CN102864106A - Ocean catalase production bacteria and directional screening method - Google Patents
Ocean catalase production bacteria and directional screening method Download PDFInfo
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Abstract
The invention relates to a method for directionally screening catalase high-production bacterial strain by a self-creative semi-solid culture medium and ocean catalase high-production bacteria obtained by the method. The method includes that hydrogen peroxide is filled into a lower semi-solid agar layer and slowly permeates into an upper liquid culture medium layer and stably supplies a certain oxidation pressure to microbes in the upper liquid culture medium layer, and thereby the purpose that directional enrichment of the catalase high-production bacteria is achieved and the ocean catalase production bacteria have the advantages of high selectivity and high efficiency. A mobility bacillus CGMCC (China General Microbiological Culture Collection Center) No.6043 can be screened from ocean water by means of directional enrichment of the semi-solid culture medium, has the advantages of short growth period, high enzymatic activity and low cost of fermenting catalase production, and is widely applicable to aspects of the textile industry, food industry and waste water treatment and the like. Accordingly, the method for directionally screening catalase high-production bacterial strain and the catalase strain have high industrial utilization value and evident economic benefit prospect.
Description
Technical field
The invention belongs to the marine biotechnology technical field, be specifically related to a kind of ocean Catalase-Producing Strain and adopt the certainly method of the semisolid medium directed screening Catalase-Producing Strain strain of wound.
Background technology
Textile industry is China's conventional industries and mainstay industry, occupies critical role in total output value and the foreign export total value at home.Current textile industry produces seriously polluted, and especially in the dyeing and printing process process, traditional technology expends a large amount of water and chemical, and not only consumes resources also causes environmental pollution simultaneously, destroys the eubiosis.Change traditional extensive economy growth pattern of textile industry highly energy-consuming, high pollution, must substitute, transform with the green bio skilled industry textile industry process, promote the textile industry industrial upgrading, solve the problems such as the shortage of resources of textile industry process and environmental pollution from the source.
The enzyme treatment process that adopts the high-performance bio catalyzer to participate in substitutes the traditional chemical treatment process and has good economic benefit and environmental benefit.Oxygen take catalase as key problem in technology floats biological purifying process and bleachinges and dyeing a bath process is one of hot technology wherein.Fabric oxygen floats rear interpolation catalase and replaces traditional chemical reducing agent and washing processing, reaches and removes remaining H
2O
2The time can directly carry out follow-up dyeing.Utilizing catalase to decompose and float remaining hydrogen peroxide in the bath, is one of the most efficiently biological enzyme reaction of finding up to now, and its major advantage has: effect can be saved mass energy in cold water; Hydrolysate is water and oxygen, to zero environmental; Shorten the flush time between bleaching and the dyeing; An enzyme is washed the deoxidation effect that just can reach three washings behind the fabric bleaching.
Except textile industry, it is very general to adopt hydrogen peroxide to bleach in slurrying and the paper industry, and such as chlorine-free bleaching (ECF) and total chlorine free bleaching (TCF) (TCF) technique, needing catalase in the follow-up deoxyprocess is the oxygen scavenger participation of representative.In recent years hydrogen peroxide also be used to trade effluent processing in, the annual hydrogen peroxide consumption in the whole world is just with 8 ~ 10% speed increase, a large amount of high dense hydrogen peroxide waste water have destroyed the active sludge in the water treatment procedure, bring enormous pressure to water treatment, so catalase also has larger application prospect in Wastewater Pretreatment.In addition, catalase also can be used for other need to remove the hydrogen peroxide occasion safely and fast, such as: food and medical field etc.
Adopting microbial fermentation to obtain catalatic method compares with other method and has some superiority.Catalase mainly extracted from animal livers in the past, crude product is processed through pasteurization and diatomite drainage method, enzyme liquid and sample loss are large, and the virus that animal is self-contained is often felt simply helpless, and there is certain hidden danger in downstream zymin user of service health.In addition, traditional method is limited by starting material, and the enzyme demand that day by day increases also be can't bear the heavy load.Adopt microbial fermentation to obtain catalase, can utilize the cheap substrates such as stalk, starch, agar, production of enzyme is large, can turn waste into wealth, and is minimum to environmental influence.Microbial fermentation large-scale production catalase all has some superiority aspect environment and economy.
Catalase extensively is present in the aerobic microorganism, has adopted at present the strain fermentation of different genera to produce catalase abroad, commercialization of part.According to the United States Patent (USP) report, lower with the hydrogen peroxide production of enzyme that fermentation mode was obtained.The related bacterial strain of domestic fermentative Production catalase is less.The employed catalase of industrial community is having larger improvement space no matter be aspect source and the preparation method at present.
Summary of the invention
An object of the present invention is to provide a kind of energy and produce catalatic high yield bacterial strain.
Bacterial strain of the present invention is the catalase zymogenic bacteria that a strain adopts the screening of semisolid medium screening method to obtain, Classification And Nomenclature is the moving property bacillus (Planomicrobium chinense) of China, separation is from the ocean seawater sample, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number is CGMCC No.6043, No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation address (100101), preservation date is on April 24th, 2012.
Through identification of morphology and biological characteristic research, preserving number is that the bacterial strain of CGMCC No.6043 has following characteristics:
(1) colonial morphology: bacterium colony is rounded, smooth surface, and the edge is smooth, microprotrusion, yellow;
(2) cellular form: gram-positive microorganism, (Olympus, BX40) presents mobility under the microcytoscope, and form is spherical (0.8 * 1.0 μ m) in the majority;
(3) physiological and biochemical property: aerobic growth; Gelatin hydrolysate, not hydrolyzed starch, casein, tween 80 and aesculin; Can utilize glucose to produce acid, can not utilize sucrose, raffinose, xylan, lactose, pectinose, cellobiose, wood sugar, rhamnosyl, melibiose, seminose or N.F,USP MANNITOL to produce acid.
Be that the 16S rRNA gene of CGMCC No.6043 bacterial strain carries out pcr amplification and sequencing to preserving number, find that 16S rRNA Gene Partial fragment length is 569bp(Seq ID No.1), the 16S rRNA gene order similarity of moving property bacillus reference culture (P.chinense) with known China is 99%.The concrete visible sequence table of the 16S rRNA sequence of CGMCC No.6043 bacterial strain.
Therefore, content with reference to " Bergey ' s Manual of Systematic Bacteriology " second edition, according to bacterial strain limiting factor characteristics, morphological specificity and physiological and biochemical index, in conjunction with 16S rRNA gene order similarity, identify that bacterial strain CGMCC No.6043 is the moving property bacillus (P.chinense) of China.Bacterial strain CGMCC No.6043 and the moving property bacillus reference culture P.Chinense CGMCC 1.3454 of China
TCompare, the former has oxydase (oxidase) vigor, and latter's oxydase reaction is negative, and latter's activity of catalase not as good as the former 1/100.
Another object of the present invention has provided a kind ofly utilizes described CGMCC No.6043 bacterial strain to produce catalatic method.
Adopt P.chinense CGMCC No.6043 to produce catalase, for the bacterial classification that sets out, obtain catalase through seed culture and liquid fermenting with CGMCC No.6043 bacterial strain, may further comprise the steps:
(1) with CGMCC No.6043 inoculation to the natural sea-water liquid nutrient medium, adding hydrogen peroxide after cultivating stimulates and cultivates as seed liquor;
(2) seed liquor is forwarded to carries out fermentation culture in the fermention medium, then adding hydrogen peroxide stimulates and cultivates the fermented liquid that can obtain having activity of catalase.
In the aforesaid method step (1), the prescription of described natural sea-water substratum comprises the 0.1-0.5% peptone, 0.1-0.5%Casamino acid, and the 0.005-0.05% yeast extract, all the other are natural sea-water, adjusting pH is 6.8-7.5.In the preferred embodiment, the natural sea-water liquid nutrient medium comprises 0.25% peptone, 0.25%Casamino acid, and 0.01% yeast extract, all the other are natural sea-water, regulating pH is 7.2.
The microbial culture time is 8-40 hour in the step (1), preferred 20-30 hour; Culture temperature is 20-40 ℃, preferred 28-35 ℃.Cultivate the hydrogen peroxide stimulation that adds 1-10mM after finishing and cultivated 1-10 hour, the hydrogen peroxide that preferably adds 2-5mM stimulates to be cultivated 2-5 hour
In the aforesaid method step (2), described fermentative medium formula comprises: the 1-10g/L yeast extract, and the 0.5-5g/L peptone, all the other are natural sea-water, pH is 6.5-8.0; Preferred prescription comprises the 3-6g/L yeast extract, the 1-3g/L peptone, and all the other are natural sea-water, pH is 7.0-7.5; Incubation time is 8-40 hour, preferred 20-30 hour; Culture temperature is 20-40 ℃, preferred 28-35 ℃.Cultivate the hydrogen peroxide stimulation that adds 1-10mM after finishing and cultivated 1-10 hour, the hydrogen peroxide that preferably adds 2-5mM stimulates to be cultivated 2-5 hour.
Can obtain the fermented liquid that activity of catalase is 100-1000U/mL according to aforesaid method, enzyme activity is 754U/mL in a preferred embodiment.
As required, can choose wantonly the fermented liquid with enzyme activity is processed, therefrom separation and purification goes out catalase, preferred purification condition such as ion exchange chromatography, reversed phase chromatography, hydrophobic chromatography, sieve chromatography or its combination.
Another object of the present invention provides a kind of the employing from the method for creating the strain of semisolid medium directed screening Catalase-Producing Strain.The method is that hydrogen peroxide is included in the semi-solid agar of lower floor, lets alone slowly to be penetrated into the method for supernatant liquid substratum.The method continues and stably awards the certain oxidative pressure of microorganism in the supernatant liquid substratum, thereby reaches the purpose of orienting enriching Catalase-Producing Strain strain.
The method of directed screening Catalase-Producing Strain of the present invention strain comprises the steps:
(1), seawater sample is added in the semisolid medium under aseptic condition, and add carbon and the nitrogenous source of in advance filtration sterilization, jolt enrichment culture;
(2), enrichment culture liquid is carried out aseptic dilution spread, selecting mono-clonal carries out the inclined-plane and preserves;
(3), will separate the bacterial strain that obtains and be forwarded to the natural sea-water liquid nutrient medium and carry out shake flask fermentation, adopt spectrophotometry to carry out catalase activity and sieve again, finishing screen is selected the catalase relatively high bacterial strain of living.
In the aforesaid method step (1), described semisolid medium prepares by the following method: natural sea-water is adopted NaOH or HCl fine setting pH to 6.8-7.5, adding mass percent is the agar moist heat sterilization of 0.5-1%, treat that temperature is reduced to the 0.5-10mM hydrogen peroxide that adds filtration sterilization about 40-60 ℃ under aseptic condition, cooled and solidified is made semisolid medium behind the mixing.
In the preferred embodiment, described semisolid medium prepares by the following method: the 4.5mL natural sea-water is adopted NaOH or HCl fine setting pH to 7.2, added mass percent and be 121 ℃ of moist heat sterilizations of agar of 0.75% 20 minutes, treat that temperature is reduced to 30% hydrogen peroxide, the 4 μ L that add filtration sterilization about 50 ℃ under aseptic condition, cooled and solidified is made semisolid medium behind the mixing.
In the step (1), to add in the semisolid medium under aseptic condition from the seawater sample of ocean, and add carbon and the nitrogenous source of in advance filtration sterilization, its final concentration is the 0.1-0.5% peptone, 0.1-0.5%Casamino acid, the 0.005-0.05% yeast extract.In a preferred embodiment, its final concentration is 0.25% peptone, 0.25%Casamino acid, 0.01% yeast extract.25-37 ℃ (preferred 28 ℃) jolt (preferred about 4 days) about enrichment culture 2-5 days.
Enrichment culture liquid is carried out aseptic dilution spread, select mono-clonal and carry out the inclined-plane preservation.
In the aforesaid method step (3), the bacterial strain that separates acquisition is forwarded to the natural sea-water liquid nutrient medium carries out shake flask fermentation, adopt spectrophotometry to carry out catalase activity and sieve again.Wherein the natural sea-water liquid nutrient medium comprises the 0.1-0.5% peptone, 0.1-0.5%Casamino acid, and the 0.005-0.05% yeast extract, all the other are natural sea-water, adjusting pH is 6.8-7.5.In the preferred embodiment, the natural sea-water liquid nutrient medium comprises 0.25% peptone, 0.25%Casamino acid, and 0.01% yeast extract, all the other are natural sea-water, regulating pH is 7.2.
Spectrophotometry is big or small according to hydrogen peroxide light absorption value under 240nm and its concentration is linear and measure enzyme concn.Method is as follows: (1) is broken born of the same parents' processing with enchylema and is obtained crude enzyme liquid; (2) reaction system is 300 μ L, comprises crude enzyme liquid 5 μ L, phosphoric acid buffer 195 μ L, 10mM H
2O
2Solution 100 μ L substitute H with water
2O
2Solution is as blank; With H
2O
2The adding starting enzymatic reaction of solution; (3) condition determination is the 1cm cuvette, 30 ℃, weighs the enzymatic degradation speed of hydrogen peroxide with the decline of absorbancy under the 240nm; (4) read absorbance one time every 5s, stop behind the 1min measuring; (6) rate of descent of light absorption value is as the size of living according to the enzyme that calculates crude enzyme liquid.Standard enzyme unit (1U) that lives is defined as: under 30 ℃, and the per minute 1 μ mol H that degrades
2O
2(1 μ mol/min) required enzyme amount.Enzyme work is calculated: (Δ OD
240/ Δ t) * and 1000 * extension rate * reaction system volume/(43.6 * enzyme liquid is long-pending), 43.6M
-1Cm
-1Be H
2O
2Molar extinction coefficient under 240nm.
The bacterial strain that multiple sieve is obtained carries out the hydrogen peroxide stimulation, improves enzyme and lives and the enzyme amount.Bacterial strain access natural sea-water liquid nutrient medium carries out shake flask fermentation, cultivates 24h to logarithmic growth latter stage for 28 ℃.The adding final concentration is that the hydrogen peroxide of 4mM stimulates jolting after 1 hour, and it is big or small to adopt its enzyme of spectrophotometry to live.Consider that enzyme is lived and enzymatic productivity promotes amplitude, final screening obtains a strain catalase relatively high bacterial strain of living, and for example the fermentation broth enzyme vigor reaches 100U/mL and may be defined as the relatively high bacterial strain of activity, and the preferred enzyme vigor reaches the above person of 500U/mL.CGMCC No.6043 bacterial strain namely obtains by the above-mentioned steps screening, and enzyme activity can reach 754U/mL in the fermented liquid.
The invention provides a kind of method and employing the method from creating the strain of semisolid medium directed screening Catalase-Producing Strain and obtain a kind of ocean Catalase-Producing Strain CGMCC No.6043 bacterial strain.Bacterial screening method of the present invention is compared with other conventional screening methods, has highly selective and high efficiency characteristics.The P.chinense CGMCC No.6043 Catalase-Producing Strain growth cycle that the present invention finds is short, and enzymic activity is high, and is with low cost, is fit to be widely used in the aspects such as foodstuffs industry and Industrial Wastewater Treatment.Therefore method of the present invention and enzymatic production bacterial strain have widely industrial application value and significant economic benefit prospect.
Embodiment
Embodiment 1 ordinary method, liquid nutrient medium screening method and semisolid medium screening method
Gather seawater sample, coat natural sea-water oligotrophic culture medium flat plate after getting 150 μ L gradient dilutions, cultivate 7 days left and right sides observationss for 28 ℃, picking list bacterium colony carries out the inclined-plane and preserves.Utilize 3% hydrogen peroxide Bubbling method preliminary screening catalase activity bacterial strain.Natural sea-water oligotrophic substratum is the 0.5g/L yeast extract, and 0.1g/L peptone, pH are 7.2, and all the other are natural sea-water.
Getting the 4.5mL seawater sample adds in the aseptic old seawater of 4.5mL, under aseptic condition, add successively 30% hydrogen peroxide, the 4 μ L of filtration sterilization and the carbon and nitrogen sources of in advance filtration sterilization (final concentration is 0.25% peptone, 0.25%Casamino acid and 0.01% yeast extract).28 ℃ of concussion enrichment culture were carried out aseptic dilution suitable multiple to pregnant solution about 4 days, and spread plate is selected mono-clonal and carried out the inclined-plane preservation.
Get the 4.5mL seawater sample and add in the semisolid medium under aseptic condition, and add the carbon and nitrogen sources of in advance filtration sterilization, final concentration is 0.25% peptone, 0.25%Casamino acid and 0.01% yeast extract.28 ℃ of concussion enrichment culture were carried out aseptic dilution suitable multiple to pregnant solution about 4 days, and spread plate is selected mono-clonal and carried out the inclined-plane preservation.
The bacterial strain that adopts above-mentioned three kinds of methods screening to obtain is forwarded to the natural sea-water liquid nutrient medium and carries out shake flask fermentation, adopts spectrophotometry to carry out catalase activity and sieves again.
Adopt semisolid medium to provide the directed screening method that continues oxidative pressure most effective.Multiple sieve obtains some catalase high reactivity bacterial strains, is semisolid medium directed screening method and obtains.Adopt liquid nutrient medium to provide the screening method efficient of direct oxidation pressure to take second place, the method efficient that adopts common minute bacterium and hydrogen peroxide primary dcreening operation to combine is extremely low.
The separation screening of moving property bacillus (P.chinense) the CGMCC No.6043 of embodiment 2 China
Gather seawater sample, add the 4.5mL seawater sample under the aseptic condition in the semisolid medium, and the carbon and nitrogen sources of in advance filtration sterilization, final concentration is 0.25% peptone, 0.25%Casamino acid and 0.01% yeast extract.28 ℃ of concussion enrichment culture were carried out aseptic dilution suitable multiple to pregnant solution about 4 days, and spread plate is selected mono-clonal and carried out the inclined-plane preservation.
The bacterial strain that the aforesaid method separation obtains is forwarded to the natural sea-water liquid nutrient medium and carries out shake flask fermentation, adopts spectrophotometry to carry out catalase activity and sieves again.The natural sea-water substratum is: aseptic old seawater, final concentration are 0.25% peptone, 0.25%Casamino acid and 0.01% yeast extract, and pH 7.2.
The high catalase activity bacterial strain that separation is obtained carries out the optimization of hydrogen peroxide stimulus method.Inoculation to natural sea-water liquid nutrient medium carries out shake flask fermentation, cultivates 24h to logarithmic growth latter stage for 28 ℃.Adding final concentration is the hydrogen peroxide of 4mM, stimulates after the jolting 1h, with the size of its enzyme of spectrophotometry ratio alive.Final screening obtains the relatively high bacterial strain of a strain activity of catalase, is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and culture presevation number is CGMCC No.6043.
Identification of morphology and the biological characteristics of moving property bacillus (P.chinense) the CGMCC No.6043 of embodiment 3 China
Moving property bacillus (P.chinense) the CGMCC No.6043 of China is inoculated in the natural sea-water liquid nutrient medium.The preparation solid medium can add the agar of 15g/L again.121 ℃ of moist heat sterilizations 20 minutes.After the inoculation culture, through evaluation, this bacterium has following characteristics: (1) colonial morphology: bacterium colony is rounded, smooth surface, and the edge is smooth, microprotrusion, yellow.(2) cellular form: gram-positive microorganism, (Olympus, BX40) presents mobility under the microcytoscope, and form is spherical (0.8 * 1.0 μ m) in the majority.(3) physiological and biochemical property: aerobic growth; Gelatin hydrolysate, not hydrolyzed starch, casein, POLYSORBATE 80 and aesculin; Can utilize glucose to produce acid, can not utilize sucrose, raffinose, xylan, lactose, pectinose, cellobiose, wood sugar, rhamnosyl, melibiose, seminose or N.F,USP MANNITOL to produce acid.
Pcr amplification and the sequencing of the 16S rRNA gene of moving property bacillus (P.chinense) the CGMCC No.6043 of embodiment 4 China
Moving property bacillus (P.chinense) the CGMCC No.6043 of China is inoculated in the natural sea-water solid medium, direct picking one ring thalline from the inclined-plane adds 400 μ L sterilized water mixings, 100 ℃ of water-baths 5 minutes, centrifugal 2 minutes of 12000rpm, supernatant is directly used in PCR.The a pair of universal primer of amplification 16S rRNA gene is as follows:
Forward primer: 5'-AGAGTTTGATCCTGGCTCAG-3';
Reverse primer: 5'-GGTTACCTTGTTACGACTT-3';
Above-mentioned primer is 8-27 and the 1510-1492 bit base of corresponding colibacillary 16S rRNA gene respectively.PCR reaction system (50 μ L) is: 10 * buffer, 5 μ L, 10mM dNTPs1 μ L, each 1 μ L of 4mM primer, sterile pure water 42 μ L, Taq enzyme 0.5 μ L, template DNA 0.5 μ L.The PCR reaction conditions is: 94 ℃ of sex change 45s; 55 ℃ of annealing 45s; 72 ℃ are extended 90s, 30 rear 72 ℃ of extension 10min of circulation.PCR product purification and sequencing are finished by Sinogenomax Co., Ltd..The 16S rRNA Gene Partial fragment length of finishing mensuration is 569bp, with bacterial strain P.chinense JCM 12466
T16S rRNA gene order similarity be 99%.The concrete visible sequence table of the 16S rRNA sequence of bacterial strain P.chinense CGMCC No.6043.
Therefore, with reference to " Bergey ' s Manual of Systematic Bacteriology " the second edition content, limiting factor characteristics, morphological specificity and physiological and biochemical index according to bacterial strain, in conjunction with 16S rRNA gene order similarity, identify that CGMCC No.6043 is the moving property bacillus (P.chinense) of China.
Bacterial strain CGMCC No.6043 and the moving property bacillus reference culture P.Chinense CGMCC 1.3454 of China
TCompare, the former has oxydase (oxidase) vigor, and latter's oxydase reaction is negative, latter's activity of catalase not as good as the former 1/100.
Adopt hydrogen peroxide to stimulate in embodiment 5 fermenting processs and increase the catalase expression amount
Moving property bacillus (P.chinense) the CGMCC No.6043 of China is seeded to the natural sea-water liquid nutrient medium, culture condition is 28 ℃ and cultivates 24h as seed liquor, switching enters to contain in the natural sea-water liquid nutrient medium of 0,0.25,0.5,1.0 and 2.0 hydrogen peroxide and cultivates respectively, determines that the highest concentration of hydrogen peroxide that bacterial strain can be grown is 1mM.
The P.Chinense CGMCC No.6043 bacterium liquid of growing under the 1mM concentration of hydrogen peroxide is forwarded to the 250mL triangular flask that the 50mL fermention medium is housed carries out fermentation culture, incubation time 24h is to logarithmic growth latter stage, 28 ℃ of culture temperature, shaking flask rotating speed 200r/min.The hydrogen peroxide that adds respectively sterile pure water and 4mM after cultivate finishing stimulates cultivates the mensuration of carrying out catalase activity behind the 1h.
Living without the fermentation broth enzyme of hydrogen peroxide stimulation is 596U/mL(936U/mg), stimulate the fermentation broth enzyme work that obtains to reach 754U/mL(1185U/mg through hydrogen peroxide).It is more remarkable to utilize hydrogen peroxide to stimulate to improve the method effect of bacterial strain catalase activity, and the enzyme activity increase rate reaches 26.5%(249U/mg).
Fermentative medium formula is as follows: the 5g/L yeast extract, and the 1g/L peptone, all the other are natural old seawater, pH is 7.2.
Embodiment 6 fermention mediums and time-optimized
Moving property bacillus (P.chinense) the CGMCC No.6043 of China is seeded to the natural sea-water liquid nutrient medium, cultivates 24h for 28 ℃ and reaches logarithmic growth latter stage.The hydrogen peroxide that adds 4mM stimulates cultivates 2h as seed liquor.The 250mL triangular flask that is forwarded to dress 50mL fermention medium carries out fermentation culture.Culture condition is pH 7.2, and incubation time is 12,24 and 48h, 28 ℃ of culture temperature, shaking flask rotating speed 200r/min.The hydrogen peroxide that adds 4mM stimulates cultivates 1h.Fermention medium has three kinds: (1) HS fermention medium: the 5g/L yeast extract, and the 1g/L peptone, all the other are natural old seawater, pH 7.2.(2) NDJ substratum: 0.5% extractum carnis, 1% peptone, 0.5% yeast extract, 2.3% sodium-chlor, pH 7.2.(3) GP substratum: 2% casein food grade, 4% glucose, 0.1% potassium primary phosphate, 0.01% sal epsom, 2.3% sodium-chlor, pH 7.2.By the thalline that three kinds of different fermentations substratum and time cultivation obtain, behind broken born of the same parents, measure the catalase enzyme activity, concrete outcome sees Table 1.
The activity of catalase that table 1 fermention medium and fermentation time optimization are measured afterwards
Moving property bacillus (P.chinense) the CGMCC No.6043 of China is through HS substratum fermentation culture 12-36h, and its activity of catalase value variation presents the two ends height, and middle low, numerical value changes inapparent characteristics relatively; Mean value is 369.7U/ml.In contrast, through NDJ substratum fermentation culture 12-36h, it is low that the variation of P.Chinense CGMCC No.6043 activity of catalase value presents two ends, middle high characteristics; Maximum value appears at cultivates 24h, reaches 748.6U/ml.Through GP substratum fermentation culture, P.Chinense CGMCC No.6043 activity of catalase value presents the trend that enzyme is lived and risen gradually with the increase of yeast culture time, reaches maximum value (878.0U/ml) behind yeast culture 36h.In view of fermentation costs and production efficiency, moving property bacillus (P.chinense) the CGMCC No.6043 catalase fermentative production of China is preferentially selected NDJ culture medium culturing 24h, is GP culture medium culturing 36h secondly, is HS culture medium culturing 12h again.
Sequence table
Claims (10)
1. catalase zymogenic bacteria, Classification And Nomenclature are the moving property bacillus (Planomicrobium chinense) of China, and deposit number is CGMCC No.6043.
2. catalase zymogenic bacteria according to claim 1, it is characterized in that: described bacterial strain has following characteristics:
(1) colonial morphology: bacterium colony is rounded, smooth surface, and the edge is smooth, microprotrusion, yellow;
(2) cellular form: present mobility under the gram-positive microorganism, microcytoscope, form is spherical (0.8 * 1.0 μ m) in the majority;
(3) physiological and biochemical property: aerobic growth; Gelatin hydrolysate, not hydrolyzed starch, casein, tween 80 and aesculin; Can utilize glucose to produce acid, can not utilize sucrose, raffinose, xylan, lactose, pectinose, cellobiose, wood sugar, rhamnosyl, melibiose, seminose or N.F,USP MANNITOL to produce acid.
3. catalase zymogenic bacteria according to claim 1, it is characterized in that: the 16S rRNA gene of described bacterial strain comprises the nucleotide sequence shown in Seq ID No.1.
4. one kind is utilized bacterial strain claimed in claim 1 to produce catalatic method, may further comprise the steps:
(1) with CGMCC No.6043 inoculation to the natural sea-water liquid nutrient medium, adding hydrogen peroxide after cultivating stimulates and cultivates as seed liquor;
(2) seed liquor is forwarded to carries out fermentation culture in the fermention medium, then adding hydrogen peroxide stimulates and cultivates the fermented liquid that can obtain having activity of catalase.
5. method according to claim 4, it is characterized in that: the prescription of the natural sea-water substratum in the described method steps (1) comprises the 0.1-0.5% peptone, 0.1-0.5%Casamino acid, 0.005-0.05% yeast extract, all the other are natural sea-water, and adjusting pH is 6.8-7.5.
6. method according to claim 4, it is characterized in that: the fermentative medium formula in the described method steps (2) comprises the 1-10g/L yeast extract, the 0.5-5g/L peptone, all the other are natural sea-water, pH is 6.5-8.0.
7. each described method according to claim 4-6, it is characterized in that: as required, can choose wantonly the fermented liquid with enzyme activity is processed, therefrom separation and purification goes out catalatic product.
8. the method for directed screening Catalase-Producing Strain strain comprises the steps:
(1), seawater sample is added in the semisolid medium under aseptic condition, and add carbon and the nitrogenous source of in advance filtration sterilization, jolt enrichment culture;
(2), enrichment culture liquid is carried out aseptic dilution spread, selecting mono-clonal carries out the inclined-plane and preserves;
(3), will separate the bacterial strain that obtains and be forwarded to the natural sea-water liquid nutrient medium and carry out shake flask fermentation, adopt spectrophotometry to carry out catalase activity and sieve again, finishing screen is selected the catalase relatively high bacterial strain of living.
9. method according to claim 8, it is characterized in that: semisolid medium prepares by the following method in the described method steps (1): natural sea-water is adopted NaOH or HCl fine setting pH to 6.8-7.5, adding mass percent is the agar moist heat sterilization of 0.5-1%, treat that temperature is reduced to the 0.5-10mM hydrogen peroxide that adds filtration sterilization about 40-60 ℃ under aseptic condition, cooled and solidified is made semisolid medium behind the mixing.
10. method according to claim 9, it is characterized in that: in the described method steps (3), the natural sea-water liquid nutrient medium comprises the 0.1-0.5% peptone, 0.1-0.5%Casamino acid, the 0.005-0.05% yeast extract, all the other are natural sea-water, adjusting pH is 6.8-7.5.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103060244A (en) * | 2013-01-21 | 2013-04-24 | 国家海洋局第二海洋研究所 | Bacillus marinus and method for producing catalase by using same |
| CN104513800A (en) * | 2013-10-08 | 2015-04-15 | 镇江拜因诺生物科技有限公司 | Screening identification method for difficultly-cultured marine microorganisms |
| CN103789225A (en) * | 2013-11-08 | 2014-05-14 | 浙江大学 | Marine catalase production strain and method for producing catalase from strain |
| CN105483061A (en) * | 2016-01-20 | 2016-04-13 | 福建省农业科学院土壤肥料研究所 | Ureibacillus thermosphaericus and method for producing high-temperature catalase from same |
| CN105483061B (en) * | 2016-01-20 | 2019-04-12 | 福建省农业科学院土壤肥料研究所 | A method of solution urea bacillus and its production high temperature catalase |
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