Gene cloning, characterization, and heterologous expression of levansucrase from Bacillus amyloliquefaciens

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 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.

Purification and Characterization of Levansucrases fromBacillus amyloliquefaciensin Intra- and Extracellular Forms Useful for the Synthesis of Levan and Fructooligosaccharides

“…A molecular mass of 49-52 kDa has been similarly reported for LS from B. amyloliquefaciens. 13,14) The molecular weight of B. amyloliquefaciens LS falls within the range reported for other LSs from Bacillus species of 52-55 kDa 9,24,28) and for LSs from gram-negative bacteria, including Z. mobilis 29) and G. diazotrophicus, 10) varying from 48 to 58 kDa.…”

“…The overall pH activity profiles of intra-and extra-cellular LSs showed only a slight difference. Mantsala and Puntala 13) and Rairakhwada et al 14) have similarly reported an optimum pH of 6.0-6.2 for extracellular LS from B. amyloliquefaciens. The pH activity profile of B. amyloliquefaciens LSs also showed features common to those of other LSs from bacillus species with an optimum pH in the range of 6.0-6.6, 9,28,32) with the exception of LS from B. circulans which exhibited optimum activity in the pH range of 5-7.…”

“…Bacillus amyloliquefaciens, having the ''generally recognized GRAS'' status, is one of the dominant bacterial workhorses with high potential for enzyme production. B. amyloliquefaciens cultivated on media containing sucrose 13) and xylose 14) has been reported to produce extracellular LS. Little information about the catalytic properties and the donor/acceptor specificity of LS from B. amyloliquefaciens is available.…”

Recent novel applications of levansucrases

“…The enzymes from Zymomonas mobilis (Sangiliyandi et al 1999) and M. laevaniformans ATCC 15953 (Park et al 2003) show maximal polymerization activity at 30°C but exhibit sucrose hydrolysis activity at 45-50°C. B. amyloliquefaciens levansucrase displays the highest activity at temperatures of 4 and 30°C for levan formation and sucrose hydrolysis, respectively (Rairakhwada et al 2010). …”

“…Generally, levan formation increases with an increasing concentration of sucrose (Ben Ammar et al 2002;Rairakhwada et al 2010;Tian et al 2011;Waldherr et al 2008;Zhang et al 2014), and the sucrose concentration usually plays an important role in controlling the molecular weight of the products.…”