微波诱变L-乳酸高产细菌的选育与表征
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摘要
乳酸是目前世界上公认的三大有机酸之一,其广泛应用于食品、医药、化工、制革、纺织、环保和农业等诸多领域。可生物降解的聚L-乳酸产品作为有可能取代造成环境污染的白色热塑产品也越来越受到人们的关注。本研究采用干酪乳杆菌作为L-乳酸生产菌种,发酵产物光学L-构型较高,符合工业化生产的要求。
本课题通过自行设计一种功率连续可调水循环冷却式微波辐照育种装置,抵消微波的热效应,对干酪乳杆菌进行连续低功率较长时间的诱变。在微波剂量为功率400W,辐照时间3min时,存活率为24.1%时,诱变效果最好。筛选得到遗传稳定性较好的高产L-乳酸的突变株W4-3-9、W4-3-28、W2-3-7,L-乳酸产量分别为115.1g/L、114.8g/L和114.1g/L,较出发菌株的L-乳酸产量提高了56.2%、56.6%和55.7%。
从形态学、生理生化机理和分子生物学等方面对微波辐照前后的乳酸细菌进行表征,发现微波辐照后菌落个体变小,透明圈变大,菌落厚度变薄,突变株菌体易凝集且表面有油状物。微波辐照前后乳酸菌在生理生化特性上有部分差异,主要表现在辐照后出现部分革兰氏阴性菌和突变株不发酵纤维二糖和七叶苷。采用原子力显微镜(AFM)对出发菌株和突变株的表面形貌和DNA进行观察对比,首次获得了微波辐照前后干酪乳杆菌的细胞表面形貌和遗传物质DNA的AFM图像。为研究开发微波诱变微生物育种技术,深入探讨微波辐照对乳酸细菌的作用,提供了技术支持和理论依据。同时通过对比出发菌株和突变株DNA的AFLP指纹图谱,可以断定微波辐照对乳酸细菌有诱发突变作用。
Lactate is one of the three major organic acids, widely used in the fields of food, pharmaceutical, chemical, leather, textiles industries etc. Biodegradable polylactide products are being of concern as a result of the serious white forth pollution caused by the white thermoplastic products. It is focused on a strain of Lactobacillus rhamnosus in this paper, by which the L-lactic acid produced has optimal purity and meets the requirements of industrial production.
The mutagenesis of Lactobacillus rhamnosus was carried out by the continuous low-power microwave irradiation exploded by a power-adjustable breeding equipment, which has the cooling water cycle to offset the heat of the microwave. Under the microwave power of 400W and irradiation length of 3min, the survival rate of Lactobacillus rhamnosus was 24.1% and the mutagenic effects were good. Three mutant strains W4-3-9, W4-3-28 and W2-3-7 of genetic stability and high-yield L-lactic acid were found by screening, the L-lattic acid production of which were 115.1g/L, 114.8g/L and 114.1g/L, respectively. Comparing with the parent mutant, the improvement of L-lactic acid produced were up to 56.2%、56.6% and 55.7%.
It was indicated that some different microbial morphology, physiological biochemical characters and molecular biological aspects existed after microwave radiation between the parent strains and the mutant ones, of which the individual colonies became smaller, the transparent zone was larger and the thickness of colonies became thinner. Also the mutants were easy to assemble and some oil-like substance appeared on the surface of the colonies. The differences of physiological and biochemical characteristics between the parent and mutant strains caused by the microwave radiation mainly manifested in the appearance of gram-negative bacteria and some mutants which can’t utilize cellobiose and esculoside. The parent and mutant images of surface morphology and AFM of DNA were observed through the atomic force microscopy for the first time. It provided technical support and theoretical basis for further study of the effect of microwave irradiation on Lactobacillus rhamnosus and the innovation of mutation techniques. And it was firmly showed that microwave irradiation had induced mutation by comparing amplified fragment length polymorphism (AFLP) fingerprints of DNA of the parent strain and the mutant one.
本课题通过自行设计一种功率连续可调水循环冷却式微波辐照育种装置,抵消微波的热效应,对干酪乳杆菌进行连续低功率较长时间的诱变。在微波剂量为功率400W,辐照时间3min时,存活率为24.1%时,诱变效果最好。筛选得到遗传稳定性较好的高产L-乳酸的突变株W4-3-9、W4-3-28、W2-3-7,L-乳酸产量分别为115.1g/L、114.8g/L和114.1g/L,较出发菌株的L-乳酸产量提高了56.2%、56.6%和55.7%。
从形态学、生理生化机理和分子生物学等方面对微波辐照前后的乳酸细菌进行表征,发现微波辐照后菌落个体变小,透明圈变大,菌落厚度变薄,突变株菌体易凝集且表面有油状物。微波辐照前后乳酸菌在生理生化特性上有部分差异,主要表现在辐照后出现部分革兰氏阴性菌和突变株不发酵纤维二糖和七叶苷。采用原子力显微镜(AFM)对出发菌株和突变株的表面形貌和DNA进行观察对比,首次获得了微波辐照前后干酪乳杆菌的细胞表面形貌和遗传物质DNA的AFM图像。为研究开发微波诱变微生物育种技术,深入探讨微波辐照对乳酸细菌的作用,提供了技术支持和理论依据。同时通过对比出发菌株和突变株DNA的AFLP指纹图谱,可以断定微波辐照对乳酸细菌有诱发突变作用。
Lactate is one of the three major organic acids, widely used in the fields of food, pharmaceutical, chemical, leather, textiles industries etc. Biodegradable polylactide products are being of concern as a result of the serious white forth pollution caused by the white thermoplastic products. It is focused on a strain of Lactobacillus rhamnosus in this paper, by which the L-lactic acid produced has optimal purity and meets the requirements of industrial production.
The mutagenesis of Lactobacillus rhamnosus was carried out by the continuous low-power microwave irradiation exploded by a power-adjustable breeding equipment, which has the cooling water cycle to offset the heat of the microwave. Under the microwave power of 400W and irradiation length of 3min, the survival rate of Lactobacillus rhamnosus was 24.1% and the mutagenic effects were good. Three mutant strains W4-3-9, W4-3-28 and W2-3-7 of genetic stability and high-yield L-lactic acid were found by screening, the L-lattic acid production of which were 115.1g/L, 114.8g/L and 114.1g/L, respectively. Comparing with the parent mutant, the improvement of L-lactic acid produced were up to 56.2%、56.6% and 55.7%.
It was indicated that some different microbial morphology, physiological biochemical characters and molecular biological aspects existed after microwave radiation between the parent strains and the mutant ones, of which the individual colonies became smaller, the transparent zone was larger and the thickness of colonies became thinner. Also the mutants were easy to assemble and some oil-like substance appeared on the surface of the colonies. The differences of physiological and biochemical characteristics between the parent and mutant strains caused by the microwave radiation mainly manifested in the appearance of gram-negative bacteria and some mutants which can’t utilize cellobiose and esculoside. The parent and mutant images of surface morphology and AFM of DNA were observed through the atomic force microscopy for the first time. It provided technical support and theoretical basis for further study of the effect of microwave irradiation on Lactobacillus rhamnosus and the innovation of mutation techniques. And it was firmly showed that microwave irradiation had induced mutation by comparing amplified fragment length polymorphism (AFLP) fingerprints of DNA of the parent strain and the mutant one.
引文
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2杨文玲,李海霞,马沛生.白色污染的治理技术.化学工业与工程. 2000(17):49~55
3 Lee S-Y. Bacterial Polyhydroxyalkanoates. Biotechnology Bioengineering. 1996(49):1~14
4汪进玉.一种可生物降解纤维-聚乳酸纤维.化工新型材料. 2000(3):25~26
5李豪,车振明.微波诱变微生物育种研究.山西食品工业. 2005(2):5~14
6凌天庭.食品添加剂.第2版.北京:化学工业出版社, 638~639
7化工百科全书编委会.化工百科全书.北京:化学工业出版社, 1998:233~341
8王博彦,金其荣.发酵有机酸生产与应用手册.北京:中国轻工业出版社, 2000, 2~3 337~389
9金其荣,张继民,徐勤.有机酸发酵工艺学.北京:中国轻工业出版社, 1989:339~406
10 Datta R., Tsai S.R., Bonsignore P.. Technological and Economic Potential of Poly(Lactic Acid) and Lactic Acid Derivatives. FEMS Microbiol. Rev. 1995 (16):221~231
11 Lichfield J. H.. Microbiological Production of Lactic Acid. In Advances in Applied Microbiology. Vol.4.San Diego Academic Press, 1996:245~295
12 Datta R.,Sai P T., Patric B., Moon S.H.,Frank J.R. International Congress on Chemicals from Biotechnology. Hannover.1993:1~8
13 Garvie E. Bacterial Lactate Dehydrogenase. Microbiological Reviews. 1980(44): 106~139
14钱志良,胡军,雷肇祖.乳酸的工业化生产、应用和市场.工业微生物. 2001,31:49~53
15 Anon. "Ecochem Completes Lactic Acid Plant" Chem Eng. News, 6. 1992. August 6
16 Vick-Roy TB. Comprehensive Biotechnology. Oxford: Peryamon Press, 1985, 23~41
17黄量,戴众信.性药物的化学与生物学.北京:化学工业出版社, 2002:204~205
18金其荣,金丰秋.乳酸衍生物发展应用新动向.山西食品工业. 2002. 32~37
19 Niju N., Roychoudhury P.K., Srivastava A. L-lactic Acid Fermentation and Its Product Polymerization. Electronic Journal of Biotechnology. 2004(7): 167~179
20 Pein Y., Maokinishina Y. O.. Enhence Production of L-lactic Acid From Corn Starch in a Culture of Rhizopus oryzae Using an Air-Lift Bioreactor . J. Ferment. Bioeng.1997(84):249~253
21曹本昌,徐建林,匡群. L-乳酸研究综述.食品与发酵工业. 1993(3):56~61.
22化工百科全书编委会,化工百科全书,北京:化学工业出版社, 1998:434~446
23 Hofvendahl K., Hagerdal B., Factors Affecting the Fermentative Lactic Acid Production from Renewable Resources, Enzyme and Microbial Technology. 2000,26:87~107
24 Bai D. M., Dai H. X., Zhao X. M., et al. The Effect of Na+ and K+ on the Morphology and L-Lactic Acid Productivity of Rhizopus oryzae SOI106. Journal of Chemical Industry and Engineering(China). 2000, 51(5):583~585
25白冬梅,赵学明,胡宗定等.米根霉发酵生产L(+)-乳酸研究进展.现代化工. 2002,22(6):9~14
26 David P. M., Plastics from Microbes, Hanser/Gardner Publications Inc, Cincinnatic Munich Vienna.New York,1994: 93~137
27杨洁彬.乳酸菌-生物学基础及应用.北京:中国轻工业出版社, 1996:98~116
28 Sunhoon K., Lee P.C., Lee E.G.. Production of Lactic Acid by Lactobacillus rhamnosus with Vitamin-Supplemented Soybean Hydrolysate. Enzyme and Microbial Technology. 2000(26):209~215
29 Rincon J., Fuertes J., Optimization of the Fermentation of Whey by Latcobacillus casei. Acta Biotechnol. 1993(13):323~331
30 Hujanen M., Linko Y-Y.Effect of Temperature and Various Nitrogen Sources on L-lactic Acid Production by Lactobacillus casei. Appl. Microbiol. Technol. 1996(45):307~313
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