Purification and biochemical properties of a glucose-stimulated β-D-glucosidase produced by Humicola grisea var. thermoidea grown on sugarcane bagasse

Nascimento, César Vanderlei; Souza, Flávio Henrique Moreira; Masui, Douglas Chodi; Leone, Francisco de Assis; Peralta, Rosane Marina; Jorge, João Atı́lio; Furriel, Rosa dos Prazeres Melo

doi:10.1007/s12275-009-0159-x

Cited by 60 publications

(15 citation statements)

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“…For example, the β-glucosidases from H. insolens [ 12 ], S.thermophilum [ 13 ], and H. grisea var. thermoidea [ 31 ] were stimulated more than 2-fold by 0.05–0.2 M glucose; the β-glucosidases from Bacillus halodurans C-125 [ 9 ], uncultured bacterium [ 16 ], and Neotermes koshunensis [ 32 ] were stimulated about 1.6-, 1.3-, and 1.3-fold by 0.2 M glucose, respectively. The phenomenon of stimulation by glucose seems to be a unique property of GH1 β-glucosidases, which is not observed among the GH3 β-glucosidases until now [ 33 ].…”

Section: Resultsmentioning

confidence: 99%

Engineering a novel glucose-tolerant β-glucosidase as supplementation to enhance the hydrolysis of sugarcane bagasse at high glucose concentration

Cao

Wang

Ren

et al. 2015

Biotechnol Biofuels

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BackgroundMost β-glucosidases reported are sensitive to the end product (glucose), making it the rate limiting component of cellulase for efficient degradation of cellulose through enzymatic route. Thus, there are ongoing interests in searching for glucose-tolerant β-glucosidases, which are still active at high glucose concentration. Although many β-glucosidases with different glucose-tolerance levels have been isolated and characterized in the past decades, the effects of glucose-tolerance on the hydrolysis of cellulose are not thoroughly studied.ResultsIn the present study, a novel β-glucosidase (Bgl6) with the half maximal inhibitory concentration (IC50) of 3.5 M glucose was isolated from a metagenomic library and characterized. However, its poor thermostability at 50 °C hindered the employment in cellulose hydrolysis. To improve its thermostability, random mutagenesis was performed. A thermostable mutant, M3, with three amino acid substitutions was obtained. The half-life of M3 at 50 °C is 48 h, while that of Bgl6 is 1 h. The Kcat/Km value of M3 is 3-fold higher than that of Bgl6. The mutations maintained its high glucose-tolerance with IC50 of 3.0 M for M3. In a 10-h hydrolysis of cellobiose, M3 completely converted cellobiose to glucose, while Bgl6 reached a conversion of 80 %. Then their synergistic effects with the commercial cellulase (Celluclast 1.5 L) on hydrolyzing pretreated sugarcane bagasse (SCB) were investigated. The supplementation of Bgl6 or mutant M3 to Celluclast 1.5 L significantly improved the SCB conversion from 64 % (Celluclast 1.5 L alone) to 79 % (Bgl6) and 94 % (M3), respectively. To further evaluate the application potential of M3 in high-solids cellulose hydrolysis, such reactions were performed at initial glucose concentration of 20–500 mM. Results showed that the supplementation of mutant M3 enhanced the glucose production from SCB under all the conditions tested, improving the SCB conversion by 14–35 %.ConclusionsThese results not only clearly revealed the significant role of glucose-tolerance in cellulose hydrolysis, but also showed that mutant M3 may be a potent candidate for high-solids cellulose refining.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0383-z) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Engineering a novel glucose-tolerant β-glucosidase as supplementation to enhance the hydrolysis of sugarcane bagasse at high glucose concentration

Cao

Wang

Ren

et al. 2015

Biotechnol Biofuels

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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: 81%

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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“…yunnanensis 258 ± 5.1182 ± 3.42.2 and 1.4 for glucose and xylose, respectively842 ± 15.560~10 h at 60 °C;BacteriumPresent work S. thermophilum 204 ± 12191 ± 140.7 and 0.5 for glucose and xylose, respectively8.96020 min at 55 °CFungi[8] H. insolens 180 ± 9200 ± 110.436.46044 min at 55 °CFungi[7] H. grisea var. thermoidea 1882020.529.5507 min at 60 °CFungi[23] B. halodurans 135.0 ± 4.3131.0 ± 0.2~0.8-0.9935030 min at 50 °CBacterium[24]Uncultured bacterium1201500.3 for glucose183.950 ND Soil metagenome[5] Marinomonas sp. MWYL1~132~1450.638940~11 min at 45 °CBacterium[9]

ND not determined

a One hundred percent specific activity (control) was estimated in the absence of carbohydrates

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Section: Discussionmentioning

confidence: 99%

Increased enzymatic hydrolysis of sugarcane bagasse by a novel glucose- and xylose-stimulated β-glucosidase from Anoxybacillus flavithermus subsp. yunnanensis E13T

Liu

Wang

et al. 2017

BMC Biochem

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Backgroundβ-Glucosidase is claimed as a key enzyme in cellulose hydrolysis. The cellulosic fibers are usually entrapped with hemicelluloses containing xylose. So there is ongoing interest in searching for glucose- and xylose-stimulated β-glucosidases to increase the efficiency of hydrolysis of cellulosic biomass.ResultsA thermostable β-glucosidase gene (Bglp) was cloned from Anoxybacillus flavithermus subsp. yunnanensis E13T and characterized. Optimal enzyme activity was observed at 60 °C and pH 7.0. Bglp was relatively stable at 60 °C with a 10-h half-life. The kinetic parameters V max and K m for p-nitrophenyl-β-D-glucopyranoside (pNPG) were 771 ± 39 μmol/min/mg and 0.29 ± 0.01 mM, respectively. The activity of Bglp is dramatically stimulated by glucose or xylose at concentrations up to 1.4 M. After Bglp was added to Celluclast® 1.5 L, the conversion of sugarcane bagasse was 48.4 ± 0.8%, which was much higher than of Celluclast® 1.5 L alone. Furthermore, Bglp showed obvious advantages in the hydrolysis when initial concentrations of glucose and xylose are high.ConclusionsThe supplementation of BglP significantly enhanced the glucose yield from sugarcane bagasse, especially in the presence of high concentrations of glucose or xylose. Bglp should be a promising candidate for industrial applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s12858-017-0079-z) contains supplementary material, which is available to authorized users.

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Purification and biochemical properties of a glucose-stimulated β-D-glucosidase produced by Humicola grisea var. thermoidea grown on sugarcane bagasse

Cited by 60 publications

References 65 publications

Engineering a novel glucose-tolerant β-glucosidase as supplementation to enhance the hydrolysis of sugarcane bagasse at high glucose concentration

Engineering a novel glucose-tolerant β-glucosidase as supplementation to enhance the hydrolysis of sugarcane bagasse at high glucose concentration

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

Increased enzymatic hydrolysis of sugarcane bagasse by a novel glucose- and xylose-stimulated β-glucosidase from Anoxybacillus flavithermus subsp. yunnanensis E13T

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