Dissertation
Dissertation > Industrial Technology > Chemical Industry > Pharmaceutical chemical industry > Antibiotics manufacture

Study on Breeding of Prolific Micromonospora Carbonacea in Antibiotics Production

Author LiZuo
Tutor LongZhongEr
School Jiangxi Normal University
Course Biochemical Engineering
Keywords Micromonospora cabonacea Nucleoside antibiotic Mutationbreeding Protoplast fusion breeding Resistance screening
CLC TQ465
Type Master's thesis
Year 2013
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With the resistant strains emerging increasingly and the antibiotics used widely,to research and develop new antibiotics becomes the necessary request for thedevelopment of human health. Micromonospora is an important source of newantibiotics. Micromonospora carbonacea JXNU-1, which was obtained by ourresearch group, can produce a single-component antibiotic. However, the antibiotic isnew nucleoside antibiotic, the low yield of which restrict the industrializedproduction.The paper selected Micromonospora carbonacea JXNU-1as the researchobject. At first, the minial inhibitory concentration of these antibiotics forMicromonospora carbonacea were determined by filtrating sensitive antibiotics.Then, the overproducing strain, which was screened by natural selection, was mutatedby4mutation methods, combined the resistance screening and including heatmutagenesis,Microwave irradiation, the nitrous acid mutagenesis, heating-HNO2complex mutagenesis. Finally, protoplats fusion of JXNU-1-16-R4andJXNU-1-16-W12was to select high antibitic-producing fusant. The results were asfollows:(1) Micromonospora carbonacea was sensitive to many antibiotics.Thesensitive degree were, in the descending order, ampicillin, ciprofloxacin, ofloxacin,tetracycline, gentamycin, rifampicin. Besides, the minial inhibitory concentration ofthese antibiotics for Micromonospora carbonacea were1μg/mL,16μg/mL,32μg/mL,40μg/mL,80μg/mL,128μg/mL respectively.(2) The overproducing strain JXNU-1-16was screened from Micromonosporacarbonacea JXNU-1by natural selection, the inhibition zone diameter of whichreached26.00mm. Thus, the strain JXNU-1-16was screened as an original strain ofmutation breeding.(3) After heat mutagenesis and gentamicin resistance screening,a mutant namedMicromonospora carbonacea JXNU-1-16-R4was obtained from the strain ofMicromonospora carbonacea JXNU-1-16. The inhibition zone diameter of theoverproducing strain JXNU-1-16-R4reached29.15mm. The production of antibioticsis98.53%more than that of the original strain. The temperatuer of76℃and heat treatment time of10min were optimum conditions of heat mutagenesis.(4) As an original strain, Micromonospora carbonacea JXNU-1-16was mutatedby microwave irradition. Combined with ciprofloxacin resistance screening,anoverproducing mutant was screened under the microwave irradition time of9s, theinhibition zone diameter of which reached27.80mm. Comparing with the originalstrain, the production of antibiotics was47.98%.(5) Because the concentration of sodium nitrite and nitrous acid treatment timehad the obvious dosage-effect relationship with the lethality rate of Micromonosporacarbonacea, optimum mutagenic conditions sodium nitrite of0.1mol/L and treatmenttime of20min were determined. After nitrous acid treatment,a mutant was screenedon the plate including rifampicin of128ug/mL.The inhibition zone diameter of theoverproducing strain JXNU-1-16-Y65reached31.96mm. The production ofantibiotics is266%more than that of the original strain.(6) A method of heating-HNO2complex mutation treatment onMicromonospora carbonacea JXNU-1-16was introduced. Under optimum conditions(heat treatment temperatuer of76℃, heat treatment time of10min, sodium nitriteof0.1mol/L and treatment time of20min), a mutant, Micromonospora carbonaceaJXNU-1-16-F28, the inhibition zone diameter of which reached32.06mm, wasobtained on the plate of gentamycin and ciprofloxacin. Relatively, the production ofantibiotics was274.1%higher than the original strain.(7) Firstly, protoplasts of Micromonospora carbonacea JXNU-1-16-R4andMicromonospora carbonacea JXNU-1-16-W12were gained under the condition ofthe concentration of enzyme3mg/mL, enzymatic hydrolysis time90min, enzymatichydrolysis temperature33℃. Then, PEG-induced protoplasts fusion method andgentamycin and ciprofloxacin double resistant screening were used to select highantibiotic-producing strain. At last, the fusant Micromonospora carbonaceaJXNU-1-Y6was screened from40fusants. After shaking flask culture, the inhibitionzone diameter of the fusant reached26.38mm, lower than the parent stain.

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