CN110157623B - Fusarium strain and method for producing D-pantolactone hydrolase by fermenting same - Google Patents
Fusarium strain and method for producing D-pantolactone hydrolase by fermenting same Download PDFInfo
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Abstract
The invention discloses Fusarium sp CH1701 which is preserved in China general microbiological culture collection center (CGMCC) in 2018 and 12 months and 07 days, and the preservation number is CGMCC No. 16967. The Fusarium sp CH1701 of the present invention is a novel strain that produces D-pantolactone hydrolase by fermentation in a fermentation medium containing assimilable carbon, nitrogen and trace element salts under aerobic conditions.
Description
Technical Field
The invention relates to a fusarium strain and a method for producing D-pantolactone hydrolase by fermenting the fusarium strain, belonging to the technical field of microbial strain screening and fermentation.
Background
The D-calcium pantothenate is a vitamin B group compound, is widely applied to food, feed and pharmaceutical industries, and has large market demand. Generally, isobutyraldehyde, formaldehyde and sodium cyanide are adopted to synthesize DL-pantoic acid lactone, D-pantoic acid lactone is obtained by chemical resolution or biological enzymatic resolution, and finally, calcium beta-alanine and D-pantoic acid lactone are directly condensed to obtain the calcium D-pantothenate.
The disadvantages of the chemical resolution of DL-pantoic acid lactone are: chemical resolving agents such as quinine compounds, chiral amines and the like have high cost and difficult impurity separation, and are not beneficial to environmental protection.
D-pantoic acid lactone hydrolase selective resolution: firstly, D-pantoic acid lactone hydrolase selectively hydrolyzes racemic DL-pantoic acid lactone to generate D-pantoic acid, then the D-pantoic acid and L-pantoic acid lactone are separated by utilizing different solubilities, and the D-pantoic acid is lactonized to form D-pantoic acid lactone with high optical purity. Recycling the L-pantoic acid lactone, racemizing to obtain DL-pantoic acid lactone, and reusing the DL-pantoic acid lactone for resolution. The enzymatic resolution cost is low, the process is simple, the environment is friendly, the industrial production of D-calcium pantothenate, D-pantothenic acid and D-panthenol is realized at present, and the specific steps are shown in figure 1.
In 2001, Chinese patent CN 1313402A discloses a microorganism strain Fusarium moniliforme SW-902 (CGGG 0536) capable of producing D-pantolactone hydrolase, and the enzyme activity of the fermentation liquid reaches 3-6U/l, thereby promoting the industrialization of producing D-pantolactone by enzyme-method selective resolution.
The enzyme activity of the production strain has influence on the successful implementation of the enzyme method resolution process and whether the enzyme has relevant bonds of economic competitiveness. Therefore, there is a need to screen microbial strains capable of producing D-pantolactone hydrolase at a high yield and to establish a method for efficiently producing the enzyme.
Disclosure of Invention
The invention aims to provide a fusarium strain and a method for producing D-pantolactone hydrolase by fermenting the fusarium strain.
In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: a Fusarium (Fusarium sp.) CH1701 is preserved in China general microbiological culture collection center (CGMCC) in 2018 at 12 months and 07 days, and the preservation number is CGMCC No. 16967.
In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: a method for producing D-pantolactone hydrolase by utilizing fusarium fermentation comprises a culture medium, wherein the culture medium contains an assimilable carbon source, an assimilable nitrogen source and assimilable trace elements, and then fusarium is inoculated in the culture medium for aerobic fermentation.
The preferable technical scheme is as follows: the assimilable carbon source is at least one of glycerol, glucose, sucrose, starch, industrial molasses, lactose, maltose, trehalose, xylan, mannitol, sorbitol and dextrin.
The preferable technical scheme is as follows: the assimilable nitrogen source is at least one of soybean cake powder, corn steep liquor dry powder, peptone, yeast extract, tryptone, cottonseed cake powder, peanut cake powder, yeast powder, beef extract, gluten powder, bran, urea and ammonium salt.
The preferable technical scheme is as follows: the assimilable trace elements are derived from MgSO4·7H2O、ZnSO4·7H2O、FeSO4·7H2O、MnCl2·4H2O、CoCl2·6H2O、CuSO4·5H2O、CaCl2、Na2MoO4·2H2O and FeCl3·6H2At least one of O.
The preferable technical scheme is as follows: the Fusarium is a high-yield mutant strain obtained by mutation or genetic engineering transformation of Fusarium (Fusarium sp.) CH1701, a mutant strain spontaneously generated by Fusarium (Fusarium sp.) CH1701, or a Fusarium sp CH 1701; the Fusarium (Fusarium sp.) CH1701 is preserved in the China general microbiological culture collection center (CGMCC) in 2018 at 12 months and 07, and the preservation number is CGMCC No. 16967.
The preferable technical scheme is as follows: the pH value of the culture medium is 6.5-7.5, the temperature of aerobic fermentation is 20-40 ℃, and the time of aerobic fermentation is 24-72 hours; the oxygen introduction amount is 0.5-1.5 vvm.
In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: the application of Fusarium sp CH1701 is characterized in that the Fusarium sp CH1701 is preserved in China general microbiological culture collection center (CGMCC) in 2018 at 12 months and 07 with the preservation number of CGMCC No. 16967; the Fusarium (Fusarium sp.) CH1701 has the ability to produce D-pantolactone hydrolase.
Due to the application of the technical scheme, compared with the prior art, the invention has the advantages that:
1. the enzyme activity is improved. The D-pantolactone hydrolase produced by the strain has greatly improved enzyme activity compared with the reported strain, and is favorable for realizing industrial production.
2. The production cost is reduced. The strain is favorable for improving the production efficiency of vitamin B group products such as D-calcium pantothenate, D-pantothenic acid, D-panthenol and the like, reducing the production cost and having economic competitiveness.
Drawings
FIG. 1 is a scheme showing the selective resolution of D-pantolactone hydrolase.
FIG. 2 shows the colony morphology of the strain of the present invention.
FIG. 3 shows the appearance of the mycelium enzyme source of the fermentation broth of the present invention.
FIG. 4 is an HPLC chromatogram of the reaction solution before and after 30min of enzymatic hydrolysis of the present invention.
Detailed Description
The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will be apparent to those skilled in the art from the present disclosure.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
In describing the preferred embodiments, specific terminology may be employed for the sake of clarity; however, it is not intended that the disclosure herein be limited to the specific terminology so selected; and it is to be understood that each specific element includes all equivalent techniques that perform the same function, operate in a similar manner, and achieve a similar result.
The enzyme activity of the D-pantolactone hydrolase of the embodiment of the invention is detected by the following method:
1. preparing an enzyme source: 5ml of fermentation liquor is taken for filtration or centrifugation, and wet thalli are left as an enzyme source.
2. Preparing a substrate solution: preparing Tris-HCl buffer solution (containing 50mmol/L CaCl) with pH of 7.5 and 2% DL-pantoic acid lactone2)。
3. Enzymatic reaction: the above reaction system was reacted at 28 ℃ for 30min on a shaker at 150rpm, and the mycelium was removed by filtration.
4. And (3) measuring enzyme activity: the hydrolysis rate of DL-pantolactone was measured by HPLC (formula shown below).
The enzyme activity was calculated by hydrolysis rate, and 1. mu. mol of substrate was converted into product per minute as one enzyme activity unit.
5. HPLC conditions: mobile phase: acetonitrile 0.02mol/L KH2PO41: 9; a chromatographic column: c18 column, 5 μm, 4.6X 250 mm; the flow rate is 1 ml/min; the detection wavelength is 215 nm; detecting the temperature: room temperature; sample introduction amount: 10 μ l.
Example 1: fusarium strain and method for producing D-pantolactone hydrolase by fermenting same
First, the source of the strain
Fusarium strain Fusarium sp.CH1701 is an original strain isolated from soil in sugarcane orchard in Huangshan City, Anhui, N+Ion beam injection mutagenesis breeding. Culturing the strain in PDA slant culture medium at 28 + -1 deg.C for 2-4 days, scraping off aerial mycelium with sterile inoculating loop, shaking in test tube containing deionized water and glass beads to break mycelium to obtain mycelium suspension, and culturing in N+Ion beam implantation mutagenesis treatment, which comprises the following steps:
(1) the mycelium suspension of the above starting strain Fusarium sp.ch1701 was diluted appropriately.
(2) 100 mul of mycelium diluent is evenly coated on a blank glass plate with the diameter of 90mm by a glass coating rod, and the blank glass plate is placed on an ultra-clean working table and dried by blowing at even wind speed to prepare a bacterial membrane.
(3)N+The ion implantation experiment is carried out on ion beam bioengineering devices in key laboratories of intense magnetic field and ion beam physics biology of Chinese academy of sciences, and low-energy N with different energies and dosages is selected+And (5) ion implantation.
(4) After ion implantation, the treated sample was scraped off uniformly with 1ml of sterile water, sterile glass coated rod, 100. mu.l of the sample was aspirated and cultured in solid PDA medium, and after 3 days, single colony counting was performed and the lethality was calculated. Picking single colony to PDA slant culture medium, culturing at 28 + -1 deg.C for 3 days, and storing grown slant strain at 4 deg.C for use.
(5) Selecting the process N+Injecting the mutagenized single colony of 2000 strains by ion beams, performing shake flask fermentation, and detecting the enzyme activity of the D-pantolactone hydrolase produced by the fermentation liquor. As a result, a high-producing mutant strain Fusarium sp.CH1701 was selected.
Fusarium (Fusarium sp.) as a microbial strain CH1701 is preserved in the China general microbiological culture Collection center (address: West Lu No.1 Hospital, China academy of sciences) of China general microbiological culture Collection management Committee in 2018, 12 months and 7 days, and the preservation number is CGMCC No.16967, and the Fusarium sp is registered in a registration to prove survival.
The main biological characteristics of the strain are as follows: the colony color is white or grey white, the colony shape is round, the surface is raised and steamed bread-shaped, no spores exist, and aerial hyphae are developed and are easy to pick up.
The present invention describes the morphological and molecular level characteristics of Fusarium sp.CH1701, and compared with the morphological and molecular level of known D-pantoate lactonohydrolase producing bacteria, Fusarium sp.CH1701 can be identified as belonging to the genus Fusarium, but it is different from the known D-pantoate lactonohydrolase producing strain Fusarium moniliforme SW-902 (CGGG 0536) but is a completely new strain.
Secondly, identifying strains
The main biological characteristics of the strain are as follows: the colony color is white or grey white, the colony shape is round and round, the colony is steamed bread-shaped, the diameter is large and medium, no spores exist, aerial hyphae are developed, and the colony is easy to pick up.
Universal primers based on conserved sequences of fungal rDNA:
a forward primer: 5'-TCCGTAGGTGAACCTGCGG-3'
Reverse primer: 5'-TCCTCCGCTTATTGATATGC-3'
The rDNA sequence of the strain Fusarium sp.CH1701 was PCR amplified using the primers described above. PCR cycling conditions: pre-denaturation at 95 deg.C for 5min, cycle parameters of denaturation at 95 deg.C for 1min, renaturation at 52 deg.C for 45s, extension at 72 deg.C for 1.5min, repeating 35 cycles, extension at 72 deg.C for 10min, and final heat preservation at 4 deg.C. The PCR product was detected by 0.8% agarose gel electrophoresis. And selecting a product with clear bands for purification. And recovering the amplification product through gel electrophoresis, connecting the amplification product to a T carrier, and sequencing to obtain a sequence of 558 basic groups of the rDNA primary structure of the strain. By performing blast in the Genebank database, it was found that the similarity of CH1701 to the gene sequence of Fusarium sp.strain XJC-02 (GenBank No. MG273757.1) was 99%.
In conclusion, CH1701 belongs to the genus Fusarium, but it is different from the known D-pantoate lactone hydrolase producing species Fusarium moniliforme SW-902 (CGGG 0536). Fusarium sp.CH1701 is a new strain producing D-pantolactone hydrolase.
Thirdly, preparing D-pantoic lactone hydrolase
(1) Preparing and culturing slant mycelium.
Preparing and culturing a slant culture medium: peeling potato 200g, cutting into blocks, adding water, boiling for 30min, squeezing and filtering with 8 layers of gauze, adding sucrose 20g and agar 20g into the filtrate, adding deionized water to reach a constant volume of 1000ml, and naturally adjusting pH. Taking 30 × 200mm glass test tubes, loading each tube with 20ml culture medium, sterilizing at 121 deg.C for 20min, and cooling to 50-60 deg.C. A Fusarium annulatum (Fusarium sp.) CH1701 mycelium is inoculated to the slant, and then the slant is cultured for 3 days at the temperature of 28 +/-1 ℃, and the mycelium is mature and ready for use.
(2) And (4) preparing and culturing seed liquid.
The seed culture medium formula comprises: 1% of glycerol, 0.6% of soybean cake powder (hot pressing), 0.6% of yeast powder, 1% of peptone, 0.3% of corn steep liquor dry powder, pH7.0-7.5 before sterilization, wherein the liquid filling amount in a shake flask is 250ml, 25ml is bottled in a triangular flask, and the sterilization is carried out for 20min at 121 ℃. The inoculation amount of the strain is aerogenic mycelium of an inoculation Fusarium circinelloides (Fusarium sp.) CH1701, the culture temperature is 28 +/-1 ℃, the rpm is 220, the shaking table is used for shaking culture for 24-26h, the pH of a culture solution is 6.5-7.5, and the concentration of the mycelium is 20-30% (v/v), so that a seed solution is obtained.
(3) Preparing and culturing an enzyme-producing fermentation culture medium.
The fermentation medium formula comprises: 2% of glycerol, 1.5% of soybean cake powder (hot pressing), 0.5% of corn steep liquor dry powder, 2% of peptone, 7.0-7.5% of pHbefore digestion, 25ml of triangular bottle with the liquid loading capacity of 250ml, and 20min of sterilization at 121 ℃. Inoculating the seed solution with an inoculum size of 5-10% (v/v), shaking and culturing at 28 + -1 deg.C and 220rpm for 46-48h, detecting D-pantolactone hydrolase activity according to standard method after fermentation, and determining the enzyme activity to be 850U/L.
Example 2: method for producing D-pantoic lactone hydrolase by fermentation
(1) And (4) preparing seed liquid in a seed tank.
Putting 8L of seed culture medium (the proportion of the seed culture medium is shown in example 1, 0.05% of antifoaming agent is added at the same time, and w/v) into a 10L seeding tank, sterilizing by steam, sterilizing at 121 deg.C for 30min, cooling, adding 80ml seed solution, culturing at 28 + -1 deg.C under stirring speed of 200rpm, and culturing for 24-26h with ventilation volume of 1vvm, pH of 6.5-7.5, and mycelium concentration of 20-30%. The strain was the same as in example 1.
(2) Preparing and culturing a culture medium of a fermentation tank.
The formula of the fermentation medium is the same as that of the previous example 1, but 0.05% (w/v) PPG is added as an antifoaming agent, the fermentation tank is 50L, the feeding volume is 40L, the pH value is 7.0 before and 6.7 after elimination, steam sterilization is carried out at 121 ℃ for 30min, after cooling, about 2L of seed tank culture solution is inoculated, the fermentation temperature is 28 +/-1 ℃, the rotation speed of a stirring paddle is 150 plus or minus 200rpm, the ventilation volume is 0.8-1.0vvm, the culture is carried out for 46-48h, the tank is placed, and the enzyme activity of the D-pantolactone hydrolase is measured by taking the fermentation liquid and is 800U/L.
Example 3: method for producing D-pantoic lactone hydrolase by fermentation
Seed culture medium: 0.5% of sucrose, 0.5% of soluble starch, 0.6% of yeast extract, 1% of gluten powder, 0.3% of beef extract and MgSO4·7H20.01 percent of O, 0.3 percent of NaCl, and the pH value before sterilization is 7.0 to 7.5, the liquid filling amount of a shake flask is 30ml/250ml of triangular flask, and the triangular flask is sterilized for 30min at the temperature of 121 ℃. A piece of mycelium was harvested from the slant of example 1 and inoculated into seed medium and cultured on a shaker at 28. + -. 1 ℃ for 24-26 h. Then 2.5ml of seed culture solution is absorbed and inoculated into a fermentation culture medium: 1% of sucrose, 1% of soluble starch and gluten1.5% of flour, 0.5% of peanut cake flour, 1% of yeast powder and MgSO4·7H2O 0.005％，NaCl 0.2％，CaCl20.005％，ZnSO4·7H2O 0.0005％，FeCl3·6H2O 0.00006％，CaCO30.3 percent, pH7.0-7.5 before disinfection, the filling amount of the fermentation medium is 25ml/250ml triangular flask, the triangular flask is sterilized at 121 ℃ for 30min, the triangular flask is placed on a shaking table for culturing for 46-48h at 28 +/-1 ℃, and the enzyme activity of the fermentation liquid is 760U/L.
Example 4: method for producing D-pantoic lactone hydrolase by fermentation
The seed culture was prepared as in example 3. Then 2.5ml of seed culture solution is inoculated into a fermentation medium: 2% of starch, 0.5% of yeast powder, 1.5% of soybean cake powder, 0.2% of NaCl, CaCO3 0.3％，FeSO4·7H2O 0.0001％，MnCl2·4H2O 0.0003％，ZnSO4·7H2O 0.0005％，CoCl2·6H2O 0.0001％，MgSO4·7H2O 0.005％，CuSO4·5H2O 0.0005％，Na2MoO4·2H2O0.0001%, pH7.0-7.5 before digestion. The fermentation medium is filled in a 25ml/250ml triangular flask, sterilized at 121 ℃ for 30min, and cultured on a shaking bed at 28 +/-1 ℃ for 46-48 h. The enzyme activity of the fermentation liquor is 640U/L.
Example 5: method for producing D-pantoic lactone hydrolase by fermentation
A Fusarium (Fusarium sp.) CH1701 is preserved in China general microbiological culture collection center (CGMCC) in 2018 at 12 months and 07 days, and the preservation number is CGMCC No. 16967.
A method for producing D-pantolactone hydrolase by utilizing fusarium fermentation comprises a culture medium, specifically a fermentation culture medium, wherein the culture medium contains an assimilable carbon source, an assimilable nitrogen source and assimilable trace elements, and then fusarium is inoculated to the culture medium for aerobic fermentation. The other media, as well as the other parts of the fermentation medium, were the same as in example 1. The fermentation conditions were the same as in example 1.
The preferred embodiment is: the assimilable carbon source is glycerol, glucose and sucrose according to the weight ratio of 1: 2: 1, and (b) a mixture of.
The preferred embodiment is: the assimilable nitrogen source is corn steep liquor dry powder, peptone and yeast extract according to the ratio of 1: 1: 1, and (b) a mixture of.
The preferred embodiment is: the assimilable trace element is derived from CuSO4·5H2O and CaCl2、Na2MoO4·2H2O。CuSO4·5H2O and CaCl2、Na2MoO4·2H2The mass ratio of O is 1: 1: 3.
the preferred embodiment is: the Fusarium is a high-yield mutant strain obtained by mutation or genetic engineering transformation of Fusarium (Fusarium sp.) CH1701, a mutant strain spontaneously generated by Fusarium (Fusarium sp.) CH1701, or a Fusarium sp CH 1701; the Fusarium (Fusarium sp.) CH1701 is preserved in the China general microbiological culture collection center (CGMCC) in 2018 at 12 months and 07, and the preservation number is CGMCC No. 16967.
The preferred embodiment is: the pH value of the culture medium is 7, the temperature of aerobic fermentation is 30 ℃, and the time of aerobic fermentation is 36 hours; the oxygen flux was 1 vvm.
Example 6: method for producing D-pantoic lactone hydrolase by fermentation
A Fusarium (Fusarium sp.) CH1701 is preserved in China general microbiological culture collection center (CGMCC) in 2018 at 12 months and 07 days, and the preservation number is CGMCC No. 16967.
A method for producing D-pantolactone hydrolase by utilizing fusarium fermentation comprises a culture medium, specifically a fermentation culture medium, wherein the culture medium contains an assimilable carbon source, an assimilable nitrogen source and assimilable trace elements, and then fusarium is inoculated to the culture medium for aerobic fermentation. The other media, as well as the other parts of the fermentation medium, were the same as in example 1. The fermentation conditions were the same as in example 1.
The preferred embodiment is: the assimilable carbon source is a method for producing D-pantolactone hydrolase by fermenting fusarium, and comprises a culture medium, specifically a fermentation culture medium, wherein the culture medium contains an assimilable carbon source, an assimilable nitrogen source and assimilable trace elements, and then the fusarium is inoculated in the culture medium for aerobic fermentation. The other media, as well as the other parts of the fermentation medium, were the same as in example 1. The fermentation conditions were the same as in example 1.
The preferred embodiment is: the assimilable carbon source is a mixture consisting of industrial molasses, lactose, maltose, trehalose, xylan, mannitol, sorbitol and dextrin, and the content of the industrial molasses, the lactose, the maltose, the trehalose, the xylan, the mannitol, the sorbitol and the dextrin in the mixture is the same.
The preferred embodiment is: the assimilable nitrogen source is beef extract.
The preferred embodiment is: the assimilable trace elements are derived from Na2MoO4·2H2O and FeCl3·6H2O is as follows 1: 1 in a mass ratio.
The preferred embodiment is: the Fusarium is a high-yield mutant strain obtained by mutation or genetic engineering transformation of Fusarium (Fusarium sp.) CH1701, a mutant strain spontaneously generated by Fusarium (Fusarium sp.) CH1701, or a Fusarium sp CH 1701; the Fusarium (Fusarium sp.) CH1701 is preserved in the China general microbiological culture collection center (CGMCC) in 2018 at 12 months and 07, and the preservation number is CGMCC No. 16967.
The preferred embodiment is: the pH value of the culture medium is 6.5, the temperature of aerobic fermentation is 20 ℃, and the time of aerobic fermentation is 24 hours; the oxygen flux was 0.5 vvm.
Example 7: method for producing D-pantoic lactone hydrolase by fermentation
A Fusarium (Fusarium sp.) CH1701 is preserved in China general microbiological culture collection center (CGMCC) in 2018 at 12 months and 07 days, and the preservation number is CGMCC No. 16967.
A method for producing D-pantolactone hydrolase by utilizing fusarium fermentation comprises a culture medium, specifically a fermentation culture medium, wherein the culture medium contains an assimilable carbon source, an assimilable nitrogen source and assimilable trace elements, and then fusarium is inoculated to the culture medium for aerobic fermentation. The other media, as well as the other parts of the fermentation medium, were the same as in example 1. The fermentation conditions were the same as in example 1.
The preferred embodiment is: the assimilable carbon source is sorbitol and dextrin, and the content is equal.
The preferred embodiment is: the assimilable nitrogen source is a mixture consisting of cottonseed cake powder, peanut cake powder, yeast powder, beef extract, gluten powder, bran, urea and ammonium salt, and the content of the cottonseed cake powder, the peanut cake powder, the yeast powder, the beef extract, the gluten powder, the bran, the urea and the ammonium salt in the mixture is equal.
The preferred embodiment is: the assimilable trace element is derived from CuSO4·5H2O、CaCl2。
The preferred embodiment is: the Fusarium is a high-yield mutant strain obtained by mutation or genetic engineering transformation of Fusarium (Fusarium sp.) CH1701, a mutant strain spontaneously generated by Fusarium (Fusarium sp.) CH1701, or a Fusarium sp CH 1701; the Fusarium (Fusarium sp.) CH1701 is preserved in the China general microbiological culture collection center (CGMCC) in 2018 at 12 months and 07, and the preservation number is CGMCC No. 16967.
The preferred embodiment is: the pH value of the culture medium is 7.5, the temperature of aerobic fermentation is 40 ℃, and the time of aerobic fermentation is 72 hours; the oxygen flux was 1.5 vvm.
The foregoing is illustrative of the preferred embodiment of the present invention and is not to be construed as limiting thereof in any way, and any modifications or variations thereof that fall within the spirit of the invention are intended to be included within the scope thereof.
Claims (7)
1. Fusarium (A. sp.) (Fusarium sp.) CH1701, which has been preserved in China general microbiological culture Collection center (CGMCC) in 2018 at 12/07 th month, with the preservation number of CGMCC No.16967。
2. A method of using the Fusarium of claim 1 (see below)Fusarium sp.) A method for producing D-pantoic lactone hydrolase by CH1701 fermentation, which is characterized in that: comprises a culture medium which contains an assimilable carbon source, an assimilable nitrogen source and assimilable trace elements, and then fusarium is inoculated into the culture medium for aerobic fermentation.
3. The method for producing D-pantolactone hydrolase by fermentation using Fusarium according to claim 2, wherein the step of hydrolyzing the D-pantolactone with the acid is carried out by the following steps: the assimilable carbon source is at least one of glycerol, glucose, sucrose, starch, industrial molasses, lactose, maltose, trehalose, xylan, mannitol and sorbitol dextrin.
4. The method for producing D-pantolactone hydrolase by fermentation using Fusarium according to claim 2, wherein the step of hydrolyzing the D-pantolactone with the acid is carried out by the following steps: the assimilable nitrogen source is at least one of soybean cake powder, corn steep liquor dry powder, peptone, yeast extract, cottonseed cake powder, peanut cake powder, yeast powder, beef extract, gluten powder, bran, urea and ammonium salt.
5. The method for producing D-pantolactone hydrolase by fermentation using Fusarium according to claim 2, wherein the step of hydrolyzing the D-pantolactone with the acid is carried out by the following steps: the assimilable trace elements are derived from MgSO4·7H2O、ZnSO4·7H2O、FeSO4·7H2O、MnCl2·4H2O、CoCl2·6H2O、CuSO4·5H2O、Na2MoO4·2H2O and FeCl3·6H2At least one of O.
6. The method for producing D-pantolactone hydrolase by fermentation using Fusarium according to claim 2, which comprises the steps of: the pH value of the culture medium is 6.5-7.5, the temperature of aerobic fermentation is 20-40 ℃, and the time of aerobic fermentation is 24-72 hours; the oxygen introduction amount is 0.5-1.5 vvm.
7. Fusarium (F.) (Fusarium sp.) The application of CH1701 in the production of D-pantoic lactone hydrolase is characterized in that: the Fusarium (A), (B), (C), (Fusarium sp.) CH1701 is preserved in China general microbiological culture Collection center (CGMCC) in 2018 at 12.07.M.with the preservation number of CGMCC No. 16967.
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| CN110845453A (en) * | 2019-11-28 | 2020-02-28 | 安徽泰格生物科技有限公司 | Racemization method of L-pantoic acid lactone |
| CN112175839B (en) * | 2020-09-30 | 2022-09-13 | 遵义医科大学 | D-pantoic acid lactone hydrolase, producing strain, genetic engineering strain and application thereof |
| CN113046337B (en) * | 2021-03-18 | 2023-04-07 | 赤峰制药股份有限公司 | Pantolactone hydrolase mutant strain and application thereof |
| CN113604371B (en) * | 2021-08-03 | 2023-06-16 | 合肥学院 | Yeast strain HF-21 and method for preparing D-pantolactone by double-phase catalysis thereof |
| CN115851458B (en) * | 2023-02-20 | 2023-05-05 | 中国科学院天津工业生物技术研究所 | Fusarium venenatum producing hypha protein and application thereof |
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