Facile purification of a C‐terminal extended His‐tagged <i>Vibrio mimicus</i> arylesterase and characterization of the purified enzyme

Lee, Ya‐Lin; Chang, Rey-Chang; Shaw, Jei‐Fu

doi:10.1007/s11746-997-0239-1

Cited by 2 publications

(3 citation statements)

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“…2), fails to exhibit enzyme activity (11). Previous work revealed that a hexadecapeptide spacer between the His-tag and C-terminus is needed to restore the enzyme functions of arylesterase (11). The recombinant thioesterase I constructed with the same spacer between the His-tag and the C-terminus resulted in an opposite result, i.e., this recombinant has no enzyme activity (data not shown).…”

Section: Resultsmentioning

confidence: 92%

“…The same man-ufactured protein of V. mimicus arylesterase, which shares a high amino acid sequence identity (51.7%) with the thioesterase I (Fig. 2), fails to exhibit enzyme activity (11). Previous work revealed that a hexadecapeptide spacer between the His-tag and C-terminus is needed to restore the enzyme functions of arylesterase (11).…”

Section: Resultsmentioning

confidence: 94%

“…Enzyme extraction and purification. His-tagged thioesterase I was purified by affinity chromatography with Ni-NTA resin as previously described (11). The protein bound to the column was eluted with 1.0 M of imidazole.…”

Section: Methodsmentioning

confidence: 99%

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C‐terminal His‐tagging results in substrate specificity changes of the thioesterase I from Escherichia coli

Lee

Huang

et al. 1999

J Americ Oil Chem Soc

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The biochemical properties of Escherichia coli thioesterase I, His-tagged (HT) on the C-terminal, were systematically analyzed and compared with that without the His-tag (WT). These two types of enzymes exhibit similar optimal temperature and pH dependence, but subtle differences were detected. Kinetic studies revealed that the k cat /K m values of the HT enzyme for the substrates palmitoyl-CoA and p-nitrophenyl dodecanoate were 36-and 10-fold lower than those of the WT, respectively. In contrast, HT had a fivefold increased catalytic efficiency for p-nitrophenyl acetate, and up to fourfold increases toward phenylalanine-and tyrosine-derived ester substrates, L-NBPNPE (N-carbobenzoxy-L-phenylalanine p-nitrophenyl ester) and L-NBTNPE (N-carbobenzoxy-L-tyrosine p-nitrophenyl ester), respectively. For L-NBPNPE and L-NBTNPE, the increases were attributed to the higher k cat values with little changes in K m , whereas the increase for p-nitrophenyl acetate was mainly attributed to the lower K m value. It is concluded that addition of six hydrophilic histidine residues on the C-terminus resulted in a change in substrate specificity of E. coli thioesterase I toward more hydrophilic substrates.

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Section: Resultsmentioning

confidence: 92%

Section: Resultsmentioning

confidence: 94%