CN102827777B - Symbiotic and epiphytic aspergillus terreus of sponge and use for preparation of (+)- terrein - Google Patents
Symbiotic and epiphytic aspergillus terreus of sponge and use for preparation of (+)- terrein Download PDFInfo
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- CN102827777B CN102827777B CN201210007947.6A CN201210007947A CN102827777B CN 102827777 B CN102827777 B CN 102827777B CN 201210007947 A CN201210007947 A CN 201210007947A CN 102827777 B CN102827777 B CN 102827777B
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Abstract
The invention discloses symbiotic and epiphytic aspergillus terreus of sponge and use of aspergillus terreus for preparation of (+)- terrain. The symbiotic and epiphytic aspergillus terreus bacterial strain of the sponge is aspergillus terreus PF26CGMCC NO.5208. The step of symbiotic and epiphytic aspergillus terreus bacterial strain of the sponge for the preparation of (+)- terrain comprises a step of fermenting symbiotic and epiphytic aspergillus terreus bacterial strain of the sponge. The method concretely comprises the steps of sterilizing fermentation medium for 30 minutes at 115 DEG C; inoculating spores of aspergillus terreus PF26 into sterilized fermentation medium, wherein the inoculation amount is 4*10<6> pore/mL, and cultivating for 24 days in a shakerat 180rpm at 28 DEG C. The symbiotic and epiphytic aspergillus terreus disclosed by the invention is separated from phakellia fusca on the yongxing island on the South China Sea, and the yield of the (+)- terrain is greatly improved in comparison with the other bacterial strains at present and can be up to 3.71g/L.
Description
Technical field
The invention belongs to microbial technology field, be specifically related to grow nonparasitically upon another plant the altogether purposes of terreus and preparation (+)-terrein of a kind of silk floss.
Background technology
Terreus has been widely used in the production of fat-reducing medicament lovastatin and industrial chemicals methylene-succinic acid, but is not yet used to the production of (+)-terrein.(+)-terrein is a kind of cyclopentenone compounds, structure as shown in Figure 1, from terreus fermented liquid, separate and obtain at first, in recent years, the activity of it is found that (+)-terrein is very various, not only there is the activity of antibacterium, fungi, weeds, the secretion of all right inhibition tumor cell angiogenesis element, check melanin is synthetic, suppresses skin keratin hyperplasia, suppress pulp cells inflammatory reaction, promote scleroblast and titanium framework fusion etc.Can predict (+)-terrein has good application prospect in various fields such as medical treatment, beauty treatment, agriculturals in the future.
The technology of chemosynthesis preparation (+)-terrein often has overall yield not high, and need to adopt the extreme reaction conditionss such as High Temperature High Pressure, need expensive reaction reagent, or end product does not possess the selectivity of three-dimensional arrangement.And adopt at present the output of microorganism fermentation preparation (+)-terrein very limited, hundreds of μ g/L (Phattanawasin P, et al.Weed growth inhibitors from Aspergillus fischeri TISTR 3272.Natural Product Research 2007 at least; 21:1286-1291), hundreds of mg/L (M.Demasi, et al.Studies on (+)-terrein as a New Class of Proteasome Inhibitors.Journal of the Brazilian Chemical Society 2010 at most; 21:229-305), fail to reach the g/L magnitude that possesses productive value.
Summary of the invention
The object of the present invention is to provide grow nonparasitically upon another plant the altogether purposes of terreus and preparation (+)-terrein of a kind of sponge.The sponge of the present invention terreus of growing nonparasitically upon another plant altogether separates from South China Sea Yongxing Island Phakellia fusca sponge, and its (+)-terrein output improves a lot compared to existing other bacterial strains, can reach 3.71g/L.
The present invention realizes by following technical scheme:
The sponge of the present invention terreus bacterial strain of growing nonparasitically upon another plant is altogether that terreus (Aspergillus terreus) PF26 submits the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms on August 31st, 2011, and deposit number is CGMCC NO.5208.
This microorganism is through molecular biology 18S and ITS rDNA gene sequencing, combining form is learned and is observed, determine that its taxonomy belongs to the terreus of Aspergillus (Aspergillus) (Aspergillus terreus), strain number is PF26.
Described 18S rDNA gene order has been submitted GenBank to, sequence number is JN831364, similar sequences is terreus sequence GU227345, maximum similarity is 99%, forms Relative Fungi phylogenetic tree including bacterial strain PF26 as shown in Figure 2 take 18S rDNA gene order homology as fundamental construction.
Described ITS rDNA gene order has been submitted GenBank to, sequence number is JN831365, similar sequences is terreus sequence JN194186, maximum similarity is 100%, forms Relative Fungi phylogenetic tree including bacterial strain PF26 as shown in Figure 3 take ITS rDNA gene order homology as fundamental construction.
The qualification result of 18S rDNA gene and ITS rDNA gene coincide, similar sequences is terreus sequence, and the sequence of PF26, also all together with terreus Sequence clustering, therefore can illustrate that by molecular biological evaluation PF26 is a strain terreus on two phylogenetic trees.
The invention still further relates to above-mentioned sponge and grow nonparasitically upon another plant altogether terreus bacterial strain in the purposes of preparing in compound (+)-terrein.
Preferably, described preparation comprises the sponge step that terreus bacterial strain ferments of growing nonparasitically upon another plant altogether.
Preferably, described fermentation is specially: by fermentation culture based on 115 ℃ of sterilizing 30min; By in the described fermention medium after the spore access sterilizing of terreus PF26, inoculum size is 4 × 10
6individual spore/mL, in 28 ℃, the shaking table of 180rpm is cultivated 24 days.
Preferably, the collocation method of described fermention medium comprises the steps: to get glucose 28.41g, maltose 23.18g, N.F,USP MANNITOL 20.00g, malt extract 8.52g, Sodium Glutamate 10.00g, ammonium chloride 10.00g, be dissolved in afterwards in 1L artificial seawater, obtain described fermention medium.
Preferably, the collocation method of described artificial seawater comprises the steps: 26.518g sodium-chlor, 2.447g magnesium chloride, 3.305g magnesium sulfate, 1.141g calcium chloride, 0.725g Repone K, 0.202g sodium bicarbonate and 0.083g Sodium Bromide to be dissolved in 1L distilled water, obtain, described in the pH value of the artificial seawater for preparing be 7.0～7.2.
The beneficial effect that the present invention has is: the invention provides sponge terreus (Aspergillus terreus) the PF26CGMCC NO.5208 that grows nonparasitically upon another plant altogether; When its production for compound (+)-terrein, the output of (+)-terrein improves a lot compared to existing other bacterial strains, can reach 3.71g/L.
Accompanying drawing explanation
Fig. 1 is the structure iron of (+)-terrein;
Fig. 2 is terreus PF26 and Relative Fungi 18S rDNA gene order phylogeny tree graph;
Fig. 3 is terreus PF26 and Relative Fungi ITS rDNA gene order phylogeny tree graph;
Fig. 4 is that 400 times of terreus PF26 conidium silk amplify light microscopic photo;
Fig. 5 is the stereoscan photograph of terreus PF26 conidial head.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.These embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, for example Sambrook equimolecular clone: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
embodiment
The present embodiment is to adopt terreus (Aspergillus terreus) PF26CGMCC NO.5208 to prepare compound (+)-terrein, comprises the steps:
1, getting the sponge terreus bacterial strain of growing nonparasitically upon another plant is altogether terreus (Aspergillus terreus) PF26CGMCC NO.5208
The morphological feature of described terreus PF26 is as follows:
(a) on artificial seawater czapek's solution 28 ℃ cultivate time, bacterium colony is smooth, quality densification, bacterium colony is yellow and light brown alternate, and periphery of bacterial colonies can produce water-soluble yellow pigment, grows comparatively slow, cultivate 4 days colony diameters and reach 2.9cm, within 7 days, can reach 6.0cm;
(b) on artificial seawater PDA substratum 28 ℃ while cultivating, bacterium colony is smooth, quality densification, and bacterium colony brown, conidium is more, and periphery of bacterial colonies can produce water-soluble yellow pigment, and growth is very fast, cultivates 4 days colony diameters and reaches 5.7cm, within 7 days, can be covered with flat board;
(c) microscopic features of this bacterial strain are: mycelia has every, the top capsule semisphere of conidial head, stigma bilayer, and the first layer is short, and the second layer is long, is radial arrangement, capsule surface, distribution top 2/3, there is catenate on top, as shown in Figure 4,5;
(d) fermenting characteristic of terreus PF26 is: this bacterial strain is in the fermention medium that contains glucose 28.41g, maltose 23.18g, N.F,USP MANNITOL 20.00g, malt extract 8.52g, Sodium Glutamate 10.00g, ammonium chloride 10.00g and 1L artificial seawater, can produce (+)-terrein of single structure, when inoculum size is 4 × 10
6individual spore/mL, at 28 ℃, the shaking table of 180rpm is cultivated 24 days, and the output of (+)-terrein can reach 3.71g/L.
Artificial seawater in aforementioned, its collocation method comprises the steps: 26.518g sodium-chlor, 2.447g magnesium chloride, 3.305g magnesium sulfate, 1.141g calcium chloride, 0.725g Repone K, 0.202g sodium bicarbonate and 0.083g Sodium Bromide to be dissolved in 1L distilled water, obtain, described in the pH value of the artificial seawater for preparing be 7.0～7.2.
2, prepare fermention medium
The collocation method of described fermention medium comprises the steps: to get glucose 28.41g, maltose 23.18g, N.F,USP MANNITOL 20.00g, malt extract 8.52g, Sodium Glutamate 10.00g, ammonium chloride 10.00g, be dissolved in afterwards in 1L artificial seawater, obtain described fermention medium;
3, compound (+)-terrein is prepared in fermentation
Substratum liquid amount in 250mL triangular flask is 100mL, in 115 ℃ of sterilizing 30min; By the substratum after the spore access sterilizing of terreus PF26, making inoculum size is 4 × 10
6individual spore/mL cultivates pH nature in 28 ℃, the constant-temperature table of 180rpm; Cultivate 24 days, (+)-terrein output in fermented liquid supernatant can reach 3.71g/L.
After finishing, cultivation by fermented liquid suction filtration, separates supernatant and thalline.Supernatant is taken turns with equal-volume ethyl acetate extraction 3, and underpressure distillation is reclaimed after ethyl acetate, obtains ethyl acetate extract.This extract is through ODS post water: methyl alcohol=7: 3 (v/v) wash-outs and reversed-phase HPLC purification obtain the Primary Fermentation product of purifying.
This Primary Fermentation product is 154.1 through mass spectroscopy molecular weight, and its nuclear magnetic data is as shown in table 1, and the specific rotation light value of the aqueous solution of its 0.1% and 1% (w/v) is respectively [α]
d=+168 ° of (c=0.1, H
2and [α] O)
d=+163 ° of (c=1, H
2o), compare with data in literature, can determine that this Primary Fermentation product is (+)-terrein.
The nuclear magnetic data (400MHz, DMSO-d6) of table 1. tunning
Claims (3)
1. the sponge terreus bacterial strain of growing nonparasitically upon another plant altogether, is characterized in that, the described sponge terreus bacterial strain of growing nonparasitically upon another plant is altogether terreus (Aspergillus terreus) PF26 CGMCC NO.5208.
2. a sponge as claimed in claim 1 grows nonparasitically upon another plant terreus bacterial strain altogether in the purposes of preparing in compound (+)-terrein.
3. purposes as claimed in claim 2, is characterized in that, described preparation comprises the sponge step that terreus bacterial strain ferments of growing nonparasitically upon another plant altogether;
Described fermentation comprises the steps: fermentation culture based on 115 ℃ of sterilizing 30min; By in the described fermention medium after the spore access sterilizing of terreus PF26, inoculum size is 4 × 10
6individual spore/mL, in 28 ℃, the shaking table of 180rpm is cultivated 24 days;
The collocation method of described fermention medium comprises the steps: to get glucose 28.41g, maltose 23.18g, N.F,USP MANNITOL 20.00g, malt extract 8.52g, Sodium Glutamate 10.00g, ammonium chloride 10.00g, be dissolved in afterwards in 1L artificial seawater, obtain described fermention medium;
The collocation method of described artificial seawater comprises the steps: 26.518g sodium-chlor, 2.447g magnesium chloride, 3.305g magnesium sulfate, 1.141g calcium chloride, 0.725g Repone K, 0.202g sodium bicarbonate and 0.083g Sodium Bromide to be dissolved in 1L distilled water, obtain, described in the pH value of the artificial seawater for preparing be 7.0～7.2.
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| CN103497975B (en) * | 2013-07-02 | 2015-04-22 | 上海交通大学 | Application of vorinostat and method for promoting synthesis of (+)-Terrein by sponge-associated aspergillus terreus |
| CN103497976B (en) * | 2013-07-02 | 2015-08-19 | 上海交通大学 | To grow nonparasitically upon another plant altogether the artificial seawater of terreus fermentative production (+)-Terrein and substratum for sponge |
| CN104805147B (en) * | 2014-01-26 | 2018-01-05 | 上海医药工业研究院 | A kind of preparation method of dioxin-like compound, purification process and application |
| CN106673984B (en) * | 2015-11-11 | 2020-01-10 | 上海医药工业研究院 | (E) -2, 4-dihydroxy-6-propenylbenzaldehyde, preparation method and use thereof |
| CN112521398B (en) * | 2020-07-30 | 2022-03-15 | 上海交通大学医学院附属仁济医院 | Sponge epiphyte-derived open-loop rearrangement steroid compound and preparation method and application thereof |
| CN113846126B (en) * | 2021-10-15 | 2023-08-04 | 中国热带农业科学院热带生物技术研究所 | Preparation method and application of banana vascular wilt resistant small molecular compound |
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