Low-resolution structure, oligomerization and its role on the enzymatic activity of a sucrose-6-phosphate hydrolase from Bacillus licheniformis

Mera, Alain; Lima, Mariana Zuliani Theodoro de; Bernardes, Amanda; Garcia, Wânius; Muniz, J.R.C.

doi:10.1007/s00726-018-02690-2

Cited by 4 publications

(9 citation statements)

References 51 publications

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“…The R S value determined for Lg As32 at pH 6 was 4.5 ± 0.3 nm, which corresponds to a molecular mass of 114 ± 18 kDa, consistent with the expected molecular mass of a homodimer in solution (∼55 kDa for a monomer). The homodimer oligomerization state has been previously described in other members of this GH32 family and is strictly related to its activity and stability under a wide range of pH conditions …”

Section: Resultsmentioning

confidence: 85%

“…The homodimer oligomerization state has been previously described in other members of this GH32 family and is strictly related to its activity and stability under a wide range of pH conditions. 16 3.3. Crystal Structure of LgAs32.…”

Section: Resultsmentioning

confidence: 99%

“…gasseri (DSM 20243/ATCC 33323/LGAS_1779 gene) belonging to the GH32 family (here named Lg As32). As previously reported by our research group, this class of enzymes can be found in many organisms . Crystal structures of apo-Lg As32 and Lg As32 in complex with β- d -fructose were determined at high resolution.…”

Section: Introductionmentioning

confidence: 82%

“…In this study, we report for the first time the cloning, expression, purification, crystallization, and crystal structure determination of a recombinant sucrose-6-phosphate hydrolase (β-fructofuranosidase, E.C. 3.2.1.26) 16 from L. gasseri (DSM 20243/ATCC 33323/LGAS_1779 gene) belonging to the GH32 family (here named LgAs32). As previously reported by our research group, this class of enzymes can be found in many organisms.…”

Section: Introductionmentioning

confidence: 99%

“…As previously reported by our research group, this class of enzymes can be found in many organisms. 16 Crystal structures of apo-LgAs32 and LgAs32 in complex with β-D-fructose were determined at high resolution. LgAs32 showed hydrolytic activity in the presence of sucrose and the production of fructooligosaccharides nystose and 1kestose, as shown by HPAEC/PAD and spectrometry assays by transfructosylation activity.…”

Section: Introductionmentioning

confidence: 99%

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Crystal Structure of a Sucrose-6-phosphate Hydrolase from Lactobacillus gasseri with Potential Applications in Fructan Production and the Food Industry

Lima

Almeida

Mera

et al. 2021

J. Agric. Food Chem.

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Fructooligosaccharides (FOSs) are polymers of fructose with a prebiotic activity because of their production and fermentation by bacteria that inhabit the gastrointestinal tract and are widely used in the industry and new functional foods. Lactobacillus gasseri stands out as an important homofermentative microorganism related to FOS production, and its potential applications in the industry are undeniable. In this study, we report the production and characterization of a sucrose-6-phosphate hydrolase from L. gasseri belonging to the GH32 family. Apo-LgAs32 and LgAs32 complexed with β-d-fructose structures were determined at a resolution of 1.94 and 1.84 Å, respectively. The production of FOS, fructans, 1-kestose, and nystose by the recombinant LgAs32, using sucrose as a substrate, shown in this study is very promising. When compared to its homologous enzyme from Lactobacillus reuteri, the production of 1-kestose by LgAs32 is increased; thus, LgAs32 can be considered as an alternative in fructan production and other industrial applications.

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Section: Resultsmentioning

confidence: 85%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 82%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Crystal Structure of a Sucrose-6-phosphate Hydrolase from Lactobacillus gasseri with Potential Applications in Fructan Production and the Food Industry

Lima

Almeida

Mera

et al. 2021

J. Agric. Food Chem.

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Structure-based design of a soluble human cytomegalovirus glycoprotein B antigen stabilized in a prefusion-like conformation

Sponholtz,

Byrne,

Lee

et al. 2024

Preprint

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Human cytomegalovirus (HCMV) glycoprotein B (gB) is a class III membrane fusion protein required for viral entry. HCMV vaccine candidates containing gB have demonstrated moderate clinical efficacy, but no HCMV vaccine has been FDA-approved. Here, we used structure-based design to identify and characterize amino acid substitutions that stabilize gB in its metastable prefusion conformation. One variant containing two engineered interprotomer disulfide bonds and two cavity-filling substitutions (gB-C7), displayed increased expression and thermostability. A 2.8 Å resolution cryo-electron microscopy structure shows that gB-C7 adopts a prefusion-like conformation, revealing additional structural elements at the membrane-distal apex. Unlike previous observations for several class I viral fusion proteins, mice immunized with postfusion or prefusion-stabilized forms of soluble gB protein displayed similar neutralizing antibody titers, here specifically against an HCMV laboratory strain on fibroblasts. Collectively, these results identify initial strategies to stabilize class III viral fusion proteins and provide tools to probe gB-directed antibody responses.TeaserA structure-based design campaign leads to stabilization of the class III viral fusion protein from HCMV in a prefusion-like conformation.

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Heterologous Expression of Inulinase Gene in Bacillus licheniformis 24 for 2,3-Butanediol Production from Inulin

et al. 2023

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Inulin is a renewable and cheap carbon source used in microbial fermentations. Bacillus licheniformis 24 is known as an excellent 2,3-butanediol (2,3-BD) producer from fructose; therefore, the cloning and expression of a robust heterologous inulinase could enhance its 2,3-BD production from inulin. The inu gene of Lacticaseibacillus paracasei DSM 23505 encoding fructan-β-fructosidase (EC 3.2.1.80) was chosen for the purpose. PCR fragments containing the complete inu (3.6 kb) and its truncated variant inu-tr (2.2 kb, lacking Big3 cell wall attachment domains) were cloned into Escherichia coli StellarTM and B. licheniformis 24. The high quality of the recombinant constructs was confirmed by restriction analysis, PCR, sequencing, and phenotypic tests. The results showed that the inulinase activity of B. licheniformis cells harboring the full-length inu variant (T26) was eightfold higher compared to the wild type, retaining cell wall attachment in the B. licheniformis host. In contrast, the truncated variant inu-tr (T14) showed mostly extracellular but weak activity, thus suggesting that the Big3 domains are also important for the enzyme’s function. During flask-batch fermentation of 100 g/L raw chicory flour (containing 90% inulin), T26 produced acetoin and 2,3-BD from inulin. Contrariwise, T14 and the wild type formed products only from the mono- and disaccharides naturally found in the chicory flour. In the fermenter, from 200 g/L of raw chicory flour, the recombinant T26 degraded approximately 140 g/L of the inulin. However, the final concentrations of the produced 2,3-BD and acetoin were 18.5 g/L and 8.2 g/L, respectively, because of the accumulation of unconverted sucrose. To conclude, further strain improvement is necessary to make the process efficient for obtaining 2,3-BD from inulin by simultaneous saccharification and fermentation (SSF).

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Low-resolution structure, oligomerization and its role on the enzymatic activity of a sucrose-6-phosphate hydrolase from Bacillus licheniformis

Cited by 4 publications

References 51 publications

Crystal Structure of a Sucrose-6-phosphate Hydrolase from Lactobacillus gasseri with Potential Applications in Fructan Production and the Food Industry

Crystal Structure of a Sucrose-6-phosphate Hydrolase from Lactobacillus gasseri with Potential Applications in Fructan Production and the Food Industry

Structure-based design of a soluble human cytomegalovirus glycoprotein B antigen stabilized in a prefusion-like conformation

Heterologous Expression of Inulinase Gene in Bacillus licheniformis 24 for 2,3-Butanediol Production from Inulin

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