Effect of the single mutation N9Y on the catalytical properties of xylanase Xyn11A from Cellulomonas uda: a biochemical and molecular dynamic simulation analysis

Cellulomonas uda produces Xyn11A, moderately thermostable xylanase, with optimal activity at 50 °C and pH 6.5. An improvement in the biochemical properties of Xyn11A was achieved by site-directed mutagenesis approach. Wild-type xylanase, Xyn11A-WT, and its mutant Xyn11A-N9Y were expressed in Escherichia coli, and then both enzymes were purified and characterized. Xyn11A-N9Y displayed optimal activity at 60 °C and pH 7.5, an upward shift of 10 ºC in the optimum temperature, and an upward shift of one unit in optimum pH; also, it manifested an 11-fold increase in thermal stability at 60 ºC, compared to that displayed by Xyn11A-WT. Molecular dynamics (MD) simulations of Xyn11A-WT and Xyn11A-N9Y suggest the substitution N9Y leads to an array of secondary structure changes at the N-terminal end and an increase in the number of hydrogen bonds in Xyn11A-N9Y. Based on the significant improvements, Xyn11A-N9Y may be considered as a candidate for several biotechnological applications.

Monitoring growth of Cellulomonas uda in solid state fermentations with Brewers’ spent grain and optimization of the bioprocess

Brewers’ spent grain (BSG) is a low-value by-product of the brewing process, which is produced in large quantities every year. In this study, the lignocellulosic feedstock was used to run solid state fermentations with Cellulomonas uda. For aerobic processes, maximum cellulase activities of 0.98 nkat∙ml, maximum xylanase activities of 5.00 nkat∙ml and cell yields of 0.22 g∙g were achieved. Under anaerobic conditions, enzyme activities and cell yields were lower, but valuable liquid products (organic acids, ethanol) were produced with a yield of 0.41 g∙g. The growth phase of the organisms was monitored by measuring extracellular concentrations of two fluorophores pyridoxin (aerobic) and tryptophan (anaerobic) and by cell count. By applying reductive conditions to the anaerobic approach, the ratio of ethanol to acetate was increased from 1.08 to 1.59 mol∙mol. This ratio was further improved to 9.2 mol∙mol by lowering the pH from 7.4 to 5.0 without decreasing the final ethanol concentration. A scale up fermentation with 15w% BSG instead of 5w% BSG quadrupled the acetate concentration, whilst ethanol was removed by gas stripping. This study provides various ideas for optimizing and monitoring solid state fermentations, which can support feasibility and incorporation into holistic biorefining approaches in the future.

Treatment of Waste Paper Using Ultrasound and Sodium Hydroxide for Bioethanol Production

Bioethanol produced from lignocellulose feedstock is a renewable substitute to declining fossil fuels. Pretreatment using ultrasound assisted alkaline was investigated to enhance the enzyme digestibility of waste paper. The pretreatment was conducted over a wide range of conditions including waste paper concentrations of 1-5%, reaction time of 10-30 min and temperatures of 30-70°C. The optimum conditions were 4 % substrate loading with 25 min treatment time at 60°C where maximum reducing sugar obtained was 1.89 g/L. Hydrolysis process was conducted with a crude cellulolytic enzymes produced by Cellulomonas uda (PTCC 1259).The maximum amount of sugar released and hydrolysis efficiency were 20.92 g/L and 78.4 %, respectively. Sugars released from waste paper were fermented into bioethanol with Saccharomyces cerevisiae. The maximum concentration of bioethanol estimated was 9.5 g/L after 48h of cultivation, the yield and volumetric productivity were 0.454 g/g glucose and 0.2g bioethanol/ L h. respectively. This study of ultrasound and sodium hydroxide treatment may be (we think) it will be a promising technique to develop bioethanol production from waste paper.

Cellulomonas composti sp. nov., a cellulolytic bacterium isolated from cattle farm compost

A bacterial strain, TR7-06T, which has cellulase and β-glucosidase activities, was isolated from compost at a cattle farm near Daejeon, Republic of Korea. It was a Gram-positive, aerobic or facultatively anaerobic, non-motile, rod-shaped bacterium. Phylogenetic analysis based on 16S rRNA gene sequences showed that this strain belongs to the genus Cellulomonas, with highest sequence similarity to Cellulomonas uda DSM 20107T (98.5 %). Cell wall analysis revealed the presence of type A4β, l-orn–d-Glu peptidoglycan. The cell-wall sugars detected were mannose and glucose. The predominant menaquinone was MK-9(H4); MK-8(H4) was detected in smaller quantities. The major fatty acids were anteiso-C15 : 0, C16 : 0, C14 : 0 and C18 : 0. The polar lipids detected were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. The results of DNA–DNA hybridization and physiological and biochemical tests clearly demonstrated that TR7-06T represents a novel species. The combined genotypic and phenotypic data show that strain TR7-06T (=KCTC 19030T=NBRC 100758T) merits description as the type strain of a novel Cellulomonas species, Cellulomonas composti sp. nov.