CN103789225A - Marine catalase production strain and method for producing catalase from strain - Google Patents
Marine catalase production strain and method for producing catalase from strain Download PDFInfo
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Abstract
The invention provides high-yield catalase stenotrophomonasmaltophilia from the ocean, which is stored in the China General Microbiological Culture Collection Center with the storage number of CGMCC No.8294 on Oct. 8th, 2013. A strain grows quickly, conditions are simple, and a substrate range is large. The strain is a base strain; the highest catalase activity and the specific activity of a coarse enzyme product prepared by performing fermentation culture on the strain are respectively up to 50,000U/mL and 200,000U/mg. The technical steps are simple, and the cost is low; the enzyme activity of an obtained product is high; catalase can be widely applied to the fields of food, textile, papermaking, industrial wastewater treatment and the like and belongs to the field of marine biotechnologies.
Description
Technical field
The invention belongs to marine biotechnology technical field, be specifically related to a strain from the Catalase-Producing Strain strain of ocean and utilize the catalatic method of this bacterium fermentative production.
Background technology
Catalase (Catalase, EC1.11.1.6) is a kind of enzyme scavenging agent, is called again catalase, is the desmoenzyme take iron porphyrin as prothetic group.It can impel hydrogen peroxide to be decomposed into molecular oxygen and water, is the enzyme that catalyzing hydrogen peroxide resolves into oxygen and water, is present in the peroxidase precursor of cell.Because hydrogen peroxide has the function of stronger oxidation and bleaching, therefore catalase and hydrogen peroxide have larger research and using value as fields such as being combined in printing and dyeing, papermaking, environmental protection and electronic industry.
Research is found, is all contained catalase in nearly all biomass cells.But at present for the production of catalatic be mainly liver and the microorganism fermentation of animal.The method that the broken born of the same parents of animal livers extract, owing to being subject to starting material restriction, causes high cost.And the method for utilizing microorganism to ferment it is advantageous that cost is lower, and production of enzyme is larger.
The ecotope that ocean is special, has bred a large amount of microorganisms and enzyme resource.The bacterial strain majority that the fermentation using bacteria of authorizing at present produces catalase patent is located away from pedotheque.And only Chinese patent 200810057969.7 provides two kinds of catalase bacterium producing multi enzyme preparations that separate from ocean with 201310022577.8, and its catalase enzyme activity and alive all lower than enzyme, be respectively 1200U/mg and 10000U/mg.
Summary of the invention
An object of the present invention is to provide one can the catalatic bacterial strain of high yield.
Bacterial strain provided by the invention, taxonomy called after germ oligotrophy unit cell (Stenotrophomonas maltophilia), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number is: CGMCC No.8294, preservation date on October 8th, 2013.
Bacterial strain CGMCC No.8294 characteristic of the present invention is as follows:
(1) growth limiting factor: temperature 20-40 ℃ (the suitableeest 30 ℃), pH7.0-9.0 (the suitableeest 7.5), salinity 0-3.5% (w/v, the suitableeest 2.0%);
(2) colonial morphology: bacterium colony is rounded, smooth surface, edge is smooth, and microprotrusion is looked for transparent;
(3) cellular form: gram negative bacillus, presents mobility, the raw single flagellum of end under the microscope;
(4) physio-biochemical characteristics: aerobic growth; Catalyst activity, oxidase activity are positive; To Multiple Classes of Antibiotics sensitivities such as penicillin, vancomycin, Streptomycin sulphate, erythromycin, paraxin, kantlex, Liu Suanyan NEOMYCIN SULPHATEs.
(5) lipid acid mainly containing is saturated fatty acid iso-C
15:0, anteiso-C
15:0, C
16:0.
Utilize universal primer to increase and sequencing to the 16S rRNA gene of bacterial strain CGMCC No.8294, obtain the gene fragment that length is 737bp (Seq ID No.1), its sequence is specifically shown in sequence table.Itself and bacterial 16 S rRNA geneseq database are compared, the similarity that obtains the reference culture S.maltophilia that itself and known germ oligotrophy unit cell belong to is 99.32%, and growth limiting factor, cellular form and the main fatty acid of bacterial strain CGMCC No.8294 are all consistent with its character pair, therefore, this two strains bacterium is different strains of the same race.With reference to the content of " 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.8294 is germ oligotrophy unit cell (S.maltophilia).
The bacterial strain CGMCC No.8294 that another object of the present invention is to provide described in a kind of utilization produces catalatic method.
Adopt S.maltophilia CGMCC No.8294 to produce catalase, for the bacterial classification that sets out, obtain catalase through seed liquor cultivation and liquid fermenting with CGMCC No.8294 bacterial strain, comprise the following steps:
(1) bacterial strain CGMCC No.8294 is seeded to seed liquor substratum, the bacterium liquid after cultivating is as seed liquor;
(2) seed liquor is forwarded in fermention medium and carries out fermentation culture, cultivate the fermented liquid that obtains having afterwards catalase activity for 2 days.
In aforesaid method step (1), the formula of described natural sea-water substratum is peptone, the yeast extract of 0.01-0.1% and the casamino acids of 0.1-0.5% that adds 0.1-0.5% in natural sea-water, and adjusting pH is 6.8-7.5.
In a preferred embodiment, natural sea-water liquid nutrient medium is in natural sea-water, to add 0.5% peptone, 0.1% yeast extract, and 0.5% casamino acids, regulating pH is 7.5.
In step (1), the microbial culture time is 36-48 hour, preferably 40-45 hour; Culture temperature is 20-40 ℃, preferably 30-35 ℃.
Fermentative medium formula in aforesaid method step (2) is in natural sea-water, to add the glucose of 0.1-2% and the peptone of 0.5-5%, and pH is 6.5-7.5.In a preferred embodiment, fermentative medium formula is in natural sea-water, to add the glucose of 1-2% and the peptone of 1-2%, and pH is 7.0-7.5.Incubation time is 36-48 hour, preferably 40-48 hour; Culture temperature is 20-40 ℃, preferably 30-35 ℃.
Can obtain activity of catalase and be respectively the fermented liquid of 10000-50000U/mL and 100000-200000U/mg than enzyme work according to aforesaid method, enzyme activity reaches respectively 53228U/mL and 181987.22U/mg in a preferred embodiment.
As required, can optionally process the fermented liquid with enzyme activity, therefrom separation and purification goes out catalatic product, and preferred purification condition is as ion exchange chromatography, reversed phase chromatography, hydrophobic chromatography, sieve chromatography or its combination.
Accompanying drawing explanation
Fig. 1 is single carbon source experimental result of germ oligotrophy unit cell (S.maltophilia) CGMCC No.8294.
Fig. 2 is the sole nitrogen source experimental result of germ oligotrophy unit cell (S.maltophilia) CGMCC No.8294.
Embodiment
The separation screening of embodiment 1 germ oligotrophy unit cell (S.maltophilia) CGMCC No.8294 and the mensuration of enzyme activity.
The wastewater sample that collection contains hydrogen peroxide, first adopts raw bacterial context soup 2216 liquid nutrient mediums in sea to cultivate and obtains logarithmic phase bacterium liquid.The formula of sea raw bacterial context soup 2216 liquid nutrient mediums is in 1L deionized water, to add magnesium sulfate 6.64g, sodium-chlor 19.45g, magnesium chloride 12.6g, Repone K 0.55g, calcium chloride 1.8g, Sodium Fluoride 2.4mg, water glass 9.3mg, ammonium nitrate 2.4mg, Sodium phosphate dibasic 8mg, potassium primary phosphate 0.4g, sodium bicarbonate 0.16g, Potassium Bromide 0.08mg, boric acid 22mg, strontium chloride 57mg, ironic citrate 0.1g, yeast extract (Difco) 1g, peptone 5g, regulating pH is 7.5.Again logarithmic phase bacterium liquid is accessed in the liquid nutrient medium that contains hydrogen peroxide and cultivated, observe its upgrowth situation.If well-grown, is seeded in the liquid nutrient medium of higher concentration of hydrogen peroxide and cultivates; Bad if grow, be seeded in the liquid nutrient medium of lower concentration of hydrogen peroxide, then observe again its upgrowth situation, to determine that its concentration of hydrogen peroxide of substratum of next inoculation is reduce or raise.So move in circles, until the content of hydrogen peroxide of substratum arrives 20-200mM.Separate through dull and stereotyped dilution spread, picking mono-clonal is preserved again.
The thalline preserving is accessed respectively in the shaking flask containing sea raw bacterial context soup 2216 liquid nutrient mediums, cultivate 40 hours for 30 ℃.The bacterium liquid 12000rpm that cultivation is obtained collects and obtains thalline for centrifugal 5 minutes, with pH7.0, and the resuspended thalline of 50mM phosphate buffered saline buffer ultrasonication in ice bath.Liquid after fragmentation centrifugal 20 minutes with 6500g, getting supernatant liquor is crude enzyme liquid, and it is carried out to activity of catalase mensuration, obtains the enzyme activity size (U/mL) of this crude enzyme liquid.
Activity of catalase adopts metric measurement, has utilized the light absorption value of hydrogen peroxide under 240nm and the linear relationship of its concentration.Concrete operations obtain crude enzyme liquid for enchylema being broken to born of the same parents' processing, and while utilizing crude enzyme liquid and hydroperoxidation, the rate of descent of light absorption value is as the size alive of the enzyme according to calculating crude enzyme liquid, specifically formula: Δ OD
240× 1000 × extension rate × reaction system volume/(Δ t × 43.6 × enzyme liquid is long-pending), 43.6M
-1cm
-1for H
2o
2molar extinction coefficient under 240nm.
Protein concn is quantitative: the making of (1) typical curve: get 6 5mL centrifuge tubes, add respectively 0,5,10,20,30,40,50 μ L and quantitative Xylene Brilliant Cyanine G G-250 reagent.Place colorimetric estimation under 595nm wavelength after 6 minutes.Utilize Excel form software, take protein content as X-coordinate, take absorbancy as ordinate zou, draw typical curve, and obtain the formula of typical curve.(2) protein content determination: get 1 5mL centrifuge tube, accurately add 50 μ L crude enzyme liquid samples, then add quantitative Xylene Brilliant Cyanine G G-250 reagent, place colorimetric after 2 minutes, record absorbancy, calculate the protein content of crude enzyme liquid sample according to typical curve formula.(3) enzyme activity calculates: the enzyme obtaining is lived to (U/mL) divided by protein content (μ g/mL) × 1000, obtain the ratio enzyme size alive (U/mg) of this crude enzyme liquid.
By above method, obtain a plant height and produce catalatic bacterial strain, its thick enzyme activity and than enzyme live best result can not reach 53228U/mL and 181987.22U/mg.Its culture presevation is in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is: CGMCC No.8294.
Embodiment 2 oligotrophy unit cells belong to identification of morphology and the biological characteristics of bacterial strain (S.maltophilia) CGMCC No.8294
Oligotrophy unit cell belongs to bacterial strain (S.maltophilia) CGMCC No.8294 and is inoculated in natural sea-water liquid nutrient medium, if prepare the agar that solid medium can add 20g/L again.Cultivate after 30 hours, identify, this bacterium has following characteristics: (1) colonial morphology: bacterium colony is rounded, and smooth surface, edge is smooth, microprotrusion, water white transparency; (2) cellular form: Gram-negative bacteria, under microcytoscope, (Olympus, BX40) presents mobility, and form is shaft-like; (3) physiological and biochemical property: aerobic growth; Oxidase positive; Can utilize the compound organic matters such as the multiple simple organic such as glucose, maltose, sucrose and extractum carnis, peptone, casein food grade and fish meal; To Multiple Classes of Antibiotics sensitivities such as penicillin, vancomycin, Streptomycin sulphate, erythromycin, paraxin, kantlex, Liu Suanyan NEOMYCIN SULPHATEs.
Embodiment 3 oligotrophy unit cells belong to pcr amplification and the sequencing of the 16S rRNA gene of bacterial strain (S.maltophilia) CGMCC No.8294
Oligotrophy unit cell belongs to bacterial strain (S.maltophilia) CGMCC No.8294 and is inoculated in natural sea-water solid medium, direct picking thalline from inclined-plane, add 200 μ L sterilized waters abundant suspension thalline, boiling water bath 30 minutes, ice bath 30 minutes immediately, then centrifugal 2 minutes of 12000rpm, supernatant liquor carries out PCR as template.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 × buffer5 μ L, 10mM dNTPs1 μ L, the each 1 μ L of 4 μ M primer, Taq enzyme 0.5 μ L, DNA profiling 0.5 μ L, sterile pure water 42 μ L.PCR reaction conditions is: 94 ℃ of denaturations 10 minutes; 94 ℃ of sex change 45 seconds, 55 ℃ of annealing 45 seconds; 72 ℃ are extended 90 seconds, 35 circulations; After 72 ℃, extend 10 minutes.PCR product purification and sequencing are completed by Shanghai Ying Jun Bioisystech Co., Ltd.The 16S rRNA Gene Partial fragment length that completes mensuration is 737bp, with bacterial strain S.maltophilia ATCC13637
t16S rRNA gene order similarity the highest.The concrete visible sequence table Seq ID No.1 of 16S rRNA sequence of bacterial strain S.maltophilia CGMCC No.8294.
Therefore, with reference to " Bergey ' s Manual ofSystematic Bacteriology " second edition content, according to the limiting factor of bacterial strain, morphological specificity and physiological and biochemical index feature, in conjunction with 16S rRNA gene order similarity, identify that CGMCC No.8294 is germ oligotrophy unit cell (S.maltophilia).
Embodiment 4 fermention medium optimizations
Germ oligotrophy unit cell (S.maltophilia) CGMCC No.8294 is seeded to natural sea-water liquid nutrient medium, cultivates for 30 ℃ and within 30 hours, reaches logarithmic growth and obtain seed liquor latter stage.Be forwarded to and the basic medium that 50mL only contains single carbon source or sole nitrogen source is housed cultivates with 1% switching amount.30 ℃ of culture temperature, shaking speed 200rpm, after cultivating 24 hours, measures with spectrophotometer, utilizes the light absorption value of bacterium liquid under 600nm wavelength and the linear relationship of cell concn, reflects the upgrowth situation of bacterial strain with the size of light absorption value.The formula of basic medium is in deionized water, to add 0.01% yeast extract.The carbon source of testing has glucose, sucrose, malt extract, starch and maltose; Nitrogenous source has yeast extract, extractum carnis, casein food grade, peptone, urea and fish meal.Concrete outcome is shown in Fig. 1,2.Result demonstration, germ oligotrophy unit cell CGMCC No.8294 is take glucose, maltose and sucrose as carbon source, and the upgrowth situation of bacterial strain is the best.The price factor that adds raw material considers, and determines that selecting sucrose is the sole carbon source of strain fermentation substratum.Aspect the utilizing of nitrogenous source, bacterial strain CGMCC No.8294 utilizes the ability of organic nitrogen source to be far better than inorganic nitrogen-sourced, grow preferably using fish meal and peptone during as nitrogen source, this may be because in organic nitrogen source except containing rich in protein, polypeptide and free amino acid, contain a small amount of carbohydrate, fat, inorganic salt, VITAMIN and some somatomedin toward contact, thereby with respect to the inorganic nitrogen-sourced thalli growth that is more conducive to.Due to the unstable of fish meal composition, consider and determine to adopt the only nitrogen source of peptone as strain fermentation substratum.
Claims (10)
1. a strain oligotrophy unit cell belongs to bacterial strain (S.maltophilia) and mutant thereof, has been preserved in Chinese common micro-organisms culture presevation administrative center, and deposit number is: CGMCC No.8294.
2. Catalase-Producing Strain according to claim 1, is characterized in that:
(1) colonial morphology: colony growth is rounded after being shaped, smooth surface, edge is smooth, microprotrusion, water white transparency;
(2) cellular form: Gram-negative bacteria, cellular form is shaft-like, presents under the microscope mobility;
(3) physiological and biochemical property: aerobic growth; In 20-40 ℃, pH7.0-9.0, salinity 0-3.5% (w/v) scope, all can grow, optimal growth condition is at 30 ℃, pH7.5, salinity 2.0% left and right; Oxidase positive; Can utilize the compound organic matters such as the multiple simple organic such as glucose, maltose, sucrose and extractum carnis, peptone, casein food grade and fish meal.
3. Catalase-Producing Strain according to claim 1, is characterized in that: 16s ribosomal subunit gene (16s rRNA) sequence of described bacterial strain comprises the nucleotide sequence as shown in Seq ID No.1.
4. mutant according to claim 1, is characterized in that: described mutant is to the primary mutant that carries out the operations such as mutagenesis, domestication, gene recombination or obtain through spontaneous mutation of bacterial strain CGMCC No.8294.
5. utilize the Catalase-Producing Strain described in claim 1 to produce a catalatic method, comprise the following steps:
(1) bacterial strain CGMCC No.8294 is seeded to seed liquor substratum, the bacterium liquid after cultivating is as seed liquor;
(2) seed liquor is forwarded in fermention medium and carries out fermentation culture, obtain having the fermented liquid of catalase activity.
6. method according to claim 5, it is characterized in that: the formula of the seed liquor substratum in described method steps (1) is peptone, the yeast extract of 0.01-0.05% and the casamino acids of 0.1-0.5% that adds 0.1-0.5% in natural sea-water, and adjusting pH is 6.8-7.5.
7. method according to claim 5, is characterized in that: the fermentative medium formula in described method steps (2) is in natural sea-water, to add the glucose of 0.1-1% and the peptone of 0.5-5%, and pH is 6.5-7.5.
8. according to the method described in claim 5-7 any one, it is characterized in that: described method steps (2) finally obtains the activity of catalase in fermented liquid and is respectively 10000-50000U/mL and 100000-200000U/mg than enzyme work.
9. according to the method described in claim 5-7 any one, it is characterized in that: as required, choosing method carries out subsequent disposal to the fermented liquid with enzyme activity arbitrarily, and therefrom separation and purification goes out catalase product.
10. method according to claim 9, is characterized in that: the purification condition of described treatment process is ion exchange chromatography, reversed phase chromatography, hydrophobic chromatography, sieve chromatography or its above combination.
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| CN105567653A (en) * | 2016-03-03 | 2016-05-11 | 仇颖超 | Preparation method of catalase |
| CN108588854A (en) * | 2018-07-01 | 2018-09-28 | 李冬生 | A kind of preparation method of mulberry fibre |
| CN108823792A (en) * | 2018-07-01 | 2018-11-16 | 李万强 | A kind of preparation method of medical non-woven fabrics |
| CN111748503A (en) * | 2020-08-04 | 2020-10-09 | 华中农业大学 | Culture medium and dosage form of deep-sea Ledebouriella cladosporioides |
| CN112011489A (en) * | 2020-09-15 | 2020-12-01 | 广东省中医院（广州中医药大学第二附属医院、广州中医药大学第二临床医学院、广东省中医药科学院） | Method for enriching and separating Francisella and/or its neighboring group species, culture medium and preparation method of culture medium |
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Cited By (6)
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| CN105567653A (en) * | 2016-03-03 | 2016-05-11 | 仇颖超 | Preparation method of catalase |
| CN108588854A (en) * | 2018-07-01 | 2018-09-28 | 李冬生 | A kind of preparation method of mulberry fibre |
| CN108823792A (en) * | 2018-07-01 | 2018-11-16 | 李万强 | A kind of preparation method of medical non-woven fabrics |
| CN111748503A (en) * | 2020-08-04 | 2020-10-09 | 华中农业大学 | Culture medium and dosage form of deep-sea Ledebouriella cladosporioides |
| CN111748503B (en) * | 2020-08-04 | 2021-12-14 | 华中农业大学 | Culture medium and dosage form of deep-sea Ledebouriella cladosporioides |
| CN112011489A (en) * | 2020-09-15 | 2020-12-01 | 广东省中医院（广州中医药大学第二附属医院、广州中医药大学第二临床医学院、广东省中医药科学院） | Method for enriching and separating Francisella and/or its neighboring group species, culture medium and preparation method of culture medium |
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