Cloning and Expression of Levansucrase Gene of Bacillus velezensis BM-2 and Enzymatic Synthesis of Levan

Abstract

Levan is a versatile and valuable fructose homopolymer, and a few bacterial strains have been found to produce levan. Although levan products have numerous specific functions, their application and promotion were limited by the production capacity and production cost. Bacillus velezensis BM-2 is a levan-synthesizing strain, but its levan production is too low to apply. In this study, the levansucrase gene of B. velezensis BM-2 was cloned to plasmid pET-32a-Acma-zz, and the recombinant plasmids were transferred to Escherichia coli BL21. A transformed clone was selected to express and secrete the fusion enzymes with an Acma-tag efficiently. The expressed products were further purified by a self-developed separating material called bacterial enhancer matrix (BEM) particles. The purification efficiency was 93.4%, with a specific activity of 16.589 U/mL protein. The enzymatic reaction results indicated that the optimal reaction temperature is 50 °C, the optimal pH of the acetate buffer is 5.6, and the buffer system greatly influenced the enzyme activity. The enzyme activity was enhanced to 130% in the presence of 5 mM Ca2+, K+, Zn2+, and Mn2+, whereas it was almost abolished in the case of Cu2+ and Fe3+. The values of Km, kcat, and kcat/Km were 17.41 mM, 376.83 s−1, and 21.64 mM−1s−1, respectively. The enzyme amount of 20 U/g sucrose was added to the system containing 400 g/L sucrose, and the levan products with a concentration of 120 g/L reached after an incubation of 18 h, which was 8 times that of the yield before optimization. The results of molecular docking analysis indicated that the Asp86 might act as a nucleophilic catalytic residue for sucrose, Arg246 and Asp247 act as transition state stabilizer of transfructosylation, and Glu340 and Arg306 were recognized as general acid donors. They formed the catalytic-groups triad. The unique properties and catalytic activity of the levansucrase suggest that it deserves further research and might have good industrial application prospects.

Levan Production by Suhomyces kilbournensis Using Sugarcane Molasses as a Carbon Source in Submerged Fermentation

“…In accordance with these results, the following experiments (the effects of nitrogen and carbon sources on EPS production) were conducted with an initial pH of 6.5. The EPS production profile during SmF at different initial pH values may be because EPS synthesis depends on the action of an extracellular enzyme that has a catalytic response to pH changes, directly impacting EPS yields [ 19 ]. As shown in Figure 2 , the highest EPS concentration was obtained in the production system implemented in the present investigation when the initial pH of the SmF was adjusted to 6.5.…”

“…Likewise, the results are consistent with the results obtained by Zhang et al [ 55 ], who reported a maximum EPS yield when 300 g/L of sucrose was used as a carbon source with Bacillus methylotrophicus . Furthermore, the EPS yield decreased significantly above this sucrose concentration, probably because of the increase in viscosity and an enzymatic inhibition that consequently impacted EPS synthesis [ 19 , 41 ]. On the other hand, the use of recombinant yeasts for levan production has been reported; e.g., Ko et al [ 29 ] reported a levansucrase from Rahnella aquatilis expressed in Saccharomyces cerevisiae , obtaining yields of 3.17 g/L/h.…”

“…Microbial levan is typically obtained through fermentation or enzymatic reactions using isolated enzymes, in which sucrose serves as both a principal carbon source and substrate, respectively. The optimal sucrose concentration for achieving maximum synthesis efficiency varies not only among different species but also often among different strains of microorganisms [ 19 , 20 ]. In the literature, there are various reports that indicate the capacity of some microorganisms to produce levan using sucrose as the only carbon source; among these, we can highlight Bacillus subtilis [ 21 , 22 ], Lactobacillus reuteri [ 23 ], and Leuconostoc citreum [ 24 ] and Gram-negative bacteria such as Gluconobacter albidus [ 25 ], Brenneria goodwinii [ 26 ], Erwinia tasmaniensis [ 27 ], and Halomonas smyrnensis [ 28 ].…”

Molecular characterization of Paenibacillus antarcticus IPAC21, a bioemulsifier producer isolated from Antarctic soil

“…The main chain is branched through the β-(2 → 1) linkage of the fructofuranosyl rings ( Mendonça et al, 2021 ). The production of levan starts in the extracellular medium through the conversion of sucrose to fructose/glucose by the enzyme SacB, which synthesizes fructose polymers through a transfructosylation reaction using sucrose as a donor fructose/glucose ( Xu et al, 2021 ). The production of levan has already been described in other Paenibacillus species, such as P. polymyxa DSM 365 ( Okonkwo et al, 2020 ), Paenibacillus sp.…”

Molecular interaction of linear and branched fructans of different sizes with levansucrase and inulosucrase from Lactobacillus gasseri: modeling and computational analysis

“…Molecular dockings with the enzymes LevG and InuGB from L. gasseri had a percentage of sequence identity of 2YFS.1.A greater than 62%, and couplings with binding energies greater than -7.0 kcal/mol for agavins of a low degree of polymerization. Compared with this study, tridimensional structures of levansucrase from proteobacteria Sphingobium chungbukense DJ77, Zymomonas mobilis and Bacillus amyloliquefaciens levansucrase KK9 with 34%, 54% and 89% of sequence identity have been employed (Xu et al, 2021). When B. amyloliquefaciens KK9 levanetype fructooligosaccharides were used, affinity energy of -7.7 kcal/mol was obtained (Phengnoi et al, 2020).…”