Yoshihisa Kitaoka

Cooperativity of stabilized mRNA and enhanced translation activity in the cell-free system

Detailed analysis of cell-free translation, coupled transcription-translation in static conditions and a continuous flow system based on E. coli S30 extracts was performed. Degradation of template mRNA was the predominant trigger to terminate the protein synthesis. In a coupled system, mRNA was preserved by repeated transcription whereas the starvation of nucleotide triphosphates led to the termination of protein synthesis in less than 1 h. In the CFCF system, NTP was held at the level of initial concentration and therefore did not arrest the translation for 15 h. The accurate coupling of transcriptional rate and translational rate was also crucial to enhance the efficiency of protein synthesis.

Cell-free system derived from heat-shocked Escherichia coli: Synthesis of enzyme protein possessing higher specific activity

Abstract heat-shocked S30 extract (HS-S30 extract) was prepared from cells of Escherichia coli strain Q13 exposed to elevated temperatures (from 37°C to 42°C) for 30 min. In a cell-free system with HS-S30 extract, the synthesized CAT protein had higher specific activity than that synthesized by a cell-free system with S30 extract prepared from Q13 cells incubated at 37°C. SDS-PAGE analysis showed that the heat-shock proteins, GroEL and DnaK, which are known to be molecular chaperones, were significantly increased in the HS-S30 extract. The addition of GroEL or DnaK to the S30 extract system increased the specific activity of the synthesized CAT protein. Heat-shock induction thus offers an effective method of modifying E. coli cell extracts.

Continuous protein synthesis system with Escherichia coli S30 extract containing endogenous T7 RNA polymerase

SummaryA modified continuous-flow, cell-free (CFCF), coupled transcription and translation system was constructed using an Escherichia coli S30 extract containing endogenous T7 RNA polymerase (S30 extract-T7pol), which was prepared from IPTG induced E. coli BL21(DE3) cells. Though the plasmid pTVCT, which harbored both a bla gene and a chloramphenicol acetyl transferase (CAT) encoding gene under the control of a T7 promoter, was used as a template, only CAT protein was continuously produced by this CFCF-coupled system .

Utilization of copper ion as a ribonuclease inhibitor in a cell-free protein synthesis system

Abstract The degradation of mRNA in a cell-free coupled transcription and translation system derived from Escherichia coli was prevented by the addition of 0.5 mM copper ion. Neither the transcription nor the translation was influenced by the presence of copper ion at this concentration, whereas the transcription was inhibited by a copper ion concentration exceeding 1.0 mM, and the translation by a concentration of 1.0 mM or more. In the cell-free system conducted with mRNA as a template, the amount of protein synthesized increased markedly in the presence of 0.5 mM copper ion. These results indicate that copper ion can stabilize cell-free protein synthesis systems by preventing mRNA degradation.

Improvement of Escherichia coli cell-free system by utilization of cell extract having additional property. Problems and countermeasures.

In theEscherichia coli cell-free system, the modification of cell extract can be achieved by preparation of the strains carrying additional property or those being induced with a certain gene expression prior to harvesting. In this study, we analyzed the cell-free system with S30 extract containing T7 RNA polymerases (S30 extract-T7pol) prepared from E.coli BL21(DE3) strain, which includes T7 RNA polymerase from extrinsic genes by IPTG induction, as a model for the improvement of the cell-free system. The fact that a significant degree of mRNA degradation was observed in the cell-free system with S30 extract-T7pol indicates the increase of ribonuclease activity was an unfavorable influence derived from the cell-extract modification process. We also showed that this influence was settled by the addition of an effective ribonuclease inhibitor, such as copper (II) ion, to the reaction mixture.

Biobleaching of hardwood kraft pulp with cellulasedeficient mutant from hyper ligninolytic fungus izu- 154

Protoplasts of the monokaryotic strain from hyperligninolytic fungus IZU-154 were treated with UV irradiation, and the regenerants were screened for their inability to degrade Walseth cellulose (WC) and carboxymetylcellulose (CMC) on an agar plate. From 2700 regenerants, cellulase deficient and noncellulase mutants were isolated and designated Cel-139 and Cel-145, respectively. Cel-139 showed 15% Avicelase, 60% CMCase, and 10% β-glucosidase activities; however, it retained the same level of lignin degrading capability and manganese peroxidase (MnP) production when compared with wild-type IZU-154. On the other hand, Cel-145 showed deterioration in lignin-degrading capability, despite a substantial level of MnP production. Furthermore, biobleaching of hardwood kraft pulp (HWKP) by wild-type IZU-154 and Cel-139 in the solidstate fermentation system, without supplemental nutrients, were investigated. After 5 d of treatments with both fungi, pulp brightness increased from 33 to 65% ISO brightness, and Kappa number decreased from 13.9 to 6.0. However, yield loss of biobleached pulp was 20% lower with Cel-139 than with wild-type IZU-154.

The indicative culture parameters of manganese peroxidase production by white rot fungus IZU-154

The production of manganese peroxidase (MnP) by the hyper-ligninolytic fungus IZU-154 was performed by batch and filter-cultivation using a conventional stirred fermentor. A maximum MnP activity of 9.7 nkat/ml (nano katal/ml) was obtained after 44 h in the batch cultivation, and that of 8.1 nkat/ml was observed after 20 h in the filter-cultivation. Culture parameters, such as redox potential and pH, were investigated in the batch cultivation. We also investigated a novel parameter, the nonenzymatic 2,6-dimethoxyphenol oxidation (NEOX) level, which seems to reflect the amount of Mn(III) oxidized from Mn(II) by MnP. The swift rise of the redox potential, the synchronous gradual rise of pH, and the saturation of the NEOX level were significantly correlated to the MnP production. The production of MnP and the onset of the profiles of parameters were apparently in response to Mn(II) addition to the culture fluid. Furthermore, we investigated the content of organic acids during the MnP production in the filter-cultivation. Tartrate contained in the culture medium was consumed coincidentally at the onset of MnP production, and oxalate and formate were newly produced in the culture fluid at the same time.