利用原生质体技术选育聚β羟基丁酸高产菌株和发酵条件的研究
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摘要
PHB在自然界中是作为微生物自身能量的储存物质而存在于细胞内，除具有与化学合成塑料聚丙烯相似性外，还具有生物相容性和生物可降解性。在“白色污染”日益严重的今天，PHB作为生物降解塑料的原料倍受瞩目，成为国内外研究的热点。但是，目前发酵生产成本高，价格是普通塑料的许多倍，不利于工业化生产。解决上述问题的主要途径包括：(1)开发优良的菌种，使其高产PHB；(2)利用价廉的原料，以降低原料成本；(3)优化PHB的合成过程，以便于操作和降低原料消耗：(4)开发合适的提取工艺，是提取成本降到最低限度。其中，高产菌种的选育是其研究的关键部分。
目的：选育PHB高产菌株，提高PHB产量。方法：实验选取两株在形态上和生理特性上差别较大的产PHB菌株作为出发菌株，进行原生质体融合和激光诱变，在此基础上对其摇瓶发酵条件进行优化。结果：用溶菌酶水解细胞壁，其原生质体形成率S-8为78.0％，Q-72为96.0％；再生率S-8为19.5％，Q-72为31.4％。然后，用促融剂聚乙二醇(PEG)使两亲本菌株进行融合，获得融合子8株。融合子R-7的PHB产量明显提高，遗传性稳定。同时经激光诱变得到正突变菌株6株，其中突变菌株Y-17在发酵过程中能够积累4.86g／L的PHB，比出发菌株S-8相比，其PHB的提高率为67.0％。在最适条件下，Y-17菌株能够积累PHB达5.82g／L。采用补料发酵实验，定时流加碳源、氮源和H_2O_2，明显提高了PHB含量，即PHB达到10.93
g／k。结论：突变菌株Y-17产PHB能力强，各种性状也接近工业生产的要求，可作为良好的发酵菌株应用于发酵工业。
Poly- -hydroxybutyrate(PHB) is a microbial polyester which is accumulated in certain bacteria. It crystallizes to form a polymer with similar properties to polypropylene. PHB is also biodegradable and biocompatibile. PHB as raw material of bioplatics is investigative hot point recently. But the high fermentation cost is one of the disad-vantage facters in the industrialization production. The main solution to the above problems includes:(l) breed the high-yield strain; (2) use the low-price raw material; (3) optimize the process of PHB synthesize; (4) develop the proper distill method.
Objective: Breed selectively the high-yield strain to increase the yield of PHB. Methods: selecting two strains with great difference in physiology characteristics and appearances as starting strains; proceeding the protoplasm fusion and laser treatment. On the condition, the condition of shaking-flask was optimized. Result: By taking S-8 and Q-72 as starting strains, the two parental strains were hydrolyzed by lyticase. The rate of protoplast formation of strain S-8 was 78.0% while the rate of regeneration 19.5%; the rate of protoplast formation of strain Q-72 was 96.0% while the rate of regeneration 31.4%. The protoplast of the two parental strains were killed by heating and UV, respectively, then the protoplasts was fused by PEG. Fusant strains could be selected, the Fusant strain R-7 is stabled, its PHB yield could be improved obviously than the starting strains. At the same time, The mutant strains could be selected by laser treatment with strain S-8 as starting strain. Compared with S-8, the highest PH
B production of strainY-l 7 amount to 4.86g/L. Its PHB yield increased by 67.0% than strain S-8. In addition, the ingredient of the fermention medium of Y-17 were optimized, the Y-17 can accumulate PHB 5.82g/L under the optimum condition. Then, feeding the carbon source, nitrogen source and H2O2 in the Fed-Batch-Process, PHB production improve greatly, amount up to 10.93 g/L. Conclusion: The properties of mutated strain Y-17 could accord with the demands of industrial production, it has important industrial application prospect as good fermentation strains.
目的：选育PHB高产菌株，提高PHB产量。方法：实验选取两株在形态上和生理特性上差别较大的产PHB菌株作为出发菌株，进行原生质体融合和激光诱变，在此基础上对其摇瓶发酵条件进行优化。结果：用溶菌酶水解细胞壁，其原生质体形成率S-8为78.0％，Q-72为96.0％；再生率S-8为19.5％，Q-72为31.4％。然后，用促融剂聚乙二醇(PEG)使两亲本菌株进行融合，获得融合子8株。融合子R-7的PHB产量明显提高，遗传性稳定。同时经激光诱变得到正突变菌株6株，其中突变菌株Y-17在发酵过程中能够积累4.86g／L的PHB，比出发菌株S-8相比，其PHB的提高率为67.0％。在最适条件下，Y-17菌株能够积累PHB达5.82g／L。采用补料发酵实验，定时流加碳源、氮源和H_2O_2，明显提高了PHB含量，即PHB达到10.93
g／k。结论：突变菌株Y-17产PHB能力强，各种性状也接近工业生产的要求，可作为良好的发酵菌株应用于发酵工业。
Poly- -hydroxybutyrate(PHB) is a microbial polyester which is accumulated in certain bacteria. It crystallizes to form a polymer with similar properties to polypropylene. PHB is also biodegradable and biocompatibile. PHB as raw material of bioplatics is investigative hot point recently. But the high fermentation cost is one of the disad-vantage facters in the industrialization production. The main solution to the above problems includes:(l) breed the high-yield strain; (2) use the low-price raw material; (3) optimize the process of PHB synthesize; (4) develop the proper distill method.
Objective: Breed selectively the high-yield strain to increase the yield of PHB. Methods: selecting two strains with great difference in physiology characteristics and appearances as starting strains; proceeding the protoplasm fusion and laser treatment. On the condition, the condition of shaking-flask was optimized. Result: By taking S-8 and Q-72 as starting strains, the two parental strains were hydrolyzed by lyticase. The rate of protoplast formation of strain S-8 was 78.0% while the rate of regeneration 19.5%; the rate of protoplast formation of strain Q-72 was 96.0% while the rate of regeneration 31.4%. The protoplast of the two parental strains were killed by heating and UV, respectively, then the protoplasts was fused by PEG. Fusant strains could be selected, the Fusant strain R-7 is stabled, its PHB yield could be improved obviously than the starting strains. At the same time, The mutant strains could be selected by laser treatment with strain S-8 as starting strain. Compared with S-8, the highest PH
B production of strainY-l 7 amount to 4.86g/L. Its PHB yield increased by 67.0% than strain S-8. In addition, the ingredient of the fermention medium of Y-17 were optimized, the Y-17 can accumulate PHB 5.82g/L under the optimum condition. Then, feeding the carbon source, nitrogen source and H2O2 in the Fed-Batch-Process, PHB production improve greatly, amount up to 10.93 g/L. Conclusion: The properties of mutated strain Y-17 could accord with the demands of industrial production, it has important industrial application prospect as good fermentation strains.
引文
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