Heterologous expression and characterization of recombinant glycerol dehydratase from <i>Klebsiella pneumoniae</i> in <i>Escherichia coli</i>

Wang, Fenghuan; Qu, Huijin; Tian, Pingfang; Tan, Tianwei

doi:10.1002/biot.200600101

Cited by 12 publications

(3 citation statements)

References 23 publications

(18 reference statements)

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“…Wang et al (2007) tried to fuse the three subunits of GDH together in tandem but the resulting fusion protein showed lower activity compared to the wild-type enzyme. In this study, the N-terminal of the b-subunit was fused to the C-terminal of either a-or c-subunit with or without the insertion of a linker peptide through gene splicing.…”

Section: Discussionmentioning

confidence: 96%

Characterization of glycerol dehydratase expressed by fusing its α- and β-subunits

et al. 2009

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The gdh and gdhr genes, encoding B(12)-dependent glycerol dehydratase (GDH) and glycerol dehydratase reactivase (GDHR), respectively, in Klebsiella pneumoniae, were cloned and expressed in E. coli. Part of the beta-subunit was lost during GDH purification when co-expressing alpha, beta and gamma subunit. This was overcome by fusing the beta-subunit to alpha- or gamma-subunit with/without the insertion of a linker peptide between the fusion moieties. The kinetic properties of the fusion enzymes were characterized and compared with wild type enzyme. The results demonstrated that the fusion protein GDHALB/C, constructed by linking the N-terminal of beta-subunit to the C-terminal of alpha subunit through a (Gly(4)Ser)(4) linker peptide, had the greatest catalytic activity. Similar to the wild-type enzyme, GDHALB/C underwent mechanism-based inactivation by glycerol during catalysis and could be reactivated by GDHR.

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Section: Discussionmentioning

confidence: 96%

Characterization of glycerol dehydratase expressed by fusing its α- and β-subunits

et al. 2009

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“…Direct expression, coexpression, and fusion expression methods were used for heterologous expression and characterization of GDHt from K. pneumoniae in E. coli , and the activities of three heterologous expression products were found to be 27.4, 2.3, and 0.2 U/mg, respectively. It was demonstrated that the highest enzyme activity was almost 17 times of that in K. pneumoniae [ 92 ], while these researches had always focused on only one enzyme. These methods could be used for the coexpressions of dhaB and dhaT genes to efficiently convert glycerol to 1,3-PD, and even in other bioconversion processes involving multienzymatic coexpressions, such as coexpressions of the GDHt, PDOR, and the reactivating factor.…”

Section: Glycerol Dehydratasementioning

confidence: 99%

Key enzymes catalyzing glycerol to 1,3-propanediol

Jiang

Wang

et al. 2016

Biotechnol Biofuels

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Biodiesel can replace petroleum diesel as it is produced from animal fats and vegetable oils, and it produces about 10 % (w/w) glycerol, which is a promising new industrial microbial carbon, as a major by-product. One of the most potential applications of glycerol is its biotransformation to high value chemicals such as 1,3-propanediol (1,3-PD), dihydroxyacetone (DHA), succinic acid, etc., through microbial fermentation. Glycerol dehydratase, 1,3-propanediol dehydrogenase (1,3-propanediol-oxydoreductase), and glycerol dehydrogenase, which were encoded, respectively, by dhaB, dhaT, and dhaD and with DHA kinase are encompassed by the dha regulon, are the three key enzymes in glycerol bioconversion into 1,3-PD and DHA, and these are discussed in this review article. The summary of the main research direction of these three key enzyme and methods of glycerol bioconversion into 1,3-PD and DHA indicates their potential application in future enzymatic research and industrial production, especially in biodiesel industry.

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“…To verify the activity of our engineered enzyme, we began by determining its specific activity over a range of temperatures. The optimum temperature (T opt ) for the unlinked enzyme is 37 C. [32] Our linked variant, KpGDHt-L, was substantially more active at higher temperatures, displaying a T opt of 57 C ( Figure 1A). Consistent with the activity data, differential scanning fluorimetry showed a midpoint for unfolding (T m ) at 58 C ( Figure 1B).…”

Section: Linked Kpgdht Variant With Enhanced Thermostabilitymentioning

confidence: 99%

An Engineered Glycerol Dehydratase With Improved Activity for the Conversion of meso‐2,3‐butanediol to Butanone

Maddock

Gerth

Patrick

2017

Biotechnology Journal

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There is substantial interest in engineering microorganisms to produce industrial chemicals that are currently derived from petroleum. One of these petrochemicals is butanone, which could be produced from microbially synthesized 2,3-butanediol through the action of a suitable dehydratase enzyme. Unfortunately, however, there are no known enzymes that natively catalyze this reaction. In this work, the authors set out to engineer the B 12 -dependent glycerol dehydratase from Klebsiella pneumoniae (KpGDHt), in order to increase its activity for the conversion of meso-2,3-butanediol into butanone. The authors began by fusing the α and β subunits of the enzyme, to simplify downstream high-throughput screening protocols. Serendipitously, the fusion protein showed a 20 C increase in its temperature optimum. Using this stabilized scaffold as a starting point, the authors employed the combinatorial active site saturation test and consensus-guided mutagenesis to randomize 28 residues within 12 Åof the KpGDHt active site. By screening over 5500 variants, the authors discovered a single point mutation (T200S) that increased the catalytic efficiency of meso-2,3-butanediol dehydration by four-fold, to a value of k cat /K M ¼ 5.1 Â 10 3 M À1 s À1 . Thus the authors report what is, to date, the most comprehensive mutagenesis and the largest engineered increase in catalytic efficiency on the B 12 -dependent glycerol dehydratase scaffold.

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Heterologous expression and characterization of recombinant glycerol dehydratase from Klebsiella pneumoniae in Escherichia coli

Cited by 12 publications

References 23 publications

Characterization of glycerol dehydratase expressed by fusing its α- and β-subunits

Characterization of glycerol dehydratase expressed by fusing its α- and β-subunits

Key enzymes catalyzing glycerol to 1,3-propanediol

An Engineered Glycerol Dehydratase With Improved Activity for the Conversion of meso‐2,3‐butanediol to Butanone

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