| Endo-β-mannanase(EC3.2.1.78) is an endohydrolase with great economic importance, which can be widely applied in food processing industry and feed production.In this study, the a-factor signal peptide in constitutive expression vector pGAPZaA was replaced by five other signal peptides (Wl, MF4I, INU1A, apre, HFBI). The relative activity of W1, MF4I, apre, and HFBI signal peptides were23.5%,203.5%,79.7%, and120.3%relative to α-factor respectively. However, INU1A signal peptide failed to secrete γ-mannanase.The further gene copy number determination by the quantitative real-time PCR revealed that recombinants of a-factor and MF4I with4MAN gene copies both secreted the most MAN. The MAN activity mediated by α-factor from1to6gene copy number level were12.95U/ml,43.33U/ml,126.63U/ml,173.53U/ml,103.23U/ml, and88.63U/ml, while that mediated by MF4I were79.22U/ml,133.89U/ml,260.14U/ml,347.5U/ml,206.15U/ml, and181.89U/ml. The max MAN activity reached347.5U/ml with4gene copies mediated by MF4I.The meanMAN activity of recombinants using SMD1168as expression host strain reached62.7%～64.1%of that using X33as expression host. Furthermore, the a-factor signal peptide in expression vector pPICZa-MAN was replaced by MF4I to reconstruct new inducing recombinants of pAOXlMF4I-MAN. The max MAN activity of those inducing recombinants is2.3times of that produced by original constitutive recombinants, which can be used in the next high density fermentation.Then optimization of fermentation conditions was investigated.27g/L corn steep powder and20g/L food grade glucose was respectively chosen as nitrogen source and carbon source of culture medium. Furthermore,2g/L (NH4)SO4and0.5g/L Oleic Acid were added to the inducing supplemented culture. With optimization of signal peptide and promotor,β-mannanase activity of recombinant Pichia pastoris reached6890U/ml after high density fermentation in5L fermentor. It has improved39.7times compared with original173.35U/ml. |
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