Isolation and properties of β-xylosidase from Aspergillus niger GS1 using corn pericarp upon solid state fermentation

Díaz-Malváez, F.I.; García-Almendárez, Blanca E.; Hernández‐Arana, Andrés; Amaro-Reyes, Aldo; Regalado‐González, Carlos

doi:10.1016/j.procbio.2013.05.003

Cited by 23 publications

(8 citation statements)

References 43 publications

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“…Similarities in CD data were confirmed by deconvolution analysis, which found the same α-helix and β-strand rates (Additional file 1: Table S3). The deconvolution data were highly similar to what was found in A. niger β-xylosidase composed of 41% α-helix and 16% β-sheet; however, there was not as much similarity to the T. reesei β-xylosidase which presented 23% α-helix and 27% β-sheet composition [32, 33].…”

Section: Discussionsupporting

confidence: 67%

Redesigning N-glycosylation sites in a GH3 β-xylosidase improves the enzymatic efficiency

Rubio

Terrasan

Contesini

et al. 2019

Biotechnol Biofuels

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Backgroundβ-Xylosidases are glycoside hydrolases (GHs) that cleave xylooligosaccharides and/or xylobiose into shorter oligosaccharides and xylose. Aspergillus nidulans is an established genetic model and good source of carbohydrate-active enzymes (CAZymes). Most fungal enzymes are N-glycosylated, which influences their secretion, stability, activity, signalization, and protease protection. A greater understanding of the N-glycosylation process would contribute to better address the current bottlenecks in obtaining high secretion yields of fungal proteins for industrial applications.ResultsIn this study, BxlB—a highly secreted GH3 β-xylosidase from A. nidulans, presenting high activity and several N-glycosylation sites—was selected for N-glycosylation engineering. Several glycomutants were designed to investigate the influence of N-glycans on BxlB secretion and function. The non-glycosylated mutant (BxlBnon-glyc) showed similar levels of enzyme secretion and activity compared to the wild-type (BxlBwt), while a partially glycosylated mutant (BxlBN1;5;7) exhibited increased activity. Additionally, there was no enzyme secretion in the mutant in which the N-glycosylation context was changed by the introduction of four new N-glycosylation sites (BxlBCC), despite the high transcript levels. BxlBwt, BxlBnon-glyc, and BxlBN1;5;7 formed similar secondary structures, though the mutants had lower melting temperatures compared to the wild type. Six additional glycomutants were designed based on BxlBN1;5;7, to better understand its increased activity. Among them, the two glycomutants which maintained only two N-glycosylation sites each (BxlBN1;5 and BxlBN5;7) showed improved catalytic efficiency, whereas the other four mutants’ catalytic efficiencies were reduced. The N-glycosylation site N5 is important for improved BxlB catalytic efficiency, but needs to be complemented by N1 and/or N7. Molecular dynamics simulations of BxlBnon-glyc and BxlBN1;5 reveals that the mobility pattern of structural elements in the vicinity of the catalytic pocket changes upon N1 and N5 N-glycosylation sites, enhancing substrate binding properties which may underlie the observed differences in catalytic efficiency between BxlBnon-glyc and BxlBN1;5.ConclusionsThis study demonstrates the influence of N-glycosylation on A. nidulans BxlB production and function, reinforcing that protein glycoengineering is a promising tool for enhancing thermal stability, secretion, and enzymatic activity. Our report may also support biotechnological applications for N-glycosylation modification of other CAZymes.

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Section: Discussionsupporting

confidence: 67%

Redesigning N-glycosylation sites in a GH3 β-xylosidase improves the enzymatic efficiency

Rubio

Terrasan

Contesini

et al. 2019

Biotechnol Biofuels

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“…Scanning electron microscopy (Jeol JSM 6390 model) was used to examine the morphological modifications of CPW and CC before and after the alkaline pre-treatment according to Díaz-Malváez et al ( 2013 ) Samples were dehydrated and mounted on stubs and sputter-coated with gold for 300 s using high vacuum and a voltage acceleration of 10 kV.…”

Section: S Rrna Amplification and Partial Gene Sequencing Of The Bamentioning

confidence: 99%

Optimization of protease production from surface-modified coffee pulp waste and corncobs using Bacillus sp. by SSF

Selvam¹,

Govarthanan

Balakrishnan

et al. 2016

3 Biotech

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The aim of the study was to identify new sources of substrate from agro-industrial waste for protease production using Bacillus sp., a local bacteria isolated from an agro-waste dumping site. The strain was identified as Bacillus sp. BT MASC 3 by 16S rRNA sequence followed by phylogenic analysis. Response surface methodology-based Box–Behnken design (BBD) was used to optimize the variables such as pH, incubation time, coffee pulp waste (CPW) and corncob (CC) substrate concentration. The BBD design showed a reasonable adjustment of the quadratic model with the experimental data. Statistics-based contour and 3-D plots were generated to evaluate the changes in the response surface and understand the relationship between the culture conditions and the enzyme yield. The maximum yield of protease production (920 U/mL) was achieved after 60 h of incubation with 3.0 g/L of CPW and 2.0 g/L of CC at pH 8 and temperature 37 °C in this study. The molecular mass of the purified enzyme was 46 kDa. The highest activity was obtained at 50 °C and pH 9 for the purified enzymes.

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“…Probably, BXYL II is homotrimeric, with three 67 kDa subunits, as verified by the presence of a more intense band. The MW of BXYL II is similar to many fungal β-xylosidases which usually present MW above 100 kDa [29][30][31][32][33] but present MW higher than that of the BXYL I, whose native MW is 200 kDa [19].…”

Section: Characterization Of the Minor β-Xylosidase From P Janczewskiimentioning

confidence: 78%

Immobilization and Stabilization of Beta-Xylosidases from Penicillium janczewskii

Terrasan

Romero‐Fernández

Orrego

et al. 2016

Appl Biochem Biotechnol

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β-Xylosidases are critical for complete degradation of xylan, the second main constituent of plant cell walls. A minor β-xylosidase (BXYL II) from Penicillium janczewskii was purified by ammonium sulfate precipitation (30% saturation) followed by DEAE-Sephadex chromatography in pH 6.5 and elution with KCl. The enzyme presented molecular weight (MW) of 301 kDa estimated by size exclusion chromatography. Optimal activity was observed in pH 3.0 and 70-75 °C, with higher stability in pH 3.0-4.5 and half-lives of 11, 5, and 2 min at 65, 70, and 75 °C, respectively. Inhibition was moderate with Pb and citrate and total with Cu, Hg, and Co. Partially purified BXYL II and BXYL I (the main β-xylosidase from this fungus) were individually immobilized and stabilized in glyoxyl agarose gels. At 65 °C, immobilized BXYL I and BXYL II presented half-lives of 4.9 and 23.1 h, respectively, therefore being 12.3-fold and 33-fold more stable than their unipuntual CNBr derivatives (reference mimicking soluble enzyme behaviors). During long-term incubation in pH 5.0 at 50 °C, BXYL I and BXYL II glyoxyl derivatives preserved 85 and 35% activity after 25 and 7 days, respectively. Immobilized BXYL I retained 70% activity after 10 reuse cycles of p-nitrophenyl-β-D-xylopyranoside hydrolysis.

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Isolation and properties of β-xylosidase from Aspergillus niger GS1 using corn pericarp upon solid state fermentation

Cited by 23 publications

References 43 publications

Redesigning N-glycosylation sites in a GH3 β-xylosidase improves the enzymatic efficiency

Redesigning N-glycosylation sites in a GH3 β-xylosidase improves the enzymatic efficiency

Optimization of protease production from surface-modified coffee pulp waste and corncobs using Bacillus sp. by SSF

Immobilization and Stabilization of Beta-Xylosidases from Penicillium janczewskii

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