Purification and characterization of a β-1,3-glucomannanase expressed in Pichia pastoris

The glycoside hydrolase beta-1,3-glucomannanase is an enzyme that specifically breaks the beta-1,3 glycosidic bond of the glucomannan, the main cell wall constituent of some yeasts. In this work, a codon optimized DNA sequence of the MAN5C gene from Penicillium Iilacinum ATCC 36010 was expressed in the yeast Pichia pastoris under the control of AOX1 promoter. The recombinant protein pIMAN5C was purified from the shake flask culture and the stirred-tank bioreactor culture in yields of 30.0 mg/l and 224.0 mg/l, respectively. The purified protein had a specific activity of 14.6 U/mg at 37 degrees C, pH 4.5. Biochemical analysis showed that the optimal temperature and pH for pIMAN5C were 50 degrees C and 4.5, respectively. The recombinant pIMAN5C was efficient in lysis of the cell wall of the red yeast Rhodosporidium toruloides to form protoplast. Our work provided an effective system for heterogeneous production of beta-1,3-glucomannanase, which should facilitate a more convenient application of this enzyme in biotechnology and other related areas. (C) 2011 Elsevier Inc. All rights reserved.