Expression of Trichoderma reesei β-Mannanase in Tobacco Chloroplasts and Its Utilization in Lignocellulosic Woody Biomass Hydrolysis

Lignocellulosic ethanol offers a promising alternative to conventional fossil fuels. One among the major limitations in the lignocellulosic biomass hydrolysis is unavailability of efficient and environmentally biomass degrading technologies. Plant-based production of these enzymes on large scale offers a cost-effective solution. Cellulases, hemicellulases including mannanases and other accessory enzymes are required for conversion of lignocellulosic biomass into fermentable sugars. β-mannanase catalyzes endo-hydrolysis of the mannan backbone, a major constituent of woody biomass. In this study, the man1 gene encoding β-mannanase was isolated from Trichoderma reesei and expressed via the chloroplast genome. PCR and Southern hybridization analysis confirmed site-specific transgene integration into the tobacco chloroplast genomes and homoplasmy. Transplastomic plants were fertile and set viable seeds. Germination of seeds in the selection medium showed inheritance of transgenes into the progeny without any Mendelian segregation. Expression of endo-β-mannanase for the first time in plants facilitated its characterization for use in enhanced lignocellulosic biomass hydrolysis. Gel diffusion assay for endo-β-mannanase showed the zone of clearance confirming functionality of chloroplast-derived mannanase. Endo-β-mannanase expression levels reached up to 25 units per gram of leaf (fresh weight). Chloroplast-derived mannanase had higher temperature stability (40°C to 70°C) and wider pH optima (pH 3.0 to 7.0) than E.coli enzyme extracts. Plant crude extracts showed 6–7 fold higher enzyme activity than E.coli extracts due to the formation of disulfide bonds in chloroplasts, thereby facilitating their direct utilization in enzyme cocktails without any purification. Chloroplast-derived mannanase when added to the enzyme cocktail containing a combination of different plant-derived enzymes yielded 20% more glucose equivalents from pinewood than the cocktail without mannanase. Our results demonstrate that chloroplast-derived mannanase is an important component of enzymatic cocktail for woody biomass hydrolysis and should provide a cost-effective solution for its diverse applications in the biofuel, paper, oil, pharmaceutical, coffee and detergent industries

Disruption of Yarrowia lipolytica TPS1 Gene Encoding Trehalose-6-P Synthase Does Not Affect Growth in Glucose but Impairs Growth at High Temperature

We have cloned the Yarrowia lipolytica TPS1 gene encoding trehalose-6-P synthase by complementation of the lack of growth in glucose of a Saccharomyces cerevisiae tps1 mutant. Disruption of YlTPS1 could only be achieved with a cassette placed in the 3′half of its coding region due to the overlap of its sequence with the promoter of the essential gene YlTFC1. The Yltps1 mutant grew in glucose although the Y. lipolytica hexokinase is extremely sensitive to inhibition by trehalose-6-P. The presence of a glucokinase, insensitive to trehalose-6-P, that constitutes about 80% of the glucose phosphorylating capacity during growth in glucose may account for the growth phenotype. Trehalose content was below 1 nmol/mg dry weight in Y. lipolytica, but it increased in strains expressing YlTPS1 under the control of the YlTEF1promoter or with a disruption of YALI0D15598 encoding a putative trehalase. mRNA levels of YlTPS1 were low and did not respond to thermal stresses, but that of YlTPS2 (YALI0D14476) and YlTPS3 (YALI0E31086) increased 4 and 6 times, repectively, by heat treatment. Disruption of YlTPS1 drastically slowed growth at 35°C. Homozygous Yltps1 diploids showed a decreased sporulation frequency that was ascribed to the low level of YALI0D20966 mRNA an homolog of the S. cerevisiae MCK1 which encodes a protein kinase that activates early meiotic gene expression

EVALUATION OF ANTIOXIDANT AND ANTIACETYLCHOLINESTERASE ACTIVITIES OF THE EXTRACTS OF PISTACIA ATLANTICA DESF. LEAVES

Pistacia atlantica Desf. (Anacardiaceae), a tree that is widely distributed in the Marmara, Mediterranean and Central Anatolia regions of Turkey, has been utilized traditionally for culinary and medicinal purposes. In the present study, methanolic and ethyl acetate extracts were prepared from young leaves of P. atlantica Desf., which were collected during the spring, and their antioxidant, scavenging, chelating and anti-acetylcholinesterase activities were evaluated. Both of the extracts showed strong antioxidant activity compared to synthetic antioxidants such as butylated hydroxyanisole, butylated hydroxytoluene and alpha-tocopherol whereas they exhibited relatively lower level of anti-acetylcholinesterase activity. The acetylcholinesterase inhibitory effect of the ethylacetate extract was higher than the inhibitory effect of the methanolic extract. Therefore, the extracts of the young leaves of P. atlantica Desf. might be a potential antioxidant supplement for food and pharmaceutical products. It might also be used to stabilize the foods against oxidative deterioration

Antioxidant activities of aqueous extracts of purslane (Portulaca oleracea subsp sativa L.)

The antioxidant activities of the infusion and aqueous extracts of purslane (Portulaca oleracea subsp. sativa L.) were investigated. Different antioxidant tests were employed, namely reducing power, total antioxidant activity, free radical scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, hydroxyl radical scavenging, metal chelating activities and P-carotene bleaching effect. In addition, the results were compared with natural and synthetic antioxidants, e.g. alpha-tocopherol, butylated hydroxytoluene and butylated hydroxyanisole. The levels of anthocyanins, total flavonoids and total phenolics of the extracts were also determined. Total phenolic and total flavonoid contents of the infusion were found to be higher than those of the aqueous extract. Infusion of purslane exhibited a strong Fe2+ chelating effect, free radical scavenging activity, hydrogen peroxide scavenging ability, and hydroxyl radical scavenging activity. Antioxidant activity of the aqueous extract increased with increased concentrations, in the range of 20-100 mu g/ mL. The aqueous extract also exhibited a strong superoxide radical scavenging activity. This study showed that purslane extracts exhibited antioxidant activity in all tests and the extracts could be considered as a source of natural antioxidants

Determination of chitinase activity in different plant samples of Turkey

The chitinase activity constitutively present in fruits, vegetables and cereal grains from various healthy plant species growing in Turkey have been determined. Crude extracts from 47 different fruit species, 35 kind vegetables and 4 various cereal grains were tested for chitinase activity using colloidal chitin as a substrat. In general, fruits seem to be the best sources of chitinase activity. The highest activity was found in persimmon fruits and grape berries

Effects of a combination of niacin and chromium (III)-chloride on the skin and lungs of hyperlipemic rats

The aim of this study was to investigate the biochemical effects of niacin and chromium(III)-chloride on serum lipid peroxidation, uric and sialic acids, and the extent of lipid peroxidation and glutathione levels in skin and lung tissues of hyperlipidemic rats

PROSTHETIC COMPLICATIONS IN IMPLANT SUPPORTED FIXED PROSTHESIS: A FOUR YEAR MULTI-CENTER RETROSPECTIVE ANALYSIS

There have been many materials and application methods developed in order to overcome the complications related with implant supported restorations. Prosthetic failures still could emerge in spite of all advanced applications. One of the most common reasons of a prosthetic failure was shown to be the excessive occlusal forces. Excessive occlusal forces may cause prosthetic complications or component fractures as well as with progressive crestal bone loss. The aim of this retrospective study was to evaluate the prosthetic complications of the implant supported fixed prosthesis followed up to four years. A Cox proportional hazard regression analysis was used to evaluate possible associations. Long bridges and single tooth crowns were associated with porcelain fracture (p<0.01, HR: 4.46) and cement loosening (p<0.05 HR: 3.22) respectively. Care must be given to occlusal alignment of the implant supported crowns/bridges to overcome long-term complications. Key Words: Implant prosthesis, prosthetic failures, retrospective analysis