title: Cloning, high-level expression, purification, and properties of a novel endo-beta-1,4-mannanase from Bacillus subtilis G1 in Pichia pastoris.
abstract: A novel gene coding for an endo-beta-1,4-mannanase (manA) from Bacillus subtilis strain G1 was cloned and overexpressed in P. pastoris GS115, and the enzyme was purified and characterized. The manA gene consisted of an open reading frame of 1,092 nucleotides, encoding a 364-aa protein, with a predicted molecular mass of 41 kDa. The beta-mannanase showed an identity of 90.2-92.9% (< or =95%) with the corresponding amino acid sequences from B. subtilis strains deposited in GenBank. The purified beta- mannanase was a monomeric protein on SDS-PAGE with a specific activity of 2,718 U/mg and identified by MALDITOF mass spectrometry. The recombinant beta-mannanase had an optimum temperature of 45 degrees C and optimum pH of 6.5. The enzyme was stable at temperatures up to 50 degrees C (for 8 h) and in the pH range of 5-9. EDTA and most tested metal ions showed a slightly to an obviously inhibitory effect on enzyme activity, whereas metal ions (Hg2+, Pb2+, and Co2+) substantially inhibited the recombinant beta-mannanase. The chemical additives including detergents (Triton X- 100, Tween 20, and SDS) and organic solvents (methanol, ethanol, n-butanol, and acetone) decreased the enzyme activity, and especially no enzyme activity was observed by addition of SDS at the concentrations of 0.25-1.0% (w/v) or n-butanol at the concentrations of 20-30% (v/v). These results suggested that the beta-mannanase expressed in P. pastoris could potentially be used as an additive in the feed for monogastric animals.
authors.affiliation: Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Distr. Caugiay, 10600 Hanoi, Vietnam.
authors.forename: Thi Thu Hang
authors.initials: TT
authors.lastname: Vu
authors.forename: Dinh Thi
authors.initials: DT
authors.lastname: Quyen
authors.forename: Thi Tuyet
authors.initials: TT
authors.lastname: Dao
authors.forename: Sy Le Thanh
authors.initials: Sle T
authors.lastname: Nguyen
mesh_terms: D001412:Bacillus subtilis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D015870:Gene Expression; D007700:Kinetics; D008361:Mannosidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008361:Mannosidases; C041867:endo-1,4-beta-D-mannanase