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We have purified L-sorbose dehydrogenase (SDH) and L-sorbosone dehydrogenase (SNDH) from Gluconobacter oxydans T-100 that showed an ability to convert D-sorbitol to 2-keto-L-gulonate (2-KLGA). A genomic library of Gluconobacter oxydans T-100 was screened with a probe, a 180-bp PCR product which was obtained from degenerate oligodeoxyribonucleotides based on the elucidated sequence of the purified SDH (used as primers) and the genomic DNA of G. oxydans T-100 (used as a template). From sequencing of the DNA from a clone positive to the probe, the SNDH and the SDH were estimated to be coded in sequential open reading frames with 1,497 and 1,599 nucleotides, respectively, which was confirmed by expression of the DNA in Escherichia coli that showed both enzymatic activities. The DNA was introduced to a shuttle vector which was prepared from a plasmid of G. oxydans T-100 and pHSG298 to obtain an expression vector designated pSDH155. The production of 2-KLGA by pSDH155 in G. oxydans G624, an L-sorbose-accumulating strain, was improved to 230% compared to that of G. oxydans T-100. Chemical mutation of the host strain to suppress the L-idonate pathway and replacement of the original promoter with that of E. coli tufB resulted in improving the production of 2-KLGA. Consequently, high-level production from D-sorbitol to 2-KLGA (130 mg/ml) was achieved by simple fermentation of the recombinant Gluconobacter.

A cephalosporanic acid acylase from Pseudomonas strain N176 catalyzes hydrolysis of both glutarylcephalosporanic acid and cephalosporin C to 7-amino-cephalosporanic acid. Chemical modification of the enzyme with acidic hydrogen peroxide was performed to investigate residues which play important roles in enzymatic activity. The activity of the enzyme was reduced to 76% of the original by oxidation. From protein chemical analysis combined with site-directed point mutagenesis, modification of Met-164 was found to correspond to the reduction in activity. To study the effect of Met-164 on the enzymatic character, we prepared mutant acylases in which Met-164 was replaced with several other amino acids and obtained the following data: (i) there existed a trend of mutation to noncharged hydrophilic residues, resulting in an increase of activity against glutarylcephalosporanic acid; (ii) the mutation of Met-164 to Gly and Ala resulted in the lowering of both Km values and the optimal pHs against glutarylcephalosporanic acid; (iii) the mutation to Leu enhanced cephalosporin C acylase activity; and (iv) the mutation to Gln improved the k(cat) value for glutarylcephalosporanic acid. In particular, the mutation to Gln resulted in a high rate of conversion of glutarylcephalosporanic acid to 7-amino-cephalosporanic acid under conditions similar to those of a bioreactor system. These results may indicate that Met-164 is located in or near the cephalosporin compound binding pocket on the enzyme.

As a result of having undergone computed tomography (CT), a 75-year-old woman with type-C liver cirrhosiswas shown to have two tumors on the ventral and dorsal sides of subsegment 3 (S3). The tumor on the ventral side was diagnosed as a classic hepatocellular carcinoma (HCC), while that on the dorsal side was considered atypical for a HCC. Although the indocyanine green (ICG) findings indicated poor hepatic reserve, the prothrombin time (PT) was relatively good. An operation was performed in February 2007; however, this resulted in exploratory laparotomy. Dynamic CT performed 12 mo after the operation revealed that the tumor on the dorsal side of S3 had apparently increased. The marginal portion of the tumor was shown to be in the early and parenchymal phases, while the internal portion was found to have grown only slightly in the delayed phase. We diagnosed this tumor as a cholangiocellular carcinoma (CCC). S3 subsegmentectomy was performed in April 2008. The tumor on the ventral side was pathologically diagnosed as a moderately differentiated HCC, and that on the dorsal side was diagnosed as a CCC. We can therefore report a rare case of synchronous development of HCC and CCC in the same subsegment of the liver in a patient with type-C liver cirrhosis. We also add a literature review for all the reported cases published in Japan and around the world, and summarize the features of double cancer exhibiting both HCC and CCC.