초록 ▼

○ 연구결과
고속 스크리닝시스템에 의한 대규모 원형질융합균주 개발을 통해 대규모 발효조 배양에서도 AVM B1a 생산 안정성과 생산성이 뛰어난 산업용 균주를 개발할 수 있었다. 또한 배양공정 자동화시스템의 개발로 인해 정밀한 배양공정 scale-up 방법이 확립되었으며, 생산공정 최적화를 수행하기 위한 제어알고리듬 개발, modelling 및 전산모사작업을 통해 효율적인 공정운영 전략을 수립하였다. 이러한 컴퓨터제어 배양공정 자동화시스템의 적용으로 인해 고생산성의 원형질융합균주를 대규모 발효조에서 배양할 지라도, 전체 공정의

○ 연구결과
고속 스크리닝시스템에 의한 대규모 원형질융합균주 개발을 통해 대규모 발효조 배양에서도 AVM B1a 생산 안정성과 생산성이 뛰어난 산업용 균주를 개발할 수 있었다. 또한 배양공정 자동화시스템의 개발로 인해 정밀한 배양공정 scale-up 방법이 확립되었으며, 생산공정 최적화를 수행하기 위한 제어알고리듬 개발, modelling 및 전산모사작업을 통해 효율적인 공정운영 전략을 수립하였다. 이러한 컴퓨터제어 배양공정 자동화시스템의 적용으로 인해 고생산성의 원형질융합균주를 대규모 발효조에서 배양할 지라도, 전체 공정의 조업안정성, 생산성 및 생산재현성이 매우 우수함을 확인할 수 있었다. 한편 친환경적 청정분리정제법인 침전법과 막분리를 이용한 에버멕틴의 분리정제를 시도하였으나, 불순물의 제거에는 한계가 있었다. 이에 따라 전통적인 추출방법을 기반으로, 세포회수, 용매에 의한 세포파쇄, 세포조각회수, methylene chloride에 의한 추출, 증발에 의한 농축, NaCl 공침법에 의한 결정화, 건조, 분쇄 등 일련의 scale-up된 공장규모의 분리정제 공정을 통하여 에버멕틴을 성공적으로 분리정제 하였다.

Abstract ▼

Avermectins(AVMs) produced by Streptomyces avermitilis are secondary metabolites with high anthelminitic and insecticidal activities that were found to be considerably useful in agriculture and animal health. The AVMs are polyketides composed of eight closely related compounds, of which AVM $B_

Avermectins(AVMs) produced by Streptomyces avermitilis are secondary metabolites with high anthelminitic and insecticidal activities that were found to be considerably useful in agriculture and animal health. The AVMs are polyketides composed of eight closely related compounds, of which AVM $B_{la}$ is the most important metabolite due to its high biological potency. To obtain high-yielding producers of AVM $B_{la}$ in the initial stage of strain improvement program, we adopted traditional methodology such as mutagenesis of the producer microorganism using UV (ultraviolet light) and/or NTG (l-methyl-3-nitro-nitrosoguanidine). Since a precursor for AVM $B_{la}$ biosynthesis is isoleucine and its biosynthetic pathway resembles that of fatty acid biosynthesis, an isoleucine-antimetabolite and an inhibitor of fatty acid biosynthesis were used to obtain various kinds of resistant mutants against these agents. The traditional mutation methods with the rational screening strategy were observed to be efficient for the rapid selection of AVM $B_{la}$ high-yielding strains, but gradual decline in the productivity was observed when the production level reached a certain level with this mutation and selection method. In order to overcome such problems intrinsic in the traditional UV and/or NTG mutation methods and then to enhance AVM $B_{la}$ productivity through protoplast fusion with high-producing strains as fusion partners, we established basic protocols necessary for protoplast fusion, and then obtained various kinds of protoplast fusants by adopting a massive strain-development procedure (a miniaturized strain screening system). An alternative fusion methods using UV and/or NTG mutation of the protoplasts were developed to screen genetic recombinants without specific selectable markers. In this method, the mutants obtained by protoplast fusion after UV and/or NTG treatment (leading to more than 99% death rate) of the respective fusion partner (respective protoplast) were regarded as DNA-recombined protoplast fusants. It was demonstrated that the protoplast recombinants obtained by UV mutation method were able to biosynthesize approximately 3 times higher level of AVM $B_{la}$ in the stable manner, compared to that of the parallel high yielding mother strains. The results of the tube cultures used in the miniaturized strain-screening system, however, showed wide fluctuations in the AVM $B_{la}$ production level, even when stable and high-producing strains were cultivated. From these results, it was assumed that lack of dissolved oxygen in the tube cultures might be one of the main factors for such unstable productivity. Accordingly, for the sufficient supply of dissolved oxygen, shaking flask cultures (30ml medium/250ml flask) for the high-yielding producers were performed. Notably enhanced production of AVM $B_{la}$, almost equal to the industrial level was obtained in the shaking flask fermentations, leading to approximately three times higher productivity compared to the corresponding tube cultures. Furthermore, for the maximized production of AVM $B_{la}$ by the high-producing S. avermitilis, optimum culture conditions such as components and concentrations of the production medium were examined through application of statistical methodology such as a central composite design protocol of the response surface method (RSM).
In the process of strain-development program, it had been observed that very high-yielding mutants of S. avermitilis frequently returned to their parallel parent strains in terms of their physiological characteristics, thus leading to decline in their AVM B1a productivity. These results implied that production stability of the high producers should be checked in order for the screened high-producers to be utilized in a scale-up bioprocess. For this purpose, we investigated the effects of long-term preservation methods, medium components and the producer's sporulation capability on AVM B1a production as well as biosynthetic stability of the high-yielding mutants. Notably, it was found in the screening process that the unstable and low-yielding mutants were not able to sporulate very well, implying that the sporulation capability of the mutants might be responsible for their reduced secondary metabolic activity for AVM B1a biosynthesis. Since trace metal element such as Fe was assumed to be closely involved in the sporulation metabolism of Streptomyces, we carefully investigated the effects of addition into the production medium of these metal compounds on the AVM B1a production and also tried to obtain stably high-yielding protoplast fusants resistant against toxic concentratins of FeCl3. Approximately 18000 units/L of AVM B1a production by the Fe-resistant fusants was observed and also these screened strains showed excellent sporulation capability with increased production stability as demonstrated in the narrow production range in the production-frequency histogram. Among the high-yielding protoplast fusants, 6 strains were intensively investigated in terms of their AVM B1a productivity and stability through cultivations for four generations. 5 strains out of the six were demonstrated not only highly productive in their specific production rate, but also very stable in their production capability, maintaining approximately 94% production level even after 4 generations.
Through this screening strategy using protoplast recombinants, we were able to solve the intrinsic genetic instability phenomena frequently observed in the high-yielding mutants of S.avermitilis, and thus able to utilize these highly stable and productive strains for the mass production of AVM B1a in the scaled-up fermentors.
Fermentations were carried out under the various conditions using various-sized fermentor to investigate the AVM B1a productivity of the high-yielding mutants. It was found that AVM B1a production was very low in D.O.-limited conditions but greatly increased when D.O. was controlled around high levels using an on-line computer control system. According to increase in D.O. concentration, AVM B1a production was almost linearly increased, demonstrating that AVM B1a production is closely dependent on D.O. concentration, especially during the exponential phase of cell growth. In order to investigate oxygen transfer capability of a 9-hole sparger and a sintered steel sparger developed in our research group, oxygen transfer coefficient (kLa) was measured according to agitation speeds and aeration rates. kLa of the sintered steel sparger was observed to be significantly higher than that of the 9-hole sparger.
When fermentation was performed using the sintered steel sparger at an agitation speed of 250 rpm, maximum amount of AVM B1a was obtained. It was concluded that sufficient oxygen supply and reduction in shear stress due to low agitation speed were the major reasons for the remarkable enhancement in the production of AVM B1a.
On-line computer-controlled fermentation system was successfully developed in order to produce AVM B1a in scaled-up fermentors in a stable and reproducible manner. This on-line control and monitoring system enabled valuable fermentation parameters to be monitored and estimated on-line during the whole period of fermentation process. The algorithms for on-line calculation of oxygen uptake rate was developed to estimate the cell concentration of the producer microorganim during the fermentation. By dividing the oxygen uptake rate by the total oxygen consumped, on-line estimation of specific growth rate of this culture was also possible. Use of gas analyzer system for vent oxygen analysis coupled with computer data acquisition has made this on-line information both very accurate (the error percent was within 5%) and readily available. It was possible to control dissolved oxygen level very accurately within 5% of the control value, so the effects of dissolved oxygen (DO) concentration and DO control modes on cell growth and AVM B1a production in the scaled-up fermentor could be investigated using the computer-controlled bioprocess system. Furthermore, by applying the algorithms, oxygen mass transfer coefficient (kLa), the most important parameter for the scale-up of avermectin B1a fermentation process was accurately measured on-line during the whole fermentation period. Furthermore, for the optimization of AVM B1a fermentation bioprocess, efficient whole bioprocess scheme was established by applying the results from the modelling and simulation studies performed based on the actual AVM B1a fermentation data.
Notably, when the computer-controlled fermentation system was utilized for mass production of in the 500 liter scaled-up fermentors by use of the high-yielding protoplast fusants (running of the fermentor 2 times for each strain), most of the strains were observed to produce significant amounts of AVM B1a in a very stable manner. In summary, it was confirmed that long-term, stable and reproducibe fermentation process was possible for the mass production of AVM B1a thanks to the development of the efficient computer-controlled fermentation system as well as high-yielding protoplast fusants.
The development of an optimum separation and purification process for avermectin is keenly required, because avermectin B1a should be separated from fermentation broth which contains 8 species of avermectin derivatives. The applicability of environmentally-friendly clean separation process applying precipitation or membrane was investigated. In case of precipitation method, the final purity of avermectin B1a was high enough, but low yield of 35% was obtained. On the other hand, almost no impurity was removed by applying UF membrane, while very high yield could be secured. Accordingly, the separation and purification process for avermectin was developed based on extraction process. The optimum aqueous phase and solvent phase for avermectin extraction was found to be methanol/water(5:3)-methylene chloride and acetone/water(5:3)-chloroform system. Both systems could reach to equilibrium within 10 minute, and showed more than 99% of extraction percentage when avermectin standard solution was extracted with 1:1 solvent to aqueous phase volume. It was observed that the percentage of extraction was increased with the volume ratio of solvent to aqueous phase. However, the percentage of extraction was reduced to 50% in case of methanol/water(5:3)-methylene chloride and to 35% in case of acetone/water(5:3) -chloroform system when actual avermectin fermentation broth was extracted with 1:1 solvent to aqueous phase volume. The reduction in the percentage of extraction was caused by the fact that some components in the broth helped forming a dispersion band between two phases and avermectin was trapped in the dispersion band. It was also found that avermectin producing cells could be easily disrupted by treating with solvent, and that methanol or acetone could be used as an optimum solvent for the crystallization.
Pilot scale investigation for developing an scaled-up separation and purification process was also performed using newly developed cell strain. It was confirmed that avermectin is intracellular product in the new cell strain. The optimum filter membrane was selected for filter press. Methanol/water(5:3)-methylene chloride extraction system was chosen as an optimum pilot scale extraction system. It was observed that the percentage of extraction was also increased with the volume ratio of solvent to aqueous phase as same as lab-scale experiment. The volume ratio of solvent to aqueous phase should be at least 3:1 for guaranteeing 90% extraction yield. Avermectin was concentrated to 30g/L by using rotary evaporator, and crystallized at 4o C with NaCl addition. As the addition amount of NaCl increased, crystallization yield increased at the expense of decreasing avermectin B1a purity in crystal. When 10 % of NaCl was added, crystallization yield was 26.7%, while avermectin B1a purity was 59% and the remains was avermectin B1b in the crystal. However, the purity of total avermectin was over 99%.
An integrated separation and purification process for 3000L fermentation was developed based on the results of lab and pilot scale investigation. An integrated separation and purification process was synthesized by combining filtering for cell recovery, cell disruption by solvent, filtering for the removal of cell debris, extraction by methylene chloride, concentration by evaporator, crystallization with NaCl addition, crystal filtering by filter press, drying by spray dryer, and grinding by mill. A PFD and material balance was developed for the synthesized process based on the results of lab scale and pilot scale experiments. P&ID was developed and an integrated separation and purification process was installed according to the P&ID. The installed process was operated, and operation data were accumulated. It was confirmed that the optimum TMP(Trans-Membrane Pressure) of Pall separator for cell and cell debris filtering was 3 bar. The flux of filterate was rapidly decreased with time and a strategy of increasing temperature was suggested for solving the flux diminution problem. Ten minutes of contact time was enough to release avermectin by solvent disruption. The percentage of extraction was also increased with the volume ratio of solvent to aqueous phase as same as pilot scale experiment. Eighty eight percentage(88%) of extraction was obtained with 3:1 solvent to aqueous phase volume. Extraction equilibrium was reached within 7 to 10 minutes. The repeated operation of concentration and crystallization were performed four times.
Avermectin was concentrated to 40g/L by evaporator and crystallized at 4o C with NaCl addition. The remained supernatant was concentrated to 40g/L and crystallized again and again. As concentration and crystallization cycle proceeded, avermectin crystal was continuously formed even though the amount of crystal formed was decreased with cycle. The percentage of extraction was increased gradually to 91.3 % after four cycle of concentration and crystallization. A crystalline avermectine was produced after recovering crystals by filter press and drying. A dried crystal was ground by mill. In addition to that, a scale-up strategy was established through the analysis of design and operation data accumulated, the development of design equation, and the estimation of design parameters. The economic feasibility was also studied through the analysis of operating cost and capital investment of commercialized process.

목차 Contents

• 제출문 ... 1
• 요약문 ... 2
• SUMMARY ... 10
• CONTENTS ... 14
• 목차 ... 15
• 제 1 장. 연구개발과제의 개요 ... 16
• 제 2 장. 국내외 기술개발 현황 ... 22
• 제 3 장. 연구개발수행 내용 및 결과 ... 24
• 제 1 절. 연구수행 방법 ... 24
• 1. 제 1 세부과제: 에버멕틴 고생산성균주 개발 및 pilot-scale에서의 에버멕틴 발효공정 scale-up 연구 ... 24
• 2. 제 1 세부위탁과제: 원형질융합에 의해 안정성있는 avermectin B1a 고생산성 돌연변이주 개발 및 배양생리학적 특성 조사 ... 32
• 3. 제 2 세부과제 및 제 2 세부위탁과제: 환경친화적 에버멕틴 분리정제공정 개발 ... 44
• 제 2 절. 연구수행 내용 및 결과 ... 47
• 1. 제 1 세부과제: 에버멕틴 고생산성균주 개발 및 pilot-scale에서의 에버멕틴 발효공정 scale-up 연구 ... 47
• 2. 제 1 세부위탁과제: 원형질융합에 의해 안정성있는 avermectin B1a 고생산성 돌연변이주 개발 및 배양생리학적 특성 조사 ... 96
• 3. 제 2 세부과제 및 제 2 세부위탁과제: 환경친화적 에버멕틴 분리정제공정 개발 ... 136
• 제 4 장. 목표달성도 및 관련분야에의 기여도 ... 194
• 제 1 절. 연구개발 최종 목표 ... 194
• 1. 평가항목, 평가방법 및 연구결과 ... 195
• 제 2 절. 연차별 연구개발 내용 및 건의 ... 196
• 제 3 절. 관련분야의 기술발전에의 기여도 ... 199
• 제 5 장. 연구개발결과의 활용계획 ... 200
• 제 6 장. 연구개발 과정에서 수집한 해외과학기술정보 ... 201
• 제 7 장 참고문헌 ... 202
• 끝페이지 ... 204