CN106434504A - Recombinant gene engineering strain for expressing levansucrase and application thereof - Google Patents
Recombinant gene engineering strain for expressing levansucrase and application thereof Download PDFInfo
- Publication number
- CN106434504A CN106434504A CN201610841441.3A CN201610841441A CN106434504A CN 106434504 A CN106434504 A CN 106434504A CN 201610841441 A CN201610841441 A CN 201610841441A CN 106434504 A CN106434504 A CN 106434504A
- Authority
- CN
- China
- Prior art keywords
- levansucrase
- bacillus pumilus
- sucrose
- substrate
- genetic engineering
- 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.)
- Granted
Links
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
- 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)
- C12N9/1055—Levansucrase (2.4.1.10)
-
- 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
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/01—Hexosyltransferases (2.4.1)
- C12Y204/0101—Levansucrase (2.4.1.10)
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a recombinant gene engineering strain for expressing levansucrase and application thereof and belongs to the field of gene engineering and enzyme engineering. A recombinant plasmid pNCMO2-Lsc is constructed and bacillus pumilus is converted to obtain recombinant bacillus pumilus. The bacillus pumilus is used as a strain for fermenting and producing the levansucrase; and the produced levansucrase is applied to preparation of lactosucrose and has a relatively good effect. The recombinant gene engineering strain disclosed by the invention has the advantages that the food safety bacillus pumilus is used as an expression host and is used for recombining to produce the levansucrase; and the recombinant strain has the advantages of high enzyme production level, wide fermentation raw material source and relatively low production cost.
Description
Technical field
The present invention relates to a kind of engineering strain of recombinant expressed levansucrase and its application, belong to genetic engineering
With enzyme engineering field.
Background technology
Levansucrase is a member in glycosyl transferase family, and it turns glycosyl activity except having, and also hydrolysis is lived
Property, sucrose hydrolysis can be glucose and Fructose.Most of levansucrase has extensive receptor active, is turning glycosyl
During be substrate donor using sucrose as its prioritizing selection, with xylose, sucrose, Lactose etc. as receptor, catalysis transfer Fructose
Residue, in the carbochain of receptor, promotes carbochain to extend, thus forming the products such as oligofructose, levan and oligomeric lactulose.
Because the oligosaccharide such as oligofructose, oligomeric lactulose are beneficial to health, therefore, the production of transfructosylase is increasingly subject to
Concern to people.Have now been found that various bacteria can produce levansucrase, such as Bacillus polymyxa (Bacillus
Polymyxa), bacillus subtilises (Bacillus subtilis), zymomonas mobilis (Zymomonasmobilis), gold
Mountain lactobacilluss (Lactobacillus sanfranciscensis), bacillus amyloliquefacienses (Bacillus
Amyloliquefaciens), actinomyces viscosus (Actinomycesviscosus), Leuconostoc mesenteroides
(Leuconostocmesenteroides), pseudomonas syringae (Pseudomonassyringae), Bacillus licheniformis
(Bacillus licheniformis) etc..The ability difference that these bacterial strains produce levansucrase is larger, produced levan
The product characteristicses that saccharase obtains to specificity and the enzymatic conversion of receptor also have larger difference.
Oligomeric lactulose (lactosucrose, LS) is a kind of trisaccharide, it by β-D- galactoside, alpha-D-glucose glycosides and
Beta-D-fructofuranose glycosides residue forms, so it can be counted as the condensation substance of a molecule Lactose and a molecule Fructose, or
It is a molecule galactose and the condensation substance of a molecule sucrose, its chemical name should be β-D- galactosyl sucrose, but traditionally, people
Be referred to as oligomeric lactulose.Oligomeric lactulose is a kind of functional oligose, and it is soluble in water, and sugariness is about sucrose
70%, hardly digested by organism and absorb, be the Effective multiplication factor of bacillus bifiduss, its value-added effect is than other functions property
Oligosaccharide such as oligofructose, oligomeric galactose etc. are more preferably.Oligomeric lactulose has low in calories, low dental caries simultaneously, reduces in blood
Cholesterol, improve blood fat, promote the special physiological function such as calcium absorption, can be used as a kind of functional health care food.Domestic to low
Poly- lactulose has done preliminary study, does not also have the report of industrialized production, and it is many to come into market in Japanese oligomeric lactulose
Year.Because it has broad application prospects, the research and development of oligomeric lactulose in recent years receive and greatly pay attention to.
Content of the invention
First purpose of the present invention is to provide a kind of restructuring Bacillus pumilus producing levansucrase
(Brevibacillus brevis), is will be short and small for the channel genes of the encoding levan sucrase from Bacillus flexus
The genetic engineering bacterium that bacillus cereuss obtain.
Described encoding levan sucrase gene nucleotide series are as shown in SEQ ID NO.1.
Second purpose of the present invention is to provide a kind of restructuring Bacillus pumilus building secreting, expressing levansucrase
Method, comprise the following steps：
1) genes of interest shown in amplification SEQ ID NO.1, is connected on carrier pNCMO2, Transformed E .coli JM109,
The resistant LB plating medium of applying implenent, obtains recombiant plasmid pNCMO2-Lsc；
2) recombiant plasmid pNCMO2-Lsc is passed through electricity conversion and enter in Bacillus pumilus, the resistant TM of applying implenent puts down
Plate culture medium, obtains Bacillus pumilus genetic engineering bacterium.
Third object of the present invention is to provide a kind of production using described Bacillus pumilus genetic engineering bacterium really to gather
The method of sucrase enzyme, be by activation after Bacillus pumilus genetic engineering bacterium be inoculated in fermentation medium, in 30 DEG C,
200r/min cultivates 48 hours, the fermentation liquid bactofugation body that will obtain, and the fermented supernatant fluid of acquisition is crude enzyme liquid.
The formula of described fermentation medium is：Industrial yeast powder 1.7%, glucose or sucrose 1%.
Fourth object of the present invention is to provide one kind to prepare oligomeric breast fruit using restructuring levansucrase converted starch
The method of sugar, includes (1) and utilizes described Bacillus pumilus genetic engineering bacterium to produce levansucrase, (2) with sucrose with
Lactose converts for substrate and produces oligomeric lactulose, and substrate sucrose and lactose concn are 200g/L, and enzyme concentration is 0.5-5U/g bottom
Thing, initial pH6.0～7.0,35 DEG C of reaction temperature, the mixing speed of 150r/min.
In one embodiment of the invention, described enzyme concentration is 2U/g substrate.
Build the Bacillus pumilus fermentation product restructuring levansucrase that the restructuring obtaining obtains, fermentation using the present invention
Supernatant enzyme activity is in 43U/mL.Generate oligomeric lactulose further with this restructuring levansucrase catalysing sucrose and Lactose,
Conversion ratio is up to 39.01%.The present invention with the Bacillus pumilus of food safety as expressive host, recombinant production levan sucrose
Enzyme, this recombinant bacterial strain producing enzyme level is high, fermentation raw material wide material sources, and production cost is relatively low.
Brief description
Fig. 1 recombinant bacterium shake flask fermentation producing enzyme curve
Fig. 2 recombinant bacterium shake flask fermentation extracellular supernatant SDS-PAGE；M：Marker, 1：Recombinant bacterium fermentation supernatant
The impact to B.brevis shake flask fermentation Biomass and producing enzyme for Fig. 3 different nitrogen sources
The impact to B.brevis shake flask fermentation Biomass and producing enzyme for Fig. 4 different carbon source
The impact to oligomeric lactulose conversion ratio of Fig. 5 enzyme concentration and response time
The impact to oligomeric lactulose conversion ratio for Fig. 6 temperature
The impact to oligomeric lactulose conversion ratio for Fig. 7 pH
Specific embodiment
The clone of embodiment 1 Bacillus flexus levansucrase encoding gene and the structure of expression vector
Bacillus flexus are cultivated 2 days in LB fluid medium, and 10000rpm is collected by centrifugation thalline, aseptic water washing,
Collect precipitation and be suspended in 500 μ L Tris-EDTA buffer, add 15uL lysozyme, be incubated 30min at 37 DEG C, add 5 μ L
RNase, is incubated 30min at 37 DEG C, adds 30 μ L 10% sodium lauryl sulphate and 15 μ L E.C. 3.4.21.64s, is incubated at 37 DEG C
60min, adds the NaCl 100 μ L and cetyl trimethylammonium bromide 80 μ L of 5M, is incubated 20min, with 700 μ L's at 65 DEG C
Phenol：Chloroform：Isoamyl alcohol volume ratio is 25:24:1 MIXED SOLVENT EXTRACTION, 10000rpm is centrifuged, the supernatant chloroform of 700 μ L：
Isoamyl alcohol volume ratio is 24:1 MIXED SOLVENT EXTRACTION, 10000rpm is centrifuged, and supernatant is mixed with the ice isoamyl alcohol of 1400 μ L volumes
Close, -20 DEG C of precipitation 30min, 10000rpm are centrifuged, the ethanol purge of precipitation plus 200 μ L70% concentration, and 10000rpm is centrifuged, and sinks
Form sediment and use Tris-EDTA buffer solution, as Bacillus flexus STb gene；
According to polysaccharide saccharase gene design primer P1, P2 in data base：
P1：5’-CGGGATCCAAAGGTAAAG GTGTAGATGAAG-3’
P2：5’-GGAATTCTTATTTTTTCTTTTCATCATACGTG-3’
With original bacteria genome as template, clone contains the genes of interest of encoded signal peptide sequence, and PCR system is：10 μM are drawn
The each 1 μ L of thing P1 and P2,2mM dNTPS 5 μ L, 10 × KOD-Plus-NeoBuffer 5 μ L, the PrimeStar polymerase of 1U/ μ L
1uL, template 0.5 μ L, plus distilled water polishing 50 μ L.PCR condition：94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 58 DEG C of annealing
30s, 72 DEG C of extension 1min 30s, 30 circulations.Obtain the base of encoding levan sucrase as shown in SEQ ID NO.1 for the sequence
Because of Lsc.PCR primer and carrier pNCMO2 are carried out double digestion respectively, and glue reclaim, connect overnight in 16 DEG C, Transformed E .coli
JM109, the LB flat board containing ammonia benzyl (100 μ g/mL) resistance for the coating, 37 DEG C of culture 10-12h, 3 transformants of picking, extract weight
Group plasmid is gone forward side by side performing PCR and double digestion checking, then measures DNA sequence to verifying correct recombiant plasmid, positive colony is
pNCMO2-Lsc.
The conversion of embodiment 2 Bacillus pumilus
1) fresh TM flat board (TM culture medium：Polyprotein peptone 1%, beef extract 0.5%, yeast powder 0.2%, glucose
1%) choose Bacillus pumilus and be inoculated in 10ml TM culture for Brevibacillus brevis (CCTCC 94025) single bacterium colony
In base, incubated overnight.
2) OD in measurement shaking flask, controls inoculum concentration, makes inoculation finish the OD of wild Oryza species600Between 0.19-0.2.Training
Foster base is 50mL GM culture medium (peptone 0.5%, soy peptone 0.5%, beef extract 0.5%, yeast extract
0.25%, glucose 0.5%, α-sodium glycerophosphate 1.9%, vitamin C 0.05%, MgSO40.012%).37 DEG C, 200rpm
Cultivate to OD600About=1.0 (about 3-4 hours).
3) whole bacterium solution ice-water bath 10min are taken, then 5000rpm, 8min, 4 DEG C is collected by centrifugation thalline.
4) turn buffer ETM (Sorbitol 9%, Mannitol 9%, glycerol 10%) washing thalline with the electricity of 40ml pre-cooling,
Supernatant is removed in 5000rpm, 8min, 4 DEG C of centrifugation, so rinsing 3 times.
5) thalline after washing is resuspended in etc. in the ETM of 500 μ L, often pipe subpackage 60 μ L.
6) 1-6 μ L plasmid will be added in 60 μ l competent cells, ice bath is incubated 5min, adds the electric revolving cup (1mm) of pre-cooling
In, electric shock is once.Electroporation is arranged：2.0kv, 25uF 200 Ω, 1mm, shock by electricity 1 time. and (between persistent period 4.5ms-5ms).
7) electric shock finishes and adds 1mL recovery medium TM immediately, 37 DEG C, 200rpm, after recovery 3h, coated plate.37 DEG C, overnight
Culture, in picking colony to TM neomycin culture medium, is verified, correct after carry out next step producing enzyme.
Embodiment 3 shake flask fermentation producing enzyme
1) fermentation culture
The product levansucrase inoculation obtaining will be screened in embodiment 2 in TM culture medium, cultivate at 30 DEG C
It is forwarded in TM fermentation medium with 5% inoculum concentration after 12h, 30 DEG C of constant temperature culture 44～48h producing enzymes.After fermentation ends, centrifugation
Collect supernatant and be crude enzyme liquid.
TM culture medium (g/L)：Polyprotein peptone 10, beef powder 5, yeast powder 2, glucose 10, pH7.0.
2) enzyme activity determination
Enzyme activity determination method by the sucrose solution of 0.5mL 20% (w/v) and 0.5mL crude enzyme liquid pH 7.0,
Mix homogeneously under the phosphate buffer of 20mmol/L, reacts 20min under 30 DEG C of water-baths, and last boiling water bath boils 10min
Terminating reaction, with the growing amount of Syrups by HPLC Fructose, glucose, determines enzyme activity.Chromatographic condition is:Chromatographic column:
Shodex nh 2 column；Column temperature:30℃；Mobile phase:75% acetonitrile (through 0.45 μm of membrane filtration)；Flow velocity:0.8mL/min；Loading body
Long-pending:10μL.Under the conditions of being somebody's turn to do, the enzyme amount needed for 1 μm of ol glucose of transfer per minute is defined as a levansucrase unit of activity
(U).
Enzyme activity extended in time and is gradually increased as seen from Figure 1, and reached stable, maximum enzyme at 48 hours about
Vigor is about 17U/ml, and protein electrophoresises result is shown in a band consistent with theoretical molecular (Fig. 2) at 55kDa.
Shake flask fermentation producing enzyme experiment in embodiment 4TM culture medium
1) preparation of one-level kind：Bacillus pumilus single bacterium colony is chosen 30 DEG C in TM fluid medium, 220rpm overnight
Culture, gained strain is one-level kind.
3) shake flask fermentation：Two grades of kinds are inoculated in 50ml shaking flask, TM culture medium, 30 DEG C, cultivate about 48 hours.Fermentation liquid
Through 10000g bactofugation, supernatant is levansucrase stock solution, and final enzyme activity is 17U/mL.
Shake flask fermentation producing enzyme experiment in embodiment 5 different nitrogen sources culture medium
1) preparation of one-level kind：Bacillus pumilus single bacterium colony is chosen 30 DEG C in TM fluid medium, 220rpm overnight
Culture, gained strain is one-level kind.
3) shake flask fermentation：With 17g L-1Industrial yeast powder, industrial proteins peptone, fish meal protein peptone, soy peptone, cattle
Meat extractum, casein, cotton seed meal, polyprotein peptone, Semen Maydiss starch etc. are single nitrogen source, replace the nitrogen source in TM culture medium.Point
Not Pei Zhi fermentation medium, one-level kind is inoculated in and wherein cultivates about 48 hours.Fermentation liquid is through 10000g bactofugation, supernatant
Be levansucrase stock solution, wherein industrial yeast powder as nitrogen source enzyme activity highest, such as Fig. 3, final enzyme activity is 31.5U/mL.
Shake flask fermentation producing enzyme experiment in embodiment 6 different carbon source culture medium
1) preparation of one-level kind：Bacillus pumilus single bacterium colony is chosen 30 DEG C in TM fluid medium, 220rpm overnight
Culture, gained strain is one-level kind.
2) shake flask fermentation：With 10g L-1Starch, dextrin, Fructose, sucrose, glycerol and glucose be as unique carbon source
Prepare fermentation medium, replace the carbon source in TM culture medium, one-level kind is inoculated in wherein to carry out enzymatic production culture about 48 little
When.Fermentation liquid is levansucrase stock solution through 10000g bactofugation, supernatant, and final enzyme activity is 37U/mL.Can from Fig. 4
To find out, in carbon source used, maximum for the Biomass of thalline during carbon source with sucrose and glucose, and the promotion to producing enzyme is the brightest
Aobvious.
The impact that embodiment 7 enzyme concentration and response time produce to oligomeric lactulose
In 30 DEG C of initial reaction temperature, initial pH 7.0, in the case that sucrose and lactose concn are 200g/L, enzyme concentration
Be controlled as 0.5,1,2,3,4,5U/g substrate, setting shaking bath temperature is 30 DEG C, rotating speed 150r/min, from the beginning of 2h
Boil terminating reaction every 2h sampling, until reaction reaches balance.Product is detected with HPLC.
HPLC testing conditions are：The amount of the oligomeric lactulose in oligomeric lactulose product, sucrose, Lactose and monosaccharide adopts
HPLC is determining.Chromatographic condition is：Agilent 1260HPLC chromatograph, Agilent automatic sampler, chromatographic column
ShodexAsahipak NH2- 504E (4.6mm × 250mm), Composition distribution is Agilent G1362A；Mobile phase adopts 75%
(v/v) mixed solution of acetonitrile and water, flow velocity is 0.8mL/min, and column temperature is set as 35 DEG C.Using external standard method, during according to retaining
Between and peak area determine the concentration of corresponding oligomeric lactulose.
Result as shown in figure 5, enzyme concentration be 0.5U/g substrate when, early stage reaction rate is relatively low, reaction carry out 10h reach flat
Weighing apparatus, oligomeric lactulose conversion ratio is 31.89%.And enzyme concentration be 1,2,3,4,5U/g substrate when, in 2-6h, their conversion
Rate relatively, between 24.41%-29.98%.After 6h, enzyme concentration is 3,4, in the sample of 5U/g substrate, oligomeric breast
The conversion ratio of Fructose is slowly increased, and close to equilibrium point, and enzyme concentration be 1, the sample of 2U/g substrate oligomeric breast fruit after 6h
The conversion ratio of sugar continues to increase, and can reach balance when reaction proceeds to 8h.Wherein enzyme concentration is the sample of 2U/g substrate
The conversion ratio of middle oligomeric lactulose can reach 37%.
Embodiment 8：The impact that temperature produces to oligomeric lactulose
In initial pH 7.0, in the case that sucrose and lactose concn are 200g/L, enzyme concentration is 2U/g substrate, rotating speed
150r/min, makes reaction temperature be respectively 25,30,35,40,45,50 DEG C, and the sampling when reaction proceeds to 8h boils termination instead
Should, until reaction reaches balance.Product is detected with HPLC.
Result as shown in fig. 6, at different temperatures the conversion ratio difference of oligomeric lactulose larger.When 35 DEG C, oligomeric breast
The conversion ratio highest of Fructose, can reach 38.32%.When less than 35 DEG C, conversion ratio raises with the rising of temperature, temperature mistake
When low, enzyme activity can be suppressed.And temperature be higher than 35 DEG C afterwards, conversion ratio with temperature raise begin to decline it may be possible to due to
Temperature raises, and has part levansucrase to inactivate.Therefore (optimal reactive temperature of enzyme is 30 to select optimal reactive temperature to be 35 DEG C
℃).
Embodiment 9：The impact that pH produces to oligomeric lactulose
When initial temperature is 35 DEG C, in the case that sucrose and lactose concn are 200g/L, enzyme concentration is 2U/g substrate,
Rotating speed 150r/min, makes reaction pH be respectively 5,5.5,6,6.5,7,7.5,8, and the sampling when reaction proceeds to 8h boils termination instead
Should, until reaction reaches balance.Product is detected with HPLC.
It is one of feature of enzyme molecule to environment acid-base value sensitivity.Each enzyme only just can show under certain pH system
The maximal rate of reaction, higher or lower than this value, response speed all can decline.Result as shown in fig. 7, in pH 6.0 oligomeric breast
The conversion ratio of Fructose is 39.01%, for the peak in the case of different pH.Additionally, when pH 6.0-7.0 is in the range of this, turning
Rate all maintains high value.
Although the present invention is open as above with preferred embodiment, it is not limited to the present invention, any is familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, can do various changes and modification, therefore the protection model of the present invention
Enclosing should be by being defined that claims are defined.
Claims (6)
1. a kind of restructuring Bacillus pumilus of secreting, expressing levansucrase are it is characterised in that be by from bending bud
The genetic engineering bacterium that the channel genes Bacillus pumilus of the encoding levan sucrase of spore bacillus obtain, described encoding levan
The nucleotide sequence of saccharase gene is as shown in SEQ ID NO.1.
2. a kind of method of the restructuring Bacillus pumilus building the secreting, expressing levansucrase described in claim 1, its
It is characterised by, comprise the following steps：
1) genes of interest shown in amplification SEQ ID NO.1, is connected on carrier pNCMO2, Transformed E .coli JM109, coating
There is the LB plating medium of resistance, obtain recombiant plasmid pNCMO2-Lsc；
2) recombiant plasmid pNCMO2-Lsc is passed through electricity conversion and enter in Bacillus pumilus, the resistant TM flat board training of applying implenent
Foster base, obtains Bacillus pumilus genetic engineering bacterium.
3. the method that the Bacillus pumilus genetic engineering bacterium described in a kind of utilization claim 1 produces levansucrase, its
It is characterised by, be the Bacillus pumilus genetic engineering bacterium after activation to be inoculated in fermentation medium, in 30 DEG C, 200r/min
Culture 48 hours, the fermentation liquid bactofugation body that will obtain, the fermented supernatant fluid of acquisition is crude enzyme liquid；Described fermentation medium
Formula be：Industrial yeast powder 1.7%, glucose or sucrose 1%.
4. a kind of using levansucrase prepare oligomeric lactulose method it is characterised in that include (1) utilize claim
Bacillus pumilus genetic engineering bacterium described in 1 produces levansucrase, and (2) are produced low with sucrose and Lactose for substrate conversion
Poly- lactulose, substrate sucrose and lactose concn are 200g/L, and enzyme concentration is 0.5-5U/g substrate, initial pH6.0～7.0, instead
Answer 35 DEG C of temperature, the mixing speed of 150r/min.
5. method according to claim 4 is it is characterised in that (1) is by the short and small bud described in the claim 1 after activation
Oxydans genetic engineering bacterium is inoculated in fermentation medium spore, in 30 DEG C, 200r/min cultivate 48 hours, by obtain fermentation liquid from
Bactofugation body, the fermented supernatant fluid of acquisition is crude enzyme liquid；The formula of described fermentation medium is：Industrial yeast powder 1.7%, Portugal
Grape sugar or sucrose 1%；(2) converted for substrate with sucrose and Lactose and produce oligomeric lactulose, substrate sucrose and lactose concn are
200g/L, enzyme concentration is 0.5-5U/g substrate, initial pH6.0～7.0,35 DEG C of reaction temperature, the mixing speed of 150r/min.
6. method according to claim 5 is it is characterised in that described enzyme concentration is 2U/g substrate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610841441.3A CN106434504B (en) | 2016-09-22 | 2016-09-22 | It is a kind of recombinantly express levansucrase engineering strain and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610841441.3A CN106434504B (en) | 2016-09-22 | 2016-09-22 | It is a kind of recombinantly express levansucrase engineering strain and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106434504A true CN106434504A (en) | 2017-02-22 |
| CN106434504B CN106434504B (en) | 2019-06-07 |
Family
ID=58166773
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610841441.3A Active CN106434504B (en) | 2016-09-22 | 2016-09-22 | It is a kind of recombinantly express levansucrase engineering strain and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106434504B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1772903A (en) * | 2005-10-17 | 2006-05-17 | 中国农业科学院生物技术研究所 | New-type froctosan saccharase gene and its use |
| CN103695501A (en) * | 2013-12-10 | 2014-04-02 | 江南大学 | Method for producing lactosucrose employing levansucrase |
| CN104073456A (en) * | 2014-07-10 | 2014-10-01 | 江南大学 | Bacterial strain for producing levansucrase and method for producing lactosucrose by utilizing lavansucrase |
| CN104480164A (en) * | 2014-12-04 | 2015-04-01 | 山东百龙创园生物科技有限公司 | Enzymic method for preparing lactosucrose |
-
2016
- 2016-09-22 CN CN201610841441.3A patent/CN106434504B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1772903A (en) * | 2005-10-17 | 2006-05-17 | 中国农业科学院生物技术研究所 | New-type froctosan saccharase gene and its use |
| CN103695501A (en) * | 2013-12-10 | 2014-04-02 | 江南大学 | Method for producing lactosucrose employing levansucrase |
| CN104073456A (en) * | 2014-07-10 | 2014-10-01 | 江南大学 | Bacterial strain for producing levansucrase and method for producing lactosucrose by utilizing lavansucrase |
| CN104480164A (en) * | 2014-12-04 | 2015-04-01 | 山东百龙创园生物科技有限公司 | Enzymic method for preparing lactosucrose |
Non-Patent Citations (3)
| Title |
|---|
| NCBI: "GenBank Accession NO.: CP011974.1", 《NCBI GENBANK》 * |
| 刘伟华等: "枯草杆菌果聚糖蔗糖酶基因转化小麦的研究", 《中国农业科学》 * |
| 李润静等: "甲基营养芽孢杆菌SK21.002产果聚糖蔗糖酶的发酵条件优化", 《食品工业科技》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106434504B (en) | 2019-06-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105087456B (en) | A method of producing the recombined bacillus subtilis building of specified molecular weight hyaluronic acid | |
| CN107236696B (en) | A kind of sucrose phosphorylase recombined bacillus subtilis for expressing the source L.mesenteroides | |
| CN109750071A (en) | A kind of method of biocatalysis synthesis Rebaudiodside A M | |
| CN112375750B (en) | Glycosyltransferase mutant and method for catalytically synthesizing rebaudioside A by using same | |
| CN102676480B (en) | Method for producing extracellular pullulanase by applying auto-induction culture medium and dual-temperature control strategy | |
| CN108102995B (en) | D-psicose 3-epimerase production strain and immobilization method thereof | |
| CN103555690B (en) | A kind of Novel fruit glycosidase and encoding gene and application | |
| CN109825466A (en) | A kind of method that straight chain maltopentaose generates enzyme secreting, expressing in bacillus subtilis | |
| CN105734092B (en) | A kind of method that enzyme process prepares D-Tag | |
| Singh et al. | Microbial inulinases and pullulanases in the food industry | |
| CN104046586B (en) | One strain gene engineering bacterium and the application in producing (2R, 3R)-2,3-butanediol thereof | |
| CN101260394A (en) | Activity expression and application of thermobifida fusca xylose isomerase in wine brewing yeast | |
| CN103205391A (en) | Gene engineering strain and application thereof | |
| CN105567779A (en) | Fermentation method of high-yield and low-molecular-weight thermal gel | |
| CN107201373B (en) | A kind of maltogenic amylase and its gene, the engineering bacteria containing the gene and its application | |
| CN107189992B (en) | A kind of heparosan synthase and its application | |
| CN102703481A (en) | Method for heat resistance modification of aspergillus oryzae GH11 xylanase gene and preparation of mutant enzyme | |
| CN112695022A (en) | Enzyme system for degrading plant polysaccharide and application thereof | |
| CN109825489A (en) | A kind of beta amylase and its application with hypersecretion ability | |
| CN106434504A (en) | Recombinant gene engineering strain for expressing levansucrase and application thereof | |
| CN106434715A (en) | Maltooligosyl trehalose synthase as well as expression gene and application thereof | |
| CN103484482B (en) | Bacillus subtilis levan sucrase mutant T305A and application thereof | |
| CN106520729B (en) | Malt oligosaccharide based mycose hydrolase and its expressing gene and application | |
| CN109609540A (en) | Recombinant Saccharomyces cerevisiae bacterial strain and its preparation method and application | |
| CN107236772A (en) | A kind of method for preparing brown alga oligose |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |