Glycerol protection and purification of Bacillus subtilis glucose dehydrogenase.

Ramaley, Robert F.; Vasantha, N.

doi:10.1016/s0021-9258(17)44213-x

Cited by 44 publications

(6 citation statements)

References 47 publications

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“…In comparison to reported industrial yields of 87–95%, , this improved yield of 36% was achieved in shake flask experiments without any other strain or process optimization. The repression of hexokinase activity successfully allowed glucose flux to be redirected into the gluconate pathway, even when the reported k cat / K M of the competing native Hxk1p was more than an order of magnitude higher than the heterologous glucose dehydrogenase. − In addition, the 10-fold decrease in hexokinase activity through transcriptional control was sufficient for the redirection of glucose, even though it was reported that transcription only accounts for 30% of the regulation of Hxk1p abundance . Thus, we believe that our tTA system is generalizable to enzymes that are regulated at the transcriptional level, even if transcription may only account partially for its regulation of abundance.…”

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confidence: 89%

“…We expressed glucose dehydrogenase, gdh , from Bacillus subtilis for the production of gluconic acid in our strains. The reported k cat / K M value of B. subtilis glucose dehydrogenase, 160 M/s, is significantly lower than that of S. cerevisiae Hxk1p, 8800 M/s. , Since Hxk1p intrinsically outcompetes glucose dehydrogenase for glucose, this pathway is an ideal model system to demonstrate the utility of a glucose valve. This proof-of-concept is one of the first demonstrations in S. cerevisiae of dynamically redirecting glucose flux away from glycolysis, resulting in superior improvement in yields (50-fold) of a heterologous pathway while simultaneously reducing ethanol byproduction even in glucose excess conditions.…”

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confidence: 97%

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confidence: 99%

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Controlling Central Carbon Metabolism for Improved Pathway Yields in Saccharomyces cerevisiae

2015

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Engineering control of metabolic pathways is important to improving product titers and yields. Traditional methods such as overexpressing pathway enzymes and deleting competing ones are restricted by the interdependence of metabolic reactions and the finite nature of cellular resources. Here, we developed a metabolite valve that controls glycolytic flux through central carbon metabolism in Saccharomyces cerevisiae. In a Hexokinase 2 and Glucokinase 1 deleted strain (hxk2Δglk1Δ), glucose flux was diverted away from glycolysis and into a model pathway, gluconate, by controlling the transcription of Hexokinase 1 with the tetracycline transactivator protein (tTA). A maximum 10-fold decrease in hexokinase activity resulted in a 50-fold increase in gluconate yields, from 0.7% to 36% mol/mol of glucose. The reduction in glucose flux resulted in a significant decrease in ethanol byproduction that extended to semianaerobic conditions, as shown in the production of isobutanol. This proof-of-concept is one of the first demonstrations in S. cerevisiae of dynamic redirection of glucose from glycolysis and into a heterologous pathway.

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confidence: 89%

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confidence: 97%

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confidence: 99%

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Controlling Central Carbon Metabolism for Improved Pathway Yields in Saccharomyces cerevisiae

2015

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“…GDH from B. amyloliquefaciens [7] has been reported to withstand up to 40 °C for 6 h, whereas B. megaterium GDH-III [6] was inactivated within 20 min in absence of 2 M NaCl above 40 °C. Although GDH from Bacillus subtilis and B. megaterium exhibit a higher specificity over P. pini GDH, these enzymes require the inclusion of 20% (w/v) glycerol for the maintenance of GDH structure and activity [26]. The GDH from thermophilic Sulfolobus solfataricus [7] is active and stable at 70 °C, but its low turnover and inhibition by glucose above 25 mM could hinder its biocatalytic application.…”

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confidence: 99%

Characterization and Application of a Robust Glucose Dehydrogenase from Paenibacillus pini for Cofactor Regeneration in Biocatalysis

et al. 2019

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Glucose dehydrogenases are important auxiliary enzymes in biocatalysis, employed in the regeneration of reduced nicotinamide cofactors for oxidoreductase catalysed reactions. Here we report the identification and characterization of a novel glucose-1-dehydrogenase (GDH) from Paenibacillus pini that prefers NAD ? as cofactor over NADP ? . The purified recombinant P. pini GDH displayed a specific activity of 247.5 U/mg. The enzyme was stable in the pH range 4-8.5 and exhibited excellent thermostability till 50 °C for 24 h, even in the absence of NaCl or glycerol. Paenibacillus pini GDH was also tolerant to organic solvents, demonstrating its potential for recycling cofactors for biotransformation. The potential application of the enzyme was evaluated by coupling with a NAD ? -dependent alcohol dehydrogenase for the reduction of acetophenone and ethyl-4-chloro-3-oxo-butanoate. Conversions higher than 95% were achieved within 2 h with low enzyme loading using lyophilized cell lysate, suggesting that P. pini GDH could be highly effective for recycling NADH in redox biocatalysis.

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“…In this study, we selected FMN-dependent NADH-azoreductase (AZR, accession No. NP_391234) and glucose 1-dehydrogenase (GDH, NP_388275) of Bacillus subtilis 168, 13) 14) as a model oxidoreductase for the biosensor and a recipient of CBD fusion. AZR is an FMN-linked enzyme and catalyzes the reduction of dichloroindophenol (DCIP) using NADH as an electron donor.…”

Section: Introductionmentioning

confidence: 99%

Construction of Cellulose Binding Domain Fusion FMN-Dependent NADH-Azoreductase and Glucose 1-Dehydrogenase for the Development of Flow Injection Analysis with Fusion Enzymes Immobilized on Cellulose

et al. 2019

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The cellulose binding domain (CBD) of cellulosome-integrating protein A from Clostridium thermocellum NBRC 103400 was genetically fused to FMN-dependent NADH-azoreductase (AZR) and glucose 1-dehydrogenase (GDH) from Bacillus subtilis. The fusion enzymes, AZR-CBD and CBD-GDH, were expressed in Escherichia coli Rosetta-gami B (DE3). The enzymes were purified from cell-free extracts, and the specific activity of AZR-CBD was 15.1 U/mg and that of CBD-GDH was 22.6 U/mg. AZR-CBD and CBD-GDH bound strongly to 0.5 % swollen cellulose at approximately 95 and 98 % of the initial protein amounts, respectively. After immobilization onto the swollen cellulose, AZR-CBD and CBD-GDH retained their catalytic activity. Both enzymes bound weakly to 0.5 % microcrystalline cellulose, but the addition of a high concentration of microcrystalline cellulose (10 %) improved the binding rate of both enzymes. A reactor for flow injection analysis was filled with microcrystalline cellulose-immobilized AZR-CBD and CBD-GDH. This flow injection analysis system was successfully applied for the determination of glucose, and a linear calibration curve was observed in the range of approximately 0.16-2.5 mM glucose, with a correlation coefficient, r, of 0.998.

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Glycerol protection and purification of Bacillus subtilis glucose dehydrogenase.

Cited by 44 publications

References 47 publications

Controlling Central Carbon Metabolism for Improved Pathway Yields in Saccharomyces cerevisiae

Controlling Central Carbon Metabolism for Improved Pathway Yields in Saccharomyces cerevisiae

Characterization and Application of a Robust Glucose Dehydrogenase from Paenibacillus pini for Cofactor Regeneration in Biocatalysis

Construction of Cellulose Binding Domain Fusion FMN-Dependent NADH-Azoreductase and Glucose 1-Dehydrogenase for the Development of Flow Injection Analysis with Fusion Enzymes Immobilized on Cellulose

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