Gene cloning, characterization, and heterlogous expression of levansucrase from Bacillus amyloliquefaciens = Bacillus amyloliquefaciens levansurase 유전자의 클로닝, 특성 규명 및 이종숙주 발현

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Title
Gene cloning, characterization, and heterlogous expression of levansucrase from Bacillus amyloliquefaciens = Bacillus amyloliquefaciens levansurase 유전자의 클로닝, 특성 규명 및 이종숙주 발현
Author(s)
D Rairakhwada; Jeong Woo Seo; M Y Seo; Oh Suk Kwon; S K Rhee; Chul Ho Kim
Bibliographic Citation
Journal of Industrial Microbiology & Biotechnology, vol. 37, no. 2, pp. 195-204
Publication Year
2010
Abstract
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°C and 4°C, respectively. The K m and V max values for levansucrase were calculated to be 47.81 mM sucrose and 57.47 μmole/min mg protein, respectively. Prominent expression of levansucrase was obtained through xylose induction in Bacillus megaterium, where most of the His6-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°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.
Keyword
Bacillus amyloliquefaciens type 1Bacillus megateriumEscherichia coliGene expressionLevansucraseResponse-surface methodology
ISSN
0169-4146
Publisher
Springer
DOI
http://dx.doi.org/10.1007/s10295-009-0664-2
Type
Article
Appears in Collections:
Jeonbuk Branch Institute > Microbial Biotechnology Research Center > 1. Journal Articles
Division of Bio Technology Innovation > SME Support Center > 1. Journal Articles
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