Engineering a thermostable Halothermothrix orenii β-glucosidase for improved galacto-oligosaccharide synthesis

Hassan, Noor; Geiger, Barbara; Gandini, Rosaria; Patel, Bharat K. C.; Kittl, Roman; Haltrich, Dietmar; Nguyễn, Thu Hà; Divne, Christina; Tan, T.C.

doi:10.1007/s00253-015-7118-8

Cited by 29 publications

(23 citation statements)

References 38 publications

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“…Thermophilic fungi are well-known to produce abundant plant cell wall-degradating hydrolases with superior characters, such as great catalytic performance, high expression level and excellent stability [ 16 , 20 , 21 , 24 , 49 – 51 ]. In decades, studies have been focused on heterologous expression of fungal β-glucosidases in P. pastoris [ 52 – 55 ] and engineering of β-glucosidases for property improvement [ 31 , 56 , 57 ]. In this study, a novel GH3 β-glucosidase, Bgl3A, was identified from thermophilic T. leycettanus JCM12802, and expressed in P. pastoris GS115 at high level.…”

Section: Discussionmentioning

confidence: 99%

Engineering a highly active thermophilic β-glucosidase to enhance its pH stability and saccharification performance

Xia

Qian

et al. 2016

Biotechnol Biofuels

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Backgroundβ-Glucosidase is an important member of the biomass-degrading enzyme system, and plays vital roles in enzymatic saccharification for biofuels production. Candidates with high activity and great stability over high temperature and varied pHs are always preferred in industrial practice. To achieve cost-effective biomass conversion, exploring natural enzymes, developing high level expression systems and engineering superior mutants are effective approaches commonly used.ResultsA newly identified β-glucosidase of GH3, Bgl3A, from Talaromyces leycettanus JCM12802, was overexpressed in yeast strain Pichia pastoris GS115, yielding a crude enzyme activity of 6000 U/ml in a 3 L fermentation tank. The purified enzyme exhibited outstanding enzymatic properties, including favorable temperature and pH optima (75 °C and pH 4.5), good thermostability (maintaining stable at 60 °C), and high catalytic performance (with a specific activity and catalytic efficiency of 905 U/mg and 9096/s/mM on pNPG, respectively). However, the narrow stability of Bgl3A at pH 4.0–5.0 would limit its industrial applications. Further site-directed mutagenesis indicated the role of excessive O-glycosylation in pH liability. By removing the potential O-glycosylation sites, two mutants showed improved pH stability over a broader pH range (3.0–10.0). Besides, with better stability under pH 5.0 and 50 °C compared with wild type Bgl3A, saccharification efficiency of mutant M1 was improved substantially cooperating with cellulase Celluclast 1.5L. And mutant M1 reached approximately equivalent saccharification performance to commercial β-glucosidase Novozyme 188 with identical β-glucosidase activity, suggesting its great prospect in biofuels production.ConclusionsIn this study, we overexpressed a novel β-glucosidase Bgl3A with high specific activity and high catalytic efficiency in P. pastoris. We further proved the negative effect of excessive O-glycosylation on the pH stability of Bgl3A, and enhanced the pH stability by reducing the O-glycosylation. And the enhanced mutants showed much better application prospect with substantially improved saccharification efficiency on cellulosic materials.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0560-8) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Engineering a highly active thermophilic β-glucosidase to enhance its pH stability and saccharification performance

Xia

Qian

et al. 2016

Biotechnol Biofuels

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“…GOS are also categorized as functional food ingredients and useful dietary fibers presenting a great interest in the field of enzyme reaction engineering and industrial interest in food related biotechnology 3 . Several commercial and non-commercial β-glucosidase (β-Glu) extracted from different sources (including microbial sources) have been evaluated under different temperature conditions for their ability and efficiency to produce GOS 4 .…”

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confidence: 99%

Immobilization of β-Glucosidase from Thermatoga maritima on Chitin-functionalized Magnetic Nanoparticle via a Novel Thermostable Chitin-binding Domain

Alnadari

Xue

Zhou

et al. 2020

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Enzyme immobilization is a powerful tool not only as a protective agent against harsh reaction conditions but also for the enhancement of enzyme activity, stability, reusability, and for the improvement of enzyme properties as well. Herein, immobilization of β-glucosidase from Thermotoga maritima (tm-β-Glu) on magnetic nanoparticles (Mnps) functionalized with chitin (ch) was investigated. This technology showed a novel thermostable chitin-binding domain (Tt-ChBD), which is more desirable in a wide range of large-scale applications. This exclusive approach was fabricated to improve the Galacto-oligosaccharide (GOS) production from a cheap and abundant by-product such as lactose through a novel green synthesis route. Additionally, SDS-PAGE, enzyme activity kinetics, transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FT-IR) revealed that among the immobilization strategies for Thermotoga maritime-β-Glucosidase thermostable chitinbinding domain (tm-β-Glu-Tt-ChBD) on the attractive substrate; Ch-MNPs had the highest enzyme binding capacity and GOS production ratio when compared to the native enzyme. More interestingly, a magnetic separation technique was successfully employed in recycling the immobilized Tm-β-Glu for repetitive batch-wise GOS without significant loss or reduction of enzyme activity. This immobilization system displayed an operative stability status under various parameters, for instance, temperature, pH, thermal conditions, storage stabilities, and enzyme kinetics when compared with the native enzyme. Conclusively, the GOS yield and residual activity of the immobilized enzyme after the 10 th cycles were 31.23% and 66%, respectively. Whereas the GOS yield from native enzyme synthesis was just 25% after 12 h in the first batch. This study recommends applying Tt-ChBD in the immobilization process of tm-β-Glu on Ch-MNPs to produce a low-cost GOS as a new eco-friendly process besides increasing the biostability and efficiency of the immobilized enzyme.

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“…Because ORF1119 protein exhibited hydrolase activity toward both p NP-β-D-PvGal and p NP-β-D-Gal, we investigated which amino acid residues are responsible for the recognition of these substrates. The crystal structure of ORF1119 was determined by molecular replacement using the structure of Halothermothrix orenii BGL 28 , 29 (HoBGL, PDB code 3TA9) as a search model. Structures of the ligand-free form and PvGal-bound form were determined at 2.5 Å and 2.45 Å resolution, respectively (Table 3 ).…”

Section: Resultsmentioning

confidence: 99%

“…W420 forms a stacking interaction with the sugar ring. Figure 7B,C show the active site structures of two GH1 enzymes: 6-phospho-β-glucosidase/galactosidase Gan1D from Geobacillus stearothermophilus 30 (PDB code 4ZEN, sequence identity 44.6%), and β-glucosidase HoBGL from Halothermothrix orenii 28 , 29 (PDB code 4PTX, sequence identity 39.1%). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Identification and characterization of a novel β-D-galactosidase that releases pyruvylated galactose

Higuchi

Matsufuji

Tanuma

et al. 2018

Sci Rep

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Pyruvyl modification of oligosaccharides is widely seen in both prokaryotes and eukaryotes. Although the biosynthetic mechanisms of pyruvylation have been investigated, enzymes that metabolize and degrade pyruvylated oligosaccharides are not well known. Here, we searched for a pyruvylated galactose (PvGal)-releasing enzyme by screening soil samples. We identified a Bacillus strain, as confirmed by the 16S ribosomal RNA gene analysis, that exhibited PvGal-ase activity toward p-nitrophenyl-β-D-pyruvylated galactopyranose (pNP-β-D-PvGal). Draft genome sequencing of this strain, named HMA207, identified three candidate genes encoding potential PvGal-ases, among which only the recombinant protein encoded by ORF1119 exhibited PvGal-ase activity. Although ORF1119 protein displayed broad substrate specificity for pNP sugars, pNP-β-D-PvGal was the most favorable substrate. The optimum pH for the ORF1119 PvGal-ase was determined as 7.5. A BLAST search suggested that ORF1119 homologs exist widely in bacteria. Among two homologs tested, BglC from Clostridium but not BglH from Bacillus showed PvGal-ase activity. Crystal structural analysis together with point mutation analysis revealed crucial amino acids for PvGal-ase activity. Moreover, ORF1119 protein catalyzed the hydrolysis of PvGal from galactomannan of Schizosaccharomyces pombe, suggesting that natural polysaccharides might be substrates of the PvGal-ase. This novel PvGal-catalyzing enzyme might be useful for glycoengineering projects to produce new oligosaccharide structures.

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Engineering a thermostable Halothermothrix orenii β-glucosidase for improved galacto-oligosaccharide synthesis

Cited by 29 publications

References 38 publications

Engineering a highly active thermophilic β-glucosidase to enhance its pH stability and saccharification performance

Engineering a highly active thermophilic β-glucosidase to enhance its pH stability and saccharification performance

Immobilization of β-Glucosidase from Thermatoga maritima on Chitin-functionalized Magnetic Nanoparticle via a Novel Thermostable Chitin-binding Domain

Identification and characterization of a novel β-D-galactosidase that releases pyruvylated galactose

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