Method for improving bacitracin produced by bacillus licheniformis

A technology for Bacillus licheniformis and bacitracin, which is applied in the field of improving the production of bacitracin by Bacillus licheniformis, can solve the problems of reduced yield of target products, loss of carbon atoms, etc., and achieves the effects of reducing growth toxicity, being easy to operate, and increasing yield

Active Publication Date: 2022-08-09
JIANGNAN UNIV
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, the accumulation of acetic acid will inhibit the growth and metabolism of E. coli on the one hand, and lead to the loss of carbon atoms and the lower yield of target products on the other hand.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for improving bacitracin produced by bacillus licheniformis
  • Method for improving bacitracin produced by bacillus licheniformis
  • Method for improving bacitracin produced by bacillus licheniformis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1: Screening of Bacillus licheniformis ATH strains

[0061] (1) After incubating the feed samples in a 28°C incubator for 24 hours, the feed samples were treated in a water bath at 80-85°C for 15 minutes, cooled to room temperature and then diluted with physiological saline. The sample dilution was spread on the bacitracin screening medium plate, and after culturing at a constant temperature of 37°C for 24 hours, the colonies that grew and full on the medium were picked and cultured in LB liquid medium.

[0062] (2) The single colony after screening is such as figure 1 As shown, the colonies are round, with uneven edges, thin, rough surface, opaque, and grayish white. The results of single-stain microscopy are as follows: figure 2 It was shown that the bacterial cells were about 1.5-3.0 μm in length and 0.6-0.8 μm in width, and the strains were Gram-positive. The 16S rDNA (nucleotide sequence shown in SEQ ID NO. 3) of the screened strain was sequenced, and th...

Embodiment 2

[0063] Example 2: Bacteriostatic test of Bacillus licheniformis ATH

[0064] (1) Screening of indicator strains

[0065] In order to verify the bacteriostatic effect of Bacillus licheniformis ATH, Escherichia coli, Bacillus cereus, Corynebacterium glutamicum, and Staphylococcus aureus were selected as indicator strains in this experiment;

[0066] After culturing the above indicated strains at 37°C for 12 hours overnight, diluting OD=0.8 with sterile water on an ultra-clean workbench, taking 200 μL and spreading them on LB plates as bacteriostatic indicator plates.

[0067] (2) Bacillus licheniformis ATH was inoculated into LB liquid medium, cultured at 37°C overnight, the bacterial concentration of Bacillus licheniformis ATH was diluted to OD600=1.0, and then filtered with a 0.22 μm microporous membrane in an ultra-clean workbench. sterilized filter paper with a diameter of 2 cm to absorb the quantitative bacterial solution, respectively point and inoculate it on the antibac...

Embodiment 3

[0082] Example 3: Construction of Acetyl-CoA synthase expression vector

[0083] Specific steps are as follows:

[0084] (1) The genomic DNA of Bacillus subtilis subsp 168 was used as a template, and F and R were used as primers for PCR amplification to obtain the heterologous acetyl-CoA synthase gene Bsacs (the nucleotide sequence is shown in SEQ ID NO.1). ); the primer sequences used are as follows:

[0085] F: 5-ATGGGTCGCGGATCCGATGAACTTGAAAGCGTTAC-3';

[0086] R: 5-CGAGTGCGGCCGCAATTAATCCTCCATTGTTGACAG-3'.

[0087] PCR amplification conditions were: 95°C pre-denaturation, 3 min; 95°C denaturation, 30s, 58°C annealing, 30s, 72°C extension, 90s, 30 cycles; 72°C final extension for 10 min.

[0088] The PCR amplification system was: template 0.5 μL, upstream and downstream primers 0.5 μL each, sterilized double-distilled water 23.5 μL, PrimerStar DNA polymerase 25 μL.

[0089] (2) ligating the gene Bsacs (with pHY300PLK plasmid) encoding acetyl-CoA synthase obtained in step ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
diameteraaaaaaaaaa
diameteraaaaaaaaaa
Login to view more

Abstract

The invention discloses a method for improving bacitracin produced by bacillus licheniformis and belongs to the technical field of microorganisms. The bacillus licheniformis ATH with high yield of bacitracin is screened from a feed sample, the acetyl coenzyme A synthetase Bsacs gene derived from bacillus subtilis is expressed in the bacillus licheniformis ATH through a genetic engineering method, supply of amino acid formed by intracellular bacitracin is increased, and the titer of bacitracin is improved. After the obtained recombinant bacillus licheniformis is inoculated into a fermentation culture medium containing soybean meal for fermentation culture for 42 hours, the titer of the obtained bacitracin is 1928 IU/mL and is improved by 46.16% compared with that of the bacitracin of an original strain ATH, and meanwhile, the content of bacitracin A is 1081 IU/mL and is improved by 37.2% compared with that of the original strain. The bacitracin production efficiency in the whole fermentation process is high, the raw material cost is low, and the method is simple and easy to operate.

Description

technical field [0001] The invention relates to a method for improving bacitracin production by Bacillus licheniformis, and belongs to the field of biotechnology. Background technique [0002] Bacitracin is a polypeptide spectrum antibiotic obtained by fermentation of Bacillus subtilis and Bacillus licheniformis, including Orn, D-Phe, His, D-Asp, Asn, Lys, D-Glu, Cys , Leu and Ile, are composed of a variety of cyclic homologues, namely Bacitracin A, B1, B2, B3, C1, C2, C3, F, of which Bacitracin A and B account for 95% of the activity, and its yield is synthesized It is determined by multiple genes and has a complex metabolic network. Its main production strains are Bacillus subtilis and Bacillus licheniformis. The bacteriostatic effect of bacitracin is due to its inhibitory effect on the formation of bacterial cell walls, thereby inhibiting Gram-positive bacteria and some target bacteria. Lanseria-negative bacteria are widely used in feed addition during animal breeding. ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N1/21C07K7/58A61K35/742A61K38/12A61P31/04A23L33/18A23L33/135A23K20/147A23K10/18A01N63/22A01P1/00C12R1/10
CPCC12N1/205C12N1/20C12N9/1029C07K7/58A61K35/742A61K38/12A61P31/04A23L33/18A23L33/135A23K20/147A23K10/18A01N63/22A01P1/00C12R2001/10Y02A50/30
Inventor 徐美娟饶志明吴洁琴夏博雅杨套伟张显
Owner JIANGNAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap