CN102174442A - Bacillus licheniformis with high protease activity and applications thereof - Google Patents
Bacillus licheniformis with high protease activity and applications thereof Download PDFInfo
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- CN102174442A CN102174442A CN 201110043496 CN201110043496A CN102174442A CN 102174442 A CN102174442 A CN 102174442A CN 201110043496 CN201110043496 CN 201110043496 CN 201110043496 A CN201110043496 A CN 201110043496A CN 102174442 A CN102174442 A CN 102174442A
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Abstract
The invention belongs to the technical field of biological engineering and particularly relates to Bacillus licheniformis with high protease activity and applications thereof. The strain was preserved in the China Center for Type Culture Collection (CCTCC) with collection number of CCTCC NO:M2011037 and named as Bacillus licheniformis Pb-WC09005 on January 25, 2011. The strain is separated from the marine environment of the Hainan tropical culture zone, has higher protease activity under the conditions of different temperatures, salinities and pH values, has a better improvement function on the tropical culture aquatic environment and the like, has stable effect and is an strain with good application prospect.
Description
Technical field
The invention belongs to technical field of bioengineering, be specifically related to a plant height protease activity Bacillus licheniformis and an application.
Background technology
Along with the lasting expansion of aquaculture scale and improving constantly of intensification degree, breaking out of aquaculture creature disease is more and more frequent, and it is also increasing due to illness to do harm to the loss that breaks out and cause.Though causing the reason that aquiculture disease takes place has a lot, but because of cultivation water worsens the pathogenic micro-organism outburst and the aquatic animal immunity degradation that cause is one of principal element wherein, moreover, though aquaculture is to promoting economic development and living standards of the people improve and to have brought into play vital role, but the water pollution that causes because of aquiculture waste water discharging also more and more is subjected to showing great attention to of society in recent years, how to reduce effectively that the quantity discharged of pollutent is a major issue that presses for solution of the sustainable sound development of culture fishery in the aquiculture waste water.Therefore, the only way that to set up a kind of new nuisanceless aquaculture pattern pollution-free or less contamination be the sustainable sound development of culture fishery, wherein, the effective degraded based on the larger molecular organics of protein is a most critical link of successfully setting up this aquaculture model.
Use probiotics adjusting cultivation water and have advantages such as low, the no recontaminate of cost, the growth of inhibition pathogenic bacteria, become one of main focus in the nuisanceless aquatic health aquaculture model.Wherein, bacillus licheniformis (B.licheniformis) is one of bacterial classification the most frequently used in the present aquaculture of aquatic animal water body purification, it is a kind of aerobic or amphimicrobian Gram-positive bacillus, to human body and environmental safety, can produce the outer hydrolase of born of the same parents in a large number and organic bacterium such as the macro-molecular protein of degrading fast, also can promote H2S, nitrite-oxidizing, and to environment have stronger adaptive faculty strong, can be in water body the bacterium of growth and breeding fast.This bacterial classification mainly changes into small molecules amino acid and organic acid by the assimilation of heterotrophic organism with macromolecule protein etc., these products can further provide nutrition for photosynthetic bacterium, algae and other planktonic organisms in the water body again, suppress the growth of pathogenetic bacteria simultaneously, thereby make water body form good micro-ecological environment.In the grass carp breeding process, use Bacillus licheniformis De as researchs such as Cao Yucheng and can make NH in the water body
3-N and NO
2-N remains at lower level culturing the middle and later periods, and its mean value is respectively 0.12 and 0.015mgL
-1, Hao Guiyu etc., Liu Bo have also obtained similar result with Liu Wenbin in the correlative study of other aquatic animal; Also have research also to show, regularly use the pH value that Bacillus licheniformis helps stablizing aquaculture water in the breeding process, make it to maintain between 8.2～8.4, avoid because of water body pH changes the excessive cultivated animals that causes stress, also help forming good transparency and water colour, promote the healthy growth of aquaculture organism.But owing to be subjected to the influence of geogen (temperature, salinity, pH value), the bacillus licheniformis bacterial strain of different growing environments purifies corresponding to the aquaculture water under the local environment has effect preferably, it is poor that the strange land environment is then often shown decontamination effect improving, decontamination effect improving instability etc.
At present, 02133713.6,02114362.5,02111038.7 domesticly mainly concentrates on aspects such as the fermented bacterium (number of patent application:, 97119176.X etc.) of fertiliser production and fodder additives (number of patent application: 02115969.6,93110883.7 etc.) about the patent of Bacillus licheniformis probiotics, do not see the Bacillus licheniformis patent that relevant cultivation water purifies, more do not see relevant patent about Bacillus licheniformis Hainan Island geographical population.
Summary of the invention
The purpose of this invention is to provide the high protein enzyme Bacillus licheniformis alive that a strain can be used for water quality improvement.
Another object of the present invention provides the application of this bacterial strain in the culture water quality of tank system improvement.
In order to solve the problems of the technologies described above, the invention provides a kind of lichem bacillus strain, this Bacillus licheniformis (B.licheniformis) bacterial strain preservation name is called: Bacillus licheniformis Pb-WC09005, depositary institution: Chinese typical culture collection center, preservation day: on January 25th, 2011, preserving number: CCTCC NO:M2011037.Bacterium colony is oyster white on common sea water medium, and is flat, surface irregularity, gauffer, and the edge is irregular, and 37 ℃ of colony diameters of cultivating 24h are 3～5mm.
Utilize bacterial 16 S rDNA universal primer Pf:5 '-AGA GTT TGA TCC TGG CTC AG-3 ' and Pr:5 '-GGT TAC CTT GTT ACG ACT-3 ' that Bacillus licheniformis Pb-WC09005 is carried out pcr amplification, order-checking back obtains the fragment of 1511bp, is HM006905.1 in the accession number of GenBank.Its sequence as in the gene order table＜400〉1 as described in sequence.
The present invention also provides the purposes of this kind lichem bacillus strain simultaneously, is used for the improvement of culture water quality of tank system.
Above-mentioned breed water is meant torrid areas breed water, comprises freshwater aquiculture water or sea farming water.
Lichem bacillus strain provided by the invention, separation is from the torrid zone, Hainan breeding seawater environment, this bacterial strain under differing temps, salinity, pH value condition, have a higher protease activity, the improving effect preferably that has to water body environments such as torrid zone breed is a kind of bacterial strain with applications well prospect.
Description of drawings
Fig. 1 is the impact effect figure of salinity to Bacillus licheniformis Pb-WC09005 inulinase-producing activity.
Fig. 2 is the impact effect figure of temperature to Bacillus licheniformis Pb-WC09005 inulinase-producing activity.
Fig. 3 is the impact effect figure of pH value to Bacillus licheniformis Pb-WC09005 inulinase-producing activity.
Fig. 4 is the RAPD finger printing of Bacillus licheniformis Pb-WC09005.Illustrate: 1-10 represents respectively available from Shanghai and gives birth to worker's primer S36, S12, S376, S93, S515, S103, S240, S119, W82089, the S7 amplification to Pb-WC09005.
Fig. 5 Bacillus licheniformis Pb-WC09005 is to the degradation effect figure of COD in water body.
Embodiment
At term used in the present invention, removing has other explanation, generally has the implication of those of ordinary skills' common sense.
1, the separation of Bacillus licheniformis Pb-WC09005
Prepare the genus bacillus selective medium with reference to " manufacturing of microbiological culture media and application " method.Seawater sample is got 100 μ L and is coated the genus bacillus selective medium after shaking up, and cultivates 24h for 50 ± 1 ℃, and the single bacterium colony of the oyster white of picking surface irregularity, gauffer is the aimed strain (Bacillus licheniformis Pb-WC09005) of primary dcreening operation.
2, protease activity determination
1) preparation of nutrient solution: with the aimed strain of transfering loop picking primary dcreening operation, be inoculated in the common seawater liquid nutrient medium, 30 ℃ of 180rpm shaking culture 24h, the centrifugal 10min of 6000rpm, collect supernatant liquor 10mL, and, obtaining the aimed strain nutrient solution with 0.20 μ m membrane filtration degerming ,-20 ℃ of preservations are standby.
2) mensuration of protease activity: preparation contains the sterilization seawater agar plate of 5% germfree defatted milk, and the punch tool evenly punching on flat board with diameter 4mm adds 20 μ L aimed strain nutrient solutions in the hole.With 10 μ g/ml Proteinase Ks of equivalent with from commercially available effective microorganism preparation product, separate the positive contrast of the nutrient solution that obtains bacterial strain, the negative contrast of aseptic seawater.Hatch 24h under 30 ℃, 10% trichoroacetic acid(TCA) is 2h fixedly, removes behind the stationary liquid with Coomassie brilliant blue G-250 dyeing, and is fully painted until plate culture medium, fully decolours with destainer after removing staining fluid, makes dull and stereotyped background be blueness and the hydrolysis circle is colourless.With vernier caliper measurement hydrolysis loop diameter.Each sample is established two repetitions.
The cultivation of going down to posterity: will through the bacterial strain with high protein enzymic activity (the hydrolysis circle of strain cultured solution is bigger) of screening with the selection condition of high temperature and high salt carry out 20 generation cultured continuously, measure the 20th generation strain cultured solution the hydrolysis loop diameter.Each sample is established two repetitions.
Final determine that the protease activity of Bacillus licheniformis Pb-WC09005 is the highest, the hydrolysis loop diameter reaches 14.16 ± 0.04mm (under the equal conditions, the hydrolysis loop diameter of 10 μ g/ml Proteinase Ks only 9.12 ± 0.02).
3, the water quality environment factor pair Bacillus licheniformis Pb-WC09005 influence of white enzyme activity of laying eggs
1) influence of salinity
(1) supernatant preparation
With transfering loop picking-80 ℃ this Bacillus licheniformis that the guarantor plants, be inoculated in different salinity gradient (NaCl concentration: among the common seawater liquid nutrient medium 50mL 0 ‰, 10 ‰, 20 ‰, 30 ‰, 40 ‰, 50 ‰) respectively, 30 ℃ of 150rpm shaking culture 24h, with the centrifugal 10min of 6000rpm, collect each 10mL of supernatant liquor, and with 0.20 μ m membrane filtration degerming ,-20 ℃ of preservations are standby.
(2) skimmed milk medium preparation
Get agar 2g respectively, the sterilization seawater 95mL that adds the different salinity gradient successively, 121 ℃ of autoclaving 15min, after treating that temperature drops to 55 ℃, aseptic skimmed milk (with the 0.20 μ m membrane filtration degerming) 5mL of adding 5%, fully be poured into the aseptic flat board that diameter is 9cm, the about 20mL of every flat board behind the mixing.After cooling, use diameter 4mm punch tool after punching on the flat board.
(3) measure protease activity
Draw the nutrient solution 20 μ L of this Bacillus licheniformis of different salinity cultivation with pipettor, add in the skimmed milk substratum 4mm plate well, the flat board sealing is kept humidity, place in 30 ℃ of incubators and hatch 24h, observe and write down experimental result (Fig. 1) with sealing compound.
2) temperature is to the lay eggs influence of white enzyme activity of bacterial strain
(1) supernatant preparation
This Bacillus licheniformis that protects kind with transfering loop picking-80 ℃ is inoculated among the common seawater liquid nutrient medium 50mL, and placing 6 temperature respectively is 18 ℃, 22 ℃, 26 ℃, 30 ℃, 34 ℃, 38 ℃ incubator shaking culture, and 180rpm cultivates 24h; The centrifugal 10min of 6000rpm collects each 10mL of supernatant liquor, 0.20 μ m membrane filtration degerming, and-20 ℃ of preservations are standby behind packing, the mark.
(2) skimmed milk medium preparation
Get agar 2g respectively, add sterilization seawater 95mL, 121 ℃ of autoclaving 15min, after treating that temperature drops to 55 ℃, aseptic skimmed milk (with the 0.20 μ m membrane filtration degerming) 5mL of adding 5% fully is poured into the aseptic flat board that diameter is 9cm, the about 20mL of every flat board behind the mixing.After cooling, use diameter 4mm punch tool after punching on the flat board.
(3) measure protease activity
Draw the nutrient solution 20 μ L that differing temps is cultivated this Bacillus licheniformis with pipettor, add in the skimmed milk substratum 4mm plate well, the flat board sealing is kept humidity, place in 30 ℃ of incubators and hatch 24h, observe and record experimental result (Fig. 2) with sealing compound.
3) the pH value is to the lay eggs influence of white enzyme activity of Pb-WC09005
(1) supernatant preparation
With transfering loop picking-80 ℃ this Bacillus licheniformis that the guarantor plants, be inoculated in respectively among the common seawater liquid nutrient medium 50mL of different pH (pH 6.0, pH 6.5, pH 7.0, pH 7.5, pH 8.0, pH 8.5, pH 9.0), 30 ℃ of 150rpm shaking culture 24h, with the centrifugal 10min of 6000rpm, collect each 10mL of supernatant liquor, and with 0.20 μ m membrane filtration degerming ,-20 ℃ of preservations are standby.
(2) skimmed milk medium preparation
Get agar 2g respectively, add sterilization seawater 95mL, 121 ℃ of autoclaving 15min, after treating that temperature drops to 55 ℃, aseptic skimmed milk (with the 0.20 μ m membrane filtration degerming) 5mL of adding 5% fully is poured into the aseptic flat board that diameter is 9cm, the about 20mL of every flat board behind the mixing.After cooling, use diameter 4mm punch tool after punching on the flat board.
(3) measure protease activity
Draw the nutrient solution 20 μ L that different pH cultivate this Bacillus licheniformis with pipettor, add in the skimmed milk substratum 4mm plate well, the flat board sealing is kept humidity, place in 30 ℃ of incubators and hatch 24h, observe and record experimental result (Fig. 3) with sealing compound.
Application case: Bacillus licheniformis Pb-WC09005 is to the influence of Environment of Litopenaeus vannamei Low COD in water body
1, test method
The clean Erlenmeyer flask of getting 4 2000mL is used to measure the actual removal effect of this Bacillus licheniformis to the prawn culturing waste water COD.Every strain contains A and two test group of B, and wherein A is for adding the breeding wastewater group of corresponding bacterial strain, and B is for adding the sterilization breeding wastewater group of corresponding bacterial strain; Simultaneously, establishing the breeding wastewater and the sterilization breeding wastewater that do not add bacterium is control group.The every bottled 1000mL of going into test water body (sterilize breeding wastewater or unpasteurized breeding wastewater) specifically sees Table 1:
Table 1
Annotate: "+" expression adds Bacillus licheniformis; "-" expression does not add Bacillus licheniformis
Specific as follows:
1
#Bottle: add 1000mL test water body,, be cooled to room temperature through 126 ℃ of sterilization 60min;
2
#Bottle: add 1000mL test water body, through 126 ℃ of sterilization 60min, be cooled to room temperature, the aseptic technique platform is drawn this Bacillus licheniformis phase application quantity down and is rendered in the 1000mL test water body, guarantees that the bacterium final concentration that drops into water body reaches 3 * 10
6More than the CFU/mL, manually normally fill oxygen.
3
#Bottle: only add 1000mL test water body;
4
#Bottle: add 1000mL test water body, this Bacillus licheniformis phase application quantity is rendered in the 1000mL test water body under the aseptic technique platform, guarantees that the bacterium final concentration that drops into water body reaches 3 * 10
6More than the CFU/mL, manually normally fill oxygen.
Place 30 ℃ respectively, the 150rpm thermostat container is cultivated, and every 12h, carefully draws water sample 2mL from the middle level with pipettor under the aseptic technique platform, measures chemical oxygen demand (COD) with micro computer COD rapid determination instrument, measures 5 days altogether.The sampling and measuring time: after dropping into bacterial strain, begin sampling and measuring.
2, result
The COD value of control group almost remains unchanged in 48h, then descends slightly to some extent; In the test later stage, the COD value of control group is apparently higher than the COD value of test group.Pb-WC09005 reaches 84.60% to the COD clearance of breeding wastewater, to the sterilization water body the COD clearance reach 87.09%.Concrete outcome sees Table 2 and Fig. 5:
Table 2
Claims (3)
1. a plant height protease activity Bacillus licheniformis, it is characterized in that: this lichem bacillus strain preservation name is called: Bacillus licheniformis Pb-WC09005, depositary institution: Chinese typical culture collection center, preservation day: on January 25th, 2011, preserving number: CCTCC NO:M2011037.
2. the application of the described bacterial strain of claim 1 in the culture water quality of tank system improvement.
3. application according to claim 2 is characterized in that: above-mentioned breed water is meant torrid areas breed water, is freshwater aquiculture water or sea farming water.
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| CN103740605A (en) * | 2013-10-31 | 2014-04-23 | 华南理工大学 | Bacillus aquimaris and application thereof |
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| CN101323843A (en) * | 2008-07-25 | 2008-12-17 | 浙江大学 | Composite bacillus preparation for improving marine culture water environment and preparation thereof |
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| CN101323843A (en) * | 2008-07-25 | 2008-12-17 | 浙江大学 | Composite bacillus preparation for improving marine culture water environment and preparation thereof |
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| 《热带生物学报》 20100930 陈圣丰，林钦，周永灿，欧阳吉隆，王世锋，谢珍玉 《高蛋白酶活性的地衣芽孢杆菌热带菌种的筛选与鉴定》 第210页-第214页 1-3 第1卷, 第3期 * |
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| CN103740605A (en) * | 2013-10-31 | 2014-04-23 | 华南理工大学 | Bacillus aquimaris and application thereof |
| CN103740605B (en) * | 2013-10-31 | 2017-01-18 | 华南理工大学 | Bacillus aquimaris and application thereof |
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Application publication date: 20110907 |