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螺旋霉素生物丙酰化技术的研究
潘杰
Subtype博士
Thesis Advisor李佐虎
1998-05-01
Degree Grantor中国科学院研究生院
Abstract生米加链霉菌变株var68可以使螺旋霉素定向丙酰化为4"-O-丙酰螺旋霉素。分析了螺旋霉素丙酰化反应的机理,建立了洗涤细胞中的转化动力学和发酵过程中新的唯象动力学模型。建立与螺旋霉素丙酰化特性相适应的发酵工艺:流加葡萄糖工艺、混菌发酵和两步发酵法。混菌发酵和两步法发酵可以将螺旋霉素生产和转化两种过程有机的结合起来,其中两步法发酵不仅简化了工艺过程,而且还能达到较高的转化率。研究了有机溶剂萃取和吸附提取的规律,在双水相萃取技术研究的基础上,建立了全发酵液萃取新技术,使丙酰螺旋霉素的萃取收率由有机溶剂萃取的53.4%提高到69.2%。发现了和一般抗生素发酵不同的耗氧规律。据此建立了分段控制溶氧水平的发酵方法,使转化率有了一定的提高。
Other AbstractTo attain more effective antibiotics, the structure modification, including microbial transformation, is one of the most important methods. A lot of work on the microbial transformation of antibiotic has been carried out. There are many kinds of transformation pattern such as acetylation, isovaleryletion, deacylation, hydrocylation, etc.. There are many reports which suggested that acylation in the 4"-position of mycarose would enhance the antibacterial activity and antimycoplasma antibiotics. It was biotransformed from spiramycin(spm) by the mutant which was obtained by treatment of S.mycarofaciens at higher temperature. The pharmacology study showed that antimicrobial activity of pro-spm is higher than that of ethylspiramycin which is mostly used as oral antibiotic. More important thing is that pro-spm is protected by patent. Unfortunately, there are less study reports on the fermentation and recovery of biotransformation. The development of the technique of biotransformation from spm to pro-spm was discussed in this paper. The various effect on the biotransformation of spiramycin in the washing cell system and fermentation was studied. The experimental results show that in the washing cell system: the optimum pH is about 5.2, the optimum phosphate concentration is 0.02M, and the nitrogen source effects the biotransformation significantly. Both the reaction substrate spiramycin and the carbon resource of growth glucose inhibit the biotransformation remarkably. The glucose concentration is the most important fact for cell growth and biotransformation. The effect of inoculate volume. pH, and fermentation volume was studied also. The transformation reaction kinetic was developed on the base of serial reaction theory. The phenomenological model of cell growth was established on the experimental results. The model fit the whole process of cell culture well. Comparing with Monod model, the phenomenological model with fewer parameters is more clear and univocal. Three fermentation processes were developed: fed-batch of glucose, mixed culture and two-step sequence fermentation. Fed-batch culture could reach higher biotransformation ratio, however, the demand of raw spiramycin lead to complex process, including twice fermentation and twice recovery processes etc. Mixed culture involved only one step of fermentation and recovery, but potency and biotransformation ratio were lower because of the growth competition of two kinds of cell. The two-step sequence fermentation consists of only three steps: spiramycin fermentation, biotransformation and recovery. Three recovery processes of propionylspiramycin was studied: organic solvent extraction, absorption by macroreticular resin and the whole broth extraction. With the model of the distribution coefficient and pH, the intrinsic distribution coefficient K_0 of pro-spm was calculated to be 19.01, lower than that of spiramycin. The dissociate coefficient of pro-spm pK1 and pK2 was first to calculate as 7.76 and 5.2 by the Gauss-Newton method. The experience formula of counter-extraction was established in the phosphate solution system. When pH was 3.15, the maximum counter-extraction rate of pro-spm was obtained. The effect of temperature on the distribution coefficient was also studied. Based on the model, the extraction nomograph obtained. The absorption process is greatly effected by the flow velocity. So a new recycling absorption method was established to attain more absorptivity. The technique of extraction propionylspiramycin(pro-spm) by aqueous two-phase(ATPE) from the solution or the fermentation broth was discussed in this paper. The effect of pH, PEG molecular weight and concentration, salt concentration was investigated in the PEG/phosphate system. The suitable conditions was obtained for scale up. The results showed that the hydrophobic interaction was the dominant effect contrary to the partition protein by ATPE. The solute itself i.e. pro-spm engaged in the ATPE. Comparing with that of the organic solvent extraction(OSE), the recovery of product was 32% higher by ATPE, but the purity was somewhat lower than that of by OSE. The feasibility of repeated use of PEG was also investigated. The extraction recovery of antibiotics from the whole broth, combined with aqueous two-phase partition and organic solvent extraction, is 69.2%, meanwhile, the recovery is 53.4% by the organic extraction. The effect of dissolved oxygen (DO) on the spiramycin biotransformation by the mutant strain of S. mycarofaciens was studied. The reaction of biotransformation was a remarkable oxygen-consumed process. Two peaks of oxygen uptake rate was observed in the whole fermentation courses. The method of controlling the difference dissolved oxygen concentration in the divided phase was developed. The highest biotransformation ratio 80% was reached and total time needed for fermentation and biotransformation was cut down 1/6.
Pages133
Language中文
Document Type学位论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/8334
Collection研究所(批量导入)
Recommended Citation
GB/T 7714
潘杰. 螺旋霉素生物丙酰化技术的研究[D]. 中国科学院研究生院,1998.
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