Efficient pathway-driven scyllo-inositol production from myo-inositol using thermophilic cells and mesophilic inositol dehydrogenases: a novel strategy for pathway control.

Mesophilic enzymes, which are active at moderate temperatures, may dominate enzymatic reactions even in the presence of thermophilic crude enzymes. This study was conducted to investigate this hypothesis with mesophilic inositol dehydrogenases (IolG and IolX) produced in Geobacillus kaustophilus HTA426. To ensure the efficient production of mesophilic enzymes, we first screened for promoters induced at moderate temperatures using transcriptome analysis and identified four genes highly expressed at 30°C in the thermophile. We further characterized these promoters using fluorescent reporter assays to determine that the mti3 promoter could direct efficient gene expression at 40°C. We cloned the promoter into an Escherichia coli-Geobacillus shuttle plasmid and confirmed that the resulting vector functioned in G. kaustophilus and other thermophiles. We then used this vector for the cooperative expression of the iolG and iolX genes from Bacillus subtilis 168. G. kaustophilus cells carrying the expression vector were incubated at 60°C for cellular propagation and then at 40°C for the production of IolG and IolX. When the cells were permeabilized, IolG and IolX acted as catalysts to convert exogenous myo-inositol into scyllo-inositol at 30°C. In a scaled-up reaction, 10 g of myo-inositol was converted to 1.8 g of scyllo-inositol, which was further purified to yield 970 mg of pure powder. Notably, myo-inositol was degraded by intrinsic enzymes of G. kaustophilus at 60°C but not at 30°C, supporting our initial hypothesis. We indicate that this approach is useful for preparing enzyme cocktails without the need for purification. IMPORTANCE: Enzyme cocktails are commonly employed for cell-free chemical synthesis; however, their preparation involves cumbersome processes. This study affirms that mesophilic enzymes in thermophilic crude extracts can function as specific catalysts at moderate temperatures, akin to enzyme cocktails. The catalyst was prepared by permeabilizing cells without the need for concentration, extraction, or purification processes; hence, its preparation was considerably simpler compared with conventional methods for enzyme cocktails. This approach was employed to produce pure scyllo-inositol from an economical substrate. Notably, this marks the first large-scale preparation of pure scyllo-inositol, holding potential pharmaceutical significance as scyllo-inositol serves as a promising agent for certain diseases but is currently expensive. Moreover, this approach holds promise for application in pathway engineering within living cells. The envisioned pathway is designed without chromosomal modification and is simply regulated by switching culture temperatures. Consequently, this study introduces a novel platform for both whole-cell and cell-free synthetic systems.

Effects of egg yolk antibody against Porphyromonas gingivalis gingipains in periodontitis patients.

Porphyromonas gingivalis gingipains is suspected to be one of the most important causative agents of periodontitis. We postulated that the inhibition of gingipains may reduce the pathogenic nature of P. gingivalis. Anti-P. gingivalis egg yolk antibody (IgY-GP) was isolated from the yolks of hens immunized with purified gingipains. We applied IgY-GP gel subgingivally in periodontitis patients who harbored P. gingivalis in their subgingival flora. Five pairs of contralateral anterior single-rooted teeth were selected. One tooth in each contralateral pair was randomly treated with IgY-GP and subgingival scaling and root planing, whereas the other tooth was treated with SRP alone. The number of P. gingivalis bacteria was assessed by real-time PCR. Bacterial levels were expressed as the percentage of total bacteria. The IgY-GP group had a significant reduction in probing depth. BOP significantly decreased in the IgY-GP group compared to the control group at week 4. The levels of P. gingivalis significantly increased in the control group at week 4, whereas the reduction in the levels of P. gingivalis was sustained in the IgY-GP group. Within the limitations of the present study, IgY-GP was shown to be an effective immunotherapeutic agent in the treatment of periodontitis.

Effects of the enamel matrix derivative and beta-tricalcium phosphate on bone augmentation within a titanium cap in rabbit calvarium.

In vitro studies suggest that enamel matrix derivative (EMD) affects the early stages of osteogenic maturation by stimulating bone cell proliferation. In the present study, we evaluated the effects of EMD and beta-tricalcium phosphate (beta-TCP) on bone augmentation within a titanium cap in rabbit calvaria, using 14 adult male Japanese white rabbits. The calvarium was exposed, a circular groove prepared, the marrow penetrated, and a standard hemispherical titanium cap placed in the groove. The cap was filled with a mixture of beta-TCP and EMD at the experimental site, and was filled with beta-TCP alone at the control site. At 1 and 3 months after cap implantation, animals were euthanized, and histological sections prepared. The sections were stained with basic fuchsin and methylene blue, and were examined using light microscopy. At 1 month, EMD tended to increase the amount of bone, but there was no significant difference in the amount of new tissue and mineralized bone between the experimental and control sites. The present findings indicate that the present mixture of EMD and beta-TCP does not accelerate bone formation, compared with beta-TCP alone.

Detection of Streptococcus anginosus and 8-hydroxydeoxyguanosine in saliva.

Several studies have demonstrated a close association between Streptococcus (S.) anginosus infection and head and neck cancer. Accumulation of 8-hydroxy-deoxyguanosine (8-OHdG), which may result from the continuous generation of reactive oxygen species associated with chronic inflammation, has been reported in human preneoplastic lesions and in cancerous tissues. The purpose of the present investigation was to assess the salivary levels of S. anginosus and 8-OHdG in patients with periodontitis. Salivary levels of S. anginosus were measured by real-time PCR. S. anginosus was detected in 28 out of 38 (73.7%) of subjects. The 8-OHdG level was significantly higher in patients positive for S. anginosus than in patients negative for the bacterium. A significant decrease in S. anginosus and 8-OHdG levels was observed after initial periodontal treatment. Our findings indicate that, although the levels of S. anginosus are relatively low, there is a correlation between the salivary level of S. anginosus and 8-OHdG, and that periodontal treatment can decrease the levels of these hazard factors.