CN110656052A - Method for high yield of D-pantolactone hydrolase - Google Patents
Method for high yield of D-pantolactone hydrolase Download PDFInfo
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Abstract
The invention relates to a novel fusarium moniliforme strain. The invention further relates to a method for producing D-pantoic acid lactone hydrolase with high yield, which comprises the steps of carrying out fermentation culture on the fusarium moniliforme strain with the preservation number of CGMCC No.18129 and extracting the D-pantoic acid lactone hydrolase from a fermentation product. The specific strain is applied to fermentation production, and D-pantolactone hydrolase fermentation liquor with high enzyme activity can be obtained, so that high-quality D-pantolactone hydrolase is efficiently obtained.
Description
Technical Field
The invention belongs to the technical field of biological engineering, and particularly relates to a method for mutagenesis and high yield of D-pantoyl lactonase strains.
Background
D-pantoic acid is an important chiral intermediate for producing D-pantothenic acid, the pantothenic acid is in great demand as a food and feed additive, and the product mostly exists in the form of calcium D-pantothenate and is short in supply. D-calcium pantothenate (D-calcium pantothenate) is a vitamin drug and is widely applied to the industries of medicines, foods and feeds.
At present, the D-pantothenic acid is generally prepared by a chemical or biological resolution method. The chemical resolution method has the disadvantages of expensive resolving agent, high cost, difficult separation and the like, and also brings about the serious environmental pollution problem. The biological method is mainly a microbial enzyme method, namely, the synthesized intermediate DL-pantolactone or the analogue thereof is converted and resolved into D-pantolactone by using D-pantolactone hydrolase produced by microorganisms, and then D-calcium pantothenate is synthesized, so that the method has the advantages of little environmental pollution and toxicity and the like.
The microorganisms producing the D-pantoic lactone hydrolase are many, and the enzyme producing effects of different microorganisms are different, so that the screening of the strain with high D-pantoic lactone hydrolase yield is particularly important.
Disclosure of Invention
The first purpose of the invention is to provide fusarium moniliforme with the preservation number of CGMCC No. 18129.
The fusarium moniliforme with the preservation number of CGMCC No.18129 is preserved in the China General Microbiological Culture Collection Center (CGMCC), No. 3 of Xilu No.1 of Beijing area facing the sun, the institute of microbiology of China academy of sciences, zip code 100101, the preservation number of CGMCC No.18129, the preservation date of 2019, 8 months and 29 days, and the classification and the name are as follows: fusarium moniliforme.
Specifically, the strain for high yield of D-pantolactone hydrolase provided by the invention is obtained by adopting the following method:
(1) carrying out normal-pressure room-temperature plasma mutagenesis on strains to be mutagenized to obtain a plurality of mutagenized strains;
(2) respectively inoculating the plurality of mutagenic strains into an enzyme production culture medium for culture;
(3) respectively detecting the activity of D-pantolactone hydrolase in the product obtained by culturing each mutagenic strain, and selecting the strain with the highest enzyme activity as the target strain.
The strain to be mutagenized can be obtained by adopting the following method: taking a plurality of strains which are not mutagenized and can produce D-pantolactone hydrolase, respectively coating the strains on a primary screening culture medium for culturing, and screening the strains with the largest color-changing circle diameter as strains to be mutagenized. The primary screening culture medium preferably contains the following components per liter: 100-200 g of potato extract, 10-30 g of D-pantolactone, 0.01-0.1 g of bromothymol blue and 10-20 g of agar. The conditions for culturing on the prescreening medium are preferably: culturing at 25-30 deg.C for 3-5 days.
The invention adopts a normal pressure room temperature plasma mutagenesis method to mutate and obtain the strain of the high-yield D-pantolactone hydrolase. In practical operation, the strain to be mutagenized can be firstly inoculated into a first-stage seed culture medium, after the strain is cultured to a logarithmic growth phase, the strain liquid is centrifuged, supernatant is discarded, and the strain is collected, and is made into 10-concentration strain by using sterile normal saline6～108And (3) uniformly coating 5-15 mu L of bacterial suspension on a slide glass, and carrying out normal-pressure room-temperature plasma mutagenesis. Preferably, the primary seed culture medium contains the following components per liter: 10-15 g of glycerol, 10-15 g of soybean peptone, 1-5 g of corn steep liquor dry powder, 5-10 g of yeast extract, 5-10 g of soybean meal powder, and the pH value of a culture medium is 7.0-7.5.
The main conditions of the normal-pressure room-temperature plasma mutagenesis are as follows: and (3) carrying out plasma irradiation for 100-180 s at a distance of 1-3 mm between the sample of the strain to be mutagenized and a plasma emission source. In practice, the following specific methods can be used: uniformly coating 10 mu L of the bacterial suspension on a slide glass, and irradiating by adopting an ARTP instrument, wherein 99.99% helium is used as working gas, the gas flow is 10L/min, the power supply power is 120W, and the irradiation time is respectively 100s, 120s, 150s and 180s under the condition that the distance between a sample and a plasma emission source is 2 mm; after irradiation, the slide glass is put into an EP tube with 1mL of sterile water for shaking elution, the eluent is coated on a PDA plate at 28 ℃, the culture is carried out for 3 days, and single colonies on the plate are respectively picked and numbered.
The invention respectively inoculates a plurality of strains obtained by mutagenesis into an enzyme production culture medium for culture so as to screen out the target strains with the highest yield. The enzyme-producing culture medium preferably contains the following components per liter: 10-15 g of glycerol, 10-15 g of soybean peptone, 1-5 g of corn steep liquor dry powder, 5-10 g of yeast extract, 5-10 g of soybean meal powder, and the pH value of a culture medium is 7.0-7.5. The conditions for culturing in the enzyme-producing medium are preferably: culturing for 20-24 h in a shaking table with the temperature of 25-30 ℃ and the rotating speed of 150-180 rpm.
The second purpose of the invention is to provide the application of the fusarium moniliforme with the preservation number of CGMCC No.18129 in the production of D-pantoic acid lactone hydrolase.
Specifically, the invention provides a method for high yield of D-pantolactone hydrolase, which comprises the steps of carrying out fermentation culture on fusarium moniliforme with the preservation number of CGMCC No.18129, and extracting the D-pantolactone hydrolase from a fermentation product.
The fermentation production according to the invention preferably comprises the following steps:
(i) inoculating the strain into a first-stage seed liquid culture medium for culturing to obtain a first-stage seed liquid;
(ii) inoculating the primary seed liquid into a secondary seed liquid culture medium for culturing to obtain a secondary seed liquid;
(iii) inoculating the secondary seed liquid into a fermentation culture medium for culture, and collecting a fermentation product;
(iv) extracting D-pantoic lactone hydrolase from the fermentation product.
As a preferable scheme of the invention, each liter of the primary seed liquid culture medium contains the following components: 10-15 g of glycerol, 10-15 g of soybean peptone, 1-5 g of corn steep liquor dry powder, 5-10 g of yeast extract, 5-10 g of soybean meal powder, and the pH value of a culture medium is 7.0-7.5.
In the actual fermentation production, the step (i) may specifically be: inoculating the strain into the first-stage seed liquid culture medium in an inoculation amount of 0.5-1.5%, and culturing at 25-30 ℃ and a rotation speed of 150-180 rpm for 20-24 h to obtain a first-stage seed liquid.
As a preferable scheme of the invention, each liter of the secondary seed liquid culture medium contains the following components: 10-15 g of sucrose, 150-200 g of corn steep liquor, 5-10 g of yeast extract, 5-10 g of peptone and the pH value of a culture medium is 6.5-7.0.
In the actual fermentation production, the step (ii) may be specifically: and inoculating the primary seed liquid into the secondary seed liquid culture medium in an inoculation amount of 5-15%, and culturing for 20-24 h at the temperature of 25-30 ℃ and the rotating speed of 150-180 rpm to obtain a secondary seed liquid.
As a preferable scheme of the invention, the fermentation medium contains the following components per liter: 10-15 g of sucrose, 150-200 g of corn steep liquor, 5-10 g of yeast extract, 5-10 g of peptone and the pH value of a culture medium is 6.5-7.0.
In the actual fermentation production, the step (iii) may be specifically: inoculating the secondary seed liquid into the fermentation medium in an inoculation amount of 5-15%, culturing for 36-40h at 25-30 ℃ and at a rotating speed of 120-150 rpm, and collecting a fermentation product.
The strain provided by the invention is applied to fermentation production, and D-pantolactone hydrolase fermentation liquor with high enzyme activity can be obtained, so that high-quality D-pantolactone hydrolase is efficiently obtained.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1
The purpose of this example is to select the appropriate species to be mutagenized.
Collecting multiple strain samples from soil in the Anhui firm town fermentation industrial park, adding sterile water respectively, placing on a shaking table for oscillation, standing, taking supernatant, diluting, coating on a primary screening culture medium respectively, culturing at 28 deg.C for 3 days, and screening the strain with the largest color-changing circle diameter as the strain to be mutagenized.
Example 2
The purpose of this example was to mutagenize a strain capable of producing D-pantolactone hydrolase in high yields. The specific operation is as follows:
(a) the strain to be mutagenized obtained by primary screening in the embodiment 1 is firstly inoculated into the first-class seed liquid, and each liter of the first-class seed liquid culture medium contains the following components: 13g of glycerol, 13g of soy peptone and 3g of corn steep liquor dry powder3g of yeast extract and 6g of soybean meal, wherein the pH value is 7.0; culturing at 28 deg.C and 180rpm to logarithmic growth phase, centrifuging under aseptic condition, discarding supernatant, and making thallus into 10% concentration with sterile normal saline6～108Bacterial suspension per mL.
(b) And (2) uniformly coating 10 mu L of the bacterial suspension on a slide glass, irradiating by adopting an ARTP instrument, taking 99.99% helium as working gas, wherein the gas flow is 10L/min, the power supply power is 120W, and the irradiation time is respectively 100s, 120s, 150s and 180s under the condition that the distance between a sample and a plasma emission source is 2 mm. After irradiation, the slide glass is put into an EP tube with 1mL of sterile water for shaking elution, the eluent is coated on a PDA plate at 28 ℃, the culture is carried out for 3 days, and single colonies on the plate are respectively picked and numbered.
(c) Respectively inoculating a plurality of single colonies (mutagenic strains) obtained in the step (2) into a shake flask enzyme production culture medium for culturing, and culturing for 24 hours at the conditions of 28 ℃ and 180 rpm; the enzyme-producing medium contains the following components per liter: 13g of glycerol, 13g of soybean peptone, 3g of corn steep liquor dry powder, 8g of yeast extract, 8g of soybean meal powder and the pH value of a culture medium of 7.2.
(d) The activity of D-pantolactone hydrolase in the product obtained by culturing each mutagenic strain is respectively detected. The detection results are shown in Table 1, wherein B5-1 is the strain to be mutagenized, and B5-1-B5-1-15 is the mutagenized strain respectively. The strain B5-1-7 with the highest enzyme activity is selected as a target strain, namely fusarium moniliforme with the preservation number of CGMCC No. 18129.
Table 1: enzyme production result of strain
Example 3
The purpose of this example is to use the target strain obtained in example 2 to perform fermentation production of D-pantoic lactone hydrolase, which specifically comprises the following steps:
mutagenesis of the target obtained in example 2Inoculating the strain B5-1-7 into the first-stage seed liquid culture medium with an inoculum size of 1%, and culturing at 28 deg.C and 180rpm for 24h to obtain first-stage seed liquid; inoculating the first-stage seed liquid into a second-stage seed liquid culture medium with the inoculation amount of 10%, and culturing at 28 ℃ and 180rpm for 24 hours to obtain a second-stage seed liquid; then inoculating the second-stage seed liquid into 10m with the inoculation amount of 10%3Culturing in a fermentation culture medium at 28 ℃ and 150rpm for 36h, and measuring the enzyme activity of the fermentation liquid to be 82.3IU/g after the fermentation is finished; extracting D-pantolactone hydrolase from the fermentation liquor by a conventional method.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. Fusarium moniliforme with preservation number of CGMCC No. 18129.
2. Application of Fusarium moniliforme with preservation number of CGMCC No.18129 in production of D-pantolactone hydrolase.
3. A method for producing D-pantoic acid lactone hydrolase with high yield is characterized in that Fusarium moniliforme with the preservation number of CGMCC No.18129 is subjected to fermentation culture, and the D-pantoic acid lactone hydrolase is extracted from a fermentation product.
4. The method according to claim 3, wherein said fermentative production comprises the steps of:
(i) inoculating the strain into a first-stage seed liquid culture medium for culturing to obtain a first-stage seed liquid;
(ii) inoculating the primary seed liquid into a secondary seed liquid culture medium for culturing to obtain a secondary seed liquid;
(iii) inoculating the secondary seed liquid into a fermentation culture medium for culture, and collecting a fermentation product;
(iv) extracting D-pantoic lactone hydrolase from the fermentation product.
5. The method of claim 4, wherein each liter of said primary seed broth comprises the following: 10-15 g of glycerol, 10-15 g of soybean peptone, 1-5 g of corn steep liquor dry powder, 5-10 g of yeast extract, 5-10 g of soybean meal powder, and the pH value of a culture medium is 7.0-7.5.
6. The method according to claim 5, wherein in the step (i), the strain is inoculated into the primary seed solution culture medium in an inoculation amount of 0.5-1.5%, and the primary seed solution is obtained by culturing at 25-30 ℃ and at a rotation speed of 150-180 rpm for 20-24 h.
7. The method of claim 4, wherein each liter of said secondary seed broth comprises the following: 10-15 g of sucrose, 150-200 g of corn steep liquor, 5-10 g of yeast extract, 5-10 g of peptone and the pH value of a culture medium is 6.5-7.0.
8. The method according to claim 7, wherein in the step (ii), the primary seed solution is inoculated into the secondary seed solution culture medium in an inoculation amount of 5-15%, and the secondary seed solution is obtained by culturing at 25-30 ℃ and 150-180 rpm for 20-24 h.
9. The method of claim 4, wherein the fermentation medium comprises, per liter: 10-15 g of sucrose, 150-200 g of corn steep liquor, 5-10 g of yeast extract, 5-10 g of peptone and the pH value of a culture medium is 6.5-7.0.
10. The method according to claim 9, wherein in the step (iii), the secondary seed liquid is inoculated into the fermentation medium in an inoculum size of 5-15%, the fermentation medium is cultured for 36-40h at 25-30 ℃ and at a rotation speed of 120-150 rpm, and a fermentation product is collected.
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| CN108624513A (en) * | 2018-06-15 | 2018-10-09 | 成都本则生科技有限公司 | A kind of method of High Density Cultivation D-pantoyl lactone hydrolase producing strains and application |
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