A synthetic oligodeoxynucleotide encoding the vasopressin peptide was ligated to the 3′ terminal codon of sacB, the structural gene of levansucrase. This gene fusion was integrated into the chromosome of a Bacillus subtilis strain able to overproduce levansucrase. The extracellular production of the hybrid protein, consisting of the whole levansucrase primary sequence plus the nine amino acids of the vasopressin peptide added at the C-terminal end, represented 50–55% of that found for the wild-type levansucrase (20 mg |-1). The purified hybrid protein displayed the same conformational stability, protease insensitivity and enzymic properties as the wild-type levansucrase. However, the rate and the yield of the unfolding-folding transition at the pH and temperature used for bacterial growth were lower in the case of the hybrid protein; the latter also required a higher iron concentration to be completely folded.
The role of the major conidial-bound cellulase — cellobiohydrolase II (CBH II) — in the triggering of cellulase formation in the fungus Trichoderma reesei was investigated by comparing the mutant strain QM 9414 with a recombinant strain unable to produce CBH II. For this purpose, the cbh2 gene was isolated from a chromosomal gene bank of T. reesei, cloned into pGEM-7Zf(+), and disrupted by insertion of the homologous pyr4 gene in its coding region to yield the plasmid vector pSB3. Transformation of the auxotrophic, pyr4-negative strain T. reesei TU-6 with pSB3 yielded 23 stable prototrophs, of which three were unable to produce CBH II — assessed by means of a monoclonal antibody — during growth on lactose or in the presence of sophorose. However, they formed cellobiohydrolase I (CBH I) at a rate comparable to strain QM 9414 under these conditions. Southern analysis of DNA of some CBH II− and CBH II+ transformants confirmed that pSB3 had integrated at the cbh2 locus in the CBH II− strains. The latter displayed normal growth on glucose or maltose as carbon source. They showed retarded growth on cellulose as sole carbon source, however, and exhibited a lag in the time course of CBH I and EG I formation, although producing roughly the same final cellulase activities. It is concluded from these results that CBH II is not essential for induction of cellulase formation by cellulose, but that it contributes significantly to the formation of lower molecular mass inducers in the early phase of growth of the fungus on cellulose.
The phototrophic bacterium Rhodobacter sphaeroides strain Si4 induced ribitol dehydrogenase (EC 22.214.171.124) when grown on ribitol- or xylitol-containing medium. This ribitol dehydrogenase was purified to apparent homogeneity by ammonium sulphate precipitation, affinity chromatography on Procion red, and chromatography on Q-Sepharose. For the native enzyme an isoelectric point of pH 6·1 and an apparent M r of 50000 was determined. SDS-PAGE yielded a single peptide band of M r 25000 suggesting a dimeric enzyme structure. The ribitol dehydrogenase was specific for NAD+ but unspecific as to its polyol substrate. In order of decreasing activity ribitol, xylitol, erythritol, D-glucitol and D-arabitol were oxidized. The pH optimum of substrate oxidation was 10, and that of substrate reduction was 6·5. The equilibrium constant of the interconversion of ribitol to D-ribulose was determined to be 0·33 nM at pH 7·0 and 25 °C. The K m-values determined for ribitol, ribulose, xylitol and NAD+ (in the presence of ribitol) were 6·3, 12·5, 77 and 0·077 mM, respectively. Because of the favourable K m for ribitol, a method for quantitative ribitol determination was elaborated.