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KMID : 1234420100380010040
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Korean Journal of Microbiololgy and Biotechnology
2010 Volume.38 No. 1 p.40 ~ p.45
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High-level Secretory Expression of Recombinant β-Agarase from Zobellia galactanivorans in Pichia pastoris
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Seok Ji-Hwan
Park Hee-Gyun
Lee Sang-Hyeon
Nam Soo-Wan
Jeon Seong-Soo
Kim Jong-Hyun
Kim Yeon-Hee
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Abstract
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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.
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KEYWORD
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β-agarase, overexpression, seretion, Zobellia galactanivorans, Pichia pastoris
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