the reaction introduces a large amount of sodium into system
Ion, after acetyl group removing, it is also necessary to exchange sodium ion from solution with cation exchange resin.And use the present invention
Technology, then be not necessarily to this operation, to reduce the production cost of Sucralose.
In fact, having the report for obtaining Sucralose using enzymatic hydrolysis sucralose-6-acetic ester.Ratnam etc.
It reports during producing Sucralose, using immobilised enzymes, deacetylated rate is up to 95%, but still needs to purify.
Chaubey etc. hydrolyzes sucralose-6-acetic ester using arthrobacterium and bacillus subtilis, and concentration of substrate 1-2% hydrolyzes 48h,
Conversion ratio reaches 100%, because without following purification steps.For the present invention compared with this report, advantage is that concentration of substrate is higher,
Hydrolysis time is shorter, concentration of substrate 3.5%, hydrolyzes for 24 hours, yield 99.6%.
The best advantage is that using same strain, by once cultivating, as whole-cell catalyst, with compared with
High catalytic efficiency carries out esterification and hydrolysis in Sucralose preparation process, greatly simplifies enzymatic clarification trichlorine sugarcane
The operating procedure of sugar.
Summary of the invention
In view of the problems of the existing technology, it is an object of the invention to design provide a kind of bacillus amyloliquefaciens and its
The technical solution of application.
A kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) WZS01, deposit number is
CCTCC NO:M 2016096, preservation Time of Day are on March 8th, 2016, and depositary institution is China typical culture collection
Center, preservation address are Wuhan, China, Wuhan University, postcode 430072.
A kind of bacillus amyloliquefaciens are catalyzed answering for cane sugar-6-acetic ester synthesis in Sucralose synthesis process
With.
The application, it is characterised in that using sucrose and vinyl acetate as substrate, the tert-butyl alcohol is inhaled as reaction medium
Polyurethane foam with bacillus amyloliquefaciens carries out reaction as catalyst and generates cane sugar-6-acetic ester.
The application, it is characterised in that the polyurethane foam for being adsorbed with bacillus amyloliquefaciens passes through following step
Suddenly obtain: it is 10~50Kg/m that will to joined w/v, which be 0.6~1.0% density,3Polyurethane foam culture medium carry out
Sterilizing, it is cooling after inoculation 1% bacillus amyloliquefaciens seed liquor, 30 DEG C are dried to obtain after culture 18~24 hours.
The application, it is characterised in that the culture medium include glucose 2.0~3.5%, peptone 0.5~1.5%,
Yeast powder 0.1%.
The application, it is characterised in that the absorption after the drying of w/v 1.0~2.5% is added into reaction system
There is the polyurethane foam of bacillus amyloliquefaciens, controls 30~38 DEG C of reaction temperature, the reaction time 16~24 hours.
A kind of bacillus amyloliquefaciens are catalyzed sucralose-6-acetic ester hydrolysis in Sucralose synthesis process
Application.
The application, it is characterised in that using sucralose-6-acetic ester as substrate, methanol is as Sucralose -6-
The polyurethane foam for being adsorbed with thallus after the cosolvent of acetic acid esters, the wet thallus containing bacillus amyloliquefaciens or drying is to urge
Generation Sucralose is hydrolyzed in agent in the buffer system of pH5.1~8.0.
The application, it is characterised in that the wet thallus or weighing body of w/v 3~5% are added into reaction system
The long-pending polyurethane foam for being adsorbed with thallus than after 1.0~2.0% dryings, 30~40 DEG C of reaction temperature of control, the reaction time 12~
24 hours.
A kind of bacillus amyloliquefaciens are catalyzed cane sugar-6-acetic ester synthesis and are urged in Sucralose synthesis process
Change the application of sucralose-6-acetic ester hydrolysis.
The bacterial strain screened in the present invention can be used for being catalyzed cane sugar-6-acetic ester synthesis and trichlorine in Sucralose preparation
Cane sugar-6-acetic ester hydrolyzes two steps.And have reaction condition mild, good reaction selectivity, product is single, high conversion rate
The advantages that.The present invention prepares Sucralose for biological method and provides new approach.
Specific embodiment
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in
This.
The screening and identification of 1 cane sugar-6-acetic ester of embodiment synthesis bacterial strain
Acetaldehyde is discharged in cane sugar-6-acetic ester synthesis process, it, can be to second using the coloration method of (2014) Zheng etc.
Aldehyde is quantitative, to reflect the size of enzyme activity.0.1g soil sample is added in liquid enriched medium of the 20ml containing olive oil, is added
Enter in liquid enriched medium of the 20ml containing olive oil, 30 DEG C, 180 r/min, shaken cultivation 48 hours.It is coated on and contains after dilution
On the primary dcreening operation plate for having tributyrin and Bromothymol blue, 30 DEG C are cultivated two days.Picking colony color becomes yellow
The single colonie of different shape after Liquid Culture, draws 1ml in centrifuge tube, and centrifugation, exhaust supernatant, dries thallus, and addition contains
The DMF 0.2mL of 10% sucrose, tert-butyl alcohol 0.8mL, 0.056mL vinyl acetate, 30 DEG C are reacted 1 hour；Take 20 times it is diluted anti-
100 μ l of liquid is answered, 100 μ l, 0.1% phenol reagent is added, 10min is reacted, adds 40 μ l, 1% NH4Fe(SO4)2﹒ 12H2O examination
30min is reacted in agent；Microplate reader measures OD598.It is anti-that cane sugar-6-acetic ester catalysis is carried out by the above-mentioned strain screened, after culture
It answers, liquid phase measurement production spectra.
The bacterium powder screened is dissolved in sterile water, is brought back to life on LB and YPD plating medium.The total DNA for extracting the bacterium, makes
It is upstream and downstream primer amplification with primer (5'-CAGAGTTTGATCCTGGCT-3' and 5'-AGGAGGTGATCCAGCCGCA-3')
The 16S rDNA of bacterial strain to be identified.It is compared by BLAST program, is only capable of identifying the bacterium being bacillus.It further uses
Downstream primer (
5'-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3' and
5'-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3') expandgyrBPartial order
Column.It after sequencing, is compared by BLAST program, and combines the phylogenetic tree of the bacterium, find the bacterial strain and solution starch gemma bar
Bacteria strain h47 is closest in affiliation.It is bacillus amyloliquefaciens WZS01 by the Strain Designation, has been stored in
It is hidden in China typical culture collection center, deposit number CCTCC NO:M2016096.
The optimization of 2 culture medium of embodiment
It is the culture medium that sets out with glucose 2.0%, peptone 2.0%, yeast powder 0.1%.Respectively by 2% concentration with lactose,
Glucose, sucrose, maltose, starch, glycerol are carbon source.30 DEG C of cultures for 24 hours, are reacted after taking 0.2ml culture dry,
1ml reaction system is reacted 30 minutes, by the coloration method that above-mentioned bacterial strains screen, measures relative response initial velocity.Glucose is carbon
When source, relative response initial velocity is significantly higher than other carbon sources.Concentration of glucose has further been investigated to the producing enzyme of bacterial strain WZS01
It influences.The ratio between relative response initial velocity of concentration of glucose 2%, 2.3%, 3%, 3.5% is respectively 0.8:1:0.99:0.77.
When carbon source be 2.5% glucose when, this experimental selection addition 1% organic nitrogen source (beef extract, peptone, yeast powder,
Corn flour) or it is inorganic nitrogen-sourced (ammonium sulfate, sodium nitrate, ammonium chloride), investigate influence of the different nitrogen sources to bacterial strain WZS01 producing enzyme, hair
When existing peptone is nitrogen source, relative response initial velocity is higher than other nitrogen sources.Peptone concentration has further been investigated to bacterial strain WZS01
Producing enzyme influence.The ratio between corresponding initial velocity that peptone concentration is 0.5%, 1%, 1.5% is respectively 0.57:1:0.77.
Influence of the 3 thallus difference incubation time of embodiment to molar yield
0.8%(w/v will be joined) polyurethane foam (density 10Kg/m3) culture medium (2.5% glucose,
1% peptone, 0.5% yeast) it sterilizes, 1% bacillus amyloliquefaciens WZS01 seed liquor, 30 DEG C of cultures are inoculated with after cooling
15,18,21,24 hours.After being adsorbed with the polyurethane foam drying of thallus, the sugarcane of above-mentioned incubation time is measured using colorimetric method
The ratio between sugar -6- acetic acid ester synthesis reaction initial velocity is 0.83:1:0.92:0.64.
The polyurethane foam for adsorbing thallus is used for the synthesis of cane sugar-6-acetic ester by embodiment 4
1) in the tool plug single port reaction flask of 25mL, the dry polyurethane foam for being adsorbed with thallus of 0.2g is weighed, is contained
The DMF 2mL of 10% sucrose, tert-butyl alcohol 8mL, vinyl acetate 0.56mL, 32 DEG C are reacted 18 hours；The density for being adsorbed with thallus is
10Kg/m3、30Kg/m3、50Kg/m3Polyurethane, molar yield are respectively 83.3%, 57.1% and 21.2%.0.2g is dry to dissociate
Thallus under the same conditions, is catalyzed the reaction, and the molar yield of cane sugar-6-acetic ester is only 17.0%.
2) in the tool plug single port reaction flask of 25mL, 0.1g, 0.15g, 0.2g, 0.25g are weighed respectively and is adsorbed with thallus
Polyurethane foam, the DMF 2mL containing 10% sucrose, tert-butyl alcohol 8mL, vinyl acetate 0.56mL, 32 DEG C are reacted 18 hours, are rubbed
Your yield is respectively 36.1%, 55.2%, 83.3%, 81.9%.
3) in the tool plug single port reaction flask of 25mL, the polyurethane foam that 0.2g is adsorbed with thallus is weighed, contains 10% sucrose
DMF 2mL, tert-butyl alcohol 8mL, vinyl acetate 0.56mL, reacted 12 hours at 30,32,34,36,38 DEG C, molar yield point
It Wei 54.6%, 73.0%, 67.5%, 62.3%, 58.3%.
4) in the tool plug single port reaction flask of 25mL, the polyurethane foam that 0.2g is adsorbed with thallus is weighed respectively, contains 10%
The DMF 2mL of sucrose, tert-butyl alcohol 8mL, vinyl acetate 0.56mL react 16,18,20,22,24 hours at 32 DEG C, HPLC inspection
- 6 acetate content of sucrose is surveyed, molar yield is respectively 78.8%, 83.3%, 90.6%, 95.6%, 92.8%.
5 bacillus amyloliquefaciens WZS01 of embodiment is used for the hydrolysis of sucralose-6-acetic ester
(1) in the tool plug single port reaction flask of 25mL, wet thallus 3%, 4%, 5%, methanol content 20%, substrate trichlorine sugarcane
Sugar -6- acetic acid esters 3.5%, 25mM phosphate buffer pH7.1 detect Sucralose content in 36 DEG C of reactions 2h, HPLC, accordingly
Molar yield be respectively 31.1%, 45.5%, 44.9%.
(2) in the tool plug single port reaction flask of 25mL, wet thallus 4%, methanol content 15%, 20%, 25%, substrate trichlorine sugarcane
Sugar -6- acetic acid esters 3.5%, 25mM phosphate buffer pH7.1 detect Sucralose content in 36 DEG C of reactions 2h, HPLC, accordingly
Molar yield be respectively 44.9%, 45.5%, 39.5%.
(3) in the tool plug single port reaction flask of 25mL, wet thallus 4%, methanol content 20%, substrate Sucralose -6- second
Acid esters 3.5%, 25mM phosphate buffer pH7.1 contain in 30,32,34,36,38,40 DEG C of reaction 2h, HPLC detection Sucraloses
Amount, corresponding molar yield is respectively 31.4%, 33.2%, 37.8%, 44.7%, 41.9%, 32.1%.
(4) in the tool plug single port reaction flask of 25mL, wet thallus 4%, methanol content 20%, substrate Sucralose -6- second
Acid esters concentration 3.5%, 25mM phosphate buffer pH5.1,6.3,7.1,7.8,8.0,36 DEG C of reactions 4h, HPLC detect trichlorine sugarcane
Sugared content, corresponding molar yield are respectively 21.8%, 61.6%, 76.1%, 73.3%, 66.0%.
(5) in the tool plug single port reaction flask of 25mL, wet thallus 4%, methanol content 20%, 25mM phosphate buffer
(pH7.1), substrate sucralose-6-acetic ester concentration 1.0%, 3.5%, 5.6%, 36 DEG C of reactions 16h, HPLC detect Sucralose
Content, corresponding molar yield are respectively 96.9%, 93.8%, 64%.
(6) in the tool plug single port reaction flask of 25mL, wet thallus 4%, methanol content 20%, 25mM phosphate buffer
(pH7.1), substrate sucralose-6-acetic ester concentration 3.5%, 36 DEG C reaction 12,16,20, for 24 hours, HPLC detection Sucralose contain
Amount, corresponding molar yield is respectively 93.2%, 94.5%, 96.4%, 99.6%.
(7) in the tool plug single port reaction flask of 25mL, suction of the w/v 1.0%, 1.5%, 2.0% after dry is used
Catalyst of the polyurethane foam as sucralose-6-acetic ester hydrolysis with thallus, methanol content 20%, 25mM phosphorus
Phthalate buffer (pH7.1), substrate sucralose-6-acetic ester concentration 3.5%, 36 DEG C of reactions 16h, HPLC detect Sucralose
Content, corresponding molar yield are respectively 82.2%, 84.5%, 88.4%.
The purifying and identification of 6 cane sugar-6-acetic ester of embodiment
1g sucrose is dissolved in 50mL solvent (DMF: the tert-butyl alcohol=1:4(V/V)), 0.8g is added and is adsorbed with solution starch gemma bar
The polyurethane foam of bacterium WZS01,2.8 mL vinyl acetates, 32 DEG C of reactions are for 24 hours.It is centrifuged after reaction, takes supernatant.60 DEG C low
The pressure distillation removal tert-butyl alcohol, the methylene chloride of 4 times of volumes is added to the obtained DMF solution containing 6 ester of sucrose, it is heavy to be collected by centrifugation
It forms sediment, as cane sugar-6-acetic ester crude product, purity 86.1%.
0.4g crude product silica G column chromatography is taken, eluent is acetonitrile: methanol: water=8:1:1(V/V), flow velocity 0.8
ML/min collects efflux, is concentrated in vacuo at 60 DEG C, obtains 0.15g cane sugar-6-acetic ester, purity 96%.
Structure determination is carried out to above-mentioned product:
Infrared spectrum analysis (KI pressed disc method): 3392.81(-OH), 2924.90(alkane is saturated C-H), 1729.35(C=
O), 1253.88(ester group C-O), 1046.28(C-O-C), 927.97(α-glycosidic bond).
Nuclear magnetic resonance wave analysis13C profiling results:13CNMR(400MHz, C2D6SO): 170.85 (-COCH2),
103.91 (C2'), 91.58 (C1), 82.75 (C5'),77.12 (C3'), 74.66 (C4'), 72.88 (C3),
71.63 (C2), 70.34 (C5), 70.17 (C4), 63.95 (C'), 62.74 (C6'), 62.43 (C1'),
20.84 (-COCH2)。
Above-mentioned map meets the structure feature of cane sugar-6-acetic ester.
The purifying and identification of 7 Sucralose of embodiment
2g sucralose-6-acetic ester is dissolved in 50mL solvent (methanol: pH7.0 phosphate buffer=1:4(V/V)), it is added
Weight in wet base 1.6g bacillus amyloliquefaciens WZS01 thallus, 36 DEG C of reactions are for 24 hours.It is centrifuged after reaction, takes supernatant, use rotary evaporation
First alcohol and water is removed in vacuo in instrument at 60 DEG C, and drying obtains yellow crystalline solid, Sucralose 6 is not detected in purity 98.6%
Acetic acid esters.
Structure determination is carried out to above-mentioned product:
Infrared spectrum analysis (KI pressed disc method): 3398.61(-OH), 2924.19(alkane is saturated C-H),
1088.82(C-O-C), 919.03,862.27,739.22(C-Cl).With the infrared spectrum phase of cane sugar-6-acetic ester
Absorption peak than, carbonyl C=O, ester group C-O disappears, this at two variation confirm that the acetyl group on 6 is removed into
Function.
Nuclear magnetic resonance wave analysis13C profiling results:13C NMR (400 MHz, D2O) δ, 104.03 (C2'), 93.36
(C1), 81.81 (C5'), 77.04 (C3'), 76.17 (C4'), 71.24 (C5), 68.88 C3), 68.50
(C2), 63.99 (C4), 62.25 (C6), 58.37 (s), 50.01 (s), 45.75 (C6'), 44.59 (C1)。
Above-mentioned map meets the structure feature of Sucralose.
Claims (6)
1. a kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) WZS01, deposit number is CCTCC NO:M
2016096。
2. bacillus amyloliquefaciens described in claim 1 are catalyzed cane sugar-6-acetic ester synthesis in Sucralose synthesis process
Application, it is characterised in that using sucrose and vinyl acetate as substrate, the tert-butyl alcohol and DMF as reaction medium, be adsorbed with solution
The polyurethane foam of bacillus amyloliquefaciens carries out reaction and generates cane sugar-6-acetic ester as catalyst.
3. application as claimed in claim 2, it is characterised in that the polyurethane foam for being adsorbed with bacillus amyloliquefaciens
Obtained by following steps: it is 10~50Kg/m that will to joined w/v, which be 0.6~1.0% density,3Polyurethane foam
Culture medium sterilizes, it is cooling after inoculation 1% bacillus amyloliquefaciens seed liquor, 30 DEG C are dry after culture 18~24 hours
It arrives.
4. application as claimed in claim 3, it is characterised in that the culture medium includes glucose 2.0~3.5%, peptone
0.5~1.5%, yeast powder 0.1%.
5. application as claimed in claim 3, it is characterised in that it is dry that w/v 1.0~2.5% is added into reaction system
The polyurethane foam for being adsorbed with bacillus amyloliquefaciens afterwards controls 30~38 DEG C of reaction temperature, the reaction time 16~24 hours.
6. bacillus amyloliquefaciens described in claim 1 are catalyzed sucralose-6-acetic ester in Sucralose synthesis process
The application of hydrolysis.
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|---|---|---|---|---|
| US5445951A (en) * | 1993-02-02 | 1995-08-29 | Mcneil-Ppc, Inc. | Regioselective enzymatic deacylation of sucrose esters in anhydrous organic media |
| CN101268090A (en) * | 2005-09-22 | 2008-09-17 | 法马德医疗保险私人有限公司 | Method of producing sucrose-6-acetate by whole-cell biocatalysis |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5445951A (en) * | 1993-02-02 | 1995-08-29 | Mcneil-Ppc, Inc. | Regioselective enzymatic deacylation of sucrose esters in anhydrous organic media |
| CN101268090A (en) * | 2005-09-22 | 2008-09-17 | 法马德医疗保险私人有限公司 | Method of producing sucrose-6-acetate by whole-cell biocatalysis |
Non-Patent Citations (1)
| Title |
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| 解淀粉芽孢杆菌胞外多糖发酵条件的优化;张丽姣 等;《食品工业科技》;20141124;第36卷(第9期);214-219 * |
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