Bacillus and application thereof

A technology of Bacillus and Bacillus pumilus, applied in application, bacteria, climate change adaptation and other directions, can solve problems such as environmental damage and residual poisoning

Inactive Publication Date: 2012-10-17
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Secondly, due to the extensive use of antibiotics, the disadvantages of microbial resistance, toxicity, teratogenicity, carcinogenicity, and mutations are gradually exposed. Residues, damage to the environment, and threats to human health have become prominent problems. The call for antibiotics is getting louder

Method used

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  • Bacillus and application thereof
  • Bacillus and application thereof
  • Bacillus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: BY-1 preparation method and enzyme activity characteristics

[0038] 1. Separation and screening of BY-1

[0039] 1) The source of Tibetan pig manure

[0040] Tibetan pig feces were collected from Tianxin Tibetan Pig Farm in Zhouzhi County, Shaanxi Province.

[0041] 2) Isolation and screening of strains

[0042] Take 0.3g of fresh feces and inoculate them into 30ml of BHM liquid medium, shake and culture at 37°C and 220r / min for 48h, draw the supernatant for gradient dilution (10 1 、10 2 、10 3 、10 4 、10 5、10 6 、10 7 ), respectively spread the diluted solution on the BHM solid medium, and after incubating at 37°C for 48 hours, stain with Congo red solution (1 mg / mL) for 1 hour; wash with 1 mol / L NaCl solution for 40 minutes. Select strains according to the ratio of the diameter of the transparent circle around the colony to the diameter of the colony. Colonies with large contrast values ​​were further isolated by streaking to obtain pure cultures an...

Embodiment 2

[0044] Example 2: Identification of BY-1

[0045] 1) Morphological features

[0046] The BY-1 strain was cultured in liquid LB medium at 37°C and shaken at 220rph for 36h. After Gram staining, it was observed under an optical microscope. The bacterial cells appeared blue-purple, and the staining result was Gram-positive bacteria ( figure 1 ), and spores appeared ( figure 2 ).

[0047] 2) BY-1 molecular biology identification

[0048] (1) Determination of gene fragment sequence of BY-1 strain 16SrRNA

[0049] Stretch culture of BY-1 preserved strains in solid LB medium, cultivate overnight at 37°C, select single clones in liquid LB batch medium, culture on a shaker at 37°C for 8 hours, collect bacteria by centrifugation, and adopt spin column type bacterial genome DNA extraction kit DP302 was used to extract short BY-1 total genomic DNA (Tiangen, China). Universal primer 27F / 2492R amplifies the 16S rRNA gene of BY-1 strain (F: AGAGTTTGATCMTGGCTCAG / R: TACGGYTACCTTGTTACGACT...

Embodiment 3

[0078] Example 3: Determination of endonuclease activity and enzymatic characteristics analysis produced by screening strains

[0079] 1) Determination of endonuclease activity of screening strains

[0080] (1) Required reagents and buffers

[0081] A) NS chromogenic solution-3,5-dinitrosalicylic acid

[0082] 3,5-dinitrosalicylic acid (DNS) chromogenic solution: accurately weigh 3,5-dinitrosalicylic acid: 6.3g, NaOH: 20g, dissolve in an appropriate amount of distilled water; then weigh potassium tartrate Sodium 182.0g, dissolved in 300mL ~ 500mL distilled water, heated until fully dissolved, mix the above two solutions, add 5.0g distilled phenol and 5.0g sodium sulfite, stir to dissolve, dilute to 1000mL, store in a brown bottle and put it in the refrigerator Use after one week, filter before use.

[0083] B) Preparation of buffer solution

[0084] (a) Disodium hydrogen phosphate-potassium dihydrogen phosphate buffer

[0085] Mother liquor A: 1 / 15mol / LNa 2 HPO 4 Soluti...

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Abstract

The invention relates to a new bacillus, which is classified and designated as Bacillus pumilus BY-1. The bacillus pumilus is separated from fresh excrement of a Tibetan pig; the bacillus pumilus is capable of generating cellulase and has the ability of degrading sodium carboxymethylcellulose; after 96 h of culture at 37 DEG C and 220 rpm, the activity of the carboxymethylcellulase is 0.92 mu mol / ml.min. With the gene sequence of the endonuclease gene bglC of Bacillus pumilus SAFR-032 as reference, a designed primer is successful in amplifying the endo-beta-1,4-glucanase gene of the Bacillus pumilus BY-1, which is designated as bglC-BY; and through NCBI blast (National Center for Biotechnology Information, Basic Local Alignment Search Tool), the similarity of the gene to the bglC is 88%, and the similarity thereof to the gene of Bacillus pumilus endoglucanase A precursor(EglA) is higher, up to 96%; therefore, the expression of pET expression system is realized and induction is startedwhen the absorbance A595 of the bacterial fluid is 0.7534 at a culture temperature of 30 DEG C and 220 rpm; the activity is highest in 42 hours after the induction; and the highest activity of the extracellular enzyme is measured to be 0.5434 mu mol / ml.min.

Description

technical field [0001] The present invention relates to a kind of bacterial strain, especially a kind of Bacillus isolated from Tibetan pig feces microbial beneficial flora, this Bacillus is a kind of new Bacillus pumilus, and classification name is Bacillus pumilus (Bacillus pumilus) BY-1, It can be used for the extraction and application of cellulase, or as a drug or medicinal additive for preventing and treating digestive tract diseases. Background technique [0002] 1. Application fields and prospects of cellulase [0003] Cellulase is widely used in animal husbandry, textile industry, paper industry, feed industry, food industry and other fields, and plays a pivotal role in new energy conversion. [0004] In recent years, the improvement of people's living standards has promoted the transformation of animal husbandry production from traditional free-range farming to intensive and modern production, which has resulted in a shortage of animal feed. Therefore, in the cas...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N15/56C12N9/42C05F3/00C05F11/02C05F11/08A61K35/74A61P1/00C12R1/07A61K35/742
CPCY02A40/20
Inventor 曹斌云刘海艳杨明明肖文萍安小鹏王建刚宋宇轩
Owner NORTHWEST A & F UNIV
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