title: Purification and characterization of a β-1,3-glucomannanase expressed in Pichia pastoris.
abstract: The glycoside hydrolase β-1,3-glucomannanase is an enzyme that specifically breaks the β-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 lilacinum ATCC 36010 was expressed in the yeast Pichia pastoris under the control of AOX1 promoter. The recombinant protein plMAN5C was purified from the shake flask culture and the stirred-tank bioreactor culture in yields of 30.0mg/l and 224.0mg/l, respectively. The purified protein had a specific activity of 14.6 U/mg at 37 °C, pH 4.5. Biochemical analysis showed that the optimal temperature and pH for plMAN5C were 50 °C and 4.5, respectively. The recombinant plMAN5C 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 β-1,3-glucomannanase, which should facilitate a more convenient application of this enzyme in biotechnology and other related areas.
authors: Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, Dalian 116023, China.
authors: Fan
authors: F
authors: Yang
authors: Sufang
authors: S
authors: Zhang
authors: Guojie
authors: G
authors: Jin
authors: Xinping
authors: X
authors: Lin
authors: Zongbao K
authors: ZK
authors: Zhao
mesh_terms: D001483:Base Sequence; D002473:Cell Wall / Q000737:chemistry / Q000378:metabolism; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005800:Genes, Fungal; D005822:Genetic Vectors; D006026:Glycoside Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008351:Mannans / Q000378:metabolism*; D010407:Penicillium / Q000201:enzymology / Q000235:genetics; D010843:Pichia / Q000235:genetics; D011523:Protoplasts / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012248:Rhodotorula / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D004271:DNA, Fungal; D005656:Fungal Proteins; D008351:Mannans; D011994:Recombinant Proteins; C022901:(1-6)-alpha-glucomannan; D006026:Glycoside Hydrolases