An endoglucanase, GsCelA, from Geobacilus sp. undergoes an intriguing self- truncation process for enhancing activity and thermostability

An endoglucanase, GsCelA, was isolated and cloned from a thermophilic Geobacillus sp. 70PC53 grown in a rice straw compost in southern Taiwan. It was observed that highly purified GsCelA was able to self-truncate, removing a segment of 53 amino acid residues from its C-terminus. The purified GsCelA does not possess any protease activity and this self-truncation process is insensitive to standard protease inhibitors except EDTA and EGTA. This unique self-truncation process takes place at a temperature higher than 10C with an optimal pH between 6-7, and can be further enhanced with certain divalent ions such as Ca+2 and Mg+2. Crystal structure of GsCelA has a typical TIM-barrel configuration with 8 alpha-helices and 8 beta-strands, but with the presence of a divalent ion. Mutations of amino acids residues surrounding this metal ion do not affect the self-truncation process, but some of these mutants have enhanced enzymatic activities. Mutation of the cleavage site between K315 and G316 does not affect the self-truncation process. However, a deletion of ten amino acids near the cleavage site, i.e. from amino acid 310 to 320, slows down the truncation process but does not block it, and a truncated form around 315 amino acids in length eventually appears. This intriguing observation indicates that the self-truncation process is not site specific, but capable of measuring 315 amino acids from the N-terminus as the cleavage site. This self-truncation process also occurs in the native host of this enzyme, Geobacillus sp. 70PC53, with almost all secreted form of this enzyme being self-truncated. The 53 amino-acid-long C-terminal segment removed by this self-truncation process has binding affinity toward both crystal and amorphous cellulose as well as the s cell walls, yet its sequence bears no apparent homology to any known carbohydrate binding motifs. Various other mutation analyses and the structure-based recombination process, SCHEMA, have been carried out, and both the activity and thermostabilty of this enzyme are further improved. The truncated and improved GsCelA has almost twice the activity as the un-truncated form, and its thermostability is also further enhanced with T50 reaching 86C and TA50 higher than 100C, making this enzyme extremely useful in industrial processes carried out at high temperatures, such as the pre-treatment of cellulosic animal feeds during the final drying step. This research was supported by grants from Taiwan Ministry of Science and Technology and from Academia Sinica

(E)-2-(4-Diethyl­amino-2-hydroxy­benzyl­idene­amino)benzonitrile

The mol­ecule of the title compound, C18H19N3O, displays a trans configuration with respect to the C=N double bond. The dihedral angle between the planes of the two benzene rings is 2.62 (11)°. A strong intra­molecular O—H⋯N hydrogen bond stabilizes the mol­ecular conformation

Enhancement of activity and thermostability of a Geobacillus endoglucanase via a unique self-truncation process

The complete utilization of lignocellulosic biomass requires the hydrolysis of cellulose fibers via the synergistic action of three enzymes: exoglucanase, endoglucanase and beta-glucosidase. GsCelA is a 368-amino-acid endoglucanase secreted from a thermophilic Geobacillus sp. 70PC53 that was isolated form a rice straw compost in south Taiwan. GsCelA belongs to the glycosyl hydrolase family 5 and has a typical TIM barrel structure. This enzyme has excellent lignocellulolytic activity and high thermostability, with optimal temperature at 60℃ and pH at 5.0. The purified GsCelA is capable of carrying out a unique self-truncation process at temperature higher than 10 ℃ with optimal pH at 6-7. This self-truncation process is not due to the action of contaminating proteases and it can be suppressed by EDTA and EGTA, and enhanced by divalent metal ions. This self-truncation process also takes place in vivo in Geobacillus sp. 70PC53. The spontaneous or engineered C-terminal truncation up to 60 amino acids from the C-terminus improves GsCelA specific activity and renders the enzyme more thermostable. To investigate the importance of specific amino acids on the enzymatic activity of GsCelA, site-directed mutagenesis and protein engineering approach were employed to alter amino acid residues unique to this enzyme. It was demonstrated that point mutations Y195T , D55S, G288T and D289L replacements increase the activity of this enzyme by 30%

Rural tourism quality of services: fundamental contributive factors from tourists’ perceptions

Despite the importance of quality of services in the rural tourism sector, it is somewhat surprising that little study has been done to delve into the fundamental contributive factors that contribute to quality of services from the tourists’ perceptions. This is the first known study undertaken with a view to understand the fundamental contributive factors (i.e. climate change, carrying capacity, relaxation environment, and community support) from the environmental perspectives that link with quality of services in rural tourism destinations. A total of 400 questionnaires were distributed to the tourists who visited four study sites from Borneo Heights, Sarawak, and 215 were returned and used for analysis. To assess the developed model, SmartPLS 2.0 (M3) is applied based on path modelling and then bootstrapping. Interestingly, the findings revealed that tourists are more concerned about the changing of climate and relaxation environment at the destination. They also agreed that community support plays a significant role in the development of quality of services in the rural tourism. This study further discussed on the implications of the findings, limitations, and directions for future researc

Language policy and governmentality in businesses in Wales:a continuum of empowerment and regulation

In this paper, I examine how language policy acts as a means of both empowering the Welsh language and theminority language worker and as a means of exerting power over them. For this purpose, the study focuses on a particular site: private sector businesses in Wales. Therein, I trace two major discursive processes: first, the Welsh Government’s national language policy documents that promote corporate bilingualism and bilingual employees as value-added resources; second, the practice and discourse of company managers who sustain or appropriate such promotional discourses for creating and promoting their own organisational values. By drawing on concepts from governmentality, critical language policy and discourse studies, I show that promoting bilingualism in business is characterised by local and global governmentalities. These not only bring about critical shifts in valuing language as symbolic entities attached to ethnonational concerns or as promotional objects that bring material gain. Language governmentalities also appear to shape new forms of ‘languaging’ the minority language worker as selfgoverning, and yet, governed subjects who are ultimately made responsible for ‘owning’ Welsh

Glycosylation variants of a β-glucosidase secreted by a Taiwanese fungus, Chaetomella raphigera, exhibit variant-specific catalytic and biochemical properties

Cellulosic biomass is an abundant and promising energy source. To make cellulosic biofuels competitive against conventional fuels, conversion of rigid plant materials into sugars must become efficient and cost-effective. During cellulose degradation, cellulolytic enzymes generate cellobiose (β-(1→4)-glucose dimer) molecules, which in turn inhibit such enzymes by negative feedback. β-Glucosidases (BGLs) cleave cellobiose into glucose monomers, assisting overall cellulolytic activities. Therefore, BGLs are essential for efficient conversion of cellulosic biomass into biofuels, and it is important to characterize newly isolated BGLs for useful traits. Here, we report our discovery that the indigenous Taiwanese fungus Chaetomella raphigera strain D2 produces two molecular weight variants of a single BGL, D2-BGL (shortened to "D2"), which differ in O-glycosylation. The more extensively O-glycosylated form of native D2 (nD2L) has increased activity toward the natural substrate, cellobiose, compared to the less O-glycosylated form (nD2S). nD2L is more stable at 60°C, in acidic pH, and in the presence of the ionic detergent sodium dodecyl sulfate than nD2S. Furthermore, unlike nD2S, nD2L does not display substrate inhibition by an artificial substrate p-nitrophenyl glucopyranoside (pNPG), and the glucose feedback inhibition kinetics of nD2L is competitive (while it is non-competitive for nD2S), suggesting that these two glycovariants of D2 bind substrates differently. Interestingly, D2 produced in a heterologous system, Pichia pastoris, closely mimics properties of nD2S. Our studies suggest that O-glycosylation of D2 is important in determining its catalytic and biochemical properties

Effect of ICU Strain on Timing of Limitations in Life-Sustaining Therapy and Death

Purpose: The effect of capacity strain in an ICU on the timing of end-of-life decision making is unknown. We sought to determine how changes in strain impact timing of new DNR orders and of death. Methods: Retrospective cohort study of 9,891 patients dying in the hospital following an ICU stay ≥ 72 hours in Project IMPACT, 2001-2008. We examined the effect of ICU capacity strain (measured by standardized census, proportion of new admissions, and average patient acuity) on time to initiation of DNR orders and time to death for all ICU decedents using fixed-effects linear regression. Results: Increases in strain were associated with shorter time to DNR for patients with limitations in therapy (predicted time to DNR 6.11 days for highest versus 7.70 days for lowest quintile of acuity, p=0.02; 6.50 days for highest versus 7.77 days for lowest quintile of admissions, p<0.001), and shorter time to death (predicted time to death 7.64 days for highest versus 9.05 days for lowest quintile of admissions, p<0.001; 8.28 days for highest versus 9.06 days for lowest quintile of census, only in closed ICUs, p=0.006). Time to DNR order significantly mediated relationships between acuity and admissions and time to death, explaining the entire effect of acuity, and 65% of the effect of admissions. There was no association between strain and time to death for decedents without a limitation in therapy. Conclusions: Strains in ICU capacity are associated with end-of-life decision making, with shorter times to placement of DNR orders and death for patients admitted during high-strain days