High-level extracellular production and characterization of Candida antarctica lipase B in Pichia pastoris(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)

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The gene encoding lipase B from Candida antarctica (CalB) was expressed in Pichia pastoris after it was synthesized by the recursive PCR and cloned into the Pichia expression plasmid, pPICZαA. The CalB was successfully secreted in the recombinant P. pastoris strain X-33 with an apparent molecular weight of 34 kDa. For 140 h flask culture, the dry cell weight and the extracellular lipase activity reached at 5.4 g/l and 57.9 U/l toward p-nitrophenyl palmitate, respectively. When we performed the fed-batch fermentation using a methanol feeding strategy for 110 h, the dry cell weight and the extracellular lipase activity were increased to 135.7 g/l and 11,900 U/l; the CalB protein concentration was 1.18 g/l of culture supernatant. The characteristics of CalB recovered from the P. pastoris culture were compared with the commercial form of CalB produced in Aspergillus oryzae. The kinetic constants and specific activity, the effects of activity and stability on temperature and pH, the glycosylation extent, the degree of immobilization on macroporous resin and the yield of esterification reaction between oleic acid and n-butanol were almost identical to each other. Therefore, we successfully proved that the Pichia-based expression system for CalB in this study was industrially promising compared with one of the most efficient production systems.