A novel protease-resistant alpha-galactosidase with high hydrolytic activity from Gibberella sp. F75: gene cloning, expression, and enzymatic characterization.
A novel alpha-galactosidase gene (aga-F75) from Gibberella sp. F75 was cloned and expressed in Escherichia coli. The gene codes for a protein of 744 amino acids with a 24-residue putative signal peptide and a calculated molecular mass of 82.94 kDa. The native structure of the recombinant Aga-F75 was estimated to be a trimer or tetramer. The deduced amino acid sequence showed highest identity (69%) with an alpha-galactosidase from Hypocrea jecorina (Trichoderma reesei), a member of the glycoside hydrolase family 36. Purified recombinant Aga-F75 was optimally active at 60 degrees C and pH 4.0 and was stable at pH 3.0-12.0. The enzyme exhibited broad substrate specificity and substantial resistance to neutral and alkaline proteases. The enzyme K (m) values using pNPG, melibiose, stachyose, and raffinose as substrates were 1.06, 1.75, 54.26, and 8.23 mM, respectively. Compared with the commercial alpha-galactosidase (Aga-A) from Aspergillus niger var. AETL and a protease-resistant alpha-galactosidase (Aga-F78) from Rhizopus sp. F78, Aga-F75 released 1.4- and 4.9-fold more galactose from soybean meal alone, respectively, and 292.5- and 8.6-fold more galactose from soybean meal in the presence of trypsin, respectively. The pH and thermal stability and hydrolytic activity of Aga-F75 make it potentially useful in the food and feed industries.
Cao
Yanan
Y
Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.
Wang
Yaru
Y
Meng
Kun
K
Bai
Yingguo
Y
Shi
Pengjun
P
Luo
Huiying
H
Yang
Peilong
P
Zhou
Zhigang
Z
Zhang
Zhifang
Z
Yao
Bin
B
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D005874:Gibberella / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D000519:alpha-Galactosidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D005656:Fungal Proteins; D000519:alpha-Galactosidase; D010447:Peptide Hydrolases