Gene cloning, characterization, and heterologous expression of levansucrase from Bacillus amyloliquefaciens.
Although levan produced by Bacillus amyloliquefaciens is known to have efficient immunostimulant property which gives 100% survival of common carp when infected with Aeromonas hydrophila, no detailed reports are available describing kinetic studies of D: -glucose production and levan formation. In this study, we cloned and characterized the enzymatic kinetics using levansucrase expressed in Escherichia coli. Optimum pH for D: -glucose production and levan formation was 6.0 and 8.0, respectively, whereas optimum temperature was 30 degrees C and 4 degrees C, respectively. The K (m) and V (max) values for levansucrase were calculated to be 47.81 mM sucrose and 57.47 1mole/min mg protein, respectively. Prominent expression of levansucrase was obtained through xylose induction in Bacillus megaterium, where most of the His(6)-tagged protein was secreted into the culture broth, giving levansucrase activity of 12,906 U/l. Response-surface methodology (RSM) was further employed to optimize the fermentation conditions and improve the level of levansucrase production. Maximum levansucrase activity of 20,251 U/l was obtained in 12 h of fermentation carried out at 28 degrees C, starting induction with 0.735% xylose when A (600) was 1.2, which was 1.6- and 62-fold higher than those obtained in the nonoptimized conditions for the recombinant strain and the native strain, respectively.
Rairakhwada
Dina
D
Molecular Bioprocess Research Center, Korea Research Institute of Bioscience and Biotechnology, Jeongeup, Jeonbuk, Korea.
Seo
Jeong-Woo
JW
Seo
Mi-young
MY
Kwon
Ohsuk
O
Rhee
Sang-Ki
SK
Kim
Chul Ho
CH
D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001410:Bacillus megaterium / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005285:Fermentation; D005630:Fructans / Q000378:metabolism; D005798:Genes, Bacterial; D006602:Hexosyltransferases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D005630:Fructans; D011993:Recombinant Fusion Proteins; C072599:levan; D006602:Hexosyltransferases; C021513:levansucrase