CN104762294A - Preparation method and application of bacillus subtilis mutant strain with high protease yield - Google Patents
Preparation method and application of bacillus subtilis mutant strain with high protease yield Download PDFInfo
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- CN104762294A CN104762294A CN201510157372.XA CN201510157372A CN104762294A CN 104762294 A CN104762294 A CN 104762294A CN 201510157372 A CN201510157372 A CN 201510157372A CN 104762294 A CN104762294 A CN 104762294A
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Abstract
The invention relates to a preparation method and application of a bacillus subtilis mutant strain with high protease yield. The method comprises the following steps: (1) preparing starting strains of bacillus subtilis; (2) carrying out culturing, mutagenesis and preliminary screening on the starting strains of bacillus subtilis; (3) screening out strains with relatively high enzyme activity; (4) screening out strains with high enzyme activity; (5) carrying out continuous shake-flask fermentation passage on the screened positive mutant strains for 10 times, wherein a finally obtained strain has good genetic stability. According to the invention, since a plasma technology in novel breeding technologies is adopted for carrying out mutagenesis on the starting strains in cooperation with ultraviolet light irradiation, the mutagenesis success rate is high, effect is significant, and the activity of protease produced by the mutant strain is substantially improved. When the strain is used for treating prawn waste and degrading protein, the specificity is high and the effect is significant. Also, the strain can be applied to the industry of animal feed. The strain has a good application and popularization prospect.
Description
Technical field
The invention belongs to preparation and the application thereof of the subtilis mutagenic strain in zymetology and fermentation engineering field, particularly a kind of high proteinase yield.
Background technology
Global prawn culturing output 3,380,000 tons in 2014, China is still the main prawn producing country in the whole world, cultured output 130.3 ten thousand tons.Breed variety is based on Environment of Litopenaeus vannamei Low, and account for 73% of cultivation total amount, all the other breed variety tigar prawn, Chinese prawn and japonicus respectively account for 7%, 6% and 6%.Each kind areal distribution is obvious, and Environment of Litopenaeus vannamei Low covers the whole nation, and Chinese prawn concentrates on the ground such as Shandong, Hebei and Jiangsu, japonicus in Shandong, Hebei cultivation is more, the cultivation of tigar prawn Guangdong is more.The consumption of prawn has three approach, and one is outlet, is mainly the shrimp that decaptitates, and two is that fresh shrimp eats, and three is that processing peeled shrimp is domestic.No matter which kind of approach, all can produce a large amount of shrimp heads, shrimp shell and shrimp tail waste, account for 20% ~ 50% of whole shrimp, containing a large amount of chitins and protein in these wastes, unsaturated fatty acids, astaxanthin, the nutritive ingredients such as mineral substance, chitin deacetylatedly can change chitosan having multi-functions into, but current production the utilization of shrimp waste adopts acid-base method chitin extraction, remaining nutritive ingredient is discharged in environment along with waste liquid, both environmental pollution was exacerbated, cause again the huge waste of resource, govern the development of prawn processing industry, therefore how scientifically prawn shrimp head is utilized to environmental protection, shrimp shell, improve economic value added, become the problem in science that current China is urgently to be resolved hurrily.
We show early-stage Study, prawn processing waste is through milk-acid bacteria decalcification and subtilis deproteinated, green safety chitin and compound protein powder are produced, there is very large value of exploiting and utilizing, and institute with subtilis separation and purification in prawn culturing pond, the proteinase activity produced is strong, specific aim is good, successful, on this basis, the method that our using plasma and ultraviolet mutagenesis combine, the subtilis that mutagenesis is separated, prepare high proteinase yield bacterial strain, for the higher value application of prawn processing waste, achieve significant treatment effect.
By retrieval, find following 1 section of patent document relevant to present patent application: the screening method (CN201210560999) that 1, the invention discloses a kind of high proteinase yield subtilis, through process and the enrichment of bacterial strain, the preliminary screening of bacterial strain and the multiple sieve of bacterial strain obtain high proteinase yield subtilis.The method is strong to high proteinase yield bacterial strain selectivity; Screening process adopts mixing screening, and efficiency is high; Methodological science, time cycle are short.By comparison, technical scheme and the extraction object of above-mentioned patent document all have the different of essence from present patent application, present patent application has novelty.
Summary of the invention
The object of the invention is for the deficiencies in the prior art, and propose a kind of preparation and application thereof of subtilis mutagenic strain of high proteinase yield.
The present invention solves its technical problem and takes following technical scheme to realize:
A preparation for the subtilis mutagenic strain of high proteinase yield, comprises step as follows:
(1) acquisition of subtilis starting strain, subtilis starting strain is obtained by screening in the bottom precipitation of prawn culturing pond;
(2) cultivation of subtilis starting strain, mutagenesis and preliminary screening, subtilis starting strain is inoculated in substratum, fermentation 6h, centrifugal, precipitation stroke-physiological saline solution is washed, dilute 5 times and survey 600nm place OD value 0.6-0.8 afterwards, the bacterium liquid got after 10 μ l dilutions is added on special tinsel, utilizes atmospheric pressure at room plasma body breeding machine to carry out strain mutagenesis; After mutagenesis, specimen slides is taken out, put in the centrifuge tube that 1ml sterile saline is housed, vibration thalline, make it thoroughly be suspended in physiological saline, with normal saline dilution, get 100 μ l and coat on screening flat board, according to the circle footpath ratio that bacterium colony produces, preliminary screening plasma inducing flattens the gain mutant bacterial strain on plate;
(3) filter out enzyme higher bacterial strain alive, respectively the bacterial strain of primary dcreening operation and original strain are inoculated in substratum, after incubated overnight, inoculum size transferred species by 1% in fermention medium, the centrifugal 3min of 15h, 12000r/min, supernatant is crude enzyme liquid, filters out enzyme higher bacterial strain alive;
(4) screen live high-enzyme strain, the high enzyme live strain filtered out is inoculated on flat board, irradiation time 5-10s under ultraviolet lamp, then repeats above process, screening live high-enzyme strain;
(5) go down to posterity 10 times to the continuous shake flask fermentation of positive mutating strain that screening obtains, the genetic stability of final obtained strains is better.
And in described step (2), substratum is: g/L, glucose 20.0, peptone 10.0, sodium-chlor 5.0, extractum carnis 5.0.
And in described step (2), the temperature of fermentation is: 25-35 DEG C.
And in described step (2), the mutagenic condition of strain mutagenesis is: power is 115W, and working gas flow is 10L/min, the spacing of plasma emission source and sample is 2mm, helium is as working gas, and irradiation time is 20-40s, ultra violet lamp time 5-10s.
And optimum irradiation time is 8s under described step (5) medium ultraviolet lamp.
An application for the subtilis mutagenic strain of high proteinase yield, is characterized in that: by above-mentioned screening gained subtilis mutagenic strain for the treatment of to shrimp waste.
And, optimal pH 6-6.5, optimum temperuture 45-50 DEG C during described subtilis mutagenic strain proteinase.
Advantage of the present invention and positively effect are:
1, the starting strain of the present invention's employing is from prawn culturing pond, with its process to shrimp waste, and degrade proteins, with strong points, successful.
2, the present invention adopts the plasma technique in new technology of breeding, mutagenesis is carried out for starting strain, again in conjunction with uv irradiating, mutagenesis success ratio is high, Be very effective, the proteinase activity that mutagenic strain produces significantly increases, and both may be used for specific aim process to shrimp waste, can animal feed industries be applied in again, there is good application prospect.
Embodiment
Below in conjunction with embodiment, the present invention is further described, and following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
Embodiment 1
The preparation of the subtilis mutagenic strain of high proteinase yield, comprises the following steps:
(1) acquisition of subtilis starting strain, subtilis starting strain is obtained by screening in the bottom precipitation of prawn culturing pond;
(2) cultivation of subtilis starting strain, mutagenesis and preliminary screening, subtilis starting strain is inoculated in substratum, and wherein substratum is: g/L, glucose 20.0, peptone 10.0, sodium-chlor 5.0, extractum carnis 5.0; Fermentation 6h, leavening temperature 27 DEG C, centrifugal, precipitation stroke-physiological saline solution is washed, dilute 5 times and survey 600nm place OD value (0.69) afterwards, the bacterium liquid got after 10 μ l dilutions is added on special tinsel, atmospheric pressure at room plasma body breeding machine (ARTP) is utilized to carry out strain mutagenesis, irradiation time 30s, wherein, the mutagenic condition of starting strain is: power is 115W, working gas flow is 10L/min, and the spacing of plasma emission source and sample is 2mm, and helium is as working gas, irradiation time is 20s, ultra violet lamp time 5s.
After mutagenesis, specimen slides is taken out, put in the centrifuge tube that 1ml sterile saline is housed, vibration thalline, make it thoroughly be suspended in physiological saline, with normal saline dilution, get 100 μ l and coat on screening flat board, according to the circle footpath ratio that bacterium colony produces, preliminary screening plasma inducing flattens the gain mutant bacterial strain on plate;
(3) enzyme higher bacterial strain alive is filtered out, respectively the bacterial strain of primary dcreening operation and original strain are inoculated in substratum, after incubated overnight, inoculum size transferred species by 1% is in fermention medium, the centrifugal 3min of 15h, 12000r/min, supernatant is crude enzyme liquid, measure proteinase activity 235.63U/mL, filter out enzyme higher bacterial strain alive;
(4) screen live high-enzyme strain, the live high-enzyme strain filtered out is inoculated on flat board, irradiation time 8s under ultraviolet lamp, then repeats above process, measure proteinase activity 364.29U/mL, screening live high-enzyme strain;
(5) go down to posterity 10 times to the continuous shake flask fermentation of positive mutating strain that screening obtains, measure proteinase activity 359.15U/mL, the proteinase activity 95.18 ± 7.29U/mL of starting strain, the genetic stability of final obtained strains is better.
The application of the subtilis mutagenic strain of high proteinase yield,
By above-mentioned screening gained subtilis mutagenic strain for the treatment of to shrimp waste, deproteinizing rate reaches 95.81%.Optimal pH 6-6.5, optimum temperuture 45-50 DEG C during described subtilis mutagenic strain proteinase,
2. embodiment 2
The preparation method of the subtilis mutagenic strain of high proteinase yield, comprises the following steps:
(1) acquisition of subtilis starting strain, subtilis starting strain obtains by prawn culturing pond;
(2) cultivation of subtilis starting strain, mutagenesis and preliminary screening, subtilis starting strain is inoculated in substratum, and wherein substratum is: g/L, glucose 20.0, peptone 10.0, sodium-chlor 5.0, extractum carnis 5.0; Fermentation 6h, leavening temperature 30 DEG C, centrifugal, precipitation stroke-physiological saline solution is washed, dilute 5 times and survey 600nm place OD value (0.75) afterwards, the bacterium liquid got after 10 μ l dilutions is added on special tinsel, atmospheric pressure at room plasma body breeding machine (ARTP) is utilized to carry out strain mutagenesis, irradiation time 35s, wherein, the mutagenic condition of starting strain is: power is 115W, working gas flow is 10L/min, and the spacing of plasma emission source and sample is 2mm, and helium is as working gas, irradiation time is 40s, ultra violet lamp time 10s.
After mutagenesis, specimen slides is taken out, put in the centrifuge tube that 1ml sterile saline is housed, vibration thalline, it is made thoroughly to be suspended in physiological saline, with normal saline dilution, get 100 μ l and coat on screening flat board, according to the gain mutant bacterial strain that the circle footpath of bacterium colony generation flattens on plate than preliminary screening plasma inducing;
(3) enzyme higher bacterial strain alive is filtered out, respectively the bacterial strain of primary dcreening operation and original strain are inoculated in substratum, after incubated overnight, inoculum size transferred species by 1% is in fermention medium, the centrifugal 3min of 15h, 12000r/min, supernatant is crude enzyme liquid, measure proteinase activity 229.54U/mL, screening enzyme higher bacterial strain alive;
(4) screen live high-enzyme strain, the high enzyme live strain of screening is inoculated on flat board, irradiation time 5s under ultraviolet lamp, then repeats above process, measure proteinase activity 359.67U/mL, screening live high-enzyme strain;
(5) the continuous shake flask fermentation of positive mutating strain that screening obtains is gone down to posterity 10 times, measure proteinase activity 361.36U/mL.
The application of the subtilis mutagenic strain of high proteinase yield,
By above-mentioned screening gained subtilis mutagenic strain for the treatment of to shrimp waste, deproteinizing rate reaches 96.39%.The proteolytic enzyme of described subtilis mutagenic strain fermentative production, optimal pH 6-6.5, optimum temperuture 45-50 DEG C.
Claims (7)
1. a preparation for the subtilis mutagenic strain of high proteinase yield, is characterized in that comprising step as follows:
(1) acquisition of subtilis starting strain, subtilis starting strain is obtained by screening in the bottom precipitation of prawn culturing pond;
(2) cultivation of subtilis starting strain, mutagenesis and preliminary screening, subtilis starting strain is inoculated in substratum, fermentation 6h, centrifugal, precipitation stroke-physiological saline solution is washed, dilute 5 times and survey 600nm place OD value 0.6-0.8 afterwards, the bacterium liquid got after 10 μ l dilutions is added on special tinsel, utilizes atmospheric pressure at room plasma body breeding machine to carry out strain mutagenesis; After mutagenesis, specimen slides is taken out, put in the centrifuge tube that 1ml sterile saline is housed, vibration thalline, make it thoroughly be suspended in physiological saline, with normal saline dilution, get 100 μ l and coat on screening flat board, according to the circle footpath ratio that bacterium colony produces, preliminary screening plasma inducing flattens the gain mutant bacterial strain on plate;
(3) filter out enzyme higher bacterial strain alive, respectively the bacterial strain of primary dcreening operation and original strain are inoculated in substratum, after incubated overnight, inoculum size transferred species by 1% in fermention medium, the centrifugal 3min of 15h, 12000r/min, supernatant is crude enzyme liquid, filters out enzyme higher bacterial strain alive;
(4) screen live high-enzyme strain, the high enzyme live strain filtered out is inoculated on flat board, irradiation time 5-10s under ultraviolet lamp, then repeats above process, screening live high-enzyme strain;
(5) go down to posterity 10 times to the continuous shake flask fermentation of positive mutating strain that screening obtains, the genetic stability of final obtained strains is better.
2. the preparation of the subtilis mutagenic strain of high proteinase yield according to claim 1, is characterized in that: in described step (2), substratum is: g/L, glucose 20.0, peptone 10.0, sodium-chlor 5.0, extractum carnis 5.0.
3. the preparation of the subtilis mutagenic strain of high proteinase yield according to claim 1, is characterized in that: in described step (2), the temperature of fermentation is: 25-35 DEG C.
4. the preparation of the subtilis mutagenic strain of high proteinase yield according to claim 1, it is characterized in that: in described step (2), the mutagenic condition of strain mutagenesis is: power is 115W, working gas flow is 10L/min, the spacing of plasma emission source and sample is 2mm, helium is as working gas, irradiation time is 20-40s, ultra violet lamp time 5-10s.
5. the preparation of the subtilis mutagenic strain of high proteinase yield according to claim 1, is characterized in that: under described step (5) medium ultraviolet lamp, optimum irradiation time is 8s.
6. an application for the subtilis mutagenic strain of high proteinase yield, is characterized in that: by above-mentioned screening gained subtilis mutagenic strain for the treatment of to shrimp waste.
7. the preparation of the subtilis mutagenic strain of high proteinase yield according to claim 6, is characterized in that: during described subtilis mutagenic strain proteinase, optimal pH 6-6.5, optimum temperuture 45-50 DEG C.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103130914A (en) * | 2011-10-08 | 2013-06-05 | 天津科技大学 | Method for preparing chitin and composite protein powder by composite microbial fermentation of prawn leftovers |
| CN103865855A (en) * | 2014-03-25 | 2014-06-18 | 南京工业大学 | Bacillus subtilis strain and application thereof |
| CN104371994A (en) * | 2014-10-16 | 2015-02-25 | 江南大学 | Method for high-throughput screening of recombinase high-yield bacillus subtilis host in combination with normal pressure room temperature plasma mutation mode |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103130914A (en) * | 2011-10-08 | 2013-06-05 | 天津科技大学 | Method for preparing chitin and composite protein powder by composite microbial fermentation of prawn leftovers |
| CN103865855A (en) * | 2014-03-25 | 2014-06-18 | 南京工业大学 | Bacillus subtilis strain and application thereof |
| CN104371994A (en) * | 2014-10-16 | 2015-02-25 | 江南大学 | Method for high-throughput screening of recombinase high-yield bacillus subtilis host in combination with normal pressure room temperature plasma mutation mode |
Non-Patent Citations (4)
| Title |
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| 乐文民: "中性纤维素酶产生菌的筛选及发酵条件初步优化", 《中国优秀硕士学位论文全文数据库工程科技I辑》 * |
| 薛刚 等: "ARTP诱变选育高温蛋白酶高产菌株及其酶学性质研究", 《食品工业科技》 * |
| 马宏宇 等: "蛋白酶产生菌的化学诱变及酶学性质", 《华中农业大学学报》 * |
| 马琦亚: "DNA shuffling 提高磷脂酶A1的催化性能", 《中国优秀硕士学位论文全文数据库基础科学辑》 * |
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Application publication date: 20150708 |