CN102864106B - 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 marine biotechnology technical field, be specifically related to a kind of ocean Catalase-Producing Strain and adopt the method from 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 at home in total output value and foreign export total value.Current textile industry produces seriously polluted, and especially, in 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 with green bio skilled industry, transform textile industry process, promote textile industry industrial upgrading, from source, solve the problems such as the shortage of resources of textile industry process and environmental pollution.
The alternative traditional chemical treatment process of enzyme treatment process that adopts high-performance bio catalyzer to participate in has good economic benefit and environmental benefit.The oxygen that the catalase of take is key problem in technology floats biological purifying process and floats dyeing one-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
2time can directly carry out follow-up dyeing.Utilizing catalase to decompose and float remaining hydrogen peroxide in bath, is one of reaction of biological enzyme the most efficiently of finding up to now, and its major advantage has: in cold water, effect can be saved mass energy; Hydrolysate is water and oxygen, to zero environmental; Shorten the flush time between bleaching and dyeing; After fabric bleaching, an enzyme is washed the deoxidation effect that just can reach three washings.
Except textile industry, in slurrying and paper industry, adopt hydrogen peroxide to bleach very general, as chlorine-free bleaching (ECF) and total chlorine free bleaching (TCF) (TCF) technique, in follow-up deoxyprocess, needing catalase is the oxygen scavenger participation of representative.In recent years hydrogen peroxide be also 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 has destroyed the active sludge in water treatment procedure, to water treatment, bring enormous pressure, so catalase also has larger application prospect in Wastewater Pretreatment.In addition, catalase also can be used for other need to remove hydrogen peroxide occasion safely and fast, such as: food and medical field etc.
Adopting microorganism fermentation to obtain catalatic method compares and has some superiority with other method.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 downstream zymin user of service health is existed to certain hidden danger.In addition, traditional method is limited by starting material, and the enzyme demand day by day increasing also be can't bear the heavy load.Adopt microorganism fermentation to obtain catalase, can utilize the cheap substrates such as stalk, starch, agar, production of enzyme is large, can turn waste into wealth, minimum to environmental influence.Microorganism fermentation large-scale production catalase all has some superiority aspect environment and economy.
Catalase is extensively present in aerobic microorganism, has adopted at present the strain fermentation of different genera to produce catalase abroad, commercialization of part.According to 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 catalase that industrial community is used at present, is having larger improvement space no matter be aspect source and preparation method.
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, separated from ocean seawater sample, be preserved in China Committee for Culture Collection of Microorganisms's 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, edge is smooth, microprotrusion, yellow;
(2) cellular form: gram-positive microorganism, under microcytoscope, (Olympus, BX40) presents mobility, 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.
The 16S rRNA gene that is CGMCC No.6043 bacterial strain to preserving number carries out pcr amplification and sequencing, 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 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 feature, morphological specificity and physiological and biochemical index, in conjunction with 16S rRNA gene order similarity, identify that bacterial strain CGMCC No.6043 is for 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 the latter's oxydase reaction is negative, and the latter's activity of catalase not as good as the former 1/100.
The CGMCC No.6043 bacterial strain that another object of the present invention has been to provide described in a kind of utilization is produced catalatic method.
Adopt P.chinense CGMCC No.6043 to produce catalase, with CGMCC No.6043 bacterial strain, for the bacterial classification that sets out, through seed culture and liquid fermenting, obtain catalase, comprise the following steps:
(1) by CGMCC No.6043 inoculation to natural sea-water liquid nutrient medium, after cultivation, add hydrogen peroxide to stimulate and cultivate as seed liquor;
(2) seed liquor is forwarded in fermention medium and carries out fermentation culture, then add hydrogen peroxide to stimulate and cultivate, can obtain having the fermented liquid of activity of catalase.
In aforesaid method step (1), the formula of described natural sea-water substratum comprises 0.1-0.5% peptone, 0.1-0.5%Casamino acid, and 0.005-0.05% yeast extract, all the other are natural sea-water, adjusting pH is 6.8-7.5.In a preferred embodiment, 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.
In step (1), the microbial culture time is 8-40 hour, preferably 20-30 hour; Culture temperature is 20-40 ℃, preferably 28-35 ℃.After cultivation finishes, add the hydrogen peroxide of 1-10mM to stimulate cultivation 1-10 hour, preferably add the hydrogen peroxide of 2-5mM to stimulate cultivation 2-5 hour
In aforesaid method step (2), described fermentative medium formula comprises: 1-10g/L yeast extract, and 0.5-5g/L peptone, all the other are natural sea-water, pH is 6.5-8.0; Preferred formula comprises 3-6g/L yeast extract, 1-3g/L peptone, and all the other are natural sea-water, pH is 7.0-7.5; Incubation time is 8-40 hour, preferably 20-30 hour; Culture temperature is 20-40 ℃, preferably 28-35 ℃.After cultivation finishes, add the hydrogen peroxide of 1-10mM to stimulate cultivation 1-10 hour, preferably add the hydrogen peroxide of 2-5mM to stimulate cultivation 2-5 hour.
According to aforesaid method, can obtain the fermented liquid that activity of catalase is 100-1000U/mL, enzyme activity is 754U/mL in a preferred embodiment.
As required, can optionally to having the fermented liquid of enzyme activity, process, therefrom separation and purification goes out catalase, and preferred purification condition is as ion exchange chromatography, reversed phase chromatography, hydrophobic chromatography, sieve chromatography or its combination.
Another object of the present invention is to provide a kind of 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 to be slowly penetrated into the method for supernatant liquid substratum.The method continues and stably awards the certain oxidative pressure of microorganism in supernatant liquid substratum, thereby reaches the object 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 semisolid medium under aseptic condition, and add carbon and the nitrogenous source of filtration sterilization in advance, jolt enrichment culture;
(2), enrichment culture liquid is carried out to aseptic dilution spread, select mono-clonal and carry out inclined-plane preservation;
(3), bacterial strain that separation is obtained is forwarded to natural sea-water liquid nutrient medium and carries out shake flask fermentation, adopts spectrophotometry to carry out catalase activity and sieves again, finishing screen is selected the catalase relatively high bacterial strain of living.
In aforesaid method step (1), described semisolid medium is prepared by the following method: natural sea-water is adopted to 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 40-60 ℃ of left and right adds filtration sterilization under aseptic condition, mixes rear cooled and solidified and makes semisolid medium.
In a preferred embodiment, described semisolid medium is prepared by the following method: 4.5mL natural sea-water is adopted to NaOH or HCl fine setting pH to 7.2, adding mass percent is 121 ℃ of moist heat sterilizations of agar of 0.75% 20 minutes, treat that temperature is reduced to the 30% hydrogen peroxide 4 μ L that 50 ℃ of left and right add filtration sterilization under aseptic condition, mixes rear cooled and solidified and makes semisolid medium.
In step (1), seawater sample from ocean is added in semisolid medium under aseptic condition, and add carbon and the nitrogenous source of filtration sterilization in advance, its final concentration is 0.1-0.5% peptone, 0.1-0.5%Casamino acid, 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 ℃ (preferably 28 ℃) jolt about enrichment culture 2-5 days (preferably about 4 days).
Enrichment culture liquid is carried out to aseptic dilution spread, select mono-clonal and carry out inclined-plane preservation.
In aforesaid method step (3), the bacterial strain that separation is obtained is forwarded to natural sea-water liquid nutrient medium and carries out shake flask fermentation, adopts spectrophotometry to carry out catalase activity and sieves again.Wherein natural sea-water liquid nutrient medium comprises 0.1-0.5% peptone, 0.1-0.5%Casamino acid, and 0.005-0.05% yeast extract, all the other are natural sea-water, adjusting pH is 6.8-7.5.In a preferred embodiment, 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 by enchylema and 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
2solution add starting enzymatic reaction; (3) condition determination is 1cm cuvette, 30 ℃, weighs the enzymatic degradation speed of hydrogen peroxide with the decline of absorbancy under 240nm; (4) every 5s, read absorbance one time, after 1min, stop 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: at 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) * 1000 * extension rate * reaction system volume/(43.6 * enzyme liquid is long-pending), 43.6M
-1cm
-1for H
2o
2molar extinction coefficient under 240nm.
The bacterial strain that multiple sieve is obtained carries out hydrogen peroxide stimulation, improves enzyme and lives and 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 ℃.Adding final concentration is that the hydrogen peroxide of 4mM stimulates jolting after 1 hour, adopts its enzyme of spectrophotometry size of living.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 fermentation broth enzyme vigor reaches 100U/mL and may be defined as the bacterial strain that activity is relatively high, and preferred enzyme vigor reaches the above person of 500U/mL.CGMCC No.6043 bacterial strain is screened and is obtained by above-mentioned steps, and in fermented liquid, enzyme activity can reach 754U/mL.
The invention provides a kind of from creating the method for semisolid medium directed screening Catalase-Producing Strain strain and adopting the method to 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 feature.The P.chinense CGMCC No.6043 Catalase-Producing Strain growth cycle that the present invention finds is short, and enzymic activity is high, with low cost, is applicable to being 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 industrial application to be widely worth 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 observationss for 28 ℃, picking list bacterium colony carries out inclined-plane preservation.Utilize 3% hydrogen peroxide Bubbling method preliminary screening catalase activity bacterial strain.Natural sea-water oligotrophic substratum is 0.5g/L yeast extract, 0.1g/L peptone, and pH is 7.2, all the other are natural sea-water.
Getting 4.5mL seawater sample adds in the aseptic old seawater of 4.5mL, under aseptic condition, add successively 30% hydrogen peroxide 4 μ L of filtration sterilization and the carbon and nitrogen sources (final concentration is 0.25% peptone, 0.25%Casamino acid and 0.01% yeast extract) of filtration sterilization in advance.28 ℃ of concussion enrichment culture, about 4 days, are carried out aseptic dilution suitable multiple to pregnant solution, and spread plate, selects mono-clonal and carry out inclined-plane preservation.
Get 4.5mL seawater sample and add in semisolid medium under aseptic condition, and to add the carbon and nitrogen sources of filtration sterilization in advance, final concentration be 0.25% peptone, 0.25%Casamino acid and 0.01% yeast extract.28 ℃ of concussion enrichment culture, about 4 days, are carried out aseptic dilution suitable multiple to pregnant solution, and spread plate, selects mono-clonal and carry out inclined-plane preservation.
The bacterial strain that adopts above-mentioned three kinds of methods screening to obtain is forwarded to 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 of lasting 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 efficiency of direct oxidation pressure to take second place, the method efficiency that adopts common minute bacterium and hydrogen peroxide primary dcreening operation to combine is extremely low.
The separation screening of embodiment 2 moving property bacillus (P.chinense) the CGMCC No.6043 of China
Gather seawater sample, under aseptic condition, in semisolid medium, add 4.5mL seawater sample, and the carbon and nitrogen sources of filtration sterilization in advance, final concentration is 0.25% peptone, 0.25%Casamino acid and 0.01% yeast extract.28 ℃ of concussion enrichment culture, about 4 days, are carried out aseptic dilution suitable multiple to pregnant solution, and spread plate, selects mono-clonal and carry out inclined-plane preservation.
The bacterial strain that aforesaid method separation obtains is forwarded to natural sea-water liquid nutrient medium and carries out shake flask fermentation, adopts spectrophotometry to carry out catalase activity and sieves again.Natural sea-water substratum is: aseptic old seawater, and final concentration is 0.25% peptone, 0.25%Casamino acid and 0.01% yeast extract, 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, after stimulating jolting 1h, by 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's common micro-organisms center, and culture presevation number is CGMCC No.6043.
Identification of morphology and the biological characteristics of embodiment 3 moving property bacillus (P.chinense) the CGMCC No.6043 of China
Moving property bacillus (P.chinense) the CGMCC No.6043 of China is inoculated in natural sea-water liquid nutrient medium.Prepare the agar that solid medium can add 15g/L again.121 ℃ of moist heat sterilizations 20 minutes.After inoculation culture, through identifying, this bacterium has following characteristics: (1) colonial morphology: bacterium colony is rounded, and smooth surface, edge is smooth, microprotrusion, yellow.(2) cellular form: gram-positive microorganism, under microcytoscope, (Olympus, BX40) presents mobility, 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 embodiment 4 moving property bacillus (P.chinense) the CGMCC No.6043 of China
Moving property bacillus (P.chinense) the CGMCC No.6043 of China is inoculated in natural sea-water solid medium, direct picking one ring thalline from inclined-plane, adds 400 μ L sterilized waters to mix, 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.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 completed by Sinogenomax Co., Ltd..The 16S rRNA Gene Partial fragment length that completes mensuration is 569bp, with bacterial strain P.chinense JCM 12466
t16S rRNA gene order similarity be 99%.The concrete visible sequence table of 16S rRNA sequence of bacterial strain P.chinense CGMCC No.6043.
Therefore, with reference to < < Bergey ' s Manual of Systematic Bacteriology > > second edition content, according to limiting factor feature, morphological specificity and the physiological and biochemical index of bacterial strain, in conjunction with 16S rRNA gene order similarity, identify that CGMCC No.6043 is for 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 the latter's oxydase reaction is negative, the latter's activity of catalase not as good as the former 1/100.
In embodiment 5 fermenting processs, adopting hydrogen peroxide to stimulate increases catalase expression amount
Moving property bacillus (P.chinense) the CGMCC No.6043 of China is seeded to natural sea-water liquid nutrient medium, culture condition is 28 ℃ and cultivates 24h as seed liquor, transfer respectively in the natural sea-water liquid nutrient medium that contains 0,0.25,0.5,1.0 and 2.0 hydrogen peroxide and cultivate, determine 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 1mM concentration of hydrogen peroxide is forwarded to the 250mL triangular flask that 50mL fermention medium is housed and carries out fermentation culture, incubation time 24h is to logarithmic growth latter stage, 28 ℃ of culture temperature, shaking flask rotating speed 200r/min.Cultivation finishes the rear hydrogen peroxide that adds respectively sterile pure water and 4mM to stimulate and cultivates the mensuration of carrying out catalase activity after 1h.
The fermentation broth enzyme stimulating without hydrogen peroxide is lived as 596U/mL(936U/mg), through hydrogen peroxide, stimulate the fermentation broth enzyme work obtaining to reach 754U/mL(1185U/mg).Utilize hydrogen peroxide to stimulate to improve the method effect of bacterial strain catalase activity more remarkable, enzyme activity increase rate reaches 26.5%(249U/mg).
Fermentative medium formula is as follows: 5g/L yeast extract, and 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 natural sea-water liquid nutrient medium, cultivates 24h for 28 ℃ and reaches logarithmic growth latter stage.Add the hydrogen peroxide of 4mM to stimulate cultivation 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.Add the hydrogen peroxide of 4mM to stimulate cultivation 1h.Fermention medium has three kinds: (1) HS fermention medium: 5g/L yeast extract, and 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% magnesium sulfate, 2.3% sodium-chlor, pH 7.2.The thalline obtaining by three kinds of different fermentations substratum and time cultivation is measured catalase enzyme activity after broken born of the same parents, and concrete outcome is in 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 it is high that its activity of catalase value variation presents two ends, and middle low, numerical value changes inapparent feature 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 feature; 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) after 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 secondly GP culture medium culturing 36h, is HS culture medium culturing 12h again.
Claims (15)
1. a catalase zymogenic bacteria, Classification And Nomenclature is the moving property bacillus (Planomicrobium chinense) of China, deposit number is CGMCC No.6043.
2. catalase zymogenic bacteria according to claim 1, is characterized in that: described bacterial strain has following characteristics:
(1) colonial morphology: bacterium colony is rounded, smooth surface, edge is smooth, microprotrusion, yellow;
(2) cellular form: gram-positive microorganism, under microcytoscope, present mobility, form is spherical 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, is characterized in that: the 16S rRNA gene of described bacterial strain is as shown in Seq ID No.1.
4. utilize the bacterial strain described in claim 1 to produce a catalatic method, comprise the following steps:
(1) by CGMCC No.6043 inoculation to natural sea-water liquid nutrient medium, the formula of described natural sea-water substratum is: 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; Microbial culture temperature is 20-40 ℃, and incubation time is 8-40 hour; After finishing, cultivation add the hydrogen peroxide stimulation of 1-10mM to cultivate 1-10 hour as seed liquor;
(2) seed liquor is forwarded in fermention medium and carries out fermentation culture, described fermentative medium formula is: 1-10g/L yeast extract, and 0.5-5g/L peptone, all the other are natural sea-water, pH is 6.5-8.0; The microbial culture time is 8-40 hour, and culture temperature is 20-40 ℃; After cultivation finishes, add the hydrogen peroxide of 1-10mM to stimulate cultivation 1-10 hour, can obtain having the fermented liquid of activity of catalase.
5. method according to claim 4, is characterized in that: the natural sea-water substratum in described method steps (1) is: 0.25% peptone, and 0.25%Casamino acid, 0.01% yeast extract, all the other are natural sea-water, regulating pH is 7.2.
6. method according to claim 4, is characterized in that: in described method steps (1), the microbial culture time is 20-30 hour.
7. method according to claim 4, is characterized in that: in described method steps (1), culture temperature is 28-35 ℃.
8. method according to claim 4, is characterized in that: in described method steps (1), cultivate and after finishing, add the hydrogen peroxide of 2-5mM to stimulate cultivation 2-5 hour.
9. method according to claim 4, is characterized in that: in described method steps (2), described fermentative medium formula is: 3-6g/L yeast extract, and 1-3g/L peptone, all the other are natural sea-water, pH is 7.0-7.5.
10. method according to claim 4, is characterized in that: in described method steps (2), the microbial culture time is 20-30 hour.
11. methods according to claim 4, is characterized in that: in described method steps (2), culture temperature is 28-35 ℃.
12. methods according to claim 4, is characterized in that: in described method steps (2), cultivate and after finishing, add the hydrogen peroxide of 2-5mM to stimulate cultivation 2-5 hour.
13. according to the method described in claim 4-12 any one, it is characterized in that: described method can obtain the fermented liquid that activity of catalase is 100-1000U/mL.
14. according to the method described in claim 4-12 any one, it is characterized in that: as required, to having the fermented liquid of enzyme activity, process, therefrom separation and purification goes out catalatic product.
15. methods according to claim 14, is characterized in that: described purification condition is ion exchange chromatography, reversed phase chromatography, hydrophobic chromatography, sieve chromatography or its combination.
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