CN101333509A - Rhodobacter sphaeroides mutant of coenzyme Q10 and culturing method - Google Patents

Abstract

Disclosed is a Rhodobacter sphearoides mutant of coenzyme Q10 as well as a culture method, belonging to the microbial technique field. The mutant is named as Rhodobacter sphearoides Shenzhou6, and the Collection No. is CGMCC No.2458. The GenBank accession number of the sequence of the 16S rDNA part of the mutant is EU649703. Compared with the original strain, the mutant has the greatly increased output of coenzyme Q10 when producing the coenzyme Q10 by fermentation, the output is 1.3 times that of the original strain, and the fermentation unit reaches 0.8g/L.

Description

The Rhodobacter sphaeroides mutant strain and the cultural method of Coenzyme Q10 99.0 produced in one strain

Technical field

The invention belongs to microbial technology field, relate to Rhodobacter sphaeroides mutant strain and cultural method that Coenzyme Q10 99.0 is produced in a strain, carry the Rhodobacter sphaeroides mutant strain that the mutagenesis means obtain by the space.The red bacterium of mutant strain called after class ball of the present invention (Rhodobacter sphaeroides) Shenzhou6, deposit number is CGMCC No.2458, the preservation time: on April 16th, 2008, China Committee for Culture Collection of Microorganisms common micro-organisms center.The invention still further relates to the application of described mutant strain aspect the production Coenzyme Q10 99.0.

Background technology

Coenzyme Q10 99.0 is a kind of chemical composition that extensively is present in the biological cell, has no side effect.It is the basis that the human body cell energy is made, and has the metabolic cardiotonic, definite effect.Coenzyme Q10 99.0 is mainly used in the treatment of treatment acute and chronic hepatitis, acute severe hepatitis, cardiovascular disorder and hypertension there is extraordinary curative effect, it can also be used for the complex therapy of cancer, simultaneously in unique effects in aspect such as anti-ageing, antifatigue, fat-reducing and beauty treatments.

The method of producing the Coenzyme Q10 99.0 raw material mainly contains four kinds: biological extraction method, solanesol semi-synthesis method and microbe fermentation method, wherein microbe fermentation method is acknowledged as a kind of technical matters that has advantage and potentiality most.

Microbe fermentation method prepares Coenzyme Q10 99.0 and realizes that suitability for industrialized production mainly contains the requirement of two aspects: the one, the bacterial classification of stable large-scale production high quality Coenzyme Q10 99.0 be arranged; The 2nd, high precision separate apparatus and technology be arranged.Improving constantly the amount that bacterial strain produces Coenzyme Q10 99.0, is the most effective, one of the most direct means that reduce production costs.

Summary of the invention

The purpose of this invention is to provide a strain and produce the Rhodobacter sphaeroides mutant strain and the cultural method of Coenzyme Q10 99.0, the bacterial strain that provides can improve the fermentation unit of Coenzyme Q10 99.0 greatly, and the fermentation unit of Coenzyme Q10 99.0 can reach 0.8g/L.

The present invention is with starting strain---and space flight 15 days, screening obtained a strain Rhodobacter sphaeroides mutant strain to the red bacterium SBL-11 of class ball (laboratory numbering) from recovery sample, has reached purpose of the present invention with " putting into practice No. eight " space breeding satellite recovery capsule.

The cultural method of described Rhodobacter sphaeroides mutant strain is:

(1) solid medium quality %: glucose 0.10-0.28, extractum carnis 0.10-0.45, fish peptone 0.20-1.7, yeast extract paste 0.15-0.80, sodium-chlor 0.25-0.90, agar powder 0.5-2.0, pH5.0-8.5;

(2) seed liquor quality %: glucose 1.0-3.5, fish peptone 0.5-2.0, yeast powder 0.3-1.8,, sodium-chlor 0.20-0.80, lime carbonate 0.3-0.8, pH4.7-8.8.500 milliliters of triangular flask packing, 8 layers of gauze seal sterilization;

(3) fermented liquid quality %: glucose 1.7-3.5, white sugar 1.0-3.5, corn steep liquor 1.0-6.5, ammonium sulfate 0.2-1.5, potassium primary phosphate 0.01-0.09, dipotassium hydrogen phosphate 0.04-0.08, sal epsom 0.005-0.025.500 milliliters of triangular flask packing, 8 layers of gauze seal sterilization;

(4) solid culture condition: bacterial classification inoculation was cultivated 2-7 days in 28-37 ℃ in culture dish that has solid medium or test tube;

(5) seed liquor culture condition: it is an amount of to get thalline with the aseptic inoculation ring from culture dish or inclined-plane, is inoculated in the sterilized seed liquor, and 28-40 ℃, the 120-300rpm shaking table was cultivated 6-48 hour;

(6) fermentation culture conditions: according to the 2-10% inoculative proportion, will cultivate seed liquor and be inoculated in the fermention medium, 28-40 ℃, the 150-300rpm shaking table was cultivated 3-8 days.

The red bacterium of class ball of the present invention (Rhodobacter sphaeroides Shenzhou6) has following character:

1, morphological specificity:

Gram-negative coccus mostly is monomer and exists.

2, compare with the colony characteristics of starting strain on the special solid substratum:

Bacterium colony is rediance to the red bacterium of class ball of the present invention (Rhodobacter sphaeroides space shenzhou6) on the red bacterium special culture media of class ball, and circle has projection, and surface wettability is glossy, neat in edge.

And starting strain---the red bacterium of class ball (Rhodobacter sphaeroide SBL-11) bacterium colony on the red bacterium special culture media of class ball is creamy white.

3, physiological and biochemical property

This bacterial strain is little aerobic bacteria, Gram-negative, and the catalase positive, oxidase positive, urease negative does not produce H ₂S can not reduce nitrate.

The GenBank accession number of the sequence of its 16S rDNA part is EU649703.

4, utilization of carbon source

Can be carbon source with sucrose, glucose, pectinose etc.; Can not utilize rhamnosyl, close disaccharides, arginine, ornithine, Methionin, D-wood sugar etc. to be carbon source.

5, the comparison in Coenzyme Q10 99.0 is produced

The fermented liquid of starting strain fermentation preparation of cozymase Q 10 is a light red, and the fermented liquid of the red bacterium shenzhou6 of class ball of the present invention is a brown.During from the broth extraction Coenzyme Q10 99.0 of this bacterium, the fermentation unit of Coenzyme Q10 99.0 than the raising of starting strain 30%.

Table 1 is the concrete detection content and the result of the Microbiological Characteristics of shenzhou6 of the present invention:

Table 1 Microbiological Characteristics

Annotate: "+" expression is positive, and "-" expression is negative

The method of during with mutant bacteria fermentation preparation of cozymase Q 10 of the present invention, extracting, measure Coenzyme Q10 99.0 from fermented liquid is:

In fermented liquid, add acid, can select one or more the mixture in hydrochloric acid, sulfuric acid, the phosphoric acid for use, adjust pH is to 1-5, add alkali afterwards, can select one or more the mixture in sodium hydroxide, potassium hydroxide, the ammoniacal liquor for use, make the pH value be returned to 6-8, bacterium liquid is got thalline after centrifugal, add organic solvent and mix, solvent can be selected one or more the mixture in sherwood oil, normal hexane, ethyl acetate, acetone, the ether for use.20-50 ℃, extracted 1-10 hour under the 100-200rpm condition.Get the supernatant liquor rotary evaporation after extracting solution is centrifugal to doing.Dry-matter anhydrous alcohol solution with after the evaporation carries out according to the detection method of Coenzyme Q10 99.0 in the Pharmacopoeia of People's Republic of China.

Culture presevation information:

Title: the red bacterium of class ball (Rhodobacter sphaeroides) Shenzhou6, deposit number is CGMCC No.2458.

Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center;

The preservation time: on April 16th, 2008.

Embodiment:

The breeding of Rhodobacter sphaeroides mutant strain space mutagenesis

1, putting into practice No. nine lift-launch samples prepares

Get the red bacterium SBL-11 of well-grown class ball yeast culture thing and transfer under aseptic condition and carry in the dedicated pipe, tighten the pipe lid, interface use bassoon with sealing to pack into after film seals to carry, and it is preceding in 4 ℃ of preservations to deliver lift-launch.This carries the experimental installation that pipe is put into " putting into practice No. eight " space breeding satellite, reclaims after 15 days with satellite flight in space.

2, the space environment of Da Zaiing

In this breeding satellite flight process, the related data of space environment is: the attitude of satellite is controlled to be the three axis stabilization attitude, the I quadrant directed towards ground of satellite wherein, and satellite recovery capsule microcephaly is a heading.The breeding satellite orbit is oval inclined plane, and satellite transit is on the track of 460km for the 180km altitude of the apogee at perigee altitude, and orbital inclination is 63 °, the position, perigee is near 35 ° of north latitude, satellite flight 15 days, 236 circles that fly altogether, 236 circles are by the center, recovery zone.Test result shows that space radiation dosage is 5.893mGy to the maximum during this flight test, minimum 2.484mGy.Average per daily dose is between 0.401～0.169mGy.The maximum temperature of satellite seed point for measuring temperature during rail is 20.72 ℃, and minimum temperature is 7.21 ℃.

3, space test condition setting

The space mutagenesis factor of considering in this research is microgravity and two aspects of irradiation.So the single irradiation in design space, single microgravity and three test conditionss such as irradiation and microgravity complex factors.

In the breeding satellite, lay the 1g whizzer,, build single radiation parameter to overcome the influence of space microgravity; In satellite, place pig, mask space overwhelming majority radiation dose, build single microgravity condition; Sample directly is placed in the seed jack star jar, and it is under irradiation and the microgravity compound condition.

4, the ground of the red bacterium SBL-11 of class ball mutant strain screening, separation, extraction

1), ground screening

Under aseptic condition, get culture area and be 0.5 square centimeter lift-launch recovery sample, in aseptic centrifuge tube, inhale with pipettor and to break even thalline elutriant with 5 ml sterile water wash-out thalline, get 0.5 milliliter of this bacterium liquid in 4.5 ml sterile waters, inhale and beat evenly, by that analogy, do doubling dilution.Get 10 ^-4With 10 ^-5Diluent is applied to plate culture medium, is coated with 50-250 microlitre dilution bacterium liquid on the every flat board, and each dilution gradient is coated with the polylith flat board.Treat that bacterium liquid is absorbed back 28-45 ℃ of inversion in incubator by substratum and cultivated 2-7 days.

After treating to grow bacterium colony on the flat board, select and darken or be pink colour, and bacterium colony bigger with shake flask fermentation in a small amount, carry out primary dcreening operation.The fermented liquid of primary dcreening operation bacterial strain under the 8000rpm condition centrifugal 8 minutes is got 60-80 ℃ of oven dry of precipitation, grinds the back and press mass volume ratio (1: 100) adding dehydrated alcohol, supersound process 1-2 hour.Under the treatment solution 8000rpm condition centrifugal 10 minutes, get supernatant liquor and utilize high performance liquid chromatography to detect.Select the comparison of Coenzyme Q10 99.0 content and carry out multiple sieve according to the high dark colour bacterium colony of bacterial strain.

In plate culture, obtain 328 single bacterium colonies altogether, wherein color is dark, bacterium colony is big 73.These 73 bacterium colonies are carried out the shake flask fermentation test.

2), sample preparation, extraction and mensuration

The hydrochloric acid that in each fermented liquid, adds 6mol/L respectively, adjust pH is to 2-4, add the 6mol/L sodium hydroxide solution afterwards, make the pH value be returned to 6-8, with bacterium liquid under the 8000rpm condition centrifugal 10 minutes, get thalline, add sherwood oil according to solid-liquid ratio (1: 100), break up thalline, make it and to extract solvent even.45 ℃ of shaking tables extracted 2 hours under the 180rpm condition.Under the extracting solution 8000rpm condition centrifugal 10 minutes, get supernatant liquor and on Rotary Evaporators, carry out rotary evaporation.Dry-matter anhydrous alcohol solution with after the evaporation carries out according to the detection method of Coenzyme Q10 99.0 in the Pharmacopoeia of People's Republic of China.

Claims (4)

1, the Rhodobacter sphaeroides mutant strain of Coenzyme Q10 99.0 is produced in a strain, the red bacterium of mutant strain called after class ball (Rhodobactersphaeroides) Shenzhou6, and deposit number is CGMCC No.2458.

2, the described Rhodobacter sphaeroides mutant strain of claim 1, the GenBank accession number of the sequence of its 16S rDNA part is EU649703.

3, according to the Rhodobacter sphaeroides mutant strain of the described Coenzyme Q10 99.0 of claim 1, its morphological specificity is: Gram-negative coccus mostly is monomer and exists; Bacterium colony is rediance on the red bacterium special culture media of class ball, and circle has projection, and surface wettability is glossy, neat in edge; Bacterial strain is little aerobic bacteria, Gram-negative, and the catalase positive, oxidase positive, urease negative, gala enzyme feminine gender does not produce H ₂S can not reduce nitrate; The fermented liquid of the red bacterium shenzhou6 of class ball is a brown; During from the broth extraction Coenzyme Q10 99.0 of this bacterium, the fermentation unit of Coenzyme Q10 99.0 than the raising of starting strain 30%.

4, a kind of cultural method of cultivating the described Rhodobacter sphaeroides mutant strain of claim 1, this bacterial strain carries mutagenic processes through 15 days spaces;

(1) solid medium quality %: glucose 0.10-0.28, extractum carnis 0.10-0.45, fish peptone 0.20-1.7, yeast extract paste 0.15-0.80, sodium-chlor 0.25-0.90, agar powder 0.5-2.0, pH5.0-8.5;

(2) seed liquor quality %: glucose 1.0-3.5, fish peptone 0.5-2.0, yeast powder 0.3-1.8, sodium-chlor 0.20-0.80, lime carbonate 0.3-0.8, pH4.7-8.8; 500 milliliters of triangular flask packing, 8 layers of gauze seal sterilization;

(3) fermented liquid quality %: glucose 1.7-3.5, white sugar 1.0-3.5, corn steep liquor 1.0-6.5, ammonium sulfate 0.2-1.5, potassium primary phosphate 0.01-0.09, dipotassium hydrogen phosphate 0.04-0.08, sal epsom 0.005-0.025; 500 milliliters of triangular flask packing, 8 layers of gauze seal sterilization;

(4) solid culture condition: bacterial classification inoculation was cultivated 2-7 days in 28-37 ℃ in culture dish that has solid medium or test tube;

(5) seed liquor culture condition: it is an amount of to get thalline with the aseptic inoculation ring from culture dish or inclined-plane, is inoculated in the sterilized seed liquor, and 28-40 ℃, the 120-300rpm shaking table was cultivated 6-48 hour;

(6) fermentation culture conditions: according to the 2-10% inoculative proportion, will cultivate seed liquor and be inoculated in the fermention medium, 28-40 ℃, the 150-300rpm shaking table was cultivated 3-8 days.