Engineering lignocellulolytic enzymes for industrial applications: Engineering of laccase and endoglucanase

Energy consumption, especially electricity demand, is continually rising, while non-renewable fossil energy sources such as petroleum, natural gas, and coal are depleting. Fossil energy production also causes greenhouse gas emissions, which accelerate climate change. Efforts are being made to reduce reliance on these energy sources by increasing wind, water, and geothermal energy production. However, additional measures are still needed to fill the resulting energy gap. This shortfall can be addressed by biofuels, which can be produced from biomass. The world's most common biomass is lignocellulose, which consists of wood, forest residues, peat, straw, and agricultural biomass. Lignocellulose has a complex molecular structure, making it difficult to break down. After pretreatment, it is broken into its main components—cellulose, hemicellulose, and lignin—which are further broken down into molecular fractions or fully soluble molecules. These can then be processed to produce bio-based chemicals and products. This process is called lignocellulose valorization, where the biomass is catalytically "upgraded" into new raw materials or products. Waste streams from existing industrial processes using lignocellulose are also being repurposed more efficiently. For example, lignin waste from paper mills, previously burned for energy, is now used as raw material for new products. Enzymes are proteins that act as biological catalysts in chemical reactions. Using enzymes in industry can reduce energy and chemical consumption, making processes more sustainable, economically profitable, and environmentally friendly. Industrial processes are large-scale, so enzyme production must be as efficient as possible to keep production costs and industrial use affordable. Additionally, industrial processes require remarkably stable and resilient enzymes, especially under extreme heat and pH conditions. Enzyme engineering can improve production yield and enzyme properties to meet industrial needs. This work focused on developing two bacterial enzymes - laccase (from Bacillus wacoensis) and endoglucanase (from Spirochaeta thermophila) - and their further utilisation in industrial processes. Public databases were searched for gene sequences of enzymes that were expected to have the desired catalytic activity. These sequences were synthesised, cloned, and produced using the standard industrial microbe Escherichia coli for the first time. The enzymes were characterised to determine their specific activities and thermal and pH stability under different conditions. The laccase enzyme was stable in alkaline, high-pH conditions and retained its activity, but its soluble yield in E. coli was very low. The goal of enzyme engineering was to increase the solubility of laccase and further improve its activity. As a result of the engineering, both the yield and activity were significantly improved, allowing the enzyme to be utilised in lignin depolymerisation at high pH and to separate small molecular lignin fractions of different sizes. Thanks to the successful development process, the enzyme's industrial use became feasible, and it remains unique for its laccase properties in lignin depolymerisation. The second enzyme studied was endoglucanase, which had a good production yield in E. coli but was not stable enough at high temperatures for use in many industrial processes. Its engineering aimed to improve thermal stability and further increase activity. As a result of the engineering, its specific activity was significantly increased, and its substrate specificity was altered. However, no improvement in thermal stability was achieved. Endoglucanase is currently used as part of an enzyme cocktail for cellulosic fibre modification processes, but its engineered properties still need to be improved to replace existing alternative enzymes sustainably

European Sea Bass (Dicentrarchus labrax) immune status and disease resistance are impaired by arginine dietary supplementation

Infectious diseases and fish feeds management are probably the major expenses in the aquaculture business. Hence, it is a priority to define sustainable strategies which simultaneously avoid therapeutic procedures and reinforce fish immunity. Currently, one preferred approach is the use of immunostimulants which can be supplemented to the fish diets. Arginine is a versatile amino acid with important mechanisms closely related to the immune response. Aiming at finding out how arginine affects the innate immune status or improve disease resistance of European seabass (Dicentrarchus labrax) against vibriosis, fish were fed two arginine-supplemented diets (1% and 2% arginine supplementation). A third diet meeting arginine requirement level for seabass served as control diet. Following 15 or 29 days of feeding, fish were sampled for blood, spleen and gut to assess cell-mediated immune parameters and immune-related gene expression. At the same time, fish from each dietary group were challenged against Vibrio anguillarum and survival was monitored. Cell-mediated immune parameters such as the extracellular superoxide and nitric oxide decreased in fish fed arginine-supplemented diets. Interleukins and immune-cell marker transcripts were down-regulated by the highest supplementation level. Disease resistance data were in accordance with a generally depressed immune status, with increased susceptibility to vibriosis in fish fed arginine supplemented diets. Altogether, these results suggest a general inhibitory effect of arginine on the immune defences and disease resistance of European seabass. Still, further research will certainly clarify arginine immunomodulation pathways thereby allowing the validation of its potential as a prophylactic strategy.European Union's Seventh Framework Programme AQUAEXCEL (Aquaculture Infrastructures for Excellence in European Fish Research) [262336]; AQUAIMPROV [NORTE-07-0124-FEDER-000038]; North Portugal Regional Operational Programme (ON. 2 - O Novo Norte) , under the National Strategic Reference Framework, through the European Regional Development Fund; North Portugal Regional Operational Programme (ON. 2 - O Novo Norte), under the National Strategic Reference Framework through the COMPETE - Operational Competitiveness Programme; Fundacao para a Ciencia e Tecnologia; Fundacao para a Ciencia e Tecnologia [SFRH/BD/89457/2012, SFRH/BPD/77210/2011]; Generalitat Valenciana through the project REVIDPAQUA [ISIC/2012/003]; [PEst-C/MAR/LA0015/2013]; [UID/Multi/04423/2013]info:eu-repo/semantics/publishedVersio

Neurological symptoms and natural course of xeroderma pigmentosum

We have prospectively followed 16 Finnish xeroderma pigmentosum (XP) patients for up to 23 years. Seven patients were assigned by complementation analysis to the group XP-A, two patients to the XP-C group and one patient to the XP-G group. Six of the seven XP-A patients had the identical mutation (Arg228Ter) and the seventh patient had a different mutation (G283A). Further patients were assigned to complementation groups on the basis of their consanguinity to an XP patient with a known complementation group. The first sign of the disease in all the cases was severe sunburn with minimal sun exposure in early infancy. However, at the time the diagnosis was made in only two cases. The XP-A patients developed neurological and cognitive dysfunction in childhood. The neurological disease advanced in an orderly fashion through its successive stages, finally affecting the whole nervous system and leading to death before the age of 40 years. Dermatological and ocular damage of the XP-A patients tended to be limited. The two XP-C patients were neurologically and cognitively intact despite mild brain atrophy as seen by neuroimaging. The XP-G patients had sensorineural hearing loss, laryngeal dystonia and peripheral neuropathy. The XP-C patients had severe skin and ocular malignancies that first presented at pre-school age. They also showed immunosuppression in cell-mediated immunity. Neurological disease appears to be associated with the complementation group and the failure of fibroblasts to recover RNA synthesis following UV irradiation, but not necessarily to the severity of the dermatological symptoms, the hypersensitivity of fibroblasts to UVB killing or the susceptibility of keratinocytes to UVB-induced apoptosis

SMBOs: buying time or improving performance?

On the basis of an empirical analysis of 491 UK recent secondary management buyouts (SMBOs), we find strong evidence of a deterioration in long-run abnormal returns following SMBO deals. SMBOs also perform worse than primary buyouts in terms of profitability, labor productivity, and growth. We find no evidence for superior performance of private equity (PE) backed SMBOs, compared with their non-PE-backed counterparts. It appears that a PE firm's reputation and change in management are important determinants of improvements in profitability and labor productivity, respectively. High debt and high percentage of management equity tend to be associated with poor performance measured by profitability and labor productivity. Notably, none of the buyout mechanisms (i.e., financial, governance, operating) normally associated with performance improvements generate growth during the secondary buyout phase. The results are robust to the use of alternative performance measures, alternative benchmarks, and the possibility of sample selection bias. © 2013 The Authors. Managerial and Decision Economics published by John Wiley & Sons, Ltd

Short-term and one-year outcome of infective endocarditis in adult patients treated in a Finnish teaching hospital during 1980–2004

<p>Abstract</p> <p>Background</p> <p>Previous studies on factors predicting the prognosis of infective endocarditis have given somewhat conflicting results. Our aim was to define the factors predicting the outcome of patients treated in a Finnish teaching hospital.</p> <p>Methods</p> <p>A total of 326 episodes of infective endocarditis in 303 patients treated during 1980–2004 were evaluated for short-term and 1-year outcome and complications.</p> <p>Results</p> <p>Infection of 2 native valves and the occurrence of neurological complications, peripheral emboli, or heart failure significantly predicted both in-hospital and 1-year mortality, while age ≥65 years or the presence of a major criterion or vegetation on echocardiography predicted death within 1 year. A significant trend was observed between the level of serum C-reactive protein (CRP) on admission and both the short-term and 1-year outcome. In the patients who had CRP values ≥100 mg/l on admission, the hazard ratio for in-hospital death was 2.9-fold and the hazard ratio for 1-year death was 3.9-fold as compared to those with lower CRP values. Male sex and age < 64 years significantly predicted a need for both in-hospital and 1-year surgery, as did the development of heart failure or the presence of a major criterion or vegetation on echocardiography. Peripheral emboli were associated with a need for in-hospital surgery, while <it>Streptococcus pneumoniae </it>as the causative agent or infection of 2 native valves predicted a need for surgery within 1 year from admission.</p> <p>Conclusion</p> <p>Some of the factors (e.g. heart failure, neurological complications, peripheral emboli) predicting a poor prognosis and/or need for surgery were the same observed in previous studies. A new finding was that high CRP values (≥100 mg/l) on admission significantly predicted both short-term and 1-year mortality.</p

Gene–environment interactions in Leber hereditary optic neuropathy

Leber hereditary optic neuropathy (LHON) is a genetic disorder primarily due to mutations of mitochondrial DNA (mtDNA). Environmental factors are thought to precipitate the visual failure and explain the marked incomplete penetrance of LHON, but previous small studies have failed to confirm this to be the case. LHON has no treatment, so identifying environmental triggers is the key to disease prevention, whilst potentially revealing new mechanisms amenable to therapeutic manipulation. To address this issue, we conducted a large, multicentre epidemiological study of 196 affected and 206 unaffected carriers from 125 LHON pedigrees known to harbour one of the three primary pathogenic mtDNA mutations: m.3460G>A, m.11778G>A and m.14484T>C. A comprehensive history of exposure to smoking, alcohol and other putative environmental insults was collected using a structured questionnaire. We identified a strong and consistent association between visual loss and smoking, independent of gender and alcohol intake, leading to a clinical penetrance of 93% in men who smoked. There was a trend towards increased visual failure with alcohol, but only with a heavy intake. Based on these findings, asymptomatic carriers of a LHON mtDNA mutation should be strongly advised not to smoke and to moderate their alcohol intake

Measurement of complement receptor 1 on neutrophils in bacterial and viral pneumonia

BACKGROUND: A reliable prediction of the causative agent of community-acquired pneumonia (CAP) is not possible based on clinical features. Our aim was to test, whether the measurement of the expression of complement receptors or Fcγ receptors on neutrophils and monocytes would be a useful preliminary test to differentiate between bacterial and viral pneumonia. METHODS: Sixty-eight patients with CAP were studied prospectively. Thirteen patients had pneumococcal pneumonia; 13 patients, influenza A pneumonia; 5 patients, atypical pneumonia, and 37 patients, aetiologically undefined pneumonia. Leukocyte receptor expression was measured within 2 days of hospital admission. RESULTS: The mean expression of complement receptor 1 (CR1) on neutrophils was significantly higher in the patients with pneumococcal pneumonia than in those with influenza A pneumonia. The mean expression of CR1 was also significantly higher in aetiologically undefined pneumonia than in influenza A pneumonia, but there was no difference between pneumococcal and undefined pneumonia. CONCLUSION: Our results suggest that the expression of CR1 is higher in classical bacterial pneumonia than in viral pneumonia. Determination of the expression of CR1 may be of value as an additional rapid tool in the aetiological diagnosis, bacterial or viral infection, of CAP. These results are preliminary and more research is needed to assess the utility of this new method in the diagnostics of pneumonia