真菌产生的低分子抗菌肽的发酵生产、分离纯化及其性质研究
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摘要
抗生素在临床治疗中的广泛使用挽救了不计其数的生命，同时，抗生素的滥用也促使抗药菌群迅猛发展，某些感染性疾病成为临床治疗的难题，能够对抗耐药菌的新型抗生素的研发迫在眉睫。本文对由山东大学微生物技术国家重点实验室筛选出来的一株产生低分子抗菌肽的青霉菌Penicillium sp．M03进行研究，在其发酵液中提取到一种具有抗菌活性的的肽类物质AF2，并对其基本性质进行了研究。
本文首先进行了预试验，证明了Penicillium sp．M03可以代谢产生抗菌肽，并且这种物质可以分泌到培养基中。为提高菌种发酵产生抗菌肽的能力，论文对真菌Penicillium sp．M03产生抗菌肽的发酵条件进行了初步的优化，筛选了实验室条件下适宜的碳源、氮源、碳氮源之比、pH、温度、接种量和发酵时间。Penicillium sp．M03能够利用多种碳源和氮源合成抗菌肽，综合考虑多种因素，最后确定抗菌肽的最佳发酵培养基为：淀粉30g，K_2HPO_4 1g，KCl 0.05g，(NH_4)_2SO_4 18g，MgSO_4·7H_2O0.5g，FeSO_4 0.01g，蒸馏水1L。种子培养基为：葡萄糖30g，K_2HPO_4 1g，KCl 0.05g，(NH_4)_2SO_4 18g，MgSO_4．7H_2O 0.5g，FeSO_4 0.01g，蒸馏水1L。最佳发酵条件为：pH 6.5，温度30℃，接种量为10％，摇床培养5天。
在对培养条件进行优化后，论文对青霉菌Penicillium sp．M03进行了小型发酵罐的放大试验，与摇瓶发酵相比，使用发酵罐对真菌进行发酵可以使发酵液中的抗菌肽浓度提高大约五倍，缩短发酵时间(发酵罐上的最佳发酵时间为三天)，提高一次发酵所得到的抗菌肽的量。由于真菌生长比较缓慢，因此在发酵早期容易出现感染杂菌的现象，论文在第三章的最后对发酵中出现染菌现象后的分析和解决方法进行了总结。
发酵液经过纱布过滤、离心、超滤、冷冻干燥等一系列处理得到黄色粉末，再经Sephadex LH-20，Sepharose-DEAE FF柱层析进行分离纯化，最后经过SephadexG-15脱盐得到纯净物质，在此过程中活性峰采用平板抑菌法来确定。
通过对AF2的红外光谱、核磁共振~1H和~(13)C光谱和质谱的解析，初步判断AF2是一种低分子多肽，分子量约为1155。AF2氨基酸组成分析结果显示其含有天冬氨酸、谷氨酸、丝氨酸、甘氨酸、苏氨酸、丙氨酸、脯氨酸、胱氨酸、亮氨酸、苯丙氨酸等氨基酸残基。AF2的活性不受β-内酰胺酶的影响，对酸碱和蛋白酶稳定，对热也有较好的耐受性，可以有效抑制金黄色葡萄球菌、MRSA、绿脓假单孢菌等病原菌，有望开发成一种对抗耐药菌感染的新型抗菌药物。
The wildly use of antibiotics in clinic has saved plenty of lives. On the other hand, unreasonable application of antibiotics has caused the rapidly arise of resistant organism, which made some infective deseases to be a tough problem of clinic.Thus, the research and development of new drug which can control resistant pathogens became more essential. A kind of antibacterial peptide named AF2 was purified from the broth of fungi Penicillium sp.M03 which was screended out by state key lab of microbial technology at SDU, and some properties of AF2 was studied.
Result of preliminary test showed that, Penicillium sp.M03 had the ability to produce some antibiotics, which can diffuse into culture medium. To increase the output of AF2, the fermentation condition of Penicillium sp.M03 was optimized. Then the best carbon source, nitrogen source, proportion of them, pH, temperature, inoculate quantity and fermental time was determened. Variety of culture medium could be used by Penicillium sp.M03 to produce AF2. Allowed for many factors, The optimum fermental medium of AF2 was as the following: Starch 30g, Na_2HPO_4 1g, KC1 0.05g, (NH_4)_2SO_418g, MgSO_4 · 7H_2O 0.5g, FeSO_4 0.01g, distilled water 1L. The optimum cutrule medium was: Glucose 30g, Na_2HPO_4 lg, KC1 0.05g, (NH_4)_ 2SO_4 18g, MgSO_4 . 7H_2O 0.5g, FeSO_4 0.01g, distilled water 1L. And the optimum culture condition was : pH 6.5, inoculate quantity10%, 30℃ for 5 days.
Then Penicillium sp.M03 was fermented by fermentor (BioFlo 410 , NBS). In this condition, the output of AF2 could be increased, and the fermentation time could be reduced. Because the growth of fungi is relatively slow, fermental process of fungi is easy to be infected by other microorganism. The last part of this chapter introduced how to analysis the causation of infection and resolve the problem.
Then followed part of this thesis was the purification of AF2. The broth was dealt with
filtration, centrifuging and ultra-filtration. After freeze-dried, the crude AF2 product was
purifided by Sephadex LH-20 molecular sieve chromatography , Sepharose-DEAE FF
and Sephadex G-15, a kind of pure component AF2 was got.
Structure and molecular-weight of AF2 were studied through UV, ER, NMR and ESI-MS.
The results showed: AF2 was a kind of peptides, Its molecular weight was about
1155.The data of amino acid analysis showed that AF2 contained twelve kinds of amino
acids: Asp, Glu, Ser, Gly, Arg, Thr, Ala, Pro, Cys, Ile, Leu and Phe. AF2 was stable to
β -lactamase, pH and proteinase, its heat-resistance was also well. It could inhibit the
growth of SA, MRSA and blue pus organism, thus, it was hopefu; AF2 be used as novel
antibiotics to control resistant-bacteria.
本文首先进行了预试验，证明了Penicillium sp．M03可以代谢产生抗菌肽，并且这种物质可以分泌到培养基中。为提高菌种发酵产生抗菌肽的能力，论文对真菌Penicillium sp．M03产生抗菌肽的发酵条件进行了初步的优化，筛选了实验室条件下适宜的碳源、氮源、碳氮源之比、pH、温度、接种量和发酵时间。Penicillium sp．M03能够利用多种碳源和氮源合成抗菌肽，综合考虑多种因素，最后确定抗菌肽的最佳发酵培养基为：淀粉30g，K_2HPO_4 1g，KCl 0.05g，(NH_4)_2SO_4 18g，MgSO_4·7H_2O0.5g，FeSO_4 0.01g，蒸馏水1L。种子培养基为：葡萄糖30g，K_2HPO_4 1g，KCl 0.05g，(NH_4)_2SO_4 18g，MgSO_4．7H_2O 0.5g，FeSO_4 0.01g，蒸馏水1L。最佳发酵条件为：pH 6.5，温度30℃，接种量为10％，摇床培养5天。
在对培养条件进行优化后，论文对青霉菌Penicillium sp．M03进行了小型发酵罐的放大试验，与摇瓶发酵相比，使用发酵罐对真菌进行发酵可以使发酵液中的抗菌肽浓度提高大约五倍，缩短发酵时间(发酵罐上的最佳发酵时间为三天)，提高一次发酵所得到的抗菌肽的量。由于真菌生长比较缓慢，因此在发酵早期容易出现感染杂菌的现象，论文在第三章的最后对发酵中出现染菌现象后的分析和解决方法进行了总结。
发酵液经过纱布过滤、离心、超滤、冷冻干燥等一系列处理得到黄色粉末，再经Sephadex LH-20，Sepharose-DEAE FF柱层析进行分离纯化，最后经过SephadexG-15脱盐得到纯净物质，在此过程中活性峰采用平板抑菌法来确定。
通过对AF2的红外光谱、核磁共振~1H和~(13)C光谱和质谱的解析，初步判断AF2是一种低分子多肽，分子量约为1155。AF2氨基酸组成分析结果显示其含有天冬氨酸、谷氨酸、丝氨酸、甘氨酸、苏氨酸、丙氨酸、脯氨酸、胱氨酸、亮氨酸、苯丙氨酸等氨基酸残基。AF2的活性不受β-内酰胺酶的影响，对酸碱和蛋白酶稳定，对热也有较好的耐受性，可以有效抑制金黄色葡萄球菌、MRSA、绿脓假单孢菌等病原菌，有望开发成一种对抗耐药菌感染的新型抗菌药物。
The wildly use of antibiotics in clinic has saved plenty of lives. On the other hand, unreasonable application of antibiotics has caused the rapidly arise of resistant organism, which made some infective deseases to be a tough problem of clinic.Thus, the research and development of new drug which can control resistant pathogens became more essential. A kind of antibacterial peptide named AF2 was purified from the broth of fungi Penicillium sp.M03 which was screended out by state key lab of microbial technology at SDU, and some properties of AF2 was studied.
Result of preliminary test showed that, Penicillium sp.M03 had the ability to produce some antibiotics, which can diffuse into culture medium. To increase the output of AF2, the fermentation condition of Penicillium sp.M03 was optimized. Then the best carbon source, nitrogen source, proportion of them, pH, temperature, inoculate quantity and fermental time was determened. Variety of culture medium could be used by Penicillium sp.M03 to produce AF2. Allowed for many factors, The optimum fermental medium of AF2 was as the following: Starch 30g, Na_2HPO_4 1g, KC1 0.05g, (NH_4)_2SO_418g, MgSO_4 · 7H_2O 0.5g, FeSO_4 0.01g, distilled water 1L. The optimum cutrule medium was: Glucose 30g, Na_2HPO_4 lg, KC1 0.05g, (NH_4)_ 2SO_4 18g, MgSO_4 . 7H_2O 0.5g, FeSO_4 0.01g, distilled water 1L. And the optimum culture condition was : pH 6.5, inoculate quantity10%, 30℃ for 5 days.
Then Penicillium sp.M03 was fermented by fermentor (BioFlo 410 , NBS). In this condition, the output of AF2 could be increased, and the fermentation time could be reduced. Because the growth of fungi is relatively slow, fermental process of fungi is easy to be infected by other microorganism. The last part of this chapter introduced how to analysis the causation of infection and resolve the problem.
Then followed part of this thesis was the purification of AF2. The broth was dealt with
filtration, centrifuging and ultra-filtration. After freeze-dried, the crude AF2 product was
purifided by Sephadex LH-20 molecular sieve chromatography , Sepharose-DEAE FF
and Sephadex G-15, a kind of pure component AF2 was got.
Structure and molecular-weight of AF2 were studied through UV, ER, NMR and ESI-MS.
The results showed: AF2 was a kind of peptides, Its molecular weight was about
1155.The data of amino acid analysis showed that AF2 contained twelve kinds of amino
acids: Asp, Glu, Ser, Gly, Arg, Thr, Ala, Pro, Cys, Ile, Leu and Phe. AF2 was stable to
β -lactamase, pH and proteinase, its heat-resistance was also well. It could inhibit the
growth of SA, MRSA and blue pus organism, thus, it was hopefu; AF2 be used as novel
antibiotics to control resistant-bacteria.
引文
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