Acidic β-mannanase from Penicillium pinophilum C1: Cloning, characterization and assessment of its potential for animal feed application.
The β-mannanase gene, man5C1, was cloned from Penicillium pinophilum C1, a strain isolated from the acidic wastewater of a tin mine in Yunnan, China, and expressed in Pichia pastoris. The sequence analysis displayed the gene consists of a 1221-bp open reading frame encoding a protein of 406 amino acids (Man5C1). The deduced amino acid sequence of Man5C1 showed the highest homology of 57.8% (identity) with a characterized β-mannanase from Aspergillus aculeatus belonging to glycoside hydrolase family 5. The purified rMan5C1 had a high specific activity of 1035U mg(-1) towards locust bean gum (LBG) and showed highest activity at pH 4.0 and 70°C. rMan5C1 was adaptable to a wide range of acidity, retaining >60% of its maximum activity at pH 3.0-7.0. The enzyme was stable over a broad pH range (3.0 to 10.0) and exhibited good thermostability at 50°C. The K(m) and V(max) values were 5.6 and 4.8mgmL(-1), and 2785 and 1608μmolmin(-1)mg(-1), respectively, when LBG and konjac flour were used as substrates. The enzyme had strong resistance to most metal ions and proteases (pepsin and trypsin), and released 8.96mgg(-1) reducing sugars from LBG in the simulated gastric fluid. All these favorable properties make rMan5C1 a promising candidate for use in animal feed.
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, PR China.
Hongying
H
Cai
Pengjun
P
Shi
Huiying
H
Luo
Yingguo
Y
Bai
Huoqing
H
Huang
Peilong
P
Yang
Bin
B
Yao
D000595:Amino Acid Sequence; D000821:Animal Feed; D000818:Animals; D001483:Base Sequence; D002681:China; D003001:Cloning, Molecular; D004795:Enzyme Stability; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010407:Penicillium / Q000201:enzymology*; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D012722:Sewage / Q000382:microbiology; D044902:beta-Mannosidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D011994:Recombinant Proteins; D012722:Sewage; D044902:beta-Mannosidase