Gene cloning, expression and biochemical characterization of a glucose- and xylose-stimulated β-glucosidase from Humicola insolens RP86

Souza, Flávio Henrique Moreira; Meleiro, Luana Parras; Machado, Carla Botelho; Zimbardi, Ana Lucia Ribeiro Latorre; Maldonado, Raquel Fonseca; Souza, T.A.C.B.; Masui, Douglas Chodi; Murakami, Mário Tyago; Jorge, João Atı́lio; Ward, Richard J.; Furriel, Rosa dos Prazeres Melo

doi:10.1016/j.molcatb.2014.04.007

Cited by 34 publications

(26 citation statements)

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“…Another kinetic peculiarity often observed with glucose tolerant BGs is the activation of enzyme at lower but inhibition at higher inhibitor concentrations. This phenomenon has been often reported with the inhibition of BGs by glucose [11–36], and xylose [13, 15, 19, 21, 22, 28–31, 37], but also by other sugars [19, 28, 31]. With some of these BGs, the Michaelis–Menten saturation kinetics holds [19, 21, 26, 31], with others, it does not [32, 38].…”

Section: Introductionmentioning

confidence: 82%

When substrate inhibits and inhibitor activates: implications of β-glucosidases

Kuusk

Väljamaë

2017

Biotechnol Biofuels

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Backgroundβ-glucosidases (BGs) catalyze the hydrolysis of β-glycosidic bonds in glucose derivatives. They constitute an important group of enzymes with biotechnological interest like supporting cellulases in degradation of lignocellulose to fermentable sugars. In the latter context, the glucose tolerant BGs are of particular interest. These BGs often show peculiar kinetics, including inhibitory effects of substrates and activating effects of inhibitors, such as glucose or xylose. The mechanisms behind the activating/inhibiting effects are poorly understood. The nonproductive binding of substrate is expected in cases where enzymes with multiple consecutive binding subsites are studied on substrates with a low degree of polymerization. The effects of inhibitors to BGs exerting nonproductive binding of substrate have not been discussed in the literature before.ResultsHere, we performed analyses of different reaction schemes using the catalysis by retaining BGs as a model. We found that simple competition of inhibitor with nonproductive binding of substrate can account for the activation of enzyme by inhibitor without involving any allosteric effects. The transglycosylation to inhibitor was also able to explain the activating effect of inhibitor. For both mechanisms, the activation was caused by the increase of k cat with increasing inhibitor concentration, while k cat/K m always decreased. Therefore, the activation by inhibitor was more pronounced at high substrate concentrations. The possible contribution of the two mechanisms in the activation by inhibitor was dependent on the rate-limiting step of glycosidic bond hydrolysis as well as on whether and which glucose-unit-binding subsites are interacting.ConclusionKnowledge on the mechanisms of the activating/inhibiting effects of inhibitors helps the rational engineering and selection of BGs for biotechnological applications. Provided that the catalysis is consistent with the reaction schemes addressed here and underlying assumptions, the mechanism of activation by inhibitor reported here is applicable for all enzymes exerting nonproductive binding of substrate.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0690-z) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 82%

When substrate inhibits and inhibitor activates: implications of β-glucosidases

Kuusk

Väljamaë

2017

Biotechnol Biofuels

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“…The K M for pNPFuc was higher (0.152 mM) than that for pNPGlc, but the V max (137 μmol min -1 mg -1 ) was also higher for pNPFuc, resulting in similar overall catalytic efficiency (k cat /K M ) for the two substrates. Compared with other known glucose-tolerant BGLs (Pérez-Pons et al, 1995; Saha and Bothast, 1996; Yan and Lin, 1997; Riou et al, 1998; Decker et al, 2001; Zanoelo et al, 2004; Fang et al, 2010; Uchima et al, 2011; Uchiyama et al, 2013; Biver et al, 2014; Souza et al, 2014), the K M of Ks5A7 for pNPGlc was the lowest (0.078 mM) and the V max was relatively high (90.8 μmol min -1 mg -1 ).…”

Section: Resultsmentioning

confidence: 75%

Glucose-tolerant Î²-glucosidase retrieved from a Kusaya gravy metagenome

2015

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β-glucosidases (BGLs) hydrolyze cello-oligosaccharides to glucose and play a crucial role in the enzymatic saccharification of cellulosic biomass. Despite their significance for the production of glucose, most identified BGLs are commonly inhibited by low (∼mM) concentrations of glucose. Therefore, BGLs that are insensitive to glucose inhibition have great biotechnological merit. We applied a metagenomic approach to screen for such rare glucose-tolerant BGLs. A metagenomic library was created in Escherichia coli (∼10,000 colonies) and grown on LB agar plates containing 5-bromo-4-chloro-3-indolyl-β-D-glucoside, yielding 828 positive (blue) colonies. These were then arrayed in 96-well plates, grown in LB, and secondarily screened for activity in the presence of 10% (w/v) glucose. Seven glucose-tolerant clones were identified, each of which contained a single bgl gene. The genes were classified into two groups, differing by two nucleotides. The deduced amino acid sequences of these genes were identical (452 aa) and found to belong to the glycosyl hydrolase family 1. The recombinant protein (Ks5A7) was overproduced in E. coli as a C-terminal 6 × His-tagged protein and purified to apparent homogeneity. The molecular mass of the purified Ks5A7 was determined to be 54 kDa by SDS-PAGE, and 160 kDa by gel filtration analysis. The enzyme was optimally active at 45°C and pH 5.0–6.5 and retained full or 1.5–2-fold enhanced activity in the presence of 0.1–0.5 M glucose. It had a low KM (78 μM with p-nitrophenyl β-D-glucoside; 0.36 mM with cellobiose) and high Vmax (91 μmol min-1 mg-1 with p-nitrophenyl β-D-glucoside; 155 μmol min-1 mg-1 with cellobiose) among known glucose-tolerant BGLs and was free from substrate (0.1 M cellobiose) inhibition. The efficient use of Ks5A7 in conjunction with Trichoderma reesei cellulases in enzymatic saccharification of alkaline-treated rice straw was demonstrated by increased production of glucose.

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“…The catalytic acid/base is located at the C-terminal of β-strand 4 and the nucleophile at the C-terminal of the β-strand 7 88 . In HiBGL, glycon binding site (subsite -1) lies at the bottom of the barrel with Gln17, His120, Trp121, Asn165, Tyr308, Trp427, Glu434, Trp435 and Phe443 residues 65 . MSA showed that these residues are conserved throughout BGL evolutionary history and the side chains of which interact with glycon moiety through both hydrogen and hydrophobic bonds.…”

Section: Journal Of Pure and Applied Microbiologymentioning

confidence: 99%