Molecular cloning and expression of a novel protease-resistant GH-36 alpha-galactosidase from Rhizopus sp. F78 ACCC 30795.
A 2172-bp full-length gene (aga-F78), encoding a protease-resistant alpha-galactosidase, was cloned from Rhizopus sp. F78 and expressed in Escherichia coli. The deduced amino acid sequence shared highest identity (45.0%) with an alpha-galactosidase of glycoside hydrolase family 36 from Absidia corymbifera. After one step purification with Ni-NTA chelating column, the recombinant Aga-F78 migrated as a single band of ~82 and ~210 kDa on SDS-PAGE and non-denaturing gradient PAGE, respectively, indicating that the native structure of the recombinant Aga-F78 was trimer. Exhibiting the similar properties as the authentic protein, purified recombinant Aga-F78 was optimally active at 50 degrees and pH 4.8, highly pH stable over the pH range 5.0-10.0, more resistant to some cations and proteases, and had wide substrate specificity (pNPG, melidiose, raffinose and stachyose). The recombinant enzyme also showed good hydrolytic ability to soybean meal, releasing galactose of 415.58 microng/g soybean meal. When combined with trypsin, the enzyme remained over 90% degradability to soybean meal. These favorable properties make Aga-F78 a potential candidate for applications in the food and feed industries.
Cao
Yanan
Y
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P. R. China.
Wang
Yaru
Y
Luo
Huiying
H
Shi
Pengjun
P
Meng
Kun
K
Zhou
Zhigang
Z
Zhang
Zhifang
Z
Yao
Bin
B
D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012233:Rhizopus / Q000201:enzymology* / Q000235:genetics; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013025:Glycine max / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014357:Trypsin / Q000378:metabolism; D000519:alpha-Galactosidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D011994:Recombinant Proteins; D000519:alpha-Galactosidase; D014357:Trypsin