Extractive bioconversion (processes with simultaneous bioconversion and product removal) using aqueous two-phase systems was carried out.
Aqueous two-phase systems are formed when aqueous solutions containing two different polymers or one polymer and one salt are mixed because of their incompatibility. These systems are usually biocompatible because of high water content in both phases and possess favorable partition properties to be applicable to large scale enzyme separation and purification, extractive bioconversion, biological analysis and cofactor regeneration. First, to reduce product and substrate inhibition in enzyme reaction, aqueous two-phase system composed of PEG/potassium phosphate was used. As a model system, acrylamide production from acrylonitrile by nitrite hydratase and 6-aminopenicillanic acid production from penicillin G by penicillin acylase were studied. Second, extracellular enzyme production was carried out in aqueous two-phase system composed of PEG/dextral for extractive fermentation.
Acrylamide was produced from acrylonitrile using Brevibacterium sp. in an aqueous two-phase system composed of 17.5%(w/v) PEG 6000 and 8% (w/v) potassium phosphate. The cells were almost completely partitioned in he potassium phosphate-rich bottom phase. The inhibition of the active enzyme by both substrate and product could be reduced by favorable partition properties. Repeated experiments over 5 runs were accomplished without a significant loss of the enzyme activity. The yields of acrylamide obtained from the top phase were 0.736, 0.834, 0.865, 0.848 and 0.917 mol acrylamide/mol acrylonitrile.
The conversion of penicillin G(pen.G) to 6-aminopenicillanic acid (6APA) using a recombinant E. coil (HB 101) pPAKS2 as a whole cell enzyme was studied in aqueous two-phase systems. In a system composed of 20% (w/v) PEG 4000 and 8%(w/v) potassium phosphate the biocatalyst were almost completely partitioned to the bottom phase. Effects of ph and temperature on the enzyme activity in the aqueous two-phase system were almost the same as those in the buffer. But, product inhibitions by G-APA and PAA were reduced. Repeated batch experiment with recirculation of the enzyme in the bottom phase was performed over several times. The initial specific productivities were 0.31-1.51 μ mol 6-APA/mg biocatalyst/min in repeated experiments over 10 times during 286 hr. The yield obtained from the top phase was 0.35-0.89 mol 6APa/mol pen.G. For continuous operations, the reaction was carried out using a mixer-settler reactor. The conversion was 95% at 0.1-0.2 $h^{-1}$ of space velocity. For a successful operation, it was important to consider the setting time of phase systems related to the feed rate.
Alkaline protease production by Bacillus licheniformis was studied in an aqueous two-phase system composed of 5% (w/w) PEG 6000 and 5%(w/w) dextran T500. The top phase was continuous and rich in PEG while the bottom phase was dispersed and rich in dextral. The cells were retained in the bottom phase and the interface. The two-phase system produced more protease in total amounts than the control in the early growth phase, but after 50 hr protease production in the control system decreased while the aqueous two-phase system continued the production and finally the total enzyme activity reached 1.3 times that of the control culture. In order to elucidate the influence of PEG and dextral on protease production, the effects of molecular weight and concentration of PEG, the effect of dextral related to PEG were investigated. It seemed that PEG promoted protease production while dextral affected the stability of protease. In order to improve the productivity of protease, repeated batch cultivation was carried out. Considering that the protease synthesis in Bacillus licheniformis is susceptible to a catabolite repression, it was possible to produce protease efficiently in an aqueous two-phase system by designing a medium to be freshly added to the top phase.
본 연구는 수성이상계에서 추출 생전환을 시행한 것이다. 수성이상계는 기질과 생산물에 의한 저해가 심한 효소반응에서 그 물질들의 분리효과로 저해를 줄일 수 있으며, 수성이상계를 구성하는 PEG가 효소자체에 작용하여 안정성을 높이는데 기여하는 효과를 볼 수 있다. 또한 pH의 변화가 반응평형에 영향을 주는 penicillin acylase에 의한 6-APA의 생산에서 potassium phosphate가 많은 아래쪽 상에서의 완충효과로 반응도중 생기는 산에 의한 pH의 변화를 줄여주어 생산물쪽으로 평형을 유지할 수 있었다.
PEG/potassium phosphate의 수성이상계를 이용한 효소반응에서 whole cell enzyme으로 사용된 Brevibacterium 과 E.coli 의 분배는 아래쪽상의 potassium phosphate의 농도가 증가할수록 salting out현상에 의하여 위쪽상으로 많이 되었다. Brevibacterium 과 E.coli에 대한 salting out constant는 각각-5,97, -1.81(1/M)으로 potassium phosphate에 대한 분배의 변화는 brevibacterium의 경우가 더 민감하였다.
효소원인 Brevibacterium 과 E.coli가 아래쪽 상에 분리되도록 phase system을 정하여 한회분의 효소반응이 끝난 후 상을 분리시켜 생긴 위쪽상을 제거하고 기질이 있는 새로운 위쪽상을 넣어주는 방식으로 반연속회분 실험을 하여 생산성을 높일 수 있었다. 또한 분리기를 달아 효소반응과 분리를 동시에 하는 연속시스템에서 6-APA를 생산하였다. Space velocity 0.1-0.2 h$^{-1}$ 에서 95%의 전환을 얻었으나 0.3 h$^{-1}$이상에서는 수성이상계의 분리시간 때문에 조업에 어려움이 수반되었다.
수성이상계에서의 추출 생전환의 또 하나의 응용으로 추출발효를 실시하였다. PEG/dextran의 수성이상계에서 Bacillus licheniformis를 배양하여 protease를 생산 하였다. PEG는 균체의 세포벽에 영향을 주어 protease의 생산을 촉진시켰으며 고농도의 PEG나 dextran은 균자체 보다는 생산물인 protease의 안정성을 증가시키는데 기여한 것 같다. Catabolite repression을 받는 Bacillus licheniformis는 반연속 발효에서 새로 넣어주는 위쪽상에서 glucose 및 soy-bean meal의 농도를 catabolite repression을 줄이도록 조절하여 protease의 생산을 높일 수 있었다.
