Purification and characterization of recombinant tomato fruit (Lycopersicon esculentum Mill.) fructokinase expressed in Escherichia coli.

Fructokinase (FK; ATP:D-fructose 6-phosphotransferase, EC 2.7.1.4) cloned from a tomato fruit cDNA library has been expressed in Escherichia coli. The recombinant protein was purified 159-fold to greater than 99% purity, based on SDS-PAGE analysis. The subunit molecular mass is estimated to be 35 kDa and the nondissociated molecular mass is 72.4 kDa, indicating that the functional form is a dimer. Two-dimensional IEF/SDS-PAGE analyses combined with immunodetection show that both native and recombinant proteins exhibit the same pattern of six closely grouped peptides with pI values ranging from 5.66 to 6.17. Biochemical characterization of the purified recombinant enzyme shows properties essentially identical to those of the native fructokinase purified from young tomato fruit: the pH optimum is 8.0, the K(m) for fructose is 0.22 mM, and severe substrate inhibition is observed when fructose concentration is greater than 0.5 mM (Ki = 3.0 mM). ATP is the preferred phosphate donor (K(m) = 0.13 mM and Vmax/K(m) = 212), followed by GTP (K(m) = 0.45 mM and Vmax/K(m) = 76) and UTP (K(m) = 1.68 mM and Vmax/K(m) = 20), but Vmax values are slightly greater with GTP and UTP. Product inhibition analyses show that the inhibition by ADP with respect to ATP is dependent on fructose concentration [Ki (ADP) = 0.41 mM with 0.5 mM fructose and decreased to 0.12 mM with 3 mM fructose]. Inhibition by fructose 6-P shows weak noncompetitive inhibition with respect to fructose; however, the recombinant protein is slightly more sensitive to fructose 6-P than the native FK.

An isoamylase with neutral pH optimum from a Flavobacterium species: cloning, characterization and expression of the iam gene.

The gene encoding an isoamylase with neutral pH optimum (iam) from a Flavobacterium species was cloned using a PCR probe generated from highly conserved regions of amylolytic enzymes. Active isoamylase was expressed from a 4.9-kb Pst I fragment in Escherichia coli, and was detected in the extracellular medium by a plate assay. The iam nucleotide sequence has an open reading frame of 2334 nucleotides (778 amino acids) with a GC content of 69%. Sequence analysis suggests that transcriptional control of the Flavobacterium sp. iam gene is mediated through the product of a malT regulatory gene. The deduced amino acid sequence of iam contained an N-terminal signal peptide of 32 amino acids, and was 61% homologous with Pseudomonas amyloderamosa isoamylase. The mature enzyme, which was engineered for overexpression in E. coli and purified to homogeneity, has a relative molecular mass of 83 kDa, a pH optimum of 6-7, and a highest rate of hydrolysis for glycogen (but did not cleave pullulan). Polyclonal antiserum generated from purified donor isoamylase cross-reacted with crude and purified recombinant isoamylase from E. coli. This is the first report of the cloning, characterization, and sequence of an novel isoamylase that has a neutral pH optimum. A comparison of the sequence of Flavobacterium sp. iam with acidic isoamylase from Pseudomonas sp. identified putative residues which may be associated with the pH for optimal activity of isoamylases.

Cloning of the cyclomaltodextrinase gene from Bacillus subtilis high-temperature growth transformant H-17.

The cyclomaltodextrinase gene from Bacillus subtilis high-temperature growth transformant H-17 was cloned on separate PstI, BamHI, and EcoRI fragments into the plasmid vector pUC18, but was expressed in an inactive form in the host, Escherichia coli DH5 alpha. High level constitutive expression of the gene product was also detrimental to the E. coli host, which led to structural instability of the recombinant plasmid. The cyclomaltodextrinase gene was cloned on a 3-kb EcoRI fragment into the plasmid vector pPL708, and the fragment was structurally maintained in the host B. subtilis YB886. The cloned gene product was synthesized in an enzymatically active form in the B. subtilis host; however, expression was at a low level. Subcloning of the 3-kb EcoRI fragment into pUC18 and transformation into E. coli XL1-Blue (F' lacIq) indicated that the cyclomaltodextrinase gene was cloned with its own promoter, since expression of the gene occurred in the absence of IPTG. Subcloning of the cyclomaltodextrinase gene downstream from the Bacillus temperature phage SPO2 promoter of pPL708 may increase expression of this gene.