Mercuric Ion Stabilizes Levansucrase Secreted by Acetobacter nitrogenifigens Strain RG1T

Abstract

The purification and characterization of an extracellular levansucrase enzyme produced by novel nitrogen-fixer Acetobacter nitrogenifigens strain RG1(T) is described. Culture conditions were optimized for maximum levansucrase production. Levansucrase purified to homogeneity by tenfold purification has a molecular weight of 65 kDa, contained four cysteine residues, polymerized raffinose and was stable for 21 days at pH 6.0 when stored at 4 °C or -20 °C but was vulnerable to DTT and β-mercaptoethanol. Interestingly, this enzyme showed enhanced hydrolytic and polymerization activity in the presence of mercuric ion which, to our knowledge, is the first report for any levansucrase enzyme characterized so far. Evidences obtained from Native PAGE, tryptophan fluorescence study and activity measurements at different temperatures and in the presence of thiol modifying agents, show that mercuric ion stabilizes the enzyme. Levan, synthesized by the enzyme, has a molecular weight of 7,080 kDa and was shown to be a homopolymer of fructose.

Secretion of Bacillus amyloliquefaciens Levansucrase from Bacillus subtilis and Its Application in the Enzymatic Synthesis of Levan

“…Levansucrase exists in a wide range of Gram-positive and Gram-negative species, such as Bacillus megaterium , Zymomonas mobilis , Erwinia amylovora , and Gluconobacter oxydans , rendering different levan yields. However, it is easy to enhance the yield of levansucrase by breeding the native-type strains and optimizing the fermentation parameters.…”

Identification of a Thermostable Levansucrase from Pseudomonas orientalis That Allows Unique Product Specificity at Different Temperatures

“…The LS from Bacillus methylotrophicus SK 21.002 is the only one that produces LMW levan with an M W of 4–5 × 10 3 Da [ 7 ]. By contrast, the LSs from Acetobacter nitrogenifigens RG1 [ 8 ] and Lactobacillus reuteri LTH5448 [ 9 ] could synthesize HMW levan as the main product, with an M W of 7.1 × 10 6 and 3.9 × 10 7 Da, respectively. In particular, the LS from Clostridium acetobutylicum could exclusively synthesize levan rather than FOSs in the reaction [ 10 ].…”

Secretion of Bacillus amyloliquefaciens Levansucrase and Its Mutants from L. lactis NZ9000 and Their Applications in the Synthesis of Levan

“…6 Additionally, levan has a bacteriostatic effect on Escherichia coli and can inhibit the formation of cell membranes of Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, and the yeast Candida albicans. 7 At present, levan is mainly synthesized by enzymes produced by microorganisms such as Bacillus subtilis, Acetobacter nitrogenifigens, 8 Clostridium acetobutylicum, 9 and Pseudomonas syringae 10 among others. Levansucrase is the kind of microbial enzyme producing levan belonging to the glycoside hydrolase family 68 (GH68).…”

“…At present, levan is mainly synthesized by enzymes produced by microorganisms such as Bacillus subtilis, Acetobacter nitrogenifigens, Clostridium acetobutylicum, and Pseudomonas syringae among others. Levansucrase is the kind of microbial enzyme producing levan belonging to the glycoside hydrolase family 68 (GH68). , The heterologous expression of levansucrase from Bacillus amylosus kk9 and Clostridium acetylbutyrate has been achieved in E.…”