Pichia pastoris에서 Zobellia galactanivorans 유래 재조합 β-Agarase의 고효율 분비생산
High-level Secretory Expression of Recombinant β-Agarase from Zobellia galactanivorans in Pichia pastoris.
1
조회
0
다운로드
7
인용
0
참고문헌
키워드
초록
Agarose의 β-1,4결합을 분해하는 Zobellia galactanivorans 유래의 β-agarase 유전자(agaB)는 클로닝되었고, AOX1(alcohol oxidase 1, methanol inducible) promoter 하류에Saccharomyces cerevisiae mating factor alpha-1 secretion signal(MFα1)를 연결하여 MFα1-AgaB를 구축하였다. 구축된 plasmid pPIC-AgaB(9 kb)를 Pichia pastoris genome에HIS4 gene 위치에 integration하였고, colony PCR을 통해확인하였다. Methanol 첨가 배지에서 자란 형질전환체는 iodine solution의 첨가에 의해 red halos를 보였으며, P. pastoris에서 agaB의 효율적 분비 발현을 확인하였다. SDS-PAGE와zymographic analysis에서 β-agarase의 분자량은 약 53 kDa 으로 추정되었으며, 15% 정도의 N-linked glycosylation이일어났음을 알 수 있었다. P. pastoris GS115/pPIC-AgaB의48시간 baffled flask culture에서 세포외 β-agarase의 활성은각각 0.1, 0.5, 1% methanol의 유도에 의해 1.34, 1.42 그리고 1.53 units/mL의 활성을 보였다. 대부분의 β-agarase의 활성은 세포 외에서 관찰되었고, 분비효율은 98%였으며 분비시의 glycosylation에 의해 열안정성도 증가되었다.
Abstract
The gene encoding β-agarase (agaB) which hydrolyzes β-1,4 linkages of agarose from Zobellia galactanivorans was cloned and fused to Saccharomyces cerevisiae mating factor alpha-1 secretion signal (MFα1), in which the transcription of MFα1-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 β-agarase was estimated to be a 53 kDa and about 15% N-linked glycosylation was occurred. The activity of extracellular β-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 β-agarase was also increased by glycosylation.
참고문헌 (26)
- Araki. T, "Purification and characterization of agarases from a marine bacterium", J. Mar. Biotechnol., 6, pp.260~265 (1998)
- Cregg, J. M, "Recent advances in the expression of foreign genes in Pichia pastoris", Bio, 1993, pp.11, 905~910
- Do,J.H, "Extraction and purification of agar from Gelidium amansii", J. Korean Fish Soc., 30, pp.423~427 (1997)
- Duckworth, M, "Structure of ahar. I. Fractionation of a complex mixture of polysaccharides", Carbo. Res., 16, pp.189~197 (1971)
- Fu Xiao Ting, "Gene Cloning, Expression, and Characterization of a β-Agarase, AgaB34, from Agarivorans albus YKW-34", Journal of Microbiology and Biotechnology, 19(3), pp.257~264 (2009), 한국미생물·생명공학회
- Han, Y. J, "Secretion of Bacillus endoglucanase in Saccharomyces cerevisiae by its own signal sequence", J. Microbiol. Biotechnol., 4, pp.24~29 (1994)
- Hatada, Y, "Hyperproduction and application of alpha-agarase to enzymatic enhancement of antioxidant activity of porphyran", J. Agric. Food Chem., 54, pp.9895~ (2006)
- Jahic, M, "Process technology for production and recovery of heterologous proteins with Pichia pastoris", Biotechnol. Prog., 22, pp.1465~1473 (2006)
- Jam, M, "The endo-β-agarase AgaA and AgaB from the marine bacterium Zobellia galactanivorans : two paralogue enzymes with different molecular organizations and catalytic behaviours", Biochem. J., 385, pp.703~713 (2005)
- Joo. D. S, "Preparation condition of agar oligosaccharide with organic acids", J. Korean Fish Soc., 36, pp.6~10 (2003)
- Kato,I, "Antioxidative and antitumorigenic properties of agarooligosaccharide", Bio Industry, 17, pp.13~19 (2000)
- 김미진, "High-Level Secretory Expression of Human Procarboxypeptidase B by Fed-Batch Cultivation of Pichia pastoris and its Partial Characterization", Journal of Microbiology and Biotechnology, 18(12), pp.1938~1944 (2008), 한국미생물·생명공학회
- Kobayashi. R, "Neoagarobiose as a novel moisturizer with whitening effect", Biosci Biotechnol Biochem., 61, pp.162~163 (1997)
- Kong, J. Y, "Cloning and expression of an agarase gene form a marine bacterium Pseudomonas sp. w7", Biotechnol. Lett., 19, pp.23~26 (1997)
- Kwon, M. A, "High-level expression and characterization of Fusarium solani cutinase in Pichia pastoris", Purif, 68, pp.104~119 (2009)
- Laemmli, U. K, "Cleavage of structural proteins during the assembly of the head of bacteriophage T4", Nature, 277, pp.680~685 (1970)
- Lee, D. G, "Over-production of a glycoside hydrolase family 50 beta-agarase from Agarivorans sp. JA-1 in Bacillus subtilis and the whitening effect of its product", Biotechnol. Lett., 30, pp.911~918 (2008)
- Lee, K. W, "Expression of antimicrobial cationic peptide by Pichia pastoris (methylotrophic yeast)", Kor. J. Anim. Sci. Technol., 43, pp.465~476 (2001)
- Li, P., "Expression of recombinant proteins in Pichia pastoris", Appl. Biochem. Biotechnol., 142, pp.105~124 (2007)
- Miller,G.L, "Use of dinitrosalicylic acid reagent for determination of reducing sugar", Anal. Chem., 31, pp.426~428 (1959)
- Ohta, Y, "Purification and characterization of a Novel α-agarase from a Thalassomonas sp", Curr. Microbiol., 50, pp.212~216 (2005)
- Scotti, P. A, "The targeting of Bacillus subtilis levansucrase in yeast is correlated to both the hydrophobicity of the signal peptide and the net charge of the N-terminus mature part", Yeast, 12, pp.953~963 (1996)
- Solovicova, A, "Highyield roduction of Saccharomycopsis fibuligera glucoamylase in Escherichia coli, refolding, and comparison of the nonglycosylated and glycosylated enzyme forms", Biochem. Biophys. Res. Comm., 224, pp.790~795 (1996)
- Yano. J. K, "New understandings of thermostable and peizostable enzymes", Curr. Opin. Biotechnol., 14, pp.360~365 (2003)
- Yoshizawa, Y, "Macrophage stimulation activity of the polysaccharide fraction from a marine alga (Porphyra yezoensis): structure-function relationships and improved solubility", Biosci. Biotechnol. Biochem., 59, pp.1933~1937 (1995)
- Zhang, W. W, "Cloning, characterization, and molecular application of a beta-agarase gene from Vibrio sp. strain V134", Appl. Environ. Microbiol., 73, pp.2825~2831 (2007)