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Evaluation of cross-linked aggregates from purified Bacillus subtilis levansucrase mutants for transfructosylation reactions.

Ortiz-Soto ME, Rudiño-Piñera E, Rodriguez-Alegria ME, Munguia AL - BMC Biotechnol. (2009)

Bottom Line: CLEAs were able to catalyze the synthesis of fructosides as efficiently as soluble enzymes.In the long term, the operational stability was affected by levan synthesis.CLEAs prepared from purified LS and mutants have the highest specific activity for immobilized fructosyltransferases (FTFs) reported in the literature.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, UNAM, Cuernavaca, Morelos, 62210, México. marisoto@ibt.unam.mx

ABSTRACT

Background: Increasing attention has been focused on inulin and levan-type oligosaccharides, including fructosyl-xylosides and other fructosides due to their nutraceutical properties. Bacillus subtilis levansucrase (LS) catalyzes the synthesis of levan from sucrose, but it may also transfer the fructosyl moiety from sucrose to acceptor molecules included in the reaction medium. To study transfructosylation reactions with highly active and robust derivatives, cross-linked enzyme aggregates (CLEAs) were prepared from wild LS and two mutants. CLEAs combine the catalytic features of pure protein preparations in terms of specific activity with the mechanical behavior of industrial biocatalysts.

Results: Two types of procedures were used for the preparation of biocatalysts from purified wild type LS (WT LS) B. subtilis and the R360K and Y429N LS mutants: purified enzymes aggregated with glutaraldehyde (cross-linked enzyme aggregates: CLEAs), and covalently immobilized enzymes in Eupergit C. The biocatalysts were characterized and used for fructoside synthesis using xylose as an acceptor model. CLEAs were able to catalyze the synthesis of fructosides as efficiently as soluble enzymes. The specific activity of CLEAs prepared from wild type LS (44.9 U/mg of CLEA), R360K (56.5 U/mg of CLEA) and Y429N (1.2 U/mg of CLEA) mutants were approximately 70, 40 and 200-fold higher, respectively, than equivalent Eupergit C immobilized enzyme preparations (U/mg of Eupergit), where units refer to global LS activity. In contrast, the specific activity of the free enzymes was 160, 171.2 and 1.5 U/mg of protein, respectively. Moreover, all CLEAs had higher thermal stability than corresponding soluble enzymes. In the long term, the operational stability was affected by levan synthesis.

Conclusion: This is the first report of cross-linked transglycosidases aggregates. CLEAs prepared from purified LS and mutants have the highest specific activity for immobilized fructosyltransferases (FTFs) reported in the literature. CLEAs from R360K and Y429N LS mutants were particularly suitable for fructosyl-xyloside synthesis as the absence of levan synthesis decreases diffusion limitation and increases operational stability.

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Images of B. subtilis levansucrase R360K mutant CLEAs. A) CLEAs images obtained immediately after quenching the cross-linking reaction; B) CLEAs washed and centrifuged after cross-linking reaction and C) CLEAs after 48 h of quenching; in this last case, CLEAS were not washed. Digital images were collected in phase contrast mode with a 40×, NA 0.75, Ph 2 objective.
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Figure 2: Images of B. subtilis levansucrase R360K mutant CLEAs. A) CLEAs images obtained immediately after quenching the cross-linking reaction; B) CLEAs washed and centrifuged after cross-linking reaction and C) CLEAs after 48 h of quenching; in this last case, CLEAS were not washed. Digital images were collected in phase contrast mode with a 40×, NA 0.75, Ph 2 objective.

Mentions: In terms of particle size, the preparation procedure plays an important role, as shown in Figure 2. Indeed, although aggregates have an average size of 15 μm, they tend to form larger aggregates when centrifuged or when residual glutaraldehyde is not washed off.


Evaluation of cross-linked aggregates from purified Bacillus subtilis levansucrase mutants for transfructosylation reactions.

Ortiz-Soto ME, Rudiño-Piñera E, Rodriguez-Alegria ME, Munguia AL - BMC Biotechnol. (2009)

Images of B. subtilis levansucrase R360K mutant CLEAs. A) CLEAs images obtained immediately after quenching the cross-linking reaction; B) CLEAs washed and centrifuged after cross-linking reaction and C) CLEAs after 48 h of quenching; in this last case, CLEAS were not washed. Digital images were collected in phase contrast mode with a 40×, NA 0.75, Ph 2 objective.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2724506&req=5

Figure 2: Images of B. subtilis levansucrase R360K mutant CLEAs. A) CLEAs images obtained immediately after quenching the cross-linking reaction; B) CLEAs washed and centrifuged after cross-linking reaction and C) CLEAs after 48 h of quenching; in this last case, CLEAS were not washed. Digital images were collected in phase contrast mode with a 40×, NA 0.75, Ph 2 objective.
Mentions: In terms of particle size, the preparation procedure plays an important role, as shown in Figure 2. Indeed, although aggregates have an average size of 15 μm, they tend to form larger aggregates when centrifuged or when residual glutaraldehyde is not washed off.

Bottom Line: CLEAs were able to catalyze the synthesis of fructosides as efficiently as soluble enzymes.In the long term, the operational stability was affected by levan synthesis.CLEAs prepared from purified LS and mutants have the highest specific activity for immobilized fructosyltransferases (FTFs) reported in the literature.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, UNAM, Cuernavaca, Morelos, 62210, México. marisoto@ibt.unam.mx

ABSTRACT

Background: Increasing attention has been focused on inulin and levan-type oligosaccharides, including fructosyl-xylosides and other fructosides due to their nutraceutical properties. Bacillus subtilis levansucrase (LS) catalyzes the synthesis of levan from sucrose, but it may also transfer the fructosyl moiety from sucrose to acceptor molecules included in the reaction medium. To study transfructosylation reactions with highly active and robust derivatives, cross-linked enzyme aggregates (CLEAs) were prepared from wild LS and two mutants. CLEAs combine the catalytic features of pure protein preparations in terms of specific activity with the mechanical behavior of industrial biocatalysts.

Results: Two types of procedures were used for the preparation of biocatalysts from purified wild type LS (WT LS) B. subtilis and the R360K and Y429N LS mutants: purified enzymes aggregated with glutaraldehyde (cross-linked enzyme aggregates: CLEAs), and covalently immobilized enzymes in Eupergit C. The biocatalysts were characterized and used for fructoside synthesis using xylose as an acceptor model. CLEAs were able to catalyze the synthesis of fructosides as efficiently as soluble enzymes. The specific activity of CLEAs prepared from wild type LS (44.9 U/mg of CLEA), R360K (56.5 U/mg of CLEA) and Y429N (1.2 U/mg of CLEA) mutants were approximately 70, 40 and 200-fold higher, respectively, than equivalent Eupergit C immobilized enzyme preparations (U/mg of Eupergit), where units refer to global LS activity. In contrast, the specific activity of the free enzymes was 160, 171.2 and 1.5 U/mg of protein, respectively. Moreover, all CLEAs had higher thermal stability than corresponding soluble enzymes. In the long term, the operational stability was affected by levan synthesis.

Conclusion: This is the first report of cross-linked transglycosidases aggregates. CLEAs prepared from purified LS and mutants have the highest specific activity for immobilized fructosyltransferases (FTFs) reported in the literature. CLEAs from R360K and Y429N LS mutants were particularly suitable for fructosyl-xyloside synthesis as the absence of levan synthesis decreases diffusion limitation and increases operational stability.

Show MeSH
Related in: MedlinePlus