title: Heterologous expression and enzymatic characterization of fructosyltransferase from Aspergillus niger in Pichia pastoris.
abstract: In this work, the cDNA encoding fructosyltransferase (FTase) from Aspergillus niger YZ59 (CICIM F0901) was obtained and expressed in the methylotrophic yeast Pichia pastoris strain GS115. The yield of recombinant FTase in a 5-L fermentor reached 1020.0 U/mL after 96 h of induction, which was 1160.4 times higher that of native FTase from A. niger YZ59. The specific activity of recombinant FTase was 6.8×10(4) U/mg. The optimum temperature and pH of the recombinant FTase were 55 °C and 5.5, respectively. The recombinant FTase was stable below 40 °C and at pH from 3.0 to 10.0. Using sucrose as the substrate, the Km and Vmax values of recombinant FTase were 159.8 g/L and 0.66 g/(L min), respectively. The turnover number (kcat) and catalytic efficiency (kcat/Km) of recombinant FTase was 1.1×10(4) min(-1) and 68.8 L/(g min), respectively. The recombinant FTase was slightly activated by 5mM Ni(2+), Mg(2+), K(+), Fe(3+), or Mn(2+), but inhibited by all other metal ions (Na(+), Li(+), Ba(2+), Ca(2+), Zn(2+), and Cu(2+)). The highest yield of fructooligosaccharides for purified FTase reached approximately 343.3 g/L (w/v). This is the first study reporting the heterologous expression of FTases from A. niger in P. pastoris. This study plays an important role in the fructooligosaccharide synthesis industry by recombinant FTases.
authors.affiliation: Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China. Electronic address: 19891996@sina.com.
authors.forename: Hailin
authors.initials: H
authors.lastname: Yang
authors.affiliation: Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
authors.forename: Yitian
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China. Electronic address: shenwei_micro@163.com.
authors.forename: Wei
authors.initials: W
authors.lastname: Shen
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology*; D019149:Bioreactors / Q000382:microbiology; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D015870:Gene Expression; D005822:Genetic Vectors / Q000378:metabolism; D006602:Hexosyltransferases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D009844:Oligosaccharides / Q000096:biosynthesis; D010843:Pichia / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors; D001426:Bacterial Proteins
chemical_list: D006602:Hexosyltransferases; D007477:Ions; D008670:Metals; D009844:Oligosaccharides; D011994:Recombinant Proteins; C116580:fructooligosaccharide; C021451:inulosucrase; D001426:Bacterial Proteins