CN103865845B - A kind of streptomyces hygroscopicus and preparing the application in voglibose - Google Patents
A kind of streptomyces hygroscopicus and preparing the application in voglibose Download PDFInfo
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Abstract
Do you the invention discloses streptomyces hygroscopicus Moganshan Mountain mutation SW0501(Streptomyces? hygroscopicus? var.moganshanensis? SW0501) by China typical culture collection center preservation, does is preserving number CCTCC? NO.M2013676, preservation date was 2013 any December 19.The invention also discloses a kind of method preparing voglibose with this strain fermentation.Zymotechnique provided by the invention carries out testing through shaking flask, amplification and 10 tons of fermentor tanks and produces, its stable production capacity.After testing, the fermentation titer preparing Valiolamine with bacterial classification provided by the invention and fermentation culture method is high, thus provides good basis for follow-up suitability for industrialized production.Simultaneously fermentation byproduct of the present invention is relatively less, and reduces the difficulty of rear extraction, has advantage being applied in suitability for industrialized production.
Description
Technical field
The present invention relates to a kind of novel microorganism and application thereof, particularly relate to a kind of streptomyces hygroscopicus and preparing the application in voglibose, in particular, is preparing the application in voglibose intermediate Valiolamine.
Background technology
Voglibose (voglibose) is a kind of hypoglycemic agents, the mechanism of its hypoglycemic activity suppresses the activity of Disaccharide hydrolase, disaccharide lytic enzyme disaccharide being decomposed into monose can be suppressed in enteron aisle, postpone the absorption and digestion of carbohydrate at small intestine, thus level of postprandial blood sugar is reduced, simultaneously without the increase of endogenous insulin secretion.
Voglibose can be used for treating diabetes, and especially treatment type II diabetes is more suitable.Due to the mechanism of action of the uniqueness of voglibose, it be can be used alone, also can the conbined usage such as same sulfonylurea, biguanides or Regular Insulin.
The activity of voglibose is high, dosage is little, and higher to the selectivity of alpha-glucosidase, enteron aisle side effect is lower, and blood sugar reducing function is steady, do not stimulate the secretion of Regular Insulin, after the meal without the appearance of hyperinsulinemia, not easily there is hypoglycemic effect, be used alone or and other ofhypoglycemic medicine conbined usage effective equally, symptom is also comparatively light, and general patient all can tolerate.Therefore voglibose is safer oral antidiabetic drug.
The route of voglibose synthesis has multiple, and Valiolamine is an important intermediate.According to bibliographical information, can prepare except this voglibose except by total synthesis method, also have two routes that can obtain voglibose.Article one, route is fermented by validamycin producing strains, and be separated its product Validacin (Takeda), and obtain key intermediate valielamine through bio-transformation, then obtain voglibose by chemical synthesis process, this route is longer, complex process; An other route is that the application adopts, and utilizes validamycin producing strains to ferment, directly separation key intermediate valiolamine from its fermentating metabolism product, voglibose is obtained again by chemical synthesis process, route is shorter, and technique is simple, can be applicable to suitability for industrialized production.
Summary of the invention
The object of this invention is to provide a kind of can stably manufactured and the few voglibose intermediate Valiolamine producing strains of by product.
Other object of the present invention is to provide this bacterial strain and is preparing the application in voglibose Valiolamine, and concrete fermentation preparation.
The object of the invention is to be realized by following technical proposal:
A kind of streptomyces hygroscopicus Moganshan Mountain mutation SW0501, Classification And Nomenclature is Streptomyceshygroscopicusvar.moganshanensisSW0501, by China typical culture collection center preservation (being called for short CCTCC), address is: Luo Jia Shan, wuchang, wuhan Wuhan University, preserving number is: CCTCCNO.M2013676, and preservation date is: on December 19th, 2013.
Described preserving number is the bacterial strain of CCTCCNO.M2013676 is obtain from the soil screening of Suburb Areas of Hangzhou.
Described preserving number is the colony characteristics of the bacterial strain of CCTCCNO.M2013676: bacterium colony is circular, white, and edge or centre are white, and back end, in golden yellow, has soluble pigment.
According to the description of microbial morphology and external related data, be the various cultural characteristics of the bacterial strain of CCTCCNO.M2013676 in conjunction with preserving number, this preserving number is that the bacterial strain of CCTCCNO.M2013676 belongs to streptomyces, names as Streptomyceshygroscopicusvar.moganshanensisSW0501.
Described preserving number is that the bacterial strain of CCTCCNO.M2013676 can be applicable to fermentation for Valiolamine.This preparation method comprises the following steps:
A. bacterial strain adopts deposit number to be the bacterial strain of CCTCCNO.M2013676;
B. prepare inclined-plane according to a conventional method, inclined-plane digs block access shake-flask seed substratum, and 30 DEG C ~ 39 DEG C, 220 ~ 250rpm, cultivates 20 ~ 48 hours, obtain shake-flask seed liquid; By shake-flask seed liquid by the long-pending 0.1 ~ 0.2%(v/v of seed tank culture matrix) inoculum size be inoculated in seeding tank, 130 ~ 180rpm, 37 DEG C ~ 39 DEG C, cultivate 20 ~ 48 hours, obtain tank seed liquor; By the 5 ~ 10%(v/v of tank seed liquor by fermentation tank culture medium volume) inoculum size be inoculated in fermentation tank culture medium, 100 ~ 200rpm, 38 DEG C ~ 41 DEG C, 3-6 days, collect fermented liquid.
Wherein said shake-flask seed, each component of seed tank culture base ratio are in the medium: in every 100mL substratum, carbon source 1.2-2.3g, nitrogenous source 0.1-0.2g, inorganic salt 0-0.6g, and all the other are water; Be preferably in every 100mL substratum, carbon source 1.5-2.0g, nitrogenous source 0.12-0.19g, inorganic salt 0.2-0.5g, all the other are water.
The ratio of each component of fermentation tank culture medium is: in every 100mL substratum, carbon source 4.5-7.5g, nitrogenous source 0.1-0.6g, inorganic salt 0.3-0.9g, all the other are water.Be preferably in every 100mL substratum, carbon source 4.8-7.0g, nitrogenous source 0.25-0.5g, inorganic salt 0.5-0.8g, all the other are water.
Wherein said carbon source, the charging capacity of nitrogenous source are the proportion conversion in material of carbon in various material, nitrogen element.The carbon of partial material used in substratum of the present invention, nitrogen element ratio, as following table, exclude the understanding by those skilled in the art's routine in this table.
Wherein said: carbon source be selected from glucose, maltose, sucrose, flour, ground rice, Semen Maydis powder, Zulkovsky starch, starch, oatmeal, glycerine one or more; Preferred carbon source be selected from glucose, ground rice, Semen Maydis powder, starch, glycerine one or more;
Nitrogenous source is selected from one or more in analysis for soybean powder, raw bean powder, dried silkworm chrysalis meal, bean cake powder, yeast powder, yeast extract powder, fish meal, peptone; One or more in preferred analysis for soybean powder, bean cake powder, dried silkworm chrysalis meal, yeast powder, fish meal, peptone;
Inorganic salt are selected from one or more in sodium-chlor, magnesium sulfate, ferrous sulfate, potassium primary phosphate, dipotassium hydrogen phosphate, calcium carbonate, ammonium chloride, saltpetre, iron(ic) chloride, calcium chloride; One or more in preferably phosphoric acid potassium dihydrogen, calcium carbonate, ammonium chloride, calcium chloride.
The producing strains of preserving number of the present invention to be the bacterial strain of CCTCCNO.M2013676 the be a kind of Valiolamine be separated at present, leavening property is excellent.Fermenting process is the important step that Valiolamine produces, and its fermentation level is directly related with technique quality, and zymotechnique provided by the invention carries out testing through shaking flask, amplification and 10 tons of fermentor tanks and produces, its stable production capacity.After testing, the fermentation titer preparing Valiolamine with bacterial classification provided by the invention and fermentation culture method is high, thus provides good basis for follow-up suitability for industrialized production.Simultaneously fermentation byproduct of the present invention is relatively less, and reduces the difficulty of rear extraction, has advantage being applied in suitability for industrialized production.
Bacterial strain preservation situation: be preserved in Wuhan China typical culture collection center (being called for short CCTCC), deposit number is CCTCCNO.M2013676, and Classification And Nomenclature is Streptomyceshygroscopicusvar.moganshanensisSW0501.Preservation date is on December 19th, 2013.
Embodiment
Be that the present invention conducts further description below in conjunction with specific embodiment, but method involved in scheme and technical parameter can not be interpreted as limitation of the present invention.
Embodiment 1: preserving number is the cultivation of the bacterial strain of CCTCCNO.M2013676 and physiological and biochemical property and utilization of carbon source situation.
1, preserving number is the morphological feature of the strain culturing of CCTCCNO.M2013676
Be that inoculation substratum in table 1 of CCTCCNO.M2013676 is cultivated by preserving number, for observing in the substratum of morphological specificity, cultivate 2 weeks, observe and be described for 23 DEG C.
1) growth characteristics
CCTCCNO.M2013676 bacterial strain growth characteristics is on the above medium in table 1.
The cultural characteristic of table 1. bacterial strain CCTCCNO.M2013676
2) mycelia feature and Conidial Stage morphological specificity
The fibrillae of spores major part of CCTCCNO.M2013676 bacterial strain is tight spiral, and minority is flexible to curling, and spore is avette and oval, uneven, its smooth surface.
2, bacterial strain major physiological biochemical character
Utilization of carbon source:
CCTCCNO.M2013676 bacterial strain can extensively utilize the several kinds of carbon source such as glucose, wood sugar, fructose, sucrose, N.F,USP MANNITOL, pectinose, starch, and its utilization power is in table 2.
The utilization of carbon source experimental result of table 2. bacterial strain CCTCCNO.M2013676
| Carbon source | Growing state | Carbon source | Growing state |
| D-Glucose | ++ | Glycerine | ++ |
| D-sucrose | ++ | Maltose | ++ |
| D-Fructose | ++ | D-dextran | + |
| D-lactose | ++ | Sorbose | + |
| D-wood sugar | ++ | Tetrahydroxybutane | _ |
| L-arabinose | ++ | Galactitol | _ |
| D-semi-lactosi | ++ | Sorbyl alcohol+ | _ |
| L-rhamnosyl | ++ | Sodium acetate | _ |
| PEARLITOL 25C | ++ | Raffinose | ++ |
| Inositol | ++ |
Note: ++ well-grown ,+growth is general, _ do not grow or grow general
2, conclusion
Bacterial strain CCTCCNO.M2013676 is more similar with streptomyces hygroscopicus, but also there were significant differences (see table 3).Because it can lysochrome be deeply brown but not golden extremely shallow orange on Cha Shi inclined-plane, Gao Shi synthetic medium inclined-plane can lysochrome be golden yellow but not pale yellow, potato agar can lysochrome be pale yellow but not chromogenesis, therefore according to the description of microbial morphology and external related data, in conjunction with various cultural characteristic and the morphological specificity of CCTCCNO.M2013676, CCTCCNO.M2013676 is decided to be a new variant of streptomyces hygroscopicus---streptomyces hygroscopicus Moganshan Mountain mutation SW0501(Streptomyceshygroscopicusvar.moganshanensisSW0501)
Table 3. bacterial strain CCTCCNO.M2013676 bacterial strain compares with streptomyces hygroscopicus
Embodiment 2: fermentation culture prepares Valiolamine
Preserving number is adopted to be the bacterial strain of CCTCCNO.M2013676.
1, slant strains is cultivated
Solid medium: saltpetre 0.1g; Potassium primary phosphate 0.05g; Sodium-chlor 0.05g; Magnesium sulfate 0.05g; Ferrous sulfate 0.001g; Zulkovsky starch 2.0g; Peptone 0.25%; Agar 1.5g; Add water 100mL, pH7.0.
Solid culture method: inoculation, in culture medium slant, is cultivated 48 hours for 36 DEG C ~ 38 DEG C.
2, shake-flask seed is cultivated
Substratum: glucose 1.75g; Analysis for soybean powder 1.5g; Peptone 0.66g; Calcium carbonate 0.2g; Add water to 100ml, pH7.0
Liquid amount: fill 100mL substratum in 500mL triangular flask
Inoculum size: inclined-plane digs block inoculation
Culture temperature: 37 DEG C
Incubation time: 24h
Shaking speed: 220rpm
Shake-flask seed cultural method: inclined-plane lawn is dug in block access shake-flask seed substratum, treat that mycelia grow, have obvious wall cling phenomenon and bacterium dense be more than 15 ~ 20%, microscopy without miscellaneous bacteria, dyeing comparatively deeply and even, in branched.
3, seeding tank seed culture medium
Substratum: glucose 1.4Kg; Analysis for soybean powder 0.06Kg; Peptone 0.6Kg; Bubble enemy 2.5g, add water about 45L, pH adjust 7.0.
Loading amount: 121 DEG C of sterilizing 30min, about fill substratum 50L in seeding tank after sterilizing.
Seed tank culture method: amass 0.2%(v/v by seed tank culture matrix) access cultured shake-flask seed nutrient solution, 38 DEG C, 150rpm cultivates, 24h.
After cultivation, microscopy mycelia is sturdy, and dyeing is dark, in branched, without microbiological contamination, and bacterium dense 15 ~ 20%.
4, fermentor cultivation
Substratum: glucose 100Kg; Yeast powder 10Kg; Analysis for soybean powder 20Kg; Potassium primary phosphate 0.5Kg; Ammonium chloride 2.3Kg; Calcium carbonate 3Kg; Bubble enemy 0.05kg, adds water to 900L, pH7.0.
Loading amount: the in-built substratum of rear 2 tons of fermentor tanks 1 ton of sterilizing
Fermentor cultivation method: by fermentation tank culture medium volume 5%(v/v) access cultured seed tank culture liquid, 38 DEG C, 120rpm, cultivate 4d.
According to above-mentioned fermentation condition and technique, 2 tons of tanks carry out 3 batch fermentation tests, the Valiolamine fermentation titer obtained after testing is high, and fermentation byproduct is less.
Embodiment 3: fermentation culture prepares Valiolamine
Deposit number is adopted to be the bacterial strain of CCTCCNO.M2013676
1, shake-flask seed is cultivated
Substratum: glucose 1g; Analysis for soybean powder 3.4g; Glycerine 1g, add water 200mL, pH7.0.
Liquid amount: fill 100mL substratum in 500mL triangular flask
Culture temperature: 38 DEG C
Incubation time: 20h
Shaking speed: 220rpm
Shake-flask seed cultural method: by cultivate after inclined-plane scrape a little spore access seed culture medium in, treat that mycelia grows, have obvious wall cling phenomenon and bacterium dense be more than 18 ~ 22%, microscopy is without miscellaneous bacteria, and dyeing is comparatively dark and even, in branched, by shake-flask seed nutrient solution, in access seeding tank.
2, seeding tank seed culture medium
Substratum: glucose 1.8Kg; Analysis for soybean powder 3.4Kg; Glycerine 1.6Kg; Calcium carbonate 0.4Kg, add water 180L, pH7.0.
Loading amount: 121 DEG C of sterilizing 30min, about fill substratum 200L in seeding tank after sterilizing.
Seed tank culture method: amass 0.1%(v/v by seed tank culture matrix) access cultured shake-flask seed nutrient solution, 37 DEG C, 160rpm, cultivate 48h.
After cultivation, microscopy mycelia is sturdy, and dyeing is dark, and in branched, without microbiological contamination, bacterium is dense >=and 15%.
3, fermentor cultivation
Substratum: ground rice 197.5Kg; Glucose 31.25Kg; Fish meal 8.5Kg; Analysis for soybean powder 12.5Kg; Potassium primary phosphate 2Kg; Ammonium chloride 2.5Kg; Calcium carbonate 3Kg, adds water to 2300L, pH6.4-6.7.
Fermentor cultivation method: 5 tons of in-built substratum of fermentor tank 2.5 tons.By fermentation tank culture medium volume 8%(v/v) access cultured seed tank culture liquid, 150rpm37 DEG C, 5d.
Embodiment 4: fermentation culture prepares Valiolamine
Deposit number is adopted to be the bacterial strain of CCTCCNO.M2013676
1, shake-flask seed is cultivated:
Glucose 11.25g; Analysis for soybean powder 7.2g; Glycerine 5g; Calcium carbonate 3g, adds water to 500ml, pH7.0
Access from a little spore access seed culture medium that inclined-plane scrapes, 220rpm, cultivates 3d for 30 DEG C and obtains seed liquor.
2, seed tank culture:
Glucose 10.5Kg; Analysis for soybean powder 4.8Kg; Peptone 1.8Kg; Calcium carbonate 1.8Kg; Add water about 270L, pH7.0, about fills substratum 300L after sterilizing in seeding tank.0.15%(v/v is amassed by seed tank culture matrix) access cultured shake-flask seed nutrient solution, 180rpm, cultivate 40h for 38 DEG C.
3, fermentor cultivation:
Flour 50Kg; Glucose 150Kg; Semen Maydis powder 60Kg; Dried silkworm chrysalis meal 60Kg; Yeast powder 24Kg; Bean cake powder 75Kg; Ammonium chloride 12Kg%; Calcium chloride 3Kg; Potassium primary phosphate 3Kg; Calcium carbonate 9Kg, adds water to 2700L, pH7.0, and all the other are water.5 tons of in-built substratum of fermentor tank 3 tons.By fermentation tank culture medium volume 10%(v/v) access cultured seed tank culture liquid, 150rpm, cultivate about 6d for 39 DEG C.
Embodiment 5:
Deposit number is adopted to be the bacterial strain of CCTCCNO.M2013676
1, shake-flask seed is cultivated:
Containing glucose 2g in every 100mL substratum; Analysis for soybean powder 14g; Glycerine 5g; Calcium carbonate 2.5g, adds water to 500ml, pH nature.Access 5%(V/V) 30% glycerine mycelia freeze pipe, 250rpm, 39 DEG C cultivate 48h obtain seed liquor.
2, seed tank culture:
Glucose 2.5Kg; Analysis for soybean powder 14.3Kg; Glycerine 1Kg; Calcium carbonate 2.5Kg, adds water to 450L, pH7.0, about fills substratum 500L after sterilizing in seeding tank.0.1%(v/v is amassed by seed tank culture matrix) access cultured shake-flask seed nutrient solution, 180rpm, cultivate 36hr for 39 DEG C.
3, fermentor cultivation:
Glucose 750Kg; Yeast powder 140Kg; Analysis for soybean powder 200Kg; Potassium primary phosphate 10Kg; Ammonium chloride 15Kg; Calcium carbonate 15Kg, adds water to 4500L, pH7.0.
In-built substratum 5 tons after 10 tons of fermentor tank sterilizings.By fermentation tank culture medium volume 9%(v/v) access cultured seed tank culture liquid, 200rpm, cultivate 6 days for 37 DEG C.
The prepared Valiolamine obtained of above embodiment, its fermentation titer is high, and fermentation byproduct is less.
Claims (10)
1. a streptomyces hygroscopicus Moganshan Mountain mutation SW0501 (
streptomyceshygroscopicusvar.moganshanensissW0501) by China typical culture collection center preservation, preserving number is CCTCCNO.M2013676, and preservation date is on December 19th, 2013.
2. preserving number as claimed in claim 1 be the bacterial strain of CCTCCNO.M2013676 in fermentation for the application in voglibose intermediate Valiolamine.
3. application rights requires that preserving number described in 1 is the method that the strain fermentation of CCTCCNO.M2013676 prepares Valiolamine, it is characterized in that comprising the following steps:
A, fermentation strain adopts deposit number to be the bacterial strain of CCTCCNO.M2013676;
B, prepares inclined-plane according to a conventional method, and inclined-plane digs block and enters seed culture medium, 30 DEG C ~ 39 DEG C, 220 ~ 250rpm, cultivates 20 ~ 48 hours, obtains shake-flask seed liquid; The inoculum size that shake-flask seed liquid amasss 0.1 ~ 0.2% by seed tank culture matrix is inoculated in seeding tank, 130 ~ 180rpm, 37 DEG C ~ 39 DEG C, cultivates 20 ~ 48 hours, obtain tank seed liquor; By tank seed liquor by fermentation tank culture medium volume 5 ~ 10% inoculum size be inoculated in fermentation tank culture medium, 38 DEG C ~ 41 DEG C, cultivate 3-6 days, collect fermented liquid.
4. fermentation preparation as claimed in claim 3, is characterized in that:
Shake-flask seed, each component of seed tank culture base ratio are in the medium: in every 100mL substratum, carbon source 1.2-2.3g, nitrogenous source 0.1-0.2g, inorganic salt 0-0.6g, and all the other are water;
The each component of fermentation tank culture medium ratio is in the medium: in every 100mL substratum, carbon source 4.5-7.5g, nitrogenous source 0.1-0.6g, inorganic salt 0.3-0.9g, all the other are water.
5. fermentation preparation as claimed in claim 4, it is characterized in that described shake-flask seed, each component of seed tank culture base ratio is in the medium: in every 100mL substratum, carbon source 1.5-2.0g, nitrogenous source 0.12-0.19g, inorganic salt 0.2-0.5g, all the other are water.
6. fermentation preparation as claimed in claim 4, is characterized in that each component of described fermentation tank culture medium ratio is in the medium: in every 100mL substratum, carbon source 4.8-7.0g, nitrogenous source 0.25-0.5g, inorganic salt 0.5-0.8g, all the other are water.
7. the preparation method as described in claim as arbitrary in claim 4 to 6, each component of wherein said substratum is: carbon source be selected from glucose, maltose, sucrose, flour, ground rice, Semen Maydis powder, Zulkovsky starch, starch, oatmeal, glycerine one or more; Nitrogenous source is selected from one or more in analysis for soybean powder, raw bean powder, dried silkworm chrysalis meal, bean cake powder, yeast powder, yeast extract powder, fish meal, peptone; Inorganic salt are selected from one or more in sodium-chlor, magnesium sulfate, ferrous sulfate, potassium primary phosphate, dipotassium hydrogen phosphate, calcium carbonate, ammonium chloride, saltpetre, iron(ic) chloride, calcium chloride.
8. preparation method as claimed in claim 7, it is characterized in that described carbon source be selected from glucose, ground rice, Semen Maydis powder, starch, glycerine one or more.
9. preparation method as claimed in claim 7, is characterized in that one or more that described nitrogenous source is selected from analysis for soybean powder, bean cake powder, dried silkworm chrysalis meal, yeast powder, fish meal, peptone.
10. preparation method as claimed in claim 7, is characterized in that one or more that described inorganic salt are selected from potassium primary phosphate, dipotassium hydrogen phosphate, calcium carbonate, ammonium chloride, calcium chloride.
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| DE3260491D1 (en) * | 1981-04-13 | 1984-09-06 | Takeda Chemical Industries Ltd | Pseudo-aminosugars, their production and use |
| CA1289904C (en) * | 1985-04-24 | 1991-10-01 | Yukihiko Kameda | Valiolamine derivatives and production thereof |
| KR100761340B1 (en) * | 2005-02-28 | 2007-10-09 | 명지대학교 산학협력단 | - - A nucleotide sequence of validamycin biosynthesis gene cluster preparation of the Streptomyces strains producing alpha-glucosidase inhibitors transformed by the cosmid harboring the cluster and mass-production method of these inhibitors |
| CN101134976B (en) * | 2006-08-29 | 2010-12-08 | 武汉天惠生物工程有限公司 | Method for producing validamycin by circulating fermentation |
| JP5546539B2 (en) * | 2008-08-08 | 2014-07-09 | ▲無▼▲錫▼▲薬▼▲興▼▲医▼▲薬▼科技有限公司 | Substances and methods for the stereoselective synthesis of variolamines |
| CN103588650A (en) * | 2012-08-15 | 2014-02-19 | 中国医药集团总公司四川抗菌素工业研究所 | High-purity voglibose and preparation method thereof |
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