本文關鍵詞：轉化右旋磷霉素菌株的誘變及培養(yǎng)條件優(yōu)化　出處：《沈陽農業(yè)大學》2017年碩士論文　論文類型：學位論文

  更多相關文章： 右旋磷霉素 解淀粉芽孢桿菌 復合誘變 響應面法 金黃色葡萄球菌

【摘要】：磷霉素是一種結構簡單且分子量小的新型廣譜抗生素,對革蘭陽性和陰性菌均有殺菌作用,被國際藥學界列為二十一世紀新型抗生素。目前工業(yè)上采用化學合成法生產磷霉素的總收率不足35%,其中50%左旋磷霉素有抗生素活性,另一半的右旋磷霉素為無活性物質。這樣不但浪費了資源,提高了生產成本,同時也污染了環(huán)境。因此轉化右旋磷霉素、提高其有效利用,對工業(yè)化生產磷霉素具有重要意義。微生物轉化法具有高度專一性,發(fā)生反應的條件溫和,工藝簡單,成本較低等特點,能夠完成化學方法難以進行的反應。采用微生物轉化法,利用具有轉化能力的生產菌株,可將右旋磷霉素中的一部分轉化為有效的左旋磷霉素。本研究在實驗中篩選出1株解淀粉芽孢桿菌,并對其進行理化復合誘變。通過試驗確定了紫外線對解淀粉芽孢桿菌的最佳誘變時間是90s,經紫外誘變后,選育出1株突變株UV2,對其進行發(fā)酵培養(yǎng),發(fā)現(xiàn)突變菌株UV2的轉化能力有所提高,其轉化率為11.76%;UV2相較于原始菌株,轉化率升高3.84%。然后以UV2為出發(fā)菌株,選擇硫酸二乙酯再進行化學誘變,確定誘變的最佳時間是40min,經過誘變后選育出1株突變菌株U-D2,對其進行發(fā)酵培養(yǎng),發(fā)現(xiàn)突變菌株的轉化能力有所提高,其轉化率為13.2%,與UV2相比轉化率提高了 1.44%。U-D2與原始菌株解淀粉芽孢桿菌相比轉化率提高了5.25%。對突變菌株U-D2進行了 6代傳代培養(yǎng),以金黃色葡萄球菌為指示菌,通過發(fā)酵液的抑菌性能測定,結果表明突變菌株U-D2具有良好的遺傳穩(wěn)定性。采用單因素試驗結合響應面設計法中的CCD中心組合試驗設計方法,對U-D2的培養(yǎng)基營養(yǎng)成分進行優(yōu)化。確定最優(yōu)培養(yǎng)基濃度為:牛肉膏3.62g/L、葡萄糖12.39g/L、蛋白胨7.19 g/L、硫酸鎂0.42 g/L、磷酸氫二鉀0.85 g/L。突變菌株U-D2以優(yōu)化的培養(yǎng)基培養(yǎng),其培養(yǎng)液通過抑菌試驗,測得抑菌圈直徑為37.4mm,U-D2的轉化率為15.51%,與培養(yǎng)基優(yōu)化前相比轉化率提高了 2.31%。
[Abstract]:Fosfomycin is a new broad-spectrum antibiotics has the advantages of simple structure and low molecular weight, the gram positive and negative bacteria have bactericidal effect, by the international pharmaceutical industry listed as twenty-first Century. New antibiotic is widely used in industrial chemical synthesis, the total yield of production of phosphonomycin is less than 35%, of which 50% levo fosfomycin is known as antibiotic activity right, the other half of fosfomycin non active substances. It is not only a waste of resources, improve the cost of production, but also pollutes the environment. So the transformation of dextrofosfomysin, improve the effective use of hormone has important significance for the industrial production of fosfomycin. Microbial transformation method has high specificity, reaction conditions a mild, simple process, low cost, able to complete the chemical method is difficult to analyze reaction. By using microbial transformation method, using the production strain has the ability of converting the dextrofosfomysin in a Part of the transformation is left-handed fosfomycin effective. The study in the experiment screened 1 strains of Bacillus amyloliquefaciens, and its chemical compound mutagenesis. Through the test the UV is 90s on the best mutation time of Bacillus amyloliquefaciens, by UV induced mutation breeding, 1 strains of mutant strain UV2 that the fermentation, found that the transformation ability of mutant strain UV2 increased, the conversion rate is 11.76%; UV2 compared with the original strain, the conversion rate of 3.84%. increased and then with UV2 as the starting strain, choose two ethyl sulfate by chemical mutagenesis, to determine the optimum time of mutagenesis is 40min, after 1 after mutation breeding mutant strain U-D2, the fermentation culture, found that transformation ability mutants increased, the conversion rate is 13.2%, compared with the UV2 conversion rate was increased by 1.44%.U-D2 and the original strain of Bacillus amyloliquefaciens compared with the conversion rate was increased by 5.25% . the mutant strain U-D2 was the 6 generation of subculture, Staphylococcus aureus as the indicator bacteria, the antibacterial properties of fermentation liquid were measured, results showed that the mutant U-D2 has good genetic stability. By single factor test and response surface design method in CCD central composite experimental design method of medium nutrients optimization of the U-D2. To determine the optimal concentration of culture medium was beef extract 3.62g/L, glucose 12.39g/L, peptone 7.19 g/L, Magnesium Sulfate 0.42 g/L, two potassium hydrogen phosphate 0.85 g/L. mutant strain U-D2 to optimize the culture medium, the medium through the antibacterial test, measured the diameter of inhibition zone was 37.4mm, the conversion rate of U-D2 to 15.51%, compared with the optimal medium before the conversion rate increased by 2.31%.

【學位授予單位】：沈陽農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q93

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