维吉尼亚链霉菌物理诱变及外源添加氯化镧对VGM产量的影响
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摘要
目的利用物理诱变结合链霉素抗性筛选维吉尼亚霉素(VGM)高产菌株维吉尼亚链霉菌,并考察外源添加稀土元素氯化镧对维吉尼亚链霉菌发酵的影响。方法一方面,以链霉素为筛选剂建立孢子致死突变标志的微波及紫外诱变两种筛选模型,采用管碟法获得抑菌圈直径较大的作为初筛菌株,再通过摇瓶复筛进一步确定高产菌株。另一方面,在发酵培养基中外源添加3～30mg/L的氯化镧,考查其对链霉菌合成VGM的影响。结果比较两种筛选模型,基于链霉素的微波诱变效果较好,正突变率高达32.83%;并获得一株高产菌株MW 178(146.72±11.80)μg/mL,较出发菌株(21.24±1.34)μg/mL提高了约6倍。在发酵初始时,外源添加10mg/L氯化镧,VGM的产量由146.72μg/mL提高到180.01μg/mL,提高了约22.69%。结论采用微波诱变结合链霉素抗性筛选的方法可以获得VGM高产突变株;在发酵初始时,外源添加适量氯化镧可以显著提高VGM产量。
Objective A method of physical mutagenesis coupled with streptomycin resitance screening was applied to select virginiamycin(VGM) high-producing strain. Also, the effect of adding lanthanum chloride on the production of VGM was studied. Methods First, two streptomycin-resistance mutational labelling selection models with physical mutagenic factor(microwave and ultraviolet) were built to screen high-yield mutants. Then preliminary positive strains were picked out by measurement of the inhibitory activity to Micrococcus luteus. And the subsequent determination by HPLC were carried out for the high VGM-producing strains cultivated in the shake-flask. Finally, lanthanum chloride was added in fermentation medium of the strain with the concentration of 3～30 mg/L. Results The mutagenesis of microwave combined with streptomycin resistance was better and the positive mutation rate was up to 32.83%. Streptomycin-resistant mutant strain MW 178 was got by microwave producing VGM with yield of(146.72±11.80)μg/mL which beyond six times higher than the initial strain(21.24±1.34)μg/mL. The production of MW 178 reached up to 180.01μg/mL eventually by adding the concentration of 10 mg/L lanthanum chloride to the medium at the beginning of the fermentation. Conclusion Microwave mutation compound streptomycin resistance screening is effetive for screening high-yeilding mutants and the addition of lanthanum chloride could significantly improve the yield of VGM.
Objective A method of physical mutagenesis coupled with streptomycin resitance screening was applied to select virginiamycin(VGM) high-producing strain. Also, the effect of adding lanthanum chloride on the production of VGM was studied. Methods First, two streptomycin-resistance mutational labelling selection models with physical mutagenic factor(microwave and ultraviolet) were built to screen high-yield mutants. Then preliminary positive strains were picked out by measurement of the inhibitory activity to Micrococcus luteus. And the subsequent determination by HPLC were carried out for the high VGM-producing strains cultivated in the shake-flask. Finally, lanthanum chloride was added in fermentation medium of the strain with the concentration of 3～30 mg/L. Results The mutagenesis of microwave combined with streptomycin resistance was better and the positive mutation rate was up to 32.83%. Streptomycin-resistant mutant strain MW 178 was got by microwave producing VGM with yield of(146.72±11.80)μg/mL which beyond six times higher than the initial strain(21.24±1.34)μg/mL. The production of MW 178 reached up to 180.01μg/mL eventually by adding the concentration of 10 mg/L lanthanum chloride to the medium at the beginning of the fermentation. Conclusion Microwave mutation compound streptomycin resistance screening is effetive for screening high-yeilding mutants and the addition of lanthanum chloride could significantly improve the yield of VGM.
引文
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[24]任勇,牛春,沈晓鸿,等.链霉素与紫外诱变联合作用维生素B12菌种的研究[J].中国抗生素杂志,2017,42(8):639-642.
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[27]郭峰,程新,陈其亮,等.南昌链霉菌的微波诱变[J].生物加工过程,2004,2(4):51-53.
[28]关亚鹏,张莉,刘靓,等.Pneumocandin B0高产菌株选育[J].中国抗生素杂志,2015,40(2):88-90.
[29]夏钱华,尤朝阳,张攀,等.微波和紫外诱变选育高效石油烃降解菌株[J].水处理技术,2017,43(4):36-41.
[30]于忠臣,牛源麟,钟柳波,等.紫外分光光度法对超高浓度含油废水的测定[J].当代化工,2015,44(3):653-656.
[31]王燕,孙彩云,杨平平,等.稀土元素对微生物的生物学作用研究进展[J].齐鲁工业大学学报,2005,19(3):14-17.
[2]Gossele F,Blain P,Marneffe T,et al.High-performance liquid chromatographic determination of virginiamycin in Stafac,premixes and animal feeds[J].Analyst,1991,116(12):1373-1380.
[3]闫明.维吉尼亚霉素的研究进展[J].湖北畜牧兽医,2014,35(9):70-72.
[4]曹文卿,林黎明,张鸿伟,等.HPLC-MS/MS快速测定家禽和猪组织中维吉尼亚霉素M1和S1残留量[J].分析测试学报,2012,31(Sl):165-170.
[5]Nott K,Paquot M,Heilporn S,et al.A fast and reliable chromatographic procedure for the purification of virginiamycin M1 factor[J].Chromatograph,2002,56(5-6):331-335.
[6]王世梅,黄为一,崔凤元.阿扎霉素B产生菌吸水链霉菌NND-52的诱变筛选[J].微生物学通报,2001,28(1):64-67.
[7]Hosoya Y,Okamoto S,Muramatsu H,et al.Acquisition of certain streptomycin-resistant(str)mutations enhances antibiotic production in bacteria[J].Antimicrob Agents Chemother,1998,42(8):2041-2047.
[8]陶纯长,谌颉,郭美锦,等.链霉素抗性突变理性筛选avermectin高产菌株[J].中国抗生素杂志,2002,27(9):521-523.
[9]杨东靖,陈冠群,陈巍,等.链霉素抗性突变-纳他霉素高产菌株的选育研究[J].微生物学通报,2003,30(4):29-32.
[10]江凌,邬建国,陈伟伟,等.链霉素抗性突变-万古霉素高产菌株的选育研究[J].中国抗生素杂志,2005,30(2):70-72.
[11]卢文玉,闻建平,范晶华,等.激光诱变玫瑰孢链霉菌结合链霉素抗性筛选法选育达托霉素高产菌株[J].微生物学通报,2006,33(3):114-117.
[12]孙玉雯,崔承彬.抗生素抗性筛选在微生物菌株选育中的作用[J].国际药学研究杂志,2008,35(3):213-217.
[13]李海燕,夏治停,关丽杰.链霉素抗性突变理性筛选链霉素高产菌株[J].科学技术与工程,2015,15(1):174-177.
[14]张颖.稀土元素镧对Corynebacterium pekinense发酵赖氨酸的影响[J].安徽农业科学,2012,10(10):5741-5742.
[15]Tanaka Y,Hosaka T,Ochi K.Rare earth elements activate the secondary metabolite-biosynthetic gene clusters in Streptomyces coelicolor A3(2)[J].J Antibiot,2010,63(8):477-481.
[16]穆云龙,丁彦博,蔡超靖,等.微波诱变及添加氯化镧筛选阿扎霉素B高产菌株[J].中国抗生素杂志,2013,38(7):498-500.
[17]王怡平,肖亚中,丁毅,等.稀土元素对红酵母的生长及类胡萝卜素合成的影响[J].微生物学通报,1999,35(2):117-119.
[18]王燕,杨平平,宋香,等.稀土元素对谷氨酸发酵产酸及其谷氨酸脱氢酶的影响[J].食品与发酵工业,2004,30(9):33-36.
[19]孙燕,叶蕊芳,任雪岩,等.稀土元素对他克莫司生物合成和相关基因表达的影响[J].中国医药工业杂志,2016,47(4):402-406.
[20]金志华,林建平,梅乐和.工业微生物遗传育种学原理与应用[M].化学工业出版社教材出版中心,2006:114-115.
[21]Shioya S,Yang Y K,Shimizu H,et al.Maximum virginiamycin production strategy and its realization in batch cultivation of Streptomyces virginiae,by autoregulator addition[J].IFAC Proceed Vol,1995,28(3):243-246.
[22]Yang Y K,Morikawa M,Shimizu H,et al.Maximum virginiamycin production by optimization of cultivation conditions in batch culture with autoregulator addition[J].Biotechnol Bioeng,1996,49(4):437-444.
[23]Yang Y K,Shimizu H,Shioy a S,e t a l.Oautoregulator addition strategy for maximum virginiamycin production in batch culture of Streptomyces virginiae[J].Biotechnol Bioeng,1995,46(5):437-442.
[24]任勇,牛春,沈晓鸿,等.链霉素与紫外诱变联合作用维生素B12菌种的研究[J].中国抗生素杂志,2017,42(8):639-642.
[25]Kawai K,Wang G,Okamoto S,et al.The rare earth,scandium,causes antibiotic overproduction in Streptomyces spp[J].FEMS Microbiol Lett,2007,274(2):311-315.
[26]来彩霞,姚新生,Kozo O.严格应答在微生物中的存在以及在细胞形态分化和生理分化中的作用[J].沈阳药科大学学报,2000,17(1):65-69.
[27]郭峰,程新,陈其亮,等.南昌链霉菌的微波诱变[J].生物加工过程,2004,2(4):51-53.
[28]关亚鹏,张莉,刘靓,等.Pneumocandin B0高产菌株选育[J].中国抗生素杂志,2015,40(2):88-90.
[29]夏钱华,尤朝阳,张攀,等.微波和紫外诱变选育高效石油烃降解菌株[J].水处理技术,2017,43(4):36-41.
[30]于忠臣,牛源麟,钟柳波,等.紫外分光光度法对超高浓度含油废水的测定[J].当代化工,2015,44(3):653-656.
[31]王燕,孙彩云,杨平平,等.稀土元素对微生物的生物学作用研究进展[J].齐鲁工业大学学报,2005,19(3):14-17.