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

Abstract

Levan is a polymer of fructose synthesized by levansucrase using sucrose as the substrate, which is employed as a viscosifier, stabilizer, emulsifier, and gelling or water-binding agent for diverse industrial applications. To enable a gentle and efficient purification process, we cloned the gene of levansucrase (sacB) from Bacillus amyloliquefaciens BH072 into an inducible vector harboring the signal peptide gene amyQ and then expressed it in Bacillus subtilis 168. The optimal pH values for d-glucose production and levan formation by the recombinant enzyme were 6.0 and 7.0, respectively, whereas the optimum temperatures were 40 and 35 °C, respectively. The K m and V max values for the recombinant levansucrase were calculated to be 0.71 mM sucrose and 0.49 μmol mL–1 min–1. Finally, levan biosynthesized by the recombinant enzyme was detected by thin layer chromatography (TLC) and Fourier transform infrared spectroscopy (FT-IR). TLC chromatograms of reaction products qualitatively indicated the formation of levan that was further assessed by high-performance liquid chromatography analysis. Some important functional groups and chemical bonds of the obtained dried levan powder were determined by FT-IR.

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

“…The Ba-SacB yield obtained from the supernatant after equilibration, washing, and elution was estimated by SDS-PAGE. 16 200 μg/mL trypsin was used to remove the Usp45 signal peptide to gain active Ba-SacB.…”

“…15 Recently, our group cloned Bacillus amyloliquefaciens levansucrase (Ba-SacB) and successfully expressed it in the food-grade host B. subtilis. 16 The construction of B. subtilis recombinant strains often involves a shuttle plasmid, making the molecular construction process complicate. Therefore, a safer host strain with easier manipulation for levansucrase production is desirable for food industry.…”

Production of Sucrolytic Enzyme by Bacillus licheniformis by the Bioconversion of Pomelo Albedo as a Carbon Source

“…Km and Vmax of sleTKU004 were estimated to be 1.16 M and 5.99 µmol/min based on a Lineweaver-Burk plot (Figure 10b). Km value of sleTKU004 was higher than that of sucrolytic enzymes from other Bacilli such as B. methylotrophicus SK 21.002 (117.2 mM) [77], B. amyloliquefacien BH072 (0.71 mM) [73], B. cereus TA-11 (370 mM) [59], and B. subtilis (33.96 g/L) [69]. A high Km value indicates that high concentration of sucrose must be present to saturate sleTKU004.…”

Production and prebiotic properties of oligofructans from sugarcane juice fermentation by Bacillus subtilis TISTR 001

“…Furthermore, this microorganism can grow in many types of food, both under anaerobic or aerobic conditions (Olmos and Paniagua-Michel 2014). Past research has shown that B. subtilis can produce levansucrase, which is a fructosyltransferase type of enzyme that is considered the main enzyme used in the production of fructo-oligosaccharides and fructan (Mu et al 2021;Raga-Carbajal et al 2016;Santos et al 2013;Shi et al 2019;Tanaka et al 2014). Fructosyltransferase is formed via an enzymatic transformation of fructose sugars connected by beta bonds (β-D-fructose), that enzymes in the primary digestive tract are unable to digest.…”

“…Prebiotics help to promote the growth of probiotics with the most common probiotic being lactic acid bacteria (LAB), such as Bifidobacterium sp., Lactobacillus sp., Streptococcus sp., Enterococcus sp., and Bacillus sp. (Chlebowska-Smigiel et al 2017Jager et al 2020;Mu et al 2021;Raveschot et al 2020;Roobab et al 2020). Additionally, they can inhibit the growth of pathogenic microorganisms, such as Clostridium sp., Escherichia coli, and Samonella sp.…”