CN115786379A - Immobilized enzyme, preparation method and method for converting FR901379 by immobilized enzyme - Google Patents
Immobilized enzyme, preparation method and method for converting FR901379 by immobilized enzyme Download PDFInfo
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- 108010093096 Immobilized Enzymes Proteins 0.000 title claims abstract description 67
- 238000000034 method Methods 0.000 title claims abstract description 27
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 102000004190 Enzymes Human genes 0.000 claims abstract description 47
- 108090000790 Enzymes Proteins 0.000 claims abstract description 47
- 238000006243 chemical reaction Methods 0.000 claims abstract description 39
- 241000894006 Bacteria Species 0.000 claims abstract description 23
- 239000007788 liquid Substances 0.000 claims abstract description 16
- 241000588724 Escherichia coli Species 0.000 claims abstract description 12
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims abstract description 9
- 239000012510 hollow fiber Substances 0.000 claims abstract description 9
- 239000012528 membrane Substances 0.000 claims abstract description 9
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- 102000057234 Acyl transferases Human genes 0.000 claims description 25
- 238000010353 genetic engineering Methods 0.000 claims description 8
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 7
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 7
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 7
- 230000008569 process Effects 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 239000013604 expression vector Substances 0.000 claims description 3
- 239000013598 vector Substances 0.000 claims description 2
- 230000001580 bacterial effect Effects 0.000 claims 4
- 238000010438 heat treatment Methods 0.000 claims 1
- 238000000855 fermentation Methods 0.000 abstract description 10
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- 241001052560 Thallis Species 0.000 abstract description 8
- 238000010276 construction Methods 0.000 abstract description 5
- 238000002386 leaching Methods 0.000 abstract description 4
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- PIEUQSKUWLMALL-YABMTYFHSA-N micafungin Chemical compound C1=CC(OCCCCC)=CC=C1C1=CC(C=2C=CC(=CC=2)C(=O)N[C@@H]2C(N[C@H](C(=O)N3C[C@H](O)C[C@H]3C(=O)N[C@H](C(=O)N[C@H](C(=O)N3C[C@H](C)[C@H](O)[C@H]3C(=O)N[C@H](O)[C@H](O)C2)[C@H](O)CC(N)=O)[C@H](O)[C@@H](O)C=2C=C(OS(O)(=O)=O)C(O)=CC=2)[C@@H](C)O)=O)=NO1 PIEUQSKUWLMALL-YABMTYFHSA-N 0.000 description 6
- 108010079246 OMPA outer membrane proteins Proteins 0.000 description 5
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- 229930027917 kanamycin Natural products 0.000 description 3
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 3
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- 102000003960 Ligases Human genes 0.000 description 2
- 108090000364 Ligases Proteins 0.000 description 2
- 208000031888 Mycoses Diseases 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
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- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- WEVYAHXRMPXWCK-UHFFFAOYSA-N acetonitrile Substances CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 2
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- 206010059866 Drug resistance Diseases 0.000 description 1
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- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- 230000000843 anti-fungal effect Effects 0.000 description 1
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- JYIKNQVWKBUSNH-WVDDFWQHSA-N caspofungin Chemical compound C1([C@H](O)[C@@H](O)[C@H]2C(=O)N[C@H](C(=O)N3CC[C@H](O)[C@H]3C(=O)N[C@H](NCCN)[C@H](O)C[C@@H](C(N[C@H](C(=O)N3C[C@H](O)C[C@H]3C(=O)N2)[C@@H](C)O)=O)NC(=O)CCCCCCCC[C@@H](C)C[C@@H](C)CC)[C@H](O)CCN)=CC=C(O)C=C1 JYIKNQVWKBUSNH-WVDDFWQHSA-N 0.000 description 1
- 229960003034 caspofungin Drugs 0.000 description 1
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- 125000002669 linoleoyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])/C([H])=C([H])\C([H])([H])/C([H])=C([H])\C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
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- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
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- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
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- 230000036963 noncompetitive effect Effects 0.000 description 1
- 230000002572 peristaltic effect Effects 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
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- 239000002243 precursor Substances 0.000 description 1
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Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
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- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention discloses an immobilized enzyme, a preparation method and a method for converting FR901379 by the immobilized enzyme, and relates to the construction of escherichia coli genetically engineered bacteria, the obtaining of thalli by a fermentation method, the obtaining of an enzyme liquid by adopting an ultrasonic crushing and hexadecyl trimethyl ammonium bromide leaching method, the concentrating of the enzyme liquid by a hollow fiber membrane, the forming of the immobilized enzyme by combining with an amino-type carrier, and the conversion of FR901379 by the immobilized enzyme to obtain FR179642. The conversion efficiency of the immobilized enzyme prepared by the invention for converting FR901379 is high and reaches more than 95%, the conversion time is short, the product is easy to separate and extract, the pigment of the conversion liquid is less, the immobilized enzyme can be repeatedly used, and the method is favorable for future scale-up production.
Description
Technical Field
The invention belongs to the field of bioengineering, and relates to an immobilized enzyme, a preparation method and a method for converting FR901379 by the immobilized enzyme.
Background
Fungal infections have always been a persistent disease that afflicts human health. The reason is that the resistance of the body to fungi is reduced and the fungi can enter the body in a deficient way due to immunosuppression caused by drug resistance of the fungi, damage to the immune system and the like caused by overuse of antibiotic drugs by human beings. At present, few medicines capable of effectively treating deep fungal infection, particularly immunodeficiency combined with disseminated fungal infection exist. Therefore, the development of new safe and effective antifungal drugs is urgently needed.
Micafungin was developed by japan tenzier, marketed in japan in 12 months in 2002, and approved by the U.S. FDA in 3 months in 2005, becoming the second echinocandin-type drug to be clinically used after caspofungin 2 nd. Micafungin can noncompetitive inhibit activity of 1, 3-beta-D glucan synthetase so as to inhibit synthesis of fungal cell wall, and mammalian cells lack 1, 3-beta-D glucan synthetase, so the compound has high specificity to fungal cells, can kill fungi rapidly, and has little influence on normal cells of human body.
The micafungin is formed by removing linoleoyl side chain from FR901379 by acyltransferase to obtain cyclic hexapeptide nucleus which is chemically modified, and FR179642 is a precursor of micafungin and is obtained by converting FR901379, so that the conversion rate of FR901379 is improved, and the yield of micafungin is effectively improved. The acyltransferase is obtained by microbial fermentation and cannot be directly synthesized, and the reported acyltransferase is produced by actinoplanes through fermentation, so that the enzyme activity is lower, and the fermentation period is longer. The solution formed by converting FR901379 with acyltransferase mainly contains acyltransferase and FR179642, and FR179642 and acyltransferase are separated from the solution, so that FR179642 with high purity can be obtained on one hand, and the acyltransferase can be recycled on the other hand, but the separation is very difficult.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides an immobilized enzyme, a preparation method of the immobilized enzyme and a method for converting FR901379 by the immobilized enzyme, which can improve the yield of micafungin and facilitate the reutilization of acyltransferase.
In order to achieve the purpose, the technical scheme of the invention is as follows: constructing Escherichia coli genetic engineering bacteria, obtaining thallus by fermentation method, extracting thallus to obtain enzyme solution, concentrating enzyme, combining with carrier to form immobilized enzyme, and converting FR901379 into FR179642 by using immobilized enzyme.
The specific technical route is as follows:
a method for converting FR901379 by immobilized enzyme relates to the construction of Escherichia coli genetically engineered bacteria, obtaining thalli by a fermentation method, obtaining enzyme liquid by adopting an ultrasonic crushing and hexadecyl trimethyl ammonium bromide leaching method, concentrating the enzyme liquid by utilizing a hollow fiber membrane, then combining the enzyme liquid with an amino-type carrier to form the immobilized enzyme, and converting FR901379 by utilizing the immobilized enzyme to obtain FR179642.
The step of obtaining the crude enzyme solution from the thalli is to control the temperature of the crude enzyme solution below 10 ℃, carry out ultrasonic crushing on the thalli, then add cetyl trimethyl ammonium bromide and ammonium sulfate, heat up to 40-45 ℃, maintain for 1-3h, and extract the acyltransferase to the outside of cells.
The ultrasonic condition selection power is 450-500W, the ultrasonic time is 5-10 min, and the ultrasonic process control temperature is not more than 10 ℃; cetyl trimethyl ammonium bromide with the concentration of 0.5-1.0 percent and ammonium sulfate with the concentration of 1.0-2.0 percent, leaching temperature of 40-45 ℃ and leaching time of 1-2h, and extracting the acyltransferase to the outside of cells. Ammonium sulfate as a precipitating agent and cetyltrimethylammonium bromide as a surfactant.
The concentrated crude enzyme solution is obtained by concentrating the crude enzyme solution by 5-6 times through a hollow fiber membrane, and then combining the concentrated enzyme solution with an amino-type carrier to form the immobilized enzyme. The molecular weight cut-off of the hollow fiber membrane is more than 5 ten thousand.
When the FR901379 is converted by the immobilized enzyme, the FR901379 is dissolved by water, then the immobilized enzyme with the weight percent of 3-7 percent of the FR901379 solution is added, and the conversion is carried out for 10-20h at the temperature of 25-35 ℃.
When the immobilized enzyme is used for converting FR901379, the concentration of FR901379 in a FR901379 solution is preferably 2-3 g/L, the conversion temperature is preferably 25-35 ℃, and the conversion rate can reach more than 95% under the condition.
The carrier used for preparing the immobilized enzyme is an amino-type carrier, and the amino-type carrier can be LX-1000HA, ECR8409 or LX-1000EPHA.
The host bacterium of the escherichia coli genetic engineering bacterium for expressing acyltransferase is escherichia coli BL21, the secretory expression plasmid is Pet28a, the target gene is RBS + OmpA + alpha subunit + RBS + OmpA + beta subunit, and the gene sequence of the target gene is shown as SEQ ID NO. 1.
The invention obtains an immobilized enzyme capable of converting FR901379 into FR179642, and the preparation method comprises the steps of obtaining thalli by fermenting escherichia coli genetic engineering bacteria for expressing acyltransferase, obtaining enzyme liquid from the thalli, combining the enzyme liquid with an amino type vector to form the immobilized enzyme, wherein the expression vector of the escherichia coli genetic engineering bacteria for expressing the acyltransferase has a gene sequence shown as SEQ ID NO. 1.
Compared with the prior art, the invention has at least the following beneficial effects:
the conversion efficiency of the immobilized enzyme conversion FR901379 is high and reaches more than 95%, the conversion time is short, the product is easy to separate and extract, the pigment of the conversion liquid is less, the immobilized enzyme can be repeatedly used, and the future scale-up production is facilitated.
Drawings
FIG. 1 is a schematic diagram of construction of engineering bacteria.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention.
The process flow of the invention is as follows:
engineering bacteria construction → fermentation to obtain bacteria → enzyme extraction → enzyme immobilization → conversion of FR901379 to FR179642.
Construction of engineering bacteria
Coli BL21 is used as host bacteria, pet28a is used as secretion expression plasmid, the target gene structure is RBS + OmpA + alpha subunit + RBS + OmpA + beta subunit, RBS is ribose binding site, and OmpA is secretion signal peptide, so as to construct the escherichia coli genetic engineering bacteria, as shown in figure 1.
The sequence of the gene of interest (SEQ ID NO: 1) is as follows:
TTTGTTTAACTTTAAGAAGGAGAGAATTCATGAAGAAAACCGCGATCGCGATCGCGGTTGCGCTGGCGGGTTTCGCGACCGTTGCTCAGGCCGGTGAAGGTGAAGGCCATGATGGCGGTTATGCGGCGCTGATCCGTCGTGCGTCTTACGGCGTTCCGCACATCACCGCGGATGATTTCGGCAGCCTGGGCTTCGGCGTTGGCTACGTTCAGGCGGAAGATAACATCTGCGTTATCGCGGAAAGCGTTGTTACCGCGAACGGTGAACGTTCTCGTTGGTTCGGCGCGACCGGCCCGGATGATGCGGATGTTCGTTCTGACCTGTTCCACCGTAAAGCGATCGATGATCGTGTTGCGGAACGCCTGCTGGAAGGCCCGCGTGATGGCGTTCGTGCGCCGTCTGATGATGTTCGTGATCAGATGCGTGGTTTCGTTGCGGGCTACAACCACTTCCTGCGTCGTACCGGCGTTCACCGTCTGACCGATCCGGCGTGCCGTGGTAAAGCGTGGGTTCGTCCGCTGAGCGAAATCGATCTGTGGCGTACCAGCTGGGATAGCATGGTTCGTGCGGGTAGCGGCGCGCTGCTGGATGGCATCGTTGCGGCGACCCCGCCGACCGCGGCGGGCCCGGCGAGCGCGCCGGAAGCGCCGGATGCGTAACCATCTTAGTATTTGTTTAACTTTAAGAAGGAGACCTAGGATGAAGAAAACCGCGATCGCGATCGCGGTTGCGCTGGCGGGTTTCGCGACCGTTGCTCAGGCCAGCAACGCGTATGGTCTGGGTGCGCAGGCGACCGTGAACGGTAGCGGTATGGTTCTGGCGAACCCGCACTTCCCGTGGCAGGGTGCGGCGCGTTTTTACCGTATGCACCTGAAAGTGCCGGGTCGTTATGACGTTGAGGGTGCGGCGCTGATCGGCGATCCGATCATTGGCATTGGTCACAACCGTACCGTTGCGTGGAGCCACACCGTTAGCACCGCGCGTCGTTTCGTGTGGCATCGTCTGAGCCTGGTTCCGGGTGACCCGACCAGCTACTATGTTGATGGTCGTCCGGAACGTATGCGTGCGCGTACCGTGACCGTTCAAACCGGTAGCGGTCCGGTTAGCCGTACCTTCCACGACACCCGTTACGGTCCGGTGGCGGTTATGCCGGGCACCTTTGATTGGACCCCGGCGACCGCGTATGCGATCACCGACGTTAACGCGGGTAACAACCGTGCGTTCGATGGTTGGCTGCGTATGGGCCAGGCGAAGGACGTGCGTGCGCTGAAAGCGGTTCTGGATCGTCACCAATTTCTGCCGTGGGTGAACGTTATTGCGGCGGATGCGCGTGGTGAGGCGCTGTACGGCGATCACAGCGTGGTTCCGCGTGTGACCGGTGCGCTGGCGGCGGCGTGCATTCCGGCGCCGTTTCAGCCGCTGTATGCGAGCAGCGGTCAAGCGGTTCTGGATGGTAGCCGTAGCGATTGCGCGCTGGGTGCGGACCCGGATGCGGCGGTGCCGGGCATCCTGGGTCCGGCGAGCCTGCCGGTGCGTTTCCGTGACGATTACGTTACCAACAGCAACGACAGCCATTGGCTGGCGAGCCCGGCGGCGCCGCTGGAAGGTTTTCCGCGTATCCTGGGTAACGAGCGTACCCCGCGTAGCCTGCGTACCCGTCTGGGTCTGGACCAGATTCAGCAACGTCTGGCGGGTACCGATGGTCTGCCGGGCAAGGGTTTCACCACCGCGCGTCTGTGGCAAGTGATGTTTGGTAACCGTATGCACGGCGCGGAACTGGCGCGTGACGATCTGGTTGCGCTGTGCCGTCGTCAACCGACCGCGACCGCGAGCAACGGTGCGATCGTGGATCTGACCGCGGCGTGCACCGCGCTGAGCCGTTTCGATGAACGTGCGGACCTGGATAGCCGTGGTGCGCACCTGTTCACCGAGTTTGCGCTGGCGGGTGGCATTCGTTTCGCGGACACCTTTGAAGTGACCGATCCGGTTCGTACCCCGCGTCGTCTGAACACCACCGACCCGCGTGTGCGTACCGCGCTGGCGGATGCGGTTCAACGTCTGGCGGGTATCCCGCTGGACGCGAAACTGGGCGACATTCACACCGATAGCCGTGGTGAACGTCGTATTCCGATTCATGGTGGCCGTGGCGAGGCGGGTACCTTCAACGTTATCACCAACCCGCTGGTGCCGGGCGTTGGTTACCCGCAGGTGGTTCACGGTACCAGCTTTGTGATGGCGGTGGAGCTGGGTCCGCATGGTCCGAGCGGTCGTCAGATTCTGACCTATGCGCAAAGCACCAACCCGAACAGCCCGTGGTACGCGGACCAAACCGTGCTGTATAGCCGTAAGGGCTGGGATACCATCAAATACACCGAAGCGCAGATTGCGGCGGACCCGAACCTGCGTGTGTATCGTGTTGCGCAACGTGGTCGTAAAAAGAAAAAGAAAAAATAA。
immobilized enzyme prepared from engineering bacteria and application of immobilized enzyme
Example 1
1. Purpose of experiment
Extracting acyltransferase from engineering bacteria of Escherichia coli.
2. Experimental Material
1. Cell disruption instrument (JY 98-IIIDN), hexadecyl trimethyl ammonium bromide, ammonium sulfate, isopropyl-beta-D-thiogalactoside (IPTG) and kanamycin.
2. Seed culture medium: 10g/L of peptone, 5g/L of yeast powder, 10g/L of NaCl, 100ug/mL of kanamycin and 6.8-7.0 of pH.
3. Fermentation medium: peptone 12g/L, yeast powder 24g/L, glycerin 5g/L, KH2PO 4.31 g/L, K 2 HPO 4 12.54g/L, kanamycin 100ug/mL, pH 7.0.
3. Experimental procedure
1. And (3) subpackaging the prepared seed culture medium and fermentation culture medium into 500ml triangular flasks respectively, filling 200ml of liquid, sterilizing at 120-122 ℃ for 30min, and cooling for later use.
2. Inoculating slant seed to seed culture medium with inoculating loop, culturing at 37 deg.C and 220rpm for 12 hr, inoculating to fermentation culture at 3% seed amount, culturing at 37 deg.C and 220rpm for 5 hr, adding 0.6mmoL/L IPTG, culturing at 20 deg.C for 15 hr, and collecting mycelium.
3. Carrying out ultrasonic disruption by using a cell disruption instrument with ultrasonic power of 450W and ultrasonic time of 10min, controlling the temperature not to exceed 10 ℃ in the ultrasonic process, and collecting thalli.
4. And slowly adding 0.5wt% of hexadecyl trimethyl ammonium bromide and 1wt% of ammonium sulfate into the thalli in sequence, controlling the temperature to be 40-45 ℃, and maintaining for 1.5h.
5. After solid-liquid separation, collecting filtrate, namely crude enzyme solution.
6. The crude enzyme solution is transformed into FR901379 at 35 ℃ and 220rpm for 15-17h to complete the transformation.
4. Results and conclusions
Performing experiments twice according to the experimental operation steps to obtain crude enzyme solutions 1# and 2# samples, then converting the FR901379 solution by using the crude enzyme solutions 1# and 2# samples, wherein the solvent of the FR901379 solution is purified water, the initial concentration of the FR901379 is 2.0mg/mL, the amount of the crude enzyme solution added into the FR901379 solution is 10wt%, the conversion time is 15-17h (15 h for conversion by using the crude enzyme solution 1# and 17h for conversion by using the crude enzyme solution 2 #), after the conversion is completed, detecting by using an HPLC method to obtain the FR179642 concentration, and calculating the conversion rate of the FR901379 by using the FR901379 concentration and the FR179642 concentration, wherein the results are shown in Table 1.
FR901379 concentration detection method (HPLC method):
a chromatographic column: phenomenex Gemini C18, 250 × 4.6mm,5 μm;
mobile phase: 0.1mol/L sodium dihydrogen phosphate (pH adjusted to 3.0 with phosphoric acid) -acetonitrile =53:47;
flow rate: 1.0ml/min, wavelength: 210nm, column temperature: at 40 ℃.
FR179642 concentration detection method (HPLC method):
a chromatographic column: aichromobond AQ-C18, 250 x 4.6mm,5 μm;
mobile phase: 0.02mol/L potassium dihydrogen phosphate (pH adjusted to 3.0 with phosphoric acid) -acetonitrile =94:6;
flow rate: 1.0ml/min, wavelength: 210nm, column temperature: 35 ℃ is carried out.
TABLE 1 conversion of crude enzyme solution to FR901379
| Sample name | FR901379 concentration | FR179642 concentration | Conversion of FR901379 |
| Crude enzyme solution 1# | 2.0mg/mL | 1.308mg/mL | 65.4％ |
| Crude enzyme solution 2# | 2.0mg/mL | 1.414mg/mL | 70.7％ |
Note that: conversion (%) = (FR 179642 concentration/FR 901379 concentration) × 100%
As can be seen from Table 1, the conversion rate of the crude enzyme solution to FR901379 can reach more than 65%. Since the crude enzyme solution is dispersed in the solution after enzymolysis, it is difficult to directly separate from the product, which affects the purification of FR179642 and the reuse of acyltransferase.
Example 2: preparation of immobilized enzyme and conversion rate of immobilized enzyme to FR901379
1. Purpose of experiment
An immobilized enzyme was prepared and its transformation effect on FR901379 was examined.
2. Experimental Material
Hollow fiber membrane (5 ten thousand molecular weight), amino type carriers LX-1000HA, FR901379
3. Experimental procedure
1. The crude enzyme solution obtained in example 1 was pumped into a hollow fiber membrane (5 ten thousand molecular weight) by a peristaltic pump, and concentrated 5 times.
2. And collecting the concentrated enzyme solution.
3. Adding 0.5g of amino-type carrier into every 100ml of concentrated enzyme solution, combining the concentrated enzyme solution and the amino-type carrier overnight, and controlling the temperature to be 20-25 ℃.
4. Filtering and collecting the carrier to obtain the immobilized enzyme.
5. The immobilized enzyme was used to convert FR901379 at 35 ℃.
4. Results and conclusions
Performing experiments twice according to the experimental operation steps to obtain immobilized enzymes 1# and 2# samples, then converting the FR901379 solution by using the immobilized enzymes 1# and 2# samples, wherein the solvent of the FR901379 solution is purified water, the initial concentration of the FR901379 is 2.0mg/mL, the amount of the immobilized enzyme added in the FR901379 solution is 5wt%, the conversion time is 16h, detecting by using an HPLC method after the conversion is finished to obtain the concentration of FR179642 in the solution, and calculating the conversion rate of the FR901379 by using the concentrations of the FR901379 and the FR179642, wherein the results are shown in Table 2.
Table 2 conversion of the immobilized enzyme to FR901379
| Sample name | FR901379 concentration | FR179642 concentration | Conversion of FR901379 |
| Immobilized enzyme 1# | 2.0mg/mL | 1.912mg/mL | 95.6％ |
| Immobilized enzyme 2# | 2.0mg/mL | 1.940mg/mL | 97.0％ |
And (3) noting that: conversion (%) = (FR 179642 concentration/FR 901379 concentration) × 100%
As can be seen from the above table, the conversion rate of the immobilized enzyme to FR901379 can reach more than 95%. By fixing the enzyme on the amino-type carrier, the conversion rate of the enzyme to FR901379 is obviously improved. Meanwhile, because the acyltransferase is fixed on the amino-type carrier, the acyltransferase can be recycled by filtration after the conversion is finished.
Example 3: number of times of recycling of immobilized enzyme
1. Purpose of experiment
And (4) investigating the repeated use times of the immobilized enzyme.
2. Experimental materials
Immobilized enzyme, FR901379
3. Experimental procedures
FR901379 was transformed with the 2# immobilized enzyme obtained in example 2 at 35 ℃, 5wt% immobilized enzyme was added to the FR901379 solution, the transformation time was 16 hours each time, and the recovered immobilized enzyme was transformed 10 times in succession. After each conversion, the concentration of FR179642 in the solution was measured by HPLC method, and the conversion rate of FR901379 was calculated from the FR901379 concentration and FR179642 concentration, and the results are shown in table 3.
4. Results and conclusions
TABLE 3 conversion of immobilized enzyme for continuous conversion of FR901379
Note that: conversion (%) = (FR 179642 concentration/FR 901379 concentration) × 100%
As can be seen from Table 3, the immobilized enzyme prepared by the invention can continuously convert FR901379 for more than 10 times, the conversion rate is not reduced and is maintained for more than 95 percent, and the stability of the acyltransferase is improved by immobilizing the acyltransferase, and meanwhile, the acyltransferase is convenient to continuously recycle.
Although the invention has been described herein with reference to illustrative embodiments thereof, it should be understood that numerous other modifications and embodiments can be devised by those skilled in the art that will fall within the spirit and scope of the principles of this disclosure.
Claims (10)
1. A method for converting FR901379 by using an immobilized enzyme is characterized by comprising the steps of constructing escherichia coli genetic engineering bacteria for expressing acyltransferase, fermenting to obtain bacteria, obtaining crude enzyme liquid from the bacteria, concentrating the crude enzyme liquid, combining the concentrated enzyme liquid with an amino-type carrier to form the immobilized enzyme, and converting FR901379 by using the immobilized enzyme to obtain FR179642.
2. The method for converting FR901379 by using an immobilized enzyme according to claim 1, wherein the step of obtaining the crude enzyme solution from the bacterial cells comprises controlling the temperature of the crude enzyme solution to be below 10 ℃, ultrasonically crushing the bacterial cells at a power of 450-500W for 5-10 min, adding hexadecyl trimethyl ammonium bromide and ammonium sulfate, heating to 40-45 ℃, maintaining for 1-2h, and extracting the acyltransferase to the extracellular side.
3. The method for converting FR901379 by using an immobilized enzyme according to claim 2, wherein the amount of cetyltrimethylammonium bromide is 0.5-1wt% of the bacterial cells, and the amount of ammonium sulfate is 1.0-2.0wt% of the bacterial cells.
4. The method for converting FR901379 by using an immobilized enzyme according to claim 1, wherein said concentrating the crude enzyme solution is carried out by concentrating the crude enzyme solution 5-to 6-fold through a hollow fiber membrane, and then binding the concentrated enzyme solution to an amino-type carrier to form the immobilized enzyme.
5. The process for converting FR901379 by an immobilized enzyme according to claim 4, wherein said hollow fiber membrane is a hollow fiber membrane having a molecular weight cut-off of 5 ten thousand or more.
6. The process for converting FR901379 by immobilized enzyme according to claim 1,
adding 0.2-0.7g of amino-type carrier into every 100ml of the concentrated enzyme solution, combining the concentrated enzyme solution and the amino-type carrier overnight, and controlling the temperature to be 20-25 ℃.
7. The method for converting FR901379 by using the immobilized enzyme as defined in claim 1, wherein when the FR901379 is converted by using the immobilized enzyme, the FR901379 is dissolved by water, then 3-7wt% of the immobilized enzyme is added to the FR901379 solution, and the conversion lasts for 10-20h at 25-35 ℃.
8. The method for converting FR901379 by using immobilized enzyme according to claim 1, wherein an expression vector of the escherichia coli genetically engineered bacterium for expressing the acyltransferase has a gene sequence shown as SEQ ID NO. 1.
9. A preparation method of an immobilized enzyme is characterized in that bacteria are obtained by fermenting escherichia coli genetic engineering bacteria for expressing acyltransferase, enzyme liquid is obtained from the bacteria, the enzyme liquid is combined with an amino-type vector to form the immobilized enzyme, and the expression vector of the escherichia coli genetic engineering bacteria for expressing acyltransferase has a gene sequence shown as SEQ ID NO. 1.
10. An immobilized enzyme obtained by the production process according to claim 9.
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