Yuki Fujita

Cloning of the Thaumatin I cDNA and Characterization of Recombinant Thaumatin I Secreted by Pichia pastoris

Thaumatin is a sweet‐tasting protein comprising a mixture of some variants. The major variants are thaumatins I and II. Although the amino acid sequence of thaumatin I was known and the nucleotide sequence of cDNA of thaumatin II was elucidated, the nucleotide sequence of thaumatin I has been controversial. We have cloned two thaumatin cDNAs from the fruit of Thaumatococcus daniellii Benth. One is the same nucleotide sequence as that of thaumatin II already reported, and the other is a novel nucleotide sequence. The amino acid sequence deduced from the novel cDNA was the same amino acid sequence as that of thaumatin I, the only exception being the residue at position 113 (Asp instead of Asn), indicating that the novel thaumatin cDNA is that for thaumatin I. This thaumatin I cDNA was transformed into Pichia pastoris X‐33, and the recombinant thaumatin I expressed was purified and characterized. The threshold value of sweetness of the recombinant thaumatin I was the same as that of the plant thaumatin I, although several unexpected amino acid residues were attached to the N‐terminal of the recombinant thaumatin I. These indicate that the N‐terminal portion of thaumatin is not critical for the elicitation of sweetness.

Enzymatic polymerization to an alternating N-L-cysteinyl chitin derivative: a novel class of multivalent glycopeptidomimetics.

A novel chitin derivative with an alternating N-L-cysteinyl group was successfully prepared. The precursor chitin derivative with an alternating N-L-thioprolinyl group could be obtained via chitinase-catalyzed polymerization of the N-L-thioprolinyl chitobiose oxazoline derivative as a transition state analog substrate monomer under weak alkaline conditions. Conversion of the L-thioprolinyl group to an L-cysteinyl group in the product chitin derivative proceeded in a dose-dependent manner by methoxyamine hydrochloride, and the reaction could be completed almost quantitatively. The obtained chitin derivative is a uniformly amino acid-branching polysaccharide, which is categorized into a novel class of multivalent glycopeptidomimetics.

Oxidation of thymine derivatives with superoxide ion

Alkylated thymine and thymidine derivatives are transformed by potassium superoxide into the corresponding ring contracted imidazolone derivatives; a plausible mechanism is presented.