CN102719379A - Bacillus tequilensis and application thereof - Google Patents
Bacillus tequilensis and application thereof Download PDFInfo
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- CN102719379A CN102719379A CN2012101973752A CN201210197375A CN102719379A CN 102719379 A CN102719379 A CN 102719379A CN 2012101973752 A CN2012101973752 A CN 2012101973752A CN 201210197375 A CN201210197375 A CN 201210197375A CN 102719379 A CN102719379 A CN 102719379A
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Abstract
The invention discloses bacillus tequilensis CGX5-1 which is preserved with the number CGMCC No.5935 in China General Microbiological Culture Collection Center on March 26, 2012. The bacillus tequilensis has high capability of producing beta-1, 3-1, 4-glucanase, and the bacillus tequilensis and good genes thereof are applied to the field of production of industrial enzyme preparations and have good prospects in reduction of cost for producing the beta-1, 3-1, 4-glucanase and improvement on enzyme preparation quality.
Description
Technical field
The invention belongs to technical field of bioengineering, relate to a strain Te Jila genus bacillus and an application thereof.
Technical background
Genus bacillus is of a great variety, distributes extensively, and the enteron aisle etc. that mainly is present in soil, water, air and animal is located.And the extracellular enzyme kind is abundant, has the excellent research prospect.Because the complicacy of growing environment, genus bacillus also can secrete the generation cellulase, like beta-glucanase.Beta-glucanase has a wide range of applications in brewage: in saccharifying, use beta-glucanase, can reduce the viscosity of wheat juice, filtration velocity is accelerated, thereby cut down the consumption of energy; In addition, in wheat juice, add beta-glucanase and can also reduce glucan content, prevent that beer is muddy, thereby improve the non-biostability of beer.In the wheat class feed owing to contain higher beta-glucan; Hindered effective constituent digesting and assimilating in animal intestinal in the feed; Become a kind of ANFs, and, can cause problems such as livestock and poultry diarrhea for the breeding of living away from home of pathogenic bacterium provides abundant nutrition; In feed, adding an amount of beta-glucanase will effectively address the above problem, thereby improve the utilization ratio of feed.
China is beer prodn big country, and beer production reached 4,610 ten thousand kilolitres in 2011, and the constantly soaring of beer production means that the beta-glucanase demand also increases day by day.The present domestic zymin of using is removed a small amount of enzyme Preparations Used for Feeds and is domestic production; Most of zymin all depends on imported product; Because production technology belongs to trade secret; This has limited the development of beer and fodder industry largely, and the industrial enzyme preparation technology that development has independent intellectual property rights is significant.The mikrobe of separation screening high yield beta-glucanase is the basis of zymotechnic, and China is vast in territory, and environmental aspect is complicated, has brought up abundant Microbial resources, for carrying out of separation screening work provides multiple possibility.Producing the wild bacterium of beta-glucanase with an isolating strain is resource, in conjunction with breeding technique, obtains the industrial producing strain of high yield beta-glucanase, finally for the production of industrial enzyme preparation new thinking and resource is provided.
Summary of the invention
The invention provides strain Te Jila genus bacillus (Bacillus tequilensis) CGX5-1.
Te Jila genus bacillus provided by the present invention (Bacillus tequilensis) CGX5-1 is preserved in Chinese typical culture collection center on March 26th, 2012, and deposit number is CGMCCNo.5935.
The invention provides the application of a kind of said Te Jila genus bacillus; From cultured Te Jila genus bacillus, transfer β-1; 3-1,4-glucanase gene, acidity, neutrality and Sumizyme MP genoid etc. carry out heterogenous expression, make up the genetic engineering bacterium of industrial enzyme preparation.
Said Te Jila genus bacillus is for to screen from hornet nest relic; Its storage conditions is following: get 1 ring genus bacillus from well-grown flat board and move into the LB substratum; 37 ℃ of shake-flask culture 15h; Get the 0.75mL immigration and fill in the glycerine pipe of aseptic 40% glycerine of 0.25mL, put-70 ℃ of refrigerators and preserve.
Said LB substratum is: Tryptone 10g L
-1, Yeast Extract 5g L
-1, Nacl 10g L
-1, pH7.0.
Fermention medium is: Semen Maydis powder 40g L
-1, soybean cake powder 61.1g L
-1, barley meal 68.4g L
-1, potassium hydrogenphosphate 1g L
-1, calcium chloride 0.1g L
-1, sal epsom 1g L
-1, alpha-amylase 16 μ L/L.
Said Te Jila genus bacillus culture condition is: glycerine is guaranteed that the genus bacillus of Tibetan inserts the LB liquid nutrient medium, at 37 ℃ of 200rpm shake-flask culture 15h to the logarithm middle and later periods, as seed liquor.With 2% inoculum size seed liquor is forwarded in the fermention medium, 37 ℃ of 200rpm cultivate, respectively at 12,24,36,48,60h is sampling spot, β in the working sample-1,3-1, the vigor of 4-LSD.
The biomaterial preservation
Te Jila genus bacillus provided by the present invention (Bacillus tequilensis) CGX5-1 is preserved in Chinese typical culture collection center on March 26th, 2012, and deposit number is CGMCCNo.5935.
Embodiment
Screening of embodiment 1 Te Jila genus bacillus and enzyme activity determination method
From the relic of hornet nest, get certain sample and join the 30mL SPSS, granulated glass sphere is broken up and is processed suspension liquid, handles 10min for 80 ℃.With the suspension liquid gradient dilution, coat that to add 2% Congo red MRS dull and stereotyped, cultivate 24h in 37 ℃ of incubators.Picking makes the tangible single bacterium colony of Congo red variable color circle.Carry out plate streaking repeatedly and separate, triplicate obtains single bacterium colony of purifying, supplies next step to identify and uses.
Identify and mainly to pass through: 1) colony characteristics is observed: direct viewing with the naked eye, and the choosing colony diameter is between 1-1.5mm, and circle, smooth surface have fold slightly, and oyster whites etc. meet the bacterial strain of genus bacillus colony characteristics.2) individual morphology is observed: observe with the opticmicroscope gramstaining.Select Gram-positive, the bacterial strain of gemma is arranged.The bacterial strain that satisfies the evaluation condition is chosen glycerine guarantee the Tibetan.
Measure-70 ℃ of glycerine stock solutions with 2% inoculation and be inoculated in the LB substratum 37 ℃ of 200rpm shake-flask culture 15h.Measure 37 ℃ of 200rpm shake-flask culture things with 2% inoculation, be forwarded in the fermention medium 37 ℃ of 200rpm shake-flask culture 36 hours.Get the centrifugal 5min of fermented liquid (8000rpm, 4 ℃) and obtain supernatant.Supernatant is diluted suitable multiple with distilled water; Get the 0.1mL diluent, add blue dextran substrate (using the acetate buffer solution equal-volume of the pH5.5 of preceding and 0.2M to mix), at 40 ℃ of reaction 10min through 40 ℃ of preheatings; Add the 3.0mL reaction terminating liquid in each response sample; Shake up the centrifugal 5min of back 8000rpm, the cuvette of 1cm is measured the light absorption value of the stillness of night at the 590nm place, does blank with the distilled water replacement enzyme liquid of 0.1mL.Through measuring β-1 in the fermented liquid, 3-1, the vigor of 4-LSD; Screening a strain has and produces β-1 more by force; 3-1, the bacterial strain of 4-LSD ability can know that through the 16SrDNA sequential analysis this bacterial strain is the Te Jila genus bacillus; Be preserved in Chinese common micro-organisms center on March 26th, 2012, deposit number is CGMCCNo.5935.
Embodiment 2 fermentative prodn β-1,3-1, the technology of 4-LSD
The Te Jila fermentation of bacillus is produced β-1,3-1, and the employed substratum of 4-LSD is divided into seed culture medium and fermention medium.Wherein seed culture medium for the LB substratum is: Tryptone 10g L
-1, Yeast Extract 5g L
-1, Nacl 10g L
-1, pH7.0.The fermentation culture based component mainly contains: Semen Maydis powder 40g L
-1, soybean cake powder 61.1g L
-1, barley meal 68.4g L
-1, potassium hydrogenphosphate 1g L
-1, calcium chloride 0.1g L
-1, sal epsom 1g L
-1, alpha-amylase 16 μ L/L.Liquid amount is the every 250mL triangular flask of 30mL.The medium sterilization condition is 121 ℃, 20min.
Single bacterium colony of picking Te Jila genus bacillus is behind activation 8h on the inclined-plane from the flat board, and picking one ring bacterium is connected to the LB substratum from the inclined-plane, and 37 ℃ of 200rpm shake-flask culture 15h are to the logarithm middle and later periods, as seed liquor.With 2% inoculum size seed liquor is forwarded in the fermention medium, 37 ℃ of 200rpm cultivate, respectively at 12,24,36,48,52,56,60h is sampling spot, β in the working sample-1,3-1, the vigor of 4-LSD.The result shows, this bacterium β-1 in its fermented liquid that in fermention medium, grows behind the 52h, and 3-1, the work of 4-LSD enzyme reaches mxm., is 191.96U/mL.
Embodiment 3 is applied to make up genetic engineering bacterium
Clone wherein β-1 according to bglS design primer among the Bacillus amyloliquefaciens LL3 (NC_017190.1) among the NCBI; 3-1; The 4-glucanase gene is used for the construction expression plasmid, is expressed in to carry to use intestinal bacteria series or pichia yeast series, is used for efficient fermentation expression and produces β-1; 3-1, the 4-LSD.
It is understandable that, concerning those of ordinary skills, can be equal to replacement or change according to technical scheme of the present invention and inventive concept thereof, and all these changes or replacement all should belong to the protection domain of the appended claim of the present invention.
Claims (5)
1. strain Te Jila genus bacillus (Bacillus tequilensis) CGX5-1 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on March 26th, 2012, and deposit number is CGMCC No.5935.
2. cultivating the method for the said Te Jila genus bacillus of claim 1, is shake-flask culture Te Jila genus bacillus in LB substratum or nutrient broth medium.
3. cultural method as claimed in claim 2 is characterized in that preferred pH 7.0; 37 ℃ of preferred culture temperature.
4. the said Te Jila genus bacillus of claim 1 is applied to clone β-1,3-1,4-glucanase gene, the genetic engineering bacterium of structure industrial enzyme preparation.
5. the said Te Jila genus bacillus of claim 1 is applied to fermentative prodn β-1,3-1,4-LSD.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104862252A (en) * | 2015-05-22 | 2015-08-26 | 福建省农业科学院农业生物资源研究所 | Bacillus tequilensis strain for producing surfactant |
| CN105349462A (en) * | 2015-11-26 | 2016-02-24 | 江苏禾喜生物科技有限公司 | Bacillus tequilensis Hexil and application thereof to composting |
| CN105695368A (en) * | 2016-04-11 | 2016-06-22 | 江苏省农业科学院 | Tequila bacillus and application thereof |
| CN106342898A (en) * | 2016-08-29 | 2017-01-25 | 佛山市艳晖生物科技有限公司 | Compound microorganism preparation against sclerotinia rot of colza and preparing method thereof |
| CN109022327A (en) * | 2018-09-05 | 2018-12-18 | 福建省致青生态环保有限公司 | A kind of preparation method of microorganism mix bacterium agent and the application in During High-Temperature Composting |
| CN110257486A (en) * | 2019-06-27 | 2019-09-20 | 福建省致青生态环保有限公司 | A method of based on cellulose enzyme gene characterizing compost maturity |
| CN112195125A (en) * | 2020-10-15 | 2021-01-08 | 千禾味业食品股份有限公司 | Fermentation method for improving amino acid nitrogen, reducing sugar and flavor of brewed soy sauce |
| CN114990000A (en) * | 2022-03-29 | 2022-09-02 | 华中农业大学 | Compound microbial agent for degrading DEHP and application of compound microbial agent in preparation of organic fertilizer |
| CN115444118A (en) * | 2022-11-09 | 2022-12-09 | 四川大学 | Dried radish production method |
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| CN101285071A (en) * | 2008-05-13 | 2008-10-15 | 江南大学 | Recombination bacillus coli of high producing beta-glucanase and construction thereof |
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Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104862252B (en) * | 2015-05-22 | 2018-01-19 | 福建省农业科学院农业生物资源研究所 | A kind of Te Jila Bacillus strains for producing surfactant |
| CN104862252A (en) * | 2015-05-22 | 2015-08-26 | 福建省农业科学院农业生物资源研究所 | Bacillus tequilensis strain for producing surfactant |
| CN105349462A (en) * | 2015-11-26 | 2016-02-24 | 江苏禾喜生物科技有限公司 | Bacillus tequilensis Hexil and application thereof to composting |
| CN105349462B (en) * | 2015-11-26 | 2018-11-06 | 江苏禾喜生物科技有限公司 | One plant of American aloe bacillus Hexi1 and its application in compost |
| CN105695368B (en) * | 2016-04-11 | 2019-03-22 | 江苏省农业科学院 | One plant of Te Jila bacillus and its application |
| CN105695368A (en) * | 2016-04-11 | 2016-06-22 | 江苏省农业科学院 | Tequila bacillus and application thereof |
| CN106342898A (en) * | 2016-08-29 | 2017-01-25 | 佛山市艳晖生物科技有限公司 | Compound microorganism preparation against sclerotinia rot of colza and preparing method thereof |
| CN109022327A (en) * | 2018-09-05 | 2018-12-18 | 福建省致青生态环保有限公司 | A kind of preparation method of microorganism mix bacterium agent and the application in During High-Temperature Composting |
| CN109022327B (en) * | 2018-09-05 | 2022-03-11 | 福建省致青生态环保有限公司 | Preparation method of microbial mixed inoculant and application of microbial mixed inoculant in high-temperature composting |
| CN110257486A (en) * | 2019-06-27 | 2019-09-20 | 福建省致青生态环保有限公司 | A method of based on cellulose enzyme gene characterizing compost maturity |
| CN110257486B (en) * | 2019-06-27 | 2022-09-09 | 福建省致青生态环保有限公司 | Method for characterizing compost maturity based on cellulase gene |
| CN112195125A (en) * | 2020-10-15 | 2021-01-08 | 千禾味业食品股份有限公司 | Fermentation method for improving amino acid nitrogen, reducing sugar and flavor of brewed soy sauce |
| CN114990000A (en) * | 2022-03-29 | 2022-09-02 | 华中农业大学 | Compound microbial agent for degrading DEHP and application of compound microbial agent in preparation of organic fertilizer |
| CN115444118A (en) * | 2022-11-09 | 2022-12-09 | 四川大学 | Dried radish production method |
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| CN102719379B (en) | 2014-06-18 |
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Address after: No. 258 Wuxing Jiayuan, Liangxi District, Wuxi City, Jiangsu Province Patentee after: Jiangnan University Address before: 214122 Jiangsu Province, Wuxi City Lake Road No. 1800, Jiangnan University Institute of biological engineering Patentee before: Jiangnan University |