Overexpression, purification, and generation of a thermostable variant of Zymomonas mobilis fructokinase.
The gene encoding fructokinase (EC 2.7.1.4) from Zymomonas mobilis has been expressed at high level in Escherichia coli by modifying the ribosome binding site using the polymerase chain reaction. A simple two-step purification from extracts of the recombinant cells results in highly purified enzyme suitable for use in fructose determination. Using the polymerase chain reaction in mutagenic conditions, a variant of fructokinase was isolated which was more thermostable than the wild type, taking the 30 min half-life from 70.1 to 72.4 degrees C. The purified thermostable variant had the same specific activity as the wild type. Sequencing of the variant indicated that only one amino acid was changed, with Ser 69 becoming Ala. Searches of the mutant libraries for variants that were (a) active with glucose or (b) had reduced inhibition by glucose were unsuccessful.
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School of Biochemistry, La Trobe University, Melbourne, Victoria, Australia.
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D017931:DNA Primers / Q000737:chemistry; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005631:Fructokinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005947:Glucose / Q000378:metabolism; D006207:Half-Life; D006358:Hot Temperature; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017355:Zymomonas / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D011994:Recombinant Proteins; D005631:Fructokinases; C046173:fructokinase; D005947:Glucose