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Pichia pastoris에서 Zobellia galactanivorans 유래 재조합 $\beta$-Agarase의 고효율 분비생산
석지환,박희균,이상현,남수완,전숭종,김종현,김연희,Seok, Ji-Hwan,Park, Hee-Gyun,Lee, Sang-Hyeon,Nam, Soo-Wan,Jeon, Sung-Jong,Kim, Jong-Hyun,Kim, Yeon-Hee 한국미생물·생명공학회 2010 한국미생물·생명공학회지 Vol.38 No.1
The gene encoding $\beta$-agarase (agaB) which hydrolyzes $\beta$-1,4 linkages of agarose from Zobellia galactanivorans was cloned and fused to Saccharomyces cerevisiae mating factor alpha-1 secretion signal ($MF{\alpha}1$), in which the transcription of $MF{\alpha}1$-AgaB was under the control of AOX1 (alcohol oxidase 1, methanol inducible) promoter. The constructed plasmid pPIC-AgaB (9 kb) was integrated into HIS4 gene locus of Pichia pastoris genome. Successful integration was confirmed by performing colony PCR. The transformed cells showed red halos around its colonies in methanol agar plate by adding iodine solution, indicating the active expression of agaB in P.pastoris. By SDS-PAGE and zymographic analysis, the molecular weight of $\beta$-agarase was estimated to be a 53 kDa and about 15% N-linked glycosylation was occurred. The activity of extracellular $\beta$-agarase reached 1.34, 1.42 and 1.53 units/mL by inducing 0.1, 0.5, and 1% methanol, respectively, at baffled flask culture of P.pastoris GS115/pPIC-AgaB for 48 hr. Most of the enzyme activity was found in the extacellular fraction and the secretion efficiency showed 98%. Thermostability of recombinant $\beta$-agarase was also increased by glycosylation. Agarose의 $\beta$-1,4결함을 분해하는 Zobellia galactanivorans 유래의 $\beta$-agarase 유전자(agaB)는 클로닝 되었고, AOX1(alcohol oxidase 1, methanol inducible) promoter 하류에 Saccharomyces cerevisiae mating factor alpha-1 secretion signal($MF{\alpha}1$)를 연결하여 $MF{\alpha}1$-AgaB를 구축하였다. 구축된 plasmid pPIC-AgaB(9 kb)를 Pichia pastoris genome에 HIS4 gene 위치에 integration하였고, colony PCR을 통해 확인하였다. Methanol 첨가 배지에서 자란 형질전환체는 iodine solution의 첨가에 의해 red halos를 보였으며, P.pastoris에서 agaB의 효율적 분비 발현을 확인하였다. SDS-PAGE와 zymographic analysis에서 $\beta$-agarase의 분자량은 약 53 kDa으로 추정되었으며, 15% 정도의 N-linked glycosylation이 일어났음을 알 수 있었다. P.pastoris GS115/pPIC-AgaB의 48시간 baffled flask culture에서 세포외 $\beta$-agarase의 활성은 각각 0.1, 0.5, 1% methanol의 유도에 의해 1.34, 1.42 그리고 1.53 units/mL의 활성을 보였다. 대부분의 $\beta$-agarase의 활성은 세포 외에서 관찰되었고, 분비효율은 98%였으며 분비시의 glycosylation에 의해 열안정성도 증가되었다.
석지환(Ji-Hwan Seok),유창식(Chang-Sik Yoo) 대한전자공학회 2007 대한전자공학회 학술대회 Vol.2007 No.7
In this paper a 600㎷ low supply voltage filter is presented. The filter is designed with 2.11㎒ cut-off frequency. The 4<SUP>th</SUP> order filter consists of two 2<SUP>nd</SUP> order biquadratic cells. Input and output of biquadratic cell is VDD/2 to maximize signal swing and the input common mode voltage of opamp is VDD/4 to suit for low voltage. Capacitor array is used to tune cut-off frequency, and the filter is designed using 0.13㎛ CMOS technology.