CN108504606A

CN108504606A - A kind of unwrapping wire bacterium culture medium and its application

Info

Publication number: CN108504606A
Application number: CN201810375966.1A
Authority: CN
Inventors: 窦同意; 陈静; 刘成林
Original assignee: Dalian University of Technology
Current assignee: Dalian University of Technology
Priority date: 2018-04-20
Filing date: 2018-04-20
Publication date: 2018-09-07

Abstract

The invention belongs to industrial biotechnology field, a kind of unwrapping wire bacterium culture medium and its application are provided, the ultimate constituent of unwrapping wire bacterium culture medium is yeast extract, peptone, Ca²⁺、Zn²⁺、Cu²⁺、Fe²⁺And carbon source, the mass ratio of above each ingredient is 2~10：1~8：0.01~0.2：0.002~0.03：0.0002~0.003：2~14, distilled water dissolved dilution controls a concentration of 2~12g/L of yeast extract.Promicromonospora section and fiber monospore Cordycepps bacterial strain of the unwrapping wire bacterium culture medium for culture, generate cellulosome analog, one of which enzyme has 7 xylose glycosidic bond activity of hydrolysis paclitaxel precursor object DAXP C, and producing enzyme efficiency is relatively high, the route of synthesis of taxol can be greatly shortened, cost is reduced, environmental pollution etc. is mitigated, is excellent enzyme preparation prepared by industrial microorganism conversion medicine intermediate.

Description

A kind of unwrapping wire bacterium culture medium and its application

Technical field

The present invention relates to a kind of unwrapping wire bacterium culture medium and its applications, belong to industrial biotechnology field.

Background technology

Actinomyces are a kind of microorganisms with Important Economic value and multiple use.It is found from microorganism at present In 8000 multiple-microorganism activity goods and materials, nearly 70% is that actinomyces generate, and many of which is widely used to clinic.With evidence Commentary of the World Health Organization (WHO) about cancer, cancer has become the world's second largest cause of the death, so anticancer drug is opened Hair seems very urgent with production.

Now, there are about 60% anticancer preparation be originated from natural prodcuts, more than 30 kinds be derived from natural product compound by with It is studied in the clinical treatment of various different phase cancers.Taxol (Taxol) class compound is existing, classic, efficient One of less toxic broad-spectrum anti-cancer drug is that the first is recognized in clinical test, and treats kinds of tumors disease by chemotherapy means Bearing taxanes.But taxol belongs to secondary metabolite, synthetic quantity is few, much can not meet clinical needs. Some scholars carry out it chemically synthesized research, but complex synthetic route, and reaction condition is difficult to control, and synthetic ratio is low. Therefore, it focuses mostly in the semi-synthesis method of taxol, i.e., is extracted in taxanes in Japanese yew branches and leaves first in subsequent research Between product then obtain taxol using chemical synthesis if 10- removes acetyl Bakating III.But due to the structure of 10-DAB There is very big difference (lacking entire side chain and an acetyl group) with Paclitaxel, causes longer (including the side chain of synthetic route Synthesis), a large amount of activating agents used, there is also potential environmental threats while increasing cost.It is grown in China Chinese yew, 10- goes the content of acetyl -7- xylosides taxol (DAXP, i.e. 10-DAXT) very high, and in european yew The content of 10-DAB is suitable, and the speed of growth is also very fast, and biomass accumulation speed is nearly 2 times of european yew, is China A resource having a high potential in taxol renewable resource.DAXP only needs after removing C-7 xylosyls, C-10 acetylations Obtain taxol.In contrast, the synthetic route of DAXP will save a large amount of active chemical reagent and organic solvent, and significantly Reduce economic cost and the harm to environment.

DAXP moves towards the bottleneck i.e. removal of C-7 xylosyls of taxol.The reaction rate of chemical conversion process is very good, but It is that specificity can be weaker, causes also to have a large amount of by-product generation while wastage of material.And biotransformation method not only has There are good specificity, transformation efficiency also relatively high.This seminar researcher has found Promicromonospora section and fiber monospore Cordycepps bacterial strain can generate a variety of enzymatic activitys under given conditions, including the born of the same parents of effectively hydrolyzing DAXP C-7 xylose glycosidic bonds Outer xylobiase activity.Since the generation of the secondary metabolite of microorganism is extremely sensitive for environmental condition, same bacterial strain There may be completely different activity under different environmental conditions, and which results in common actinomycete fermentation medium cultures Bacterial strain do not have hydrolysis DAXP C-7 xylose glycosidic bonds activity.So need to be improved to unwrapping wire bacterium culture medium, bacterial strain is made to produce The activity of unboiled water solution DAXP C-7 xylose glycosidic bonds, and there is higher hydrolysing activity.

Invention content

In order to overcome the above problem existing for prior art, the object of the present invention is to provide a kind of trainings of actinomyces Based formulas is supported, Promicromonospora section is solved and fiber monospore Cordycepps bacterial strain hydrolysis DAXP C-7 xyloses glycosidic bond activity is relatively low Problem.

Technical scheme of the present invention：

A kind of unwrapping wire bacterium culture medium, steps are as follows：

The ultimate constituent of unwrapping wire bacterium culture medium is yeast extract, peptone, Ca²⁺、Zn²⁺、Cu²⁺、Fe²⁺And carbon source, with The mass ratio of upper each ingredient is 2~10：1~8：0.01~0.2：0.002~0.03：0.0002~0.003：2~14, distilled water Dissolved dilution controls a concentration of 2~12g/L of yeast extract.

Further include Mn in the basic composition of unwrapping wire bacterium culture medium²⁺And Na⁺, control Mn²⁺、Na⁺And Ca²⁺Mass ratio be 0.002~0.02：0.03~0.4：0.01~0.2.

The carbon source is the mixing of one or more of cellulose, hemicellulose, lignin.

Promicromonospora section and fiber monospore Cordycepps bacterial strain of the unwrapping wire bacterium culture medium for culture, it is similar to generate cellulosome Object, one of which enzyme have hydrolysis paclitaxel precursor object DAXP C-7 xyloses glycosidic bond activity.

Beneficial effects of the present invention：Actinomyces culture medium prescription provided by the invention is small using the original of the medium culture Monospore Cordycepps and fiber monospore Cordycepps bacterial strain can generate cellulosome analog, which has a variety of enzyme activity The features such as property, nanoscale molecular amount, monolithic stability.One of which enzyme has hydrolysis paclitaxel precursor object DAXP C-7 xylose glycosidic bonds Activity, and producing enzyme efficiency is relatively high, can greatly shorten the route of synthesis of taxol, reduces cost, mitigates environmental pollution Deng, be industrial microorganism conversion medicine intermediate prepare excellent enzyme preparation.

Description of the drawings

Fig. 1 is different metal ions to the active effects of strain F16；A is HPLC-UV result figures；B is Native-PAGE Result figure.

Fig. 2 is various concentration metal ion on the active influences of strain F16；A is various concentration Cu²⁺To the shadow of bacterial activity It rings；B is various concentration Ca²⁺Influence to bacterial activity.

Fig. 3 is various concentration influence Native-PAGE result figures active on strain F16.

Specific implementation mode

Below in conjunction with attached drawing and technical solution, the specific implementation mode that further illustrates the present invention.

Reagent, bacterium and the measuring instrument that the present invention uses：Yeast extract, peptone, dipotassium hydrogen phosphate, microcrystalline cellulose Element, CaCl₂、ZnSO₄·7H₂O、CuSO₄·5H₂O、FeSO₄·7H₂O、MnSO₄·4H₂O、Na₂SO₄、MgSO₄·7H₂O；F16； High performance liquid chromatograph (China spectrum S6000).

Embodiment 1

(1) culture medium is prepared：1. 10g yeast extracts, 4g peptones, 4g dipotassium hydrogen phosphates, 14g microcrystalline celluloses, distilled water Add to 1000ml；2. each ingredient in formula is uniformly mixed, pH to 7.0 is adjusted, culture medium is dispensed into 25mL to 100mL conical flasks In, it is separately added into 2mL 2.5g/L CaCl₂、1g/L ZnSO₄·7H₂O、0.25g/L CuSO₄·5H₂O、5g/L FeSO₄· 7H₂O、1g/L MnSO₄·4H₂O、50g/L Na₂SO₄、12.5g/LMgSO₄·7H₂O、Mixture；3. the culture that will have been dispensed Base autoclave sterilization 20min, 4 DEG C save backup.

(2) experimental implementation：1. every bottle of culture medium adds 10 μ L strains F16,30 DEG C of 150rpm/min to cultivate 48h；2. culture knot Bacterium solution after beam takes supernatant to carry out active reaction (reaction system：158 μ L Buffer (pH 7.5, Tris-HCl), 2 μ L CaCl2 (1M), 30 μ L Sample, 10 μ L DAXP；Reaction condition：The metal bath of 30 DEG C of 200rpm/min reacts 2h；After reaction, 400 μ L methanol are added and terminate reaction)；3. taking Sample supernatants after reaction, using HPLC-UV methods, (HPLC-UV methods are as follows Shown in table) and Native-PAGE (Maeker selects 5% BSA) measurement enzymatic activity.

Table HPLC-UV methods

(3) experimental result：Two kinds of methods of inspection show that the strain through above-mentioned medium culture all has different degrees of Inulinase-producing activity, experimental result are as shown in Figure 1.The visible each metal ions of Fig. 1-a all have an impact strain inulinase-producing activity.Fig. 1-b can See, in each metal ion, Ca²⁺、Zn²⁺、Cu²⁺、Mg²⁺And each trace element mixture is more apparent on the influence of strain inulinase-producing activity, Mn²⁺And Na⁺It influences relatively small.

Embodiment 2

(1) culture medium is prepared：1. 10g yeast extracts, 4g peptones, 4g dipotassium hydrogen phosphates, 14g microcrystalline celluloses, distilled water Add to 1000ml；2. each ingredient in formula is uniformly mixed, pH to 7.0 is adjusted, is dispensed in 25mL to 100mL conical flasks, respectively 2mL 0g/L, 0.001g/L, 0.005g/L, 0.01g/L, 0.05g/L, 0.1g/L, 0.5g/L, 1g/L, 5g/L, 10g/L is added CuSO₄·5H₂O solution；3. the culture medium autoclave sterilization 20min that will have been dispensed, 4 DEG C save backup.

(2) experimental implementation：1. every bottle of culture medium adds 10 μ L strains F16,30 DEG C of 150rpm/min to cultivate 48h；2. culture knot Bacterium solution after beam takes supernatant to carry out active reaction (reaction system：158 μ L Buffer (pH 7.5, Tris-HCl), 2 μ L CaCl₂ (1M), 30 μ L Sample, 10 μ L DAXP；Reaction condition：The metal bath of 30 DEG C of 200rpm/min reacts 2h；After reaction, 400 μ L methanol are added and terminate reaction)；3. taking Sample supernatants after reaction, enzymatic activity (HPLC- is measured using HPLC-UV methods In UV methods such as embodiment 1 shown in table).

(3) experimental result：There is different inulinase-producing activities, experimental result using the bacterial strain of various concentration metal ion culture As shown in Figure 2.Fig. 2-a and Fig. 2-b are as it can be seen that with Cu²⁺And Ca²⁺The increase of concentration, inulinase-producing activity gradually increase, when concentration increases After adding to a certain concentration, inulinase-producing activity starts to reduce.Fig. 3 is as it can be seen that work as Cu²⁺After concentration increase to a certain extent, strain finally will Lose inulinase-producing activity.In summary, the inulinase-producing activity of strain can increase as the concentration of metal ion increases, when increasing to one After determining degree, activity starts to reduce, until losing activity, that is to say, that Cu²⁺Concentration is too low or excessively high can all influence inulinase-producing activity.

Claims

1. a kind of unwrapping wire bacterium culture medium, which is characterized in that steps are as follows：

The ultimate constituent of unwrapping wire bacterium culture medium is yeast extract, peptone, Ca²⁺、Zn²⁺、Cu²⁺、Fe²⁺And carbon source, it is above each The mass ratio of ingredient is 2~10：1~8：0.01~0.2：0.002~0.03：0.0002~0.003：2~14, distill water dissolution Dilution, controls a concentration of 2~12g/L of yeast extract.

2. unwrapping wire bacterium culture medium according to claim 1, which is characterized in that the basic composition of the unwrapping wire bacterium culture medium In further include Mn²⁺And Na⁺, control Mn²⁺、Na⁺And Ca²⁺Mass ratio be 0.002~0.02：0.03~0.4：0.01~0.2.

3. unwrapping wire bacterium culture medium according to claim 1 or 2, which is characterized in that the carbon source is cellulose, hemicellulose The mixing of one or more of element, lignin.

4. Promicromonospora section and fiber monospore Cordycepps bacterial strain of a kind of unwrapping wire bacterium culture medium for culture, generate cellulosome class Like object.