Molecular characterization of a novel trehalose-6-phosphate hydrolase, TreA, from Bacillus licheniformis

Chuang, Tzu-Ting; Ong, Ping-Lin; Wang, Tzu-Fan; Huang, Huey W.; Chi, Meng-Chun; Lin, Long-Liu

doi:10.1016/j.ijbiomac.2012.01.011

Cited by 7 publications

(15 citation statements)

References 99 publications

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“…Investigations on Pseudoalteromonas haloplanctis α-amylase have demonstrated that chloride ion is not bound by K337R and the starchdegrading ability of this variant is consequently independent of Cl − (Feller et al 1996). Very recently, we have constructed a chimeric plasmid for the functional expression of Bacillus licheniformis trehalose-6-phosphate hydrolase (Bl TreA) in recombinant Escherichia coli (Chuang et al 2012). The recombinant enzyme is active towards its natural substrate, trehalose-6-phosphate, and quite stable in the presence of organic solvents.…”

Section: Removal Of CLmentioning

confidence: 99%

“…The treA gene encoding a recombinant Bl TreA with a His tag at its N-terminal end was employed as the DNA template (Chuang et al 2012). For site-directed mutagenesis, eight pairs of complementary mutagenic primers were used for the substitution mutation of Arg201, Asn327, and Tyr365 residues (Table 1).…”

Section: Site-directed Mutagenesismentioning

confidence: 99%

“…To overexpress Bl TreA and its variants, E. coli M15 host cells harbouring pQE-Bl TreA (Chuang et al 2012) or each of the mutated plasmids were grown in 100-mL LB medium containing ampicillin (100 µg/mL) and kanamycin (25 µg/mL) at 37…”

Section: Enzyme Expression and Purificationmentioning

confidence: 99%

“…The resuspended cells were sonicated for 5 min with 30-s rests on ice between each burst, and the lysate sample was clarified by centrifugation (12,000×g for 10 min). The crude extract was then subjected to protein purification under native conditions with high-affinity Ni-NTA resin (Chuang et al 2012). The elution fractions were pooled and dialyzed against 50 mM Hepes-NaOH buffer (pH 8.0) using a 10-kDa cutoff Ultracel-PL membrane (Amicon) for the removal of salts.…”

Section: Enzyme Expression and Purificationmentioning

confidence: 99%

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Identification of critical amino acid residues for chloride binding of Bacillus licheniformis trehalose-6-phosphate hydrolase

et al. 2013

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Based on sequence alignment of selected Cl − dependent and independent glycoside hydrolase family 13 enzymes, two invariant residues (Arg201 and Asn347) and one tyrosine (Tyr365) that might be responsible for the binding of Bacillus licheniformis trehalose-6-phosphate hydrolase (Bl TreA) to chloride ion were identified. The role of these three residues was further explored by mutational and biophysical analyses. The mutant enzymes (R201Q/E/K, N327Q/D/K, and Y365A/R) and Bl TreA were individually overexpressed in Escherichia coli M15 host cells and purified by one-step nickel affinity chromatography on Ni-NTA resin. The purified Bl TreA and Y365A had a specific activity of 236.9 and 47.6 U/mg protein, respectively. The remaining enzymes lost their hydrolase activity completely even in the presence of high salt. With the exception of Y365A, all mutant enzymes did not have the ability to bind fluoride, chloride and nitrate anions. Structural analyses showed that the circular dichroism spectra of the mutant proteins were consistent with those of Bl TreA. However, wild-type and mutant enzymes displayed a slight difference in the profiles of intrinsic tryptophan fluorescence. Collectively, these results clearly indicate that Arg201 and Agr327 residues might play an essential role in chloride binding of Bl TreA.

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Section: Removal Of CLmentioning

confidence: 99%

Section: Site-directed Mutagenesismentioning

confidence: 99%

Section: Enzyme Expression and Purificationmentioning

confidence: 99%

Section: Enzyme Expression and Purificationmentioning

confidence: 99%

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Identification of critical amino acid residues for chloride binding of Bacillus licheniformis trehalose-6-phosphate hydrolase

et al. 2013

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“…As a member of family GH13, trehalose-6-phosphate hydrolase (TreA) cleaves the α , α -1,1-glycosidic linkage of trehalose-6-phosphate to produce glucose and glucose-6-phosphate and plays a role in bacterial trehalose metabolism [ 29 ]. Recently, we have characterized the TreA protein from Bacillus licheniformis ( Bl TreA) at the molecular level [ 30 ]. The recombinant enzyme starts to unfold beyond ~0.14 M guanidine hydrochloride (GdnHCl) and reaches the unfolded intermediates, [GdnHCl] 0.5,N–I and [GdnHCl] 0.5,I–U , at 1.02 and 2.24 M, respectively.…”

Section: Introductionmentioning

confidence: 99%

Beneficial Effect of Sugar Osmolytes on the Refolding of Guanidine Hydrochloride-Denatured Trehalose-6-phosphate Hydrolase fromBacillus licheniformis

Chen

Chi

Lin

et al. 2015

BioMed Research International

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The influence of three sugar osmolytes on the refolding of guanidine hydrochloride- (GdnHCl-) denatured trehalose-6-phosphate hydrolase of Bacillus licheniformis (BlTreA) was studied by circular dichroism (CD) spectra, fluorescence emission spectra, and the recovery of enzymatic activity. These experimental results clearly indicated that sorbitol, sucrose, and trehalose at a concentration of 0.75 M improved the refolding yields of GdnHCl-denatured BlTreA, probably due to the fact that these sugars favored the formation of tertiary architectures. Far-UV CD measurements demonstrated the ability of sugar osmolytes to shift the secondary structure of GdnHCl-denatured enzyme towards near-native conformations. ANS fluorescence intensity measurements revealed a reduction of exposed hydrophobic surfaces upon the treatment of denatured enzyme with sugar osmolytes. These observations suggest that sugar osmolytes possibly play a chaperone role in the refolding of chemically denatured BlTreA.

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Survival strategies of Bacillus spp. in saline soils: Key factors to promote plant growth and health

Valencia-Marin,

Chávez-Avila,

Guzmán-Guzmán

et al. 2024

Biotechnology Advances

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Molecular characterization of a novel trehalose-6-phosphate hydrolase, TreA, from Bacillus licheniformis

Cited by 7 publications

References 99 publications

Identification of critical amino acid residues for chloride binding of Bacillus licheniformis trehalose-6-phosphate hydrolase

Identification of critical amino acid residues for chloride binding of Bacillus licheniformis trehalose-6-phosphate hydrolase

Beneficial Effect of Sugar Osmolytes on the Refolding of Guanidine Hydrochloride-Denatured Trehalose-6-phosphate Hydrolase fromBacillus licheniformis

Survival strategies of Bacillus spp. in saline soils: Key factors to promote plant growth and health

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