Optimization of induction condition for β-agarase overexpression in Pichia pastoris

β-agarase hydrolyzes β-1,4 linkages of agarose, yielding neoagarooligosaccharides. The neoagaro-oligosaccharides inhibit the growth of bacteria and slow down the degradation of starch and they can reduce the caloric value as additives. Therefore, economically feasibleproduction process of β-agarase enzyme is required. The β-agarase gene (agaB, 1 kb ORF) from Zobellia galactanivorans was subcloned into Pichia expression vector, pPIC9. The constructed plasmid pPICAgaB (9 kb) was integrated into HIS4 and AOX1 locus of P. pastorisgenome, respectively, resulting in Mut+(AOX1) and Muts (aox1Δ, methanol utilization slow) strain. The transformed cells showed red halos around its colonies in methanol agar plate by adding iodine solution, indicating the secretory expression of agaB in P. pastoris.Also, the protein and activity of secreted β-agarase were confirmed by using SDS-PAGE and zymographic analysis. When transformed cells (Mut+ and Muts) were cultured on medium containing each 0.1%, 0.5% and 1% methanol, the extracellular activity of β-agarase wereincreased from about 1.3 unit/ml to 1.6 unit/ml according to elevate methanol concentration. However, the expression level of β-agarase in Mut+ strain was indistinguishable from that of Muts strain.