Mutation design of a thermophilic Rubisco based on three-dimensional structure enhances its activity at ambient temperature.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) plays a central role in carbon dioxide fixation on our planet. Rubisco from a hyperthermophilic archaeon Thermococcus kodakarensis (Tk-Rubisco) shows approximately twenty times the activity of spinach Rubisco at high temperature, but only one-eighth the activity at ambient temperature. We have tried to improve the activity of Tk-Rubisco at ambient temperature, and have successfully constructed several mutants which showed higher activities than the wild-type enzyme both in vitro and in vivo. Here, we designed new Tk-Rubisco mutants based on its three-dimensional structure and a sequence comparison of thermophilic and mesophilic plant Rubiscos. Four mutations were introduced to generate new mutants based on this strategy, and one of the four mutants, T289D, showed significantly improved activity compared to that of the wild-type enzyme. The crystal structure of the Tk-Rubisco T289D mutant suggested that the increase in activity was due to mechanisms distinct from those involved in the improvement in activity of Tk-Rubisco SP8, a mutant protein previously reported to show the highest activity at ambient temperature. Combining the mutations of T289D and SP8 successfully generated a mutant protein (SP8-T289D) with the highest activity to date both in vitro and in vivo. The improvement was particularly pronounced for the in vivo activity of SP8-T289D when introduced into the mesophilic, photosynthetic bacterium Rhodopseudomonas palustris, which resulted in a strain with nearly two-fold higher specific growth rates compared to that of a strain harboring the wild-type enzyme at ambient temperature. Proteins 2016; 84:1339-1346. © 2016 Wiley Periodicals, Inc.

Limitations of reuse for silver-palladium alloys -Evaluating post-recasting heat treatment impact on corrosion resistance.

The aim of this study was to investigate the effect of repeated casting and heat treatment on the corrosion resistance of a commercial Ag-Pd-Cu-Au alloy as evaluated by electrochemical techniques. After repeated casting, the fifth cast of the Ag-Pd-Cu-Au alloy exhibited dramatic degradation of properties, although upon heat treatment, this corrosion resistance did improve. Despite the improvement by heat treatment, after five castings, this alloy may not have satisfactory hardness for clinical use. These results of this study demonstrate that, up to the fourth cast and heat treatment, the Ag-Pd-Cu-Au alloy has acceptable corrosion resistance and hardness.

Effect of lime pretreatment of brown midrib sorghums.

The effect of lime pretreatment of brown midrib sorghums on enzymatic saccharification was investigated. Under most of the pretreatment conditions, the saccharification yields of bmrs were higher than those of the normal counterparts. This result suggests that bmr is useful to reduce pretreatment costs, because the amount of lime necessary for the pretreatment of biomass can reduced by using bmr mutants.

Use of whole crop sorghums as a raw material in consolidated bioprocessing bioethanol production using Flammulina velutipes.

The possibility of using two kinds of sorghum as raw materials in consolidated bioprocessing bioethanol production using Flammulina velutipes was investigated. Enzymatic saccharification of sweet sorghum was not as high as in brown mid-rib (bmr) mutated sorghum, but the amount of ethanol production was higher. Ethanol production from bmr mutated sorghum significantly increased when saccharification enzymes were added to the culture.

Evaluation of Two Sets of Sorghum Bagasse Samples as the Feedstock for Fermentable Sugar Recovery via the Calcium Capturing by Carbonation (CaCCO) Process.

Sorghum bagasse samples from two sets (n6 and bmr6; n18 and bmr18) of wild-type and corresponding "brown midrib" (bmr) mutant strains of sweet sorghum were evaluated as the feedstock for fermentable sugar recovery via the calcium capturing by carbonation (CaCCO) process, which involves Ca(OH)2 pretreatment of bagasse with subsequent neutralization with CO2 for enzymatic saccharification. Saccharification tests under various pretreatment conditions of the CaCCO process at different Ca(OH)2 concentrations, temperatures or residence periods indicated that bmr strains are more sensitive to the pretreatment than their counterparts are. It is expected that variant bmr6 is more suitable for glucose recovery than its wild-type counterpart because of the higher glucan content and better glucose recovery with less severe pretreatment. Meanwhile, bmr18showed higher scores of glucose recovery than its counterpart did, only at low pretreatment severity, and did not yield higher sugar recovery under the more severe conditions. The trend was similar to that of xylose recovery data from the two bmr strains. The advantages of bmr strains were also proven by means of simultaneous saccharification and fermentation of CaCCO-pretreated bagasse samples by pentose-fermenting yeast strain Candida shehatae Cs 4R. The amounts needed for production of 1 L of ethanol from n6, bmr6, n18, and bmr18samples were estimated as 4.11, 3.46, 4.03, and 3.95 kg, respectively. The bmr strains seem to have excellent compatibility with the CaCCO process for ethanol production, and it is expected that integrated research from the feedstock to bioprocess may result in breakthroughs for commercialization.

Effect of air-drying dentin surfaces on dentin bond strength of a solvent-free one-step adhesive.

The purpose was to evaluate the effect of air-drying dentin surfaces on the microtensile bond strength (µTBS) of a solvent-free onestep adhesive (Bond 1 SF). Twelve human molars were ground with 600-grit SiC paper. Before applying bonding agent, the dentin surface was rinsed with distilled water and blot-dried with tissue paper, followed by air-drying for 0, 3, 30, and 60 s using with a dental air syringe. After applying and curing Bond 1 SF, resin composite was incrementally built up. Specimens were then stored in distilled water for 24 h and then µTBSs were measured at a cross-head speed of 1 mm/min. Higher µTBS were observed when the dentin surface was air-dried for 3 s (33.2±6.8MPa)>0 s (26.7±4.5MPa)>30 s (22.6±5.5MPa)=60 s (20.4±5.0MPa). The results suggested that prolonged air-drying of the dentin surface removed water and decreased the bond strengths of Bond 1 SF.

Low temperature alkali pretreatment for improving enzymatic digestibility of sweet sorghum bagasse for ethanol production.

A low temperature alkali pretreatment method was proposed for improving the enzymatic hydrolysis efficiency of lignocellulosic biomass for ethanol production. The effects of the pretreatment on the composition, structure and enzymatic digestibility of sweet sorghum bagasse were investigated. The mechanisms involved in the digestibility improvement were discussed with regard to the major factors contributing to the biomass recalcitrance. The pretreatment caused slight glucan loss but significantly reduced the lignin and xylan contents of the bagasse. Changes in cellulose crystal structure occurred under certain treatment conditions. The pretreated bagasse exhibited greatly improved enzymatic digestibility, with 24-h glucan saccharification yield reaching as high as 98% using commercially available cellulase and ß-glucosidase. The digestibility improvement was largely attributed to the disruption of the lignin-carbohydrate matrix. The bagasse from a brown midrib (BMR) mutant was more susceptible to the pretreatment than a non-BMR variety tested, and consequently gave higher efficiency of enzymatic hydrolysis.