Saccharification of ß-chitin from squid pen by a fermentation method using recombinant chitinase-secreting Escherichia coli.

Two strains [BL21(DE3) and HMS174(DE3)] of Escherichia coli harboring the recombinant chitinase expression plasmid pVP-Chi, which contains Vibrio parahaemolyticus chitinase gene with an attached signal sequence, were prepared. These E. coli transformants produced a large amount of recombinant chitinase, which hydrolyzes chitin to yield di-N-acetylchitobiose (GlcNAc)2, under the presence of isopropyl-1-thio-ß-D-galactopyranoside (IPTG), and secreted the enzyme into their culture fluid with the aid of the signal peptide. Cultivation of these E. coli transformants in Luria-Bertani medium containing squid pen ß-chitin and IPTG gave rise to the decomposition of this polysaccharide and the accumulation of (GlcNAc)2 in the culture fluid. Through these experiments, we confirmed that the use of strain HMS174(DE3) was preferable for the stable accumulation of (GlcNAc)2 in the culture fluid during cultivation owing to lower (GlcNAc)2 assimilation compared to BL21(DE3). Next, using E. coli HMS174(DE3) transformants, we conducted saccharification of different forms (fluffy fiber, flake, and powder) of ß-chitin samples prepared from squid pens in Bacterion-N-KS(B)K medium containing 2 % of each sample under the presence of IPTG. In these experiments, (GlcNAc)2 was isolated with a more than 20 % stoichiometric yield from each culture supernatant through charcoal column chromatography followed by recrystallization.

Expansion of the first polyalanine tract of the ARX gene in a boy presenting with generalized dystonia in the absence of infantile spasms.

Mutations in the Aristaless-related homeobox (ARX) gene are associated with pleiotropic phenotypes including infantile spasms, mental retardation and dystonia. However, relatively consistent genotype-phenotype correlations have been emphasized in prior reports. We report a boy presenting with mental retardation, tonic seizures and dystonia but without infantile spasms. Gene sequencing detected an additional seven GCG repeats in the first polyalanine tract of the ARX gene, a mutation which leads to an expansion of the normal 16 alanine residues to 23. The same ARX gene mutation has been reported in patients with infantile spasms, but was absent in the present case. This finding highlights the diverse phenotypic spectrum that may result from ARX gene mutations.