CN110157623A - A kind of method of reaping hook bacteria strain and its fermenting and producing D-pantoyl lactone hydrolase - Google Patents
A kind of method of reaping hook bacteria strain and its fermenting and producing D-pantoyl lactone hydrolase Download PDFInfo
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Abstract
The invention discloses one plant of sickle-like bacteria (Fusarium sp.) CH1701, are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on December 07th, 2018, and deposit number is CGMCC No.16967.Sickle-like bacteria (Fusarium sp.) CH1701 of the invention is a kind of completely new strain, can produce D-pantoyl lactone hydrolase by fermentation in the fermentation medium containing assimilable carbon source, nitrogen source and trace element salt, and under conditions of logical oxygen.
Description
Technical field
The present invention relates to a kind of methods of reaping hook bacteria strain and its fermenting and producing D-pantoyl lactone hydrolase, belong to micro- life
Object bacterial strain screening and fermentation technical field.
Background technique
D-VB5 calcium is vitamin B complex compound, is widely used in food, feed and medical industry, market demand
Greatly.In general, synthesizing DL- pantoic acid lactone using isobutylaldehyde, formaldehyde, Cymag, then torn open by chemical method fractionation or biological enzyme
D-pantoyl lactone is separately won to obtain, D-VB5 calcium is finally obtained by Beta-alanine calcium and D-pantoyl lactone direct polycondensation.
The shortcomings that chemical method fractionation DL- pantoic acid lactone is: the chemical resolutions agent such as quinine, Chiral Amine is at high cost,
Impurity separation is difficult, and is unfavorable for environmental protection.
D-pantoyl lactone hydrolyzes enzyme selectivity and splits: firstly, D-pantoyl lactone hydrolase optional water racemic
DL- pantoic acid lactone generate Pantothenic acid, recycle dissolubility difference Pantothenic acid is separated with L- pantoic acid lactone, D- is general
Solution acid is by lactonizing to form the D-pantoyl lactone of high-optical-purity.L- pantoic acid lactone is recycled, racemization reaction obtains
To DL- pantoic acid lactone, it is reused for splitting.Enzymatic Resolution is at low cost, simple process, environmental-friendly, has realized industry at present
Metaplasia produces D-VB5 calcium, D-VB5, D-pantothenyl aleohol, and specific steps are as shown in Figure 1.
2001, Chinese patent CN 1313402A disclosed one plant of microbial bacteria that can produce D-pantoyl lactone hydrolase
Strain Fusorium moniliforme Sheldon mould Fusarium moniliforme SW-902 (CGMGG 0536), fermentation liquid enzyme activity reach 3~6U/l,
Enzyme process has been pushed selectively to split the industrialization of production D-pantoyl lactone based on this.
Produce the enzyme activity height of bacterial strain to enzymic resolution craft it is smooth implement, whether have with economic competitiveness it is crucial
It influences.Therefore, it is necessary to the efficient preparation screened the microorganism fungus kind for capableing of high-yield D-pantolactone hydrolytic enzyme and establish enzyme
Method.
Summary of the invention
The purpose of the present invention is to provide the sides of a kind of reaping hook bacteria strain and its fermenting and producing D-pantoyl lactone hydrolase
Method.
In order to achieve the above objects and other related objects, present invention provide the technical scheme that one plant of sickle-like bacteria
It is common to be preserved in China Committee for Culture Collection of Microorganisms on December 07th, 2018 by (Fusarium sp.) CH1701
Microorganism center (CGMCC), deposit number are CGMCC No.16967.
In order to achieve the above objects and other related objects, present invention provide the technical scheme that a kind of sent out using sickle-like bacteria
The method that ferment produces D-pantoyl lactone hydrolase, an including culture medium, the culture medium contain assimilable carbon source, assimilable
Then sickle-like bacteria is inoculated in the culture medium and carries out aerobic fermentation by nitrogen source and assimilable microelement.
Preferred technical solution are as follows: the assimilable carbon source is glycerol, glucose, sucrose, starch, industrial molasses, cream
At least one of sugar, maltose, trehalose, xylan, mannitol, sorbierite and dextrin.
Preferred technical solution are as follows: the assimilable nitrogen source is soybean cake powder, Dried Corn Steep Liquor Powder, peptone, yeast pumping
In extract, tryptone, cottonseed meal, groundnut meal, yeast powder, beef extract, seitan powder, wheat bran, urea and ammonium salt extremely
Few one kind.
Preferred technical solution are as follows: the assimilable microelement derives from MgSO4·7H2O、ZnSO4·7H2O、
FeSO4·7H2O、MnCl2·4H2O、CoCl2·6H2O、CuSO4·5H2O、CaCl2、Na2MoO4·2H2O and FeCl3·6H2In O
At least one.
Preferred technical solution are as follows: the sickle-like bacteria is sickle-like bacteria (Fusarium sp.) CH1701, sickle-like bacteria
(Fusarium sp.) CH1701 spontaneous mutant strain, sickle-like bacteria (Fusarium sp.) CH1701 pass through mutagenesis or genetic engineering
Obtained enhanced variant is transformed；Sickle-like bacteria (Fusarium sp.) CH1701, in preservation on December 07 in 2018
In China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), deposit number is CGMCC No.16967.
Preferred technical solution are as follows: the pH of the culture medium is 6.5-7.5, and the temperature of aerobic fermentation is 20 DEG C -40 DEG C, is had
The time of aerobe fermentation is 24-72 hours；Oxygen-supply quantity is 0.5-1.5vvm.
In order to achieve the above objects and other related objects, present invention provide the technical scheme that one plant of sickle-like bacteria
The application of (Fusarium sp.) CH1701, sickle-like bacteria (Fusarium sp.) CH1701 are protected on December 07th, 2018
It is hidden in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), deposit number CGMCC
No.16967；Sickle-like bacteria (Fusarium sp.) CH1701 has the ability of production D-pantoyl lactone hydrolase.
Since above-mentioned technical proposal is used, the present invention has the advantage, that compared with prior art
1, enzyme activity is improved.Registration strain has the D-pantoyl lactone hydrolase enzyme activity that strain of the present invention generates more
It is greatly improved, is advantageously implemented industrialized production.
2, production cost is reduced.It is general that strain of the present invention is conducive to raising vitamin B complex product such as D-VB5 calcium, D-
The production efficiency of acid, D-pantothenyl aleohol etc. reduces production cost, and has more economic competitiveness.
Detailed description of the invention
Fig. 1 is that D-pantoyl lactone hydrolyzes enzyme selectivity fractionation flow chart.
Fig. 2 is the colonial morphology of bacterial strain of the present invention.
Fig. 3 is fermentation liquid mycelium enzyme source of the present invention appearance.
Fig. 4 is the HPLC map of reaction solution before and after enzyme hydrolysis 30min of the present invention.
Specific embodiment
Embodiments of the present invention are illustrated by particular specific embodiment below, those skilled in the art can be by this implementation
Content disclosed by example is understood other advantages and efficacy of the present invention easily.
The purpose of term used herein, which is only that, illustrates particular embodiment, it is not intended to be limited to the present invention.It removes
Non- context is explicitly shown, otherwise singular " one " used herein, "one" also include plural form.
When illustrating preferred embodiment, it is potentially based on clear purpose and uses special term；However, this specification institute
Revealer is not intended to be limited in the selected special term；And it is to be understood that each particular element includes having
Identical function, all equivalence techniques for operating in a similar manner and reaching similar effects.
The D-pantoyl lactone hydrolase enzyme activity of the embodiment of the present invention is detected by the following method:
1, prepared by enzyme source: taking 5 milliliters of filtering fermentation liquors or centrifugation, stays wet thallus as enzyme source.
2, substrate solution is prepared: preparing 2%DL- pantoic acid lactone in the Tris-HCl buffer of 0.2mol/L, pH7.5
(CaCl containing 50mmol/L2)。
3, enzymatic reaction: above-mentioned reaction system carries out reaction 30min, filtering removal mycelia in the shaking table of 28 DEG C, 150rpm
Body.
4, it measures enzyme activity: utilizing the percent hydrolysis of HPLC measurement DL- pantoic acid lactone (formula is as follows).
Enzyme activity is calculated by percent hydrolysis, 1 μm of ol substrate is converted into product as an enzyme-activity unit per minute.
5, HPLC condition: mobile phase: acetonitrile: 0.02mol/L KH2PO4=1:9；Chromatographic column: C18 column, 5 μm, 4.6 ×
250mm；Flow velocity 1ml/min；Detection wavelength 215nm；Detect temperature: room temperature；Sample volume: 10 μ l.
Embodiment 1: a kind of method of reaping hook bacteria strain and its fermenting and producing D-pantoyl lactone hydrolase
One, bacterium source
Reaping hook bacteria strain Fusarium sp.CH1701 system is isolated from the soil of one cane field of Anhui Province Huangshan
Original strain, pass through N+Ion beam mutation mutagenic and breeding obtains.The bacterial strain is cultivated in PDA slant medium in 28 ± 1 DEG C
After 2-4 days, aerial hyphae is scraped with aseptic inoculation ring, is shaken in the test tube containing deionized water, bead by mycelium
It smashes, mycelium suspended liquid, N is made+Ion beam mutation mutagenic treatment, the specific steps are as follows:
(1) the mycelium suspended liquid of above-mentioned starting strain Fusarium sp.CH1701 is suitably diluted.
(2) applying stick 100 μ l mycelium dilutions are spread evenly across diameter with glass is to set in 90mm blank glass plate
It is dried up in superclean bench face with uniform wind speed and mycoderm is made.
(3)N+Ion implanting is tested in Chinese Academy of Sciences's high-intensity magnetic field and ion beam physical biology key lab ion beam
It is carried out on bioengineering device, selects different-energy and dosage low energy N+Ion implanting.
(4) processing sample 1ml sterile water, sterile glass are applied stick and uniformly scraped after ion implanting, draw 100 μ l in solid
It is cultivated in body PDA culture medium, single colonie numeration is carried out after 3 days, and calculate lethality.Single colonie is chosen into PDA slant medium
In 28 ± 1 DEG C cultivate 3 days, 4 DEG C of the slant strains grow preservations for use.
(5) it selects by N+2000 plants of single colonie after ion beam mutation mutagenesis, carries out shake flask fermentation, and detection fermentation liquid produces
The enzyme activity of D-pantoyl lactone hydrolase.As a result enhanced variant Fusarium sp.CH1701 is filtered out.
Microorganism fungus kind sickle-like bacteria (Fusarium sp.) CH1701 is preserved in China Microbiological bacterium on December 7th, 2018
Kind preservation administration committee common micro-organisms center (address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, the micro- life of the Chinese Academy of Sciences
Object research institute), deposit number is CGMCC No.16967, and is registered on the books, it was demonstrated that survival.
Above-mentioned bacterial strains main biological property are as follows: colony colour white or canescence, colonial morphology rounding, protrusion of surface steamed bun
Head, no spore, aerial hyphae is flourishing, easily provokes.
The present invention describes the feature on the morphology and molecular level of Fusarium sp.CH1701, by with it is known
D-pantoyl lactone hydrolase producing strains carry out the comparison of morphology and molecular biology level, it can be assumed that Fusarium
Sp.CH1701 belongs to Fusarium, but it is different from known D-pantoyl lactone hydrolase producing bacterial strain Fusorium moniliforme Sheldon mould
Fusarium moniliforme SW-902 (CGMGG 0536), but one plant of completely new bacterial strain.
Two, strain idenfication
Bacterial strain main biological property are as follows: colony colour white or canescence, colonial morphology are in steamed bun type, directly compared with rounding
Diameter size is large and medium-sized, no spore, and aerial hyphae is flourishing, is easily provoked.
According to the universal primer of fungi rDNA conserved sequence:
Forward primer: 5 '-TCCGTAGGTGAACCTGCGG-3 '
Reverse primer: 5 '-TCCTCCGCTTATTGATATGC-3 '
PCR amplification is carried out using the rDNA sequence of above-mentioned primer pair bacterial strain Fusarium sp.CH1701.PCR cycle item
Part: 95 DEG C of initial denaturation 5min, loop parameter 95 DEG C of denaturation 1min, 52 DEG C of renaturation 45s, 72 DEG C of extension 1.5min repeat 35 and follow
After ring, 72 DEG C re-extend 10min, last 4 DEG C of heat preservations.PCR product is detected with 0.8% agarose gel electrophoresis.Select band clear
Clear product is purified.Amplified production is recycled through gel electrophoresis, and is sequenced after being connected to carrier T, and the bacterial strain is obtained
The sequence of rDNA primary structure is 558 bases.By carrying out blast in Genebank database, as a result, it has been found that CH1701
With gene order (GenBank no.MG273757.1) similitude of sickle-like bacteria mutation Fusarium sp.strain XJC-02
It is 99%.
In conclusion CH1701 belongs to Fusarium, but it is different from known D-pantoyl lactone hydrolase producing strains
Kind Fusorium moniliforme Sheldon mould Fusarium moniliforme SW-902 (CGMGG 0536).Fusarium sp.CH1701 is one
The new D-pantoyl lactone hydrolase producing bacterial strain of strain.
Three, D-pantoyl lactone hydrolase is prepared
(1) the mycelial preparation in inclined-plane and culture.
The preparation and culture of slant medium: peeling potatoes 200g is cut into block and adds water, boils 30min, with 8 layers of gauze
Filtering is squeezed, sucrose 20g, agar 20g is added in filtrate, deionized water constant volume is added to 1000ml, natural pH.Take 30 ×
200mm teat glass, every dress 20ml culture medium are cooled to 50-60 DEG C of pendulum inclined-plane through 121 DEG C of sterilizing 20min.Inclined-plane access
It is cultivated 3 days under the conditions of 28 ± 1 DEG C after the mycelium of one ring sickle-like bacteria (Fusarium sp.) CH1701, mycelia is mature, spare.
(2) preparation and culture of seed liquor.
Seed culture based formulas: glycerol 1%, soybean cake powder (hot moulding) 0.6%, yeast powder 0.6%, peptone 1%, corn
Paste dry powder 0.3%, pH7.0-7.5 before disappearing, shaking flask liquid amount is the bottled 25ml of 250ml triangle, through 121 DEG C of sterilizing 20min.Thallus
Inoculum concentration be an oese sickle-like bacteria (Fusarium sp.) CH1701 aerial mycelium, 28 ± 1 DEG C of cultivation temperature,
220rpm, shaking table shaken cultivation 24-26h, culture solution pH6.5-7.5, mycelial concentration are 20-30% (v/v) at this time, obtain seed
Liquid.
(3) preparation and culture of producing enzyme fermentation medium.
Fermentative medium formula: glycerol 2%, soybean cake powder (hot moulding) 1.5%, Dried Corn Steep Liquor Powder 0.5%, peptone 2%,
PH7.0-7.5 before disappearing, shaking flask liquid amount is the bottled 25ml of 250ml triangle, through 121 DEG C of sterilizing 20min.By seed liquor with 5-10%
(v/v) inoculum concentration access, in 28 ± 1 DEG C, 220rpm shaking table shaken cultivation 46-48h, is examined according to standard method after fermentation
D-pantoyl lactone hydrolytic enzyme activities are surveyed, measuring enzyme activity is 850U/L.
Embodiment 2: the method for fermenting and producing D-pantoyl lactone hydrolase
(1) preparation of seeding tank seed liquor.
(proportion of seed culture medium is shown in embodiment 1 to the seed culture medium of investment 8L, while addition disappears in 10L seeding tank
Infusion 0.05%, w/v), sterilizing uses steam sterilizing, and 30min under the conditions of 121 DEG C accesses 80ml shake-flask seed after cooling
Liquid, cultivates 24-26h, pH6.5-7.5, mycelia are dense at this time by 28 ± 1 DEG C of cultivation temperature, speed of agitator 200rpm, ventilatory capacity 1vvm
Degree is 20-30%.Strain is the same as embodiment 1.
(2) preparation and culture of fermentation tank culture medium.
The formula of fermentation medium is identical as previous embodiment 1, but to add the PPG of 0.05% (w/v) as defoaming agent,
Fermentor 50L, the volume that feeds intake are 40L, pH7.0 before disappearing, pH6.7 after disappearing, and the steam sterilizing 30min at 121 DEG C connects after cooling
Enter about 2L seed tank culture liquid, 28 ± 1 DEG C of fermentation temperature, rotating speed of agitator 150-200rpm, ventilatory capacity 0.8-1.0vvm, trains
46-48h is supported, tank is put, taking fermentation liquid to measure the enzyme activity of D-pantoyl lactone hydrolase is 800U/L.
Embodiment 3: the method for fermenting and producing D-pantoyl lactone hydrolase
Seed culture medium: sucrose 0.5%, soluble starch 0.5%, yeast extract 0.6%, seitan powder 1%, beef leaching
Cream 0.3%, MgSO4·7H2O 0.01%, NaCl 0.3%, pH7.0-7.5 before disappearing, shaking flask liquid amount are 30ml/250ml triangle
Bottle, through 121 DEG C of sterilizing 30min.A ferfas filament is dug from the inclined-plane of embodiment 1 and accesses seed culture medium, is placed in 28 ± 1 on shaking table
DEG C culture 24-26h.Then the seed culture fluid for drawing 2.5ml accesses fermentation medium: sucrose 1%, soluble starch 1%, bran
Matter powder 1.5%, groundnut meal 0.5%, yeast powder 1%, MgSO4·7H2O 0.005%, NaCl 0.2%, CaCl2
0.005%, ZnSO4·7H2O 0.0005%, FeCl3·6H2O 0.00006%, CaCO30.3%, pH7.0-7.5 before disappearing, hair
Ferment culture medium loading amount is 25ml/250ml triangular flask, and 121 DEG C of sterilizing 30min are placed in 28 ± 1 DEG C of culture 46-48h on shaking table, is surveyed
Obtaining fermentation liquid enzyme activity is 760U/L.
Embodiment 4: the method for fermenting and producing D-pantoyl lactone hydrolase
The preparation of seed culture fluid is carried out according to embodiment 3.Then the seed culture fluid of 2.5ml accesses fermentation medium:
Starch 2%, yeast powder 0.5%, soybean cake powder 1.5%, NaCl 0.2%, CaCO30.3%, FeSO4·7H2O 0.0001%,
MnCl2·4H2O 0.0003%, ZnSO4·7H2O 0.0005%, CoCl2·6H2O 0.0001%, MgSO4·7H2O
0.005%, CuSO4·5H2O 0.0005%, Na2MoO4·2H2O 0.0001%, pH7.0-7.5 before disappearing.Fermentation medium dress
Amount is 25ml/250ml triangular flask, and 121 DEG C of sterilizing 30min are placed in 28 ± 1 DEG C of culture 46-48h on shaking table.Measure fermentation broth enzyme
Living is 640U/L.
Embodiment 5: the method for fermenting and producing D-pantoyl lactone hydrolase
One plant of sickle-like bacteria (Fusarium sp.) CH1701, is preserved in Chinese microorganism strain on December 07th, 2018
Preservation administration committee common micro-organisms center (CGMCC), deposit number are CGMCC No.16967.
A method of using sickle-like bacteria fermenting and producing D-pantoyl lactone hydrolase, including a culture medium, specially send out
Ferment culture medium, which contains assimilable carbon source, assimilable nitrogen source and assimilable microelement, then by sickle-like bacteria
It is inoculated in the culture medium and carries out aerobic fermentation.The other parts of other culture mediums and fermentation medium and 1 phase of embodiment
Together.Fermentation condition is also the same as embodiment 1.
Preferred embodiment are as follows: the assimilable carbon source is glycerol, glucose, sucrose are mixed according to what 1:2:1 was constituted
Close object.
Preferred embodiment are as follows: the assimilable nitrogen source is Dried Corn Steep Liquor Powder, peptone, yeast extract according to 1:
The mixture that 1:1 is constituted.
Preferred embodiment are as follows: the assimilable microelement derives from CuSO4·5H2O and CaCl2、Na2MoO4·
2H2O。CuSO4·5H2O and CaCl2、Na2MoO4·2H2Mass ratio between O is 1:1:3.
Preferred embodiment are as follows: the sickle-like bacteria is sickle-like bacteria (Fusarium sp.) CH1701, sickle-like bacteria
(Fusarium sp.) CH1701 spontaneous mutant strain, sickle-like bacteria (Fusarium sp.) CH1701 pass through mutagenesis or genetic engineering
Obtained enhanced variant is transformed；Sickle-like bacteria (Fusarium sp.) CH1701, in preservation on December 07 in 2018
In China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), deposit number is CGMCC No.16967.
Preferred embodiment are as follows: the pH of the culture medium is 7, and the temperature of aerobic fermentation is 30 DEG C, aerobic fermentation when
Between be 36 hours；Oxygen-supply quantity is 1vvm.
Embodiment 6: the method for fermenting and producing D-pantoyl lactone hydrolase
One plant of sickle-like bacteria (Fusarium sp.) CH1701, is preserved in Chinese microorganism strain on December 07th, 2018
Preservation administration committee common micro-organisms center (CGMCC), deposit number are CGMCC No.16967.
A method of using sickle-like bacteria fermenting and producing D-pantoyl lactone hydrolase, including a culture medium, specially send out
Ferment culture medium, which contains assimilable carbon source, assimilable nitrogen source and assimilable microelement, then by sickle-like bacteria
It is inoculated in the culture medium and carries out aerobic fermentation.The other parts of other culture mediums and fermentation medium and 1 phase of embodiment
Together.Fermentation condition is also the same as embodiment 1.
Preferred embodiment are as follows: the assimilable carbon source utilizes sickle-like bacteria fermenting and producing D-pantoyl lactone to be a kind of
The method of hydrolase, including a culture medium, specially fermentation medium, the culture medium contain assimilable carbon source, assimilable
Then sickle-like bacteria is inoculated in the culture medium and carries out aerobic fermentation by nitrogen source and assimilable microelement.Other culture mediums, with
And the other parts of fermentation medium are same as Example 1.Fermentation condition is also the same as embodiment 1.
Preferred embodiment are as follows: the assimilable carbon source is poly- for industrial molasses, lactose, maltose, trehalose, wood
The mixture that sugar, mannitol, sorbierite and dextrin form, and industrial molasses, lactose, maltose, trehalose, wood in mixture
Glycan, mannitol, sorbierite and dextrin content are identical.
Preferred embodiment are as follows: the assimilable nitrogen source is beef extract.
Preferred embodiment are as follows: the assimilable microelement derives from Na2MoO4·2H2O and FeCl3·6H2O is pressed
The mixture constituted according to the mass ratio of 1:1.
Preferred embodiment are as follows: the sickle-like bacteria is sickle-like bacteria (Fusarium sp.) CH1701, sickle-like bacteria
(Fusarium sp.) CH1701 spontaneous mutant strain, sickle-like bacteria (Fusarium sp.) CH1701 pass through mutagenesis or genetic engineering
Obtained enhanced variant is transformed；Sickle-like bacteria (Fusarium sp.) CH1701, in preservation on December 07 in 2018
In China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), deposit number is CGMCC No.16967.
Preferred embodiment are as follows: the pH of the culture medium is 6.5, and the temperature of aerobic fermentation is 20 DEG C, aerobic fermentation
Time is 24 hours；Oxygen-supply quantity is 0.5vvm.
Embodiment 7: the method for fermenting and producing D-pantoyl lactone hydrolase
One plant of sickle-like bacteria (Fusarium sp.) CH1701, is preserved in Chinese microorganism strain on December 07th, 2018
Preservation administration committee common micro-organisms center (CGMCC), deposit number are CGMCC No.16967.
A method of using sickle-like bacteria fermenting and producing D-pantoyl lactone hydrolase, including a culture medium, specially send out
Ferment culture medium, which contains assimilable carbon source, assimilable nitrogen source and assimilable microelement, then by sickle-like bacteria
It is inoculated in the culture medium and carries out aerobic fermentation.The other parts of other culture mediums and fermentation medium and 1 phase of embodiment
Together.Fermentation condition is also the same as embodiment 1.
Preferred embodiment are as follows: the assimilable carbon source is sorbierite and dextrin, and content is equal.
Preferred embodiment are as follows: the assimilable nitrogen source be cottonseed meal, groundnut meal, yeast powder, beef extract,
The mixture that seitan powder, wheat bran, urea and ammonium salt form, and in mixture, cottonseed meal, groundnut meal, yeast powder, beef leaching
Cream, seitan powder, wheat bran, urea and amounts of ammonium salt are equal.
Preferred embodiment are as follows: the assimilable microelement derives from CuSO4·5H2O、CaCl2。
Preferred embodiment are as follows: the sickle-like bacteria is sickle-like bacteria (Fusarium sp.) CH1701, sickle-like bacteria
(Fusarium sp.) CH1701 spontaneous mutant strain, sickle-like bacteria (Fusarium sp.) CH1701 pass through mutagenesis or genetic engineering
Obtained enhanced variant is transformed；Sickle-like bacteria (Fusarium sp.) CH1701, in preservation on December 07 in 2018
In China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), deposit number is CGMCC No.16967.
Preferred embodiment are as follows: the pH of the culture medium is 7.5, and the temperature of aerobic fermentation is 40 DEG C, aerobic fermentation
Time is 72 hours；Oxygen-supply quantity is 1.5vvm.
As described above is only to be not intended to tool to explain the preferred embodiments of the invention to do any shape to the present invention
Limitation in formula should all wrap therefore all have any modification or change for making the related present invention under identical spirit
It includes in the scope that the invention is intended to protect.
Claims (8)
1. one plant of sickle-like bacteria (Fusarium sp.) CH1701 has been preserved in Chinese microorganism strain guarantor on December 07th, 2018
It hides administration committee's common micro-organisms center (CGMCC), deposit number is CGMCC No.16967.
2. a kind of method using sickle-like bacteria fermenting and producing D-pantoyl lactone hydrolase, it is characterised in that: including a culture medium,
The culture medium contains assimilable carbon source, assimilable nitrogen source and assimilable microelement, and sickle-like bacteria is then inoculated in institute
It states culture medium and carries out aerobic fermentation.
3. the method according to claim 2 using sickle-like bacteria fermenting and producing D-pantoyl lactone hydrolase, feature exist
It is that glycerol, glucose, sucrose, starch, industrial molasses, lactose, maltose, trehalose, wood are poly- in: the assimilable carbon source
At least one of sugar, mannitol, sorbierite and dextrin.
4. the method according to claim 2 using sickle-like bacteria fermenting and producing D-pantoyl lactone hydrolase, feature exist
In: the assimilable nitrogen source be soybean cake powder, Dried Corn Steep Liquor Powder, peptone, yeast extract, tryptone, cottonseed meal,
At least one of groundnut meal, yeast powder, beef extract, seitan powder, wheat bran, urea and ammonium salt.
5. the method according to claim 2 using sickle-like bacteria fermenting and producing D-pantoyl lactone hydrolase, feature exist
In: the assimilable microelement derives from MgSO4·7H2O、ZnSO4·7H2O、FeSO4·7H2O、MnCl2·4H2O、
CoCl2·6H2O、CuSO4·5H2O、CaCl2、Na2MoO4·2H2O and FeCl3·6H2At least one of O.
6. using the method for sickle-like bacteria fermenting and producing D-pantoyl lactone hydrolase according to claim 2 any one,
It is characterized by: the sickle-like bacteria is sickle-like bacteria (Fusarium sp.) CH1701, sickle-like bacteria (Fusarium sp.) CH1701
The high yield mutant bacteria that spontaneous mutant strain, sickle-like bacteria (Fusarium sp.) CH1701 are transformed by mutagenesis or genetic engineering
Strain；Sickle-like bacteria (Fusarium sp.) CH1701, is preserved in Chinese microorganism strain preservation on December 07th, 2018
Administration committee's common micro-organisms center (CGMCC), deposit number are CGMCC No.16967.
7. preparation method according to claim 2, it is characterised in that: the pH of the culture medium is 6.5-7.5, aerobic fermentation
Temperature be 20 DEG C -40 DEG C, time of aerobic fermentation is 24-72 hours；Oxygen-supply quantity is 0.5-1.5vvm.
8. the application of one plant of sickle-like bacteria (Fusarium sp.) CH1701, it is characterised in that: the sickle-like bacteria (Fusarium
Sp.) CH1701 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 07th, 2018
(CGMCC), deposit number is CGMCC No.16967；Sickle-like bacteria (Fusarium sp.) CH1701 has the production general solution of D-
The ability of acid lactone hydrolase.
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| CN112175839A (en) * | 2020-09-30 | 2021-01-05 | 遵义医科大学 | D-pantoic acid lactone hydrolase, producing strain, genetic engineering strain and application thereof |
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| CN113604371A (en) * | 2021-08-03 | 2021-11-05 | 合肥学院 | Yeast strain HF-21 and method for preparing D-pantolactone by two-phase catalysis of yeast strain HF-21 |
| CN115851458A (en) * | 2023-02-20 | 2023-03-28 | 中国科学院天津工业生物技术研究所 | Fusarium venenatum with high yield of hypha protein and application thereof |
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| CN113046337A (en) * | 2021-03-18 | 2021-06-29 | 赤峰制药股份有限公司 | Pantolactone hydrolase mutant strain and application thereof |
| CN113604371A (en) * | 2021-08-03 | 2021-11-05 | 合肥学院 | Yeast strain HF-21 and method for preparing D-pantolactone by two-phase catalysis of yeast strain HF-21 |
| CN115851458A (en) * | 2023-02-20 | 2023-03-28 | 中国科学院天津工业生物技术研究所 | Fusarium venenatum with high yield of hypha protein and application thereof |
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