Production of Antifreeze Protein from Antarctic Bacterium Flavobacterium frigoris PS1 by using Fed-batch Culture of Recombinant Pichia pastoris

Abstract

Antifreeze proteins (AFP) inhibit ice growth to permit the survival of polar organisms in the cold environments. The recombinant AFP from an Antarctic bacterium, Flavobacterium frigoris PS1, FfIBP (Flavobacterium frigoris icebinding protein), was produced using Pichia pastoris expression system. The optimum fermentation temperature (30 o C) and pH (5) for FfIBP production were determined using a fedbatch culture system. The maximal cell density and purified FfIBP were 112 g/L and 70 mg/L, respectively. The thermal hysteresis (TH) activity (0.85) of FfIBP obtained using a glycerol-methanol fed-batch culture system was 2-fold higher than that of the LeIBP (Leucosporidium ice-binding protein). This work allows for large-scale production of FfIBP, which could be extended to further application studies using recombinant AFPs.

Precision cellular agriculture: The future role of recombinantly expressed protein as food

“…β‐rich AFPs consist of stacked β‐sheet elements in a structure referred to as a β‐solenoid. Komagataella phaffii has been used to express fish‐sourced Type II AFPs (Li et al., 2001; Liu et al., 2018a; Nishimiya et al., 2006, 2008), and AFPs from carrot ( Daucus carota ) (Liu, Liang, Wang, et al., 2018; Liu et al., 2018a, 2018b), Flavobacterium frigoris (bacterial) (Kim et al., 2014), Tenebrio molitor (beetle) (Liu et al., 2018a), polar yeasts ( Leucosporidium [Koh et al., 2015; Lee et al., 2012, 2013; Park et al., 2012], Glaciozyma antarctica [Tab et al., 2018]), and cold‐adapted fungi (Xiao et al., 2010; Yamauchi et al., 2020). The algae C. reinhardtii was used to produce ryegrass ( Lolium perenne ) AFP (Lauersen, Berger, et al., 2013; Lauersen et al., 2015; Lauersen, Vanderveer, et al., 2013).…”

Pilot-scale Production of the Antifreeze Protein from Antarctic Bacterium Flavobacterium frigoris PS1 by Recombinant Escherichia coli with a Cold Shock Induction System

“…3(A)). 재조합 P. pastoris에서 생산된 FfIBP 의 최대 TH활성이 0.85 인 것에 비해 1.8배 높은 값이다 [13]. 결정화된 FfIBP를 이용하여 측정한 TH 활성의 최대값인 2.5 에는 미치지 못하였는데, 이는 완벽하게 정제되지 않은 시료 를 사용하여 상대적으로 낮은 농도에서 측정한 때문으로 풀 이된다 [12].…”

“…AY30으로부터 약 25kDa의 결빙방지단백질 LeIBP (Leucosporidium icebinding protein)을 분리하여 특성 및 대량생산 연구를 수행하 였다 [8][9][10][11]. 또한, 본 연구팀에서는 남극 호냉성 세균 Flavobacterium frigoris PS1으로부터 결빙방지단백질 (FfIBP)을 분 리하여 LeIBP와 얼음 결합능을 분자 구조적인 관점에서 비 교하였고 [12], 재조합 Pichia pastoris를 이용하여 유가식 배 양을 통해 생산최적화를 수행한 결과 FfIBP는 LeIBP과 유사 한 3차원 구조를 가짐에도 불구하고 LeIBP의 0.42°C 보다 두 배정도 높은 0.85°C의 TH 활성을 보였다 [13] . 본 연구에서는 재조합 FfIBP를 저온 유도 발현 시스템을 …”