Computational design of Bacillus licheniformis RN-01 levansucrase for control of the chain length of levan-type fructooligosaccharides

Kanjanatanin, Pongsakorn; Pichyangkura, Rath; Sitthiyotha, Thassanai; Charoenwongpaiboon, Thanapon; Wangpaiboon, Karan; Chunsrivirot, Surasak

doi:10.1016/j.ijbiomac.2019.08.151

Cited by 26 publications

(19 citation statements)

References 44 publications

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“…Accordingly, the mutation of K363 to alanine greatly impacted the enzymatic activity and eliminated the synthesis of LMW levans by limiting the efficient formation of oligosaccharides with a DP of up to 5, even though larger oligosaccharides were barely detected. A similar size-limiting effect was observed in alanine mutation studies targeting homologous residues in LSs from B. megaterium and B. licheniformis RN-01 (12,19), which are enzymes that natively accumulate LMW levans with DPs higher than 10 (31, 32). However, remarkably, K363A did not affect the formation of HMW levans by SacB, suggesting a nonessential role of this residue in processive levan synthesis.…”

Section: Insights Into the Ob1 Subsites +1 And +2supporting

confidence: 53%

“…R246 has, in fact, a relevant role in maintaining LS activity (22), which is most likely due to its participation in coordinating donor substrates at subsite -1. On the other hand, many reports have already highlighted the participation of N242 in the polymerization reaction of gram-positive LSs, and its mutation to alanine, histidine, tyrosine, or tryptophan invariably limited the products to tetrasaccharides (12,17,19,33). Considering that the SacB mutant N242A also showed an increased Km, its deleterious effect may be related to the impaired coordination of sucrose as an acceptor molecule, thereby greatly affecting the formation of both HMW and LMW levans at the very beginning of the elongation process.…”

Section: Insights Into the Ob1 Subsites +1 And +2mentioning

confidence: 99%

“…Furthermore, residues N242 and Y237 were identified as part of the +2 subsite, as they are responsible for only a few water-mediated interactions with the galactosyl unit from raffinose (15). Site-directed mutagenesis studies on LSs have revealed that modifications of residues N242, K363, and Y237 (numbering of B. subtilis SacB), which are located on the surface of the catalytic cavity, affect the enzyme catalytic efficiency, transfructosylation and hydrolysis ratio by interrupting the polymerization process, and thus, these residues control the chain lengths of levans (12,(16)(17)(18)(19). However, the interactions mediated by these residues have not yet been identified, and the existence of additional external acceptor binding subsites acting as structural determinants in the elongation of levans has not yet been clarified.…”

Section: Introductionmentioning

confidence: 99%

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The molecular basis of the nonprocessive elongation mechanism in levansucrases

Raga-Carbajal

Diaz-Vilchis

Rojas-Trejo

et al. 2021

Journal of Biological Chemistry

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Levansucrases (LSs) synthesize levan, a β2-6-linked fructose polymer, by successively transferring the fructosyl moiety from sucrose to a growing acceptor molecule. Elucidation of the levan polymerization mechanism is important for using LSs in the production of size-defined products for application in the food and pharmaceutical industries. For a deeper understanding of the levan synthesis reaction, we determined the crystallographic structure of Bacillus subtilis LS (SacB) in complex with a levan-type fructooligosaccharide and utilized site-directed mutagenesis to identify residues involved in substrate binding. The presence of a levanhexaose molecule in the central catalytic cavity allowed us to identify five substrate-binding subsites (−1, +1, +2, +3, and +4). Mutants affecting residues belonging to the identified acceptor subsites showed similar substrate affinity ( K m) values to the wildtype (WT) K m value but had a lower turnover number and transfructosylation/hydrolysis ratio. Of importance, compared with the WT, the variants progressively yielded smaller-sized low-molecular-weight levans, as the affected subsites that were closer to the catalytic site, but without affecting their ability to synthesized high-molecular-weight levans. Furthermore, an additional oligosaccharide-binding site 20 Å away from the catalytic pocket was identified, and its potential participation in the elongation mechanism is discussed. Our results clarify, for the first time, the interaction of the enzyme with an acceptor/product oligosaccharide and elucidate the molecular basis of the nonprocessive levan elongation mechanism of LSs.

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Section: Insights Into the Ob1 Subsites +1 And +2supporting

confidence: 53%

Section: Insights Into the Ob1 Subsites +1 And +2mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The molecular basis of the nonprocessive elongation mechanism in levansucrases

Raga-Carbajal

Diaz-Vilchis

Rojas-Trejo

et al. 2021

Journal of Biological Chemistry

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“…Previous study found that blocking the oligosaccharide binding track of levansucrase with aromatic residues (F, Y and W) was an effective strategy to block elongation of polysaccharide and increase the yields of oligosaccharides. 18 Therefore, we employed this approach in redesigning LrInu so that it could produce high yields of short to medium chain length oligosaccharides. Because R483 of LrInu is located next to N543, we hypothesized that blocking at this position might increase the yield of short FOSs like that obtained from the N543A variant.…”

Section: Rational Protein Designmentioning

confidence: 99%

Rational re-design of Lactobacillus reuteri 121 inulosucrase for product chain length control

et al. 2019

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“…Researchers have not reported on the levansucrase from B. velezensis in detail, including its gene information and enzymatic properties. Different microbial sources play distinct roles in the formation and activity of levansucrase, which could affect the size, branches, and biological activity of the polysaccharide [23][24][25]. To meet the needs of levansucrase research and its ultimate application, it is necessary to screen more microbial resources.…”

Section: Introductionmentioning

confidence: 99%

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

Zhang

Zhao

et al. 2021

Processes

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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.

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Computational design of Bacillus licheniformis RN-01 levansucrase for control of the chain length of levan-type fructooligosaccharides

Cited by 26 publications

References 44 publications

The molecular basis of the nonprocessive elongation mechanism in levansucrases

The molecular basis of the nonprocessive elongation mechanism in levansucrases

Rational re-design of Lactobacillus reuteri 121 inulosucrase for product chain length control

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

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